Homozygous human C3 deficiency. The role of C3 in antibody production, C-1s-induced vasopermeability, and cobra venom-induced passive hemolysis. Studies of the family of a patient with marked deficiency of the third component of complement (C3) demonstrated that the patient was homozygous for a blank allele at the C3 locus, C3-. Metabolic studies with purified radiolabeled C3 in the patient revealed a mildly elevated fractional catabolic rate and a markedly reduced synthesis rate, consistent with a lack of C3 synthesis as the patient's primary defect. There was also a mild increase in the rate of conversion of purified C3 added to her serum and incubated at 37 degrees C in vitro. Major blood group-compatible erythrocytes from a patient with paroxysmal nocturnal hemoglobinuria had the same shortened survival in the C3-deficient patient as in a normal control. Although no leukocytosis developed in the patient in spontaneous infection by pyogenic organisms, there was a normal leukocytosis in response to the injection of thyphoid vaccine. The intradermal injection of C-1s, which produces a marked increase in vasopermeability in the skin of normal subjects, produced no definite change in the patient, possibly implicating C3 or a protein in the alternative pathway as the normal mediator of this response. The patient's serum exhibited near-normal immune adherence activity, confirming the lack of requirement of C3 for this function. C5 inactivation and passive hemolysis of unsensitized guinea pig erythrocytes occurred normally in C3-deficient serum on incubation with cobra venom factor, indicating that C3 is not required for these reactions. The patient's humoral antibody response to both protein and carbohydrate antigens was entirely normal, making it unlikely that C3 is required for antigen processing.


Maintenance of insulin release from pancreatic islets stored in the cold for up to 5 weeks. Insulin content and release were measured from hand-dissected pancreatic islets from noninbred ob/ob mice after 1-5 wk storage in tissue culture medium 199 at various temperatures and glucose concentrations. After storage of islets for 1 wk at 37 degrees, 22 degrees, or 8 degrees C in 18 mM glucose medium and preincubation with 1 mM glucose, glucose-stimulated insulin release during the subsequent incubation was only 20-35% of that of fresh islets. The addition of a 4-h period at 37 degrees C with 18 mM glucose between the cold storage and perincubation restored glucose-stimulated insulin release from 8 degrees C stored islets to fresh-islet levels. Release throughout the 1-18 mM glucose range was strikingly parallel to that of fresh islets. Exposure of fresh islets to the same 4-h period increased basal release but did not affect maximal release. When islets were stored at 8 degrees C with 18 mM glucose for more than 1 wk, a short period at 37 degrees C every week was necessary for maintenance of release. After 5 wk of this procedure, glucose-stimulated insulin release was one-third that of fresh islets, or similar to that of islets stored for only 1 wk at 37 degrees C. Storage at 8 degrees C for 1 wk with 3 mM glucose, or continuously for 3 or 5 wk with 18 mM glucose, maintained islet insulin content, whereas release was lost. Thus, glucose-stimulated insulin release is best maintained by storage of pancreatic islets in tissue culture medium with a high concentration of glucose at 8 degrees C with short weekly periods at 37 degrees C.


Isolation of chlamydia in irradiated and non-irradiated McCoy cells. Specimens from eye and genital tract were cultured in parallel in irradiated and non-irradiated McCoy cells and the frequency of isolation of chlamydia using these culture methods was compared. There was a significant difference between the frequencies of isolation; irradiated McCoy cells produced a greater number of positive results.


Laboratory contamination of blood cultures. A prospective study of the use of a laminar flow cabinet, an exhaust-ventilated safety hood, and the open bench for the microbiological examination of blood is described. Blood samples from 1600 patients were subcultured on the open bench, 2700 in a safety hood, and 2607 in a laminar flow cabinet. Use of the laminar flow cabinet produced a significantly greater level of contamination than the other methods, and it is concluded that the exhaust-ventilated safety hood should be used for this procedure.


Controlled clinical trial of oral and parenteral nefopam hydrochloride. A novel and potent analgesic drug. The results of a controlled, double-blind clinical trial are reported demonstrating the potency of analgesia produced by orally and parenterally administered nefopam HCl in hospitalized patients with pain principally of skeletal and neuromuscular origin. The drug is an analogue of orphenadrine, consisting of a cyclization of the diphenhydramine molecule. A double-blind, crossover study was made of the analgesic effects of intramuscular doses of 20 mg nefopam HCl, 50 mg pethidine, and saline placebo in 20 patients. Nefopam and pethidine were found to be equally effective and statistically superior to placebo. A double-blind, randomized study was made of orally administered nefopam HCl, 60 mg t.i.d., for three days and of placebo t.i.d. for three days in 80 patients. Nefopam was distinctly superior to placebo in analgesic effectiveness, both in the initial single dose and in maintaining therapeutic benefit for the duration of the three-day trial. It was concluded that nefopam is a potent analgesic of novel structure and unique physiologic properties.


A double-blind comparison of loxapine succinate and trifluoperazine in newly admitted schizophrenic patients. A study was conducted evaluating the efficacy of loxapine succinate in newly admitted schizophrenic patients through a four-week double-blind comparison with trifluoperazine. Twenty-four patients received between 40 and 80 mg loxapine succinate daily and 19 patients received between 20 and 50 mg trifluoperazine daily. The two groups showed comparable significant improvement on the BPRS and CGI. The discharge and termination rates of the two groups were not significantly different and the incidence and severity of side effects, most frequently extrapyramidal signs, were similar in both groups. Loxapine succinate was judged to be an effective treatment for newly admitted schizophrenic patients.


Rapid detoxification of the narcotic addict with naloxone hydrochloride. A preliminary report. Parenteral (0.2--2.0 mg) and oral (500 mg) doses of naloxone hydrochloride were administered to 29 parolees showing evidence of increasing opiate use while participating in an aftercare abstinence program. The naloxone was found to be capable of inducing withdrawal symptoms, the intensity of which being a function of the amount of naloxone administered and of the level of physical dependence. Some patients (38%) showed a "detoxification effect" characterized by a positive abstinence reaction to initial naloxone administrations but a negative reaction to subsequent administrations. All of the 29 subjects, however, returned to illicit heroin use within several days following their release from the treatment unit. the potential of naloxone as a rapid detoxification tool is discussed in counterpoint to the apparent lack of potential the procedure has as a means of attenuating opiate-seeking behavior.


A dual approach to the evaluation of the efficacy of a new rheumatoid arthritic agent--pirprofen. Pirprofen was compared to placebo in a double-blind crossover study in 12 rheumatoid arthritis patients. Two approaches--univariate and multivariate--were used to analyze the study results which were in the form of arithmetic changes from pretreatment levels of six efficacy measurements. The univariate analysis failed to permit a single decision to be made regarding the further investigation and use of pirprofen in rheumatoid arthritis. However, the multivariate analysis which treats the efficacy variables simultaneously showed a clear differentation from placebo. Thus, multivariate analysis enabled the clinical pharmacologist to evaluate the new therapeutic agent in a complete and comprehensive manner. It allowed for a single decision to be made regarding the merits of pirprofen compared to placebo.


The topographical distribution of S-100 and GFA proteins in the adult rat brain: an immunohistochemical study using horseradish peroxidase-labelled antibodies. The cytological and topographical distribution of S-100 and glial fibrillary acidic (GFA) proteins in the adult rat brain has been compared using the horseradish peroxidase-labelled antibody technique. Both proteins are present in astrocytes and structures composed of astrocytic processes, namely the glial limitans and the perivascular membranes, but the cytological localization varies between the two proteins. S-100 is found in the nucleus and cytoplasm whereas GFA protein is confined to the cytoplasm. Neither is found in neurons, but S-100 is present in some oligodendroglia, suggesting a general regulatory role in glia. Although GFA protein in present in both protoplasmic and fibrous astrocytes, it is more prominent in the latter, confirming its association with astrocytic filaments.


Metabolism and toxicity of cadmium, mercury, and lead in animals: a review. Cadmium, mercury, and lead are toxic to humans and animals. Although cadmium and inorganic mercury toxicities occur in humans, they have not been observed in domestic livestock under practical conditions. In contrast, cattle, especially young calves, are extremely susceptible to lead toxicity. Apparently, cattle are more tolerant of cadmium than are other animal species. Due partially to higher absorption and longer retention times in the body, the alkyl mercuries, especially methyl mercury, are more toxic than inorganic mercury compounds. Inorganic forms of cadmium, mercury, and lead are poorly absorbed from the intestine. However, due to lack of effective homeostasis, after absorption retention time is long. Injected cadmium, mercury, and lead are metabolized differently from that naturally absorbed. Most cadmium and mercury are in kidney and liver (50 and 23% of total body in goats); but highest total load of methyl mercury is in muscle (72% in cows). With low to moderate body burden, most lead is retained in the skeleton. However, beyond a certain point, the kidney accumulates large quantities. Only minute amounts of cadmium and mercury are secreted into milk, but milk is only moderately well protected from dietary lead. Likewise, little cadmium and inorganic mercury pass the placental barrier whereas lead and methyl mercury pass more readily.


Sodium cloxacillin for treatment of mastitis in lactating cows. Sodium cloxacillin was used to treat 107 cows affected with acute mastitis and 138 affected with subclinical mastitis. Based on the application of conservative bacteriologic criteria, the cure rate was 53% for acute staphylococci mastitis and 65% for subclinical staphylococci mastitis. The cure rate for Streptococcus agalactiae infections was 91% in acute conditions and 92% in subclinical conditions. The cure rate was 73% for the acute infections and 88% in cases of subclinical infections when Streptococcus spp. "non ag" was the cause. Daily milk production had no effect on response rate. No adverse reactions due to treatment were reported by any investigator.


A study of three preoperative sedative combinations. The use of drugs as premedication for child patients to be treated in the office may provide the dentist with an adequate means of controlling the patient otherwise incapable of cooperation. Drug dosages for children cannot be determined alone on the basis of weight and age, especially when dental treatment is to be provided. Alphaprodine and promethazine; chloral hydrate and hydroxyzine pamoate; pentobarbital, meperidine, and scopolamine are three groups of balanced medication that fulfill the needs in pedodontic office procedures. The Pedi-Wrap is an important factor in treatment and control of movements unknowingly exhibited by the patient from one to six years of age. Its use should be required for the very young child patient, to prevent falls and bodily injury and to allow the doctor to execute his treatment effectively.


Strain typing of oral streptococci by the use of bacterial antagonism. Strain-typing methods utilizing microbial antagonism have been developed for S sanguis and for S mutans. The inhibition observed resembled that of bacteriocins. Inhibition within the species S sanguis was obtained with group D streptococci and facultative members of the genus Bacillus. Thirty-three inhibitors were paired (not in all possible combinations) with 37 isolates of S sanguis. Depending on the inhibitor, growth of between 15 and 85% of the sensitive indicators was halted. Three of the inhibitors manifested identical patterns of inhibition. Of the 37 S sanguis isolates, 34 exhibited individual patterns of inhibition. The three exceptions could be differentiated by their antagonism against sensitive indicators of S mutans. Variations in susceptibility to antagonism produced by group D streptococci allowed each of ten strains of S mutans to be typed. Present work for typing the oral streptococci is focused on computer-assisted searches for minimal typing sets. S mutans is sensitive to inhibition by some members of the species S sanguis. From a group of 58 challengers, 44 inhibited at least one of ten strains of S mutans, and 11 strains of S sanguis were able to halt the growth of half or more of the sensitive indicators. These results are useful not only for strain typing but form the basis for in vivo competition studies.


Attitudes toward and use of pit and fissure sealants. The results indicate that attitudes toward and use of sealants are generally low. There are, however, some dentists who use sealants proportionately more than other dentists, these being less recent graduates, dentists who practice in the northeast, and those dentists who are oriented generally more toward preventive procedures. The reasons selected by dentists for not using pit-and-fissure sealants were surprising. Despite considerable published evidence to the contrary and the provisional acceptance of two by the American Dental Association, Council on Dental Materials and Devices, most dentists are not yet convinced of the effectiveness of the procedure in preventing caries. In addition, many dentists were concerned that the material did not last long in the mouth, although high retention rates for periods in excess of one to three years had been reported. Also, a great concern for the possible sealing in of decay seems unsubstantiated by either clinical experience or reports from studies designed to monitor the activity of bacteria sealed in pits and fissures of teeth. Conversely, dentists infrequently selected either the cost of equipment and material or the time required for the procedure as reasons for not using pit-and-fissure sealants. The demanding technical requirements for proper application of the sealant, however, especially the avoidance of any moisture contamination, does require skilled personnel and substantial chair time. These factors are important considerations in the determination of the cost-effectiveness of this procedure in comparison to other preventive procedures for pit-and-fissure caries. These inconsistencies indicate a problem in the transmission of knowledge regarding sealants. Increased use of sealants, then, seems to be dependent on improving communication regarding their function and value to the dental profession. Data on the effectiveness of sealants over periods of three or more years have recently been reported and additional evaluations of this procedure are being reported. It would be expected that reports published since this research during the winter of 1974 will have an effect on both attitudes toward and use of sealants.


Serological identification of Actinomyces using fluorescent antibody techniques. Species-specific FITC conjugated antiserum can be prepared for each of the five species of Actinomyces and for Arachnia propionica. These serums can be used for rapid and specific identification of pure or mixed cultures of the bacteria and for identification of organisms seen in direct smears of clinical material or in tissue sections. Two serotypes each of A bovis, A odontolyticus, A israelii, A viscosus, and A propionica have been established, and A naeslundii has been tentatively divided into four serotypes.


A comparison between stainless steel crowns and multisurface amalgams in primary molars. From the findings in this study, it appears that steel crowns for primary teeth are significantly superior to multisurface amalgam fillings, in terms of the percentage of teeth requiring subsequent care: 88.7 percent of the teeth initially restored with amalgam required follow-up treatment, and only 30.3 percent of the crowned teeth required such treatment. Because both types of restorations were studied for approximately the same length of time, one could not justly conclude that the crowns were superior in terms of longevity. Steel crowns may prove to be more economical for the patient than amalgam fillings, if follow-up visits are considered. The author plans to conduct a second study on the cost factor.


Preparation of antiserums for use in the fluorescent antibody identification of certain plaque bacteria. The interaction of many factors involved in FA antiserum production determines the quality of each antiserum or conjugate prepared. Performance and physicochemical characteristics of 58 FA antiserums to S mutans currently in use suggested that many of these conjugates could have been improved by using the methods and evaluation procedures described in this symposium. Deficiencies in the conjugates included low titers, incomplete fractionation, inadequately labeled antibody, fluorochromed albumin, free fluorescein and cross-reactions. Titers ranged from 1:1 up to 1:4,000. Titers for S mutans serotype c conjugates were uniformly low. S mutans serotype c conjugates were prepared from antiserums produced using a modified immunization schedule. The schedule used both viable and killed whole cells and gave FA titers as high as 1:1,000 (adjusted to 10 mg protein/ml). Procedures presented in this paper and the report by Pittman and co-workers27 should permit direct FA serum titers of up to 1:1,000 for each of the recognized serotypes of S mutans. The availability of highly specific antiserums with adequate titers will advance the use of the FA technique to identify microorganisms directly in specimens, such as dental plaque.29,30 This technique could be used to study such phenomena as the transmission of microorganisms from person to person and the establishment of the oral flora. It could also be used in epidemiological studies and perhaps to monitor the effect of a therapeutic agent on microbial composition of dental plaque.


Computer content analysis of applied biological knowledge in dentistry. The study suggests that the Inquirer II System used by computers in content analysis of (textual) specific written material has value for longitudinal studies. The research findings indicate that the application of the computer in content analysis requires considerable effort in the preparation of materials and specifications of directions to the computer; standardizes data analysis, thus reducing subjective error in replication of studies; reduced measurement error in longitudinal studies, giving greater stability and power to statistical comparison; classified data as it is collected, quickly and reliably, while tabulating statistical information; reduced costs compared to human coders when used with large amounts of data; and competes effectively with human coders in terms of reliability and validity.


The ecology of the prosthodontic problem. Denture failures are a constant reminder that mechanical fit and good occlusion are not necessarily the only factors that contribute to denture success. Although diet and nutrition represent one of the significant factors in determining the resistance or susceptibility of oral tissues to the denture and its trauma, they receive little attention in the average dental office. This is true notwithstanding the fact that many published clinical observations attest to the importance of diet and nutrition in the assurance of denture success. A cursory review of the literature, however, revealed only two controlled studies dealing with nutrition and denture success or failure. These reports suggest that protein, trace minerals, and the reduction in refined carbohydrate intake can be used to increase denture tolerance. Such an effect increases the chance of denture success and decreases the chance of denture failure.


Bacteriological evaluation of a mobile laminar cross-flow unit for surgery, under laboratory circumstances. A mobile laminar cross-flow unit for surgery has been evaluated by the use of an experimental bacterial aerosol of Escherichia coli in different concentrations, generated at several different sites. A separate ventilation system, mounted underneath the table, produced an almost downward directed curtain of sterile air along both sides of the table. When the velocity of the air stream, discharged by the cross-flow unit, was adjusted at 0-50 m./sec. at 2 m. from the filter face (at the head end of the operation table), the surface of the table could be maintained free of bacterial settling even after aerosolization of heavily concentrated suspensions of 10(8) bacteria/ml. at different sites outside the enclosure and underneath the table as well.


Specific immunoglobulins in infants with the congenital rubella syndrome. The indirect immunofluorescent technique has been used to detect and titrate the specific immunoglobulins in serum specimens from 154 infants with confirmed or suspected congenital rubella. IgM antibody was stained more efficiently in sucrose density gradient fractions than in whole serum and was detected in this way in 27 out of 40 patients with confirmed congenital rubella at ages ranging from birth to 2 years. It was present in 48 out of 50 serum specimens during the first 6 months of life and in 11 out of 38 specimens obtained at ages between 6 1/2 months and 2 years. IgM antibody was therefore estimated to persist for about 6 months in the majority of cases and up to 2 years in a few individuals. IgM antibody was also detected by this method in 11 out of 114 infants with suspected but unconfirmed congenital rubella at ages up to 5 months. The total concentrations of IgM were above the normal range in nearly all sera taken from confirmed cases during the first 3 months of life and in half the specimens obtained between the ages of 3 and 6 months. IgG antibody was detected by fluorescent staining of whole serum in all patients with congenital rubella. Geometric mean titres increased during the first 3 months of life and then declined slowly. IgA antibody was not detected, except in two patients in whom traces were present at the age of 6 months, and the total concentrations of IgA were usually within normal limits. Fluorescent staining of fractions showed that the sedimentation characteristics of rubella IgG and IgM antibodies were the same in infants as in adults. The peak IgM fractions never contained IgG antibody, and the presence of specific IgM in these fractions could usually  have been safely inferred from their HAI titres. Fluorescent staining, however, was more sensitive and frequently detected IgM antibody in fractions which had no definite HAI activity. Fluorescent staining of whole serum for IgM antibody was less distinct, and often unsuccessful, even in specimens in which specific IgM was detected in the fractions. The addition of IgG- to IgM-containing fractions caused depression of IgM staining and suggested that failure to detect IgM antibody in whole serum was partly due to competitive inhibition by specific IgG.


Survival of Salmonella east bourne and Salmonella typhimurium in chocolate. Experiments were carried out to assess the reduction rate of two salmonella strains (S. eastbourne and S. typhimurium) in chocolate bars. After artificial contamination of chocolate, after 'conching', with about 10(6) S. eastbourne/g. this organism was still recovered after 9 months storage. The strain of S. typhimurium was less resistant. Both serotypes died off more rapidly in bitter chocolate than in milk chocolate. After contamination with a smaller dose (about 10(3)/g.) with these two serotypes, similar differences were observed.


The preparation of simulated water samples for the purpose of bacteriological quality control. As part of the bacteriological quality control programme of the Public Health Laboratory Service, we were asked to investigate the possibility of providing simulated water samples for distribution to the laboratories. For this purpose it was necessary to find some means whereby suspensions of coliform organisms and Escherichia coli could be kept relatively stable in number at room temperature for a period of 7-10 days. This, it was finally found, was best achieved by adding selected strains of the organisms to improved formate lactose glutamate medium (Gray, 1964) without the lactose but with added boric acid to a final concentration of 1-8%. The procedures adopted in the successful quality control programme are described.


Studies on the estimation of the hygienic condition of frozen broiler chickens. Various sampling techniques to determine the aerobic, E. coli and Enterobacteriaceae counts and to detect the presence of salmonellas were compared. As a simple method for the detection of salmonellas a modified Surkeiwicz procedure using both drip and rinse water is advocated. To evaluate hygiene during processing, determination of the number of Enterobacteriaceae in pieces of skin from the ventral, lateral and breast region is preferred.


Observations on environmental contamination in a microbiological laboratory. Contamination of a laboratory environment with pathogenic or non-pathogenic micro-organisms may be relevant to safety of technicians and quality of technical performance. Two widely separated incidents in 1968 and 1974 initiated a study of aspects of the laboratory environment. Water-baths, water of syneresis and portions of salmonella cultures spurting out of the sterilizing flame were examined. The water of water-baths was shown to be contaminated from the fluid cultures incubated in them. This raised questions of potential cross-contamination and reporting of false positives. Water of syneresis was sometimes contaminated with salmonellas. A few quantitative counts were made. The range of counts varied between 16 salmonellas per ml. and 13,000,000 salmonellas per ml. Five hundred portions of salmonella cultures and 571 portions of Shigella sonnei cultures which had spurted from the sterilizing flame were examined. All these samples failed to grow salmonellas or shigellas. Precautions necessary to avoid environmental contamination are briefly discussed.


The virulence of trimethoprim-resistant thymine-requiring strains of Salmonella. A thymine-requiring (thy-), trimethoprim-resistant (tmpr) mutant isolated from the faeces of chickens experimentally infected with salmonella typhimurium and treated with mixture of trimethoprim and sulphadiazine was less virulent for chickens than the parent strain and a thy+tmps revertant prepared in vitro from the mutant. The difference in chicken-virulence was more noticeable when the strains were administered orally than when they were administered subcutaneously. All tmpr mutants prepared in vitro from four other salmonella strains were also thy-; those tested were less virulent for chickens and mice than their parent strains. After oral infection, thy- salmonella organisms were found much less commonly in the alimentary tract of chickens then were thy+ organisms. This was especially so in the caeca, the principal site of colonization of both the thy+ and thy- organisms. Relatiely high concentrations of thymine or related compounds were found in the contents of all regions of the alimentary tract of chickens except the caeca; the caeca usually contained low or undetectable concentrations. The thy- salmonella strains would not grow on one brand of briliant green agar because of its deficiency in thymine; their colonial and appearance on other kinds of media used for isolating salmonellae from clinical material was often 'un-salmonella-like'.


An eosinophil chemotactic factor present in blister fluids of bullous pemphigoid patients. Tissue eosinophilia is often found in the inflammatory lesions of bullous perphigoid. A study made on naturally occurring eosinophil chemotactic activity in the blister fluids of four bullous pemphigoid patients revealed the existence of this activity in all of them. Sephadex G-25 column chromatography showed that the greater part of this eosinophil chemotactic activity was composed of low molecular substance of which the weight was close to that of vitamin B12 (m.w. 1357). The blister fluids and the sera of these patients contained elevated levels of IgE. An IgE anti-skin basement, membrane antibody was found in two if the four sera, and deposits of IgE were detected along the basement membrane zone of the involved skin in one of the patients. On the basis of these findings, we have reason to believe that an eosinophil chemotactic factor of anaphylaxis (ECF-A) participates in the accumulation of eosinophils in the lesions of bullous pemphigoid.


Partial characterization of murine migration inhibitory factor (MIF). These studies describe the production of murine migration inhibitory factor (MIF)3 in sufficient quantities to allow  its partial characterization by physiochemical and enzymatic methods. MIF was obtained from murine spleen cell cultures (C57BL/6 strain) stimulated with concanavalin A (Con A). Characterization of murine MIF was performed using Sephadex G-100 gel chromatography, isopycnic centrifugation in a CsCl density gradient, polyacrylamide disc electrophoresis, heat stability, and enzymatic treatment. MIF-containing and control fractions were assayed on normal C57BL/6 peritoneal exudate cells by using a microcapillary tube assay. Peak MIF activity was found in a Sephadex G-100 fraction containing molecules the size of albumin and slightly smaller, molecular weight 67,000 to 48,000. Murine MIF was stable to heating at 56 degrees C for 30 min but lost its activity at 80 degrees C for 30 min. Incubation of G-100 fractions containing MIF with water insoluble chymotrypsin destroyed the activity of MIF, indicating its protein nature. CsCl density gradient centrifugation revealed that murine MIF had a buoyand density greater than protein, consistent with its being a glycoprotein. Further, when subjected to disc electrophoresis on polyacylamide gels, murine MIF migrated in a region cathodal to albumin. Thus, mitogen stimulation of murine spleen cells produced MIF in quantities which allowed its partial characterization and purification, and its comparison with human and guinea pig MIF; this makes it feasible to analyze the role of murine MIF in cellular immunity and in its relationship to lymphocyte mediators which regulate humoral immune responses.


Age-dependent changes in sensitivity to antigen: its relationship with the antigen processing system. The relationship between the antigen processing or accessory cell system and the maturation, with age, of antibody-producing capability was investigated in the mouse. This was done by analyzing the antibody responses given by immunocompetent cells from neonatal and from adult male mice in an identical antigen-processing system environment. Specifically, 4 x 10(7) normal spleen cells from either 12-day-old or adult mice were challenged with varying numbers of SRC in adult irradiated syngeneic recipients. The subsequent IgM and IgG2a PFC responses for both age groups were analyzed in terms of antigen dose-antibody response curves. Analyses of these curves indicate: 1) the dose of antigen required to elicit the optimal antibody response is essentially identical for both age groups and (2) the bandwidths obtained using neonatal donors are significantly narrower than those obtained with adult donors. Whereas, intact neonatal and adult mice exhibit differences in antigen optimal doses, these differences are eliminated when the immunocompetent cells are stimulated in the presence of identical antigen-processing systems. It is concluded that maturation of the antigen-processing system results in an increased sensitivity to antigen. Examination of the bandwidths of the dose-response curves revealed that immunocompetent cells from the young mice, either in situ or in adult irradiated recipients exhibited narrower bandwidths than did their adult counter parts. Thus, the increase in bandwidth observed with age is attributed to changes in the population of immunocompetent cells--perhaps a reflection of increased diversity, and is not due to the antigen-processing system. Quantitation of the antigen-processing function using accessory cell-depleted and partially restored mice indicated that when IgG responses were compared a higher frequency of accessory cells was demonstrated in adultspleens as opposed to neonatal spleens.


Antibody responses of mice to alkaline detoxifield lipopolysaccharide. The antibody responses of outbred normal mice and nude mice injected with alkaline detoxified lipopolysaccharide (Alk-LPS) were measured. In some cases the antibody against lipopolysaccharide (LPS) and native protoplasmic polysaccharide (NPP). The kinetics of the primary responses to Alk-LPS and NPP were similar, whereas LPS stimulated a more rapid appearance of antibodies in the primary responses. Alk-LPS stimulated only primary antibody responses in both types of mice and sensitized nude mice for secondary responses which could be triggered with LPS. However, secondary antibody responses could not be triggered in normal mice primed with Alk-LPS. These data suggested that, on a functional basis, Alk-LPS possessed the specific antigenic signal associated with LPS antigens but lack the second nonspecific mitogenic signal dependent on the lipid A portion of LPS.


The effect of heat-killed streptococci on the survival of heart grafts in inbred strains of mice. Studies with inbred strains of mice revealed that exposure to type 12, Group A, beta-hemolytic streptococci affects the host response to heart grafts implanted in the ear. Intraperitoneal injection of streptococci 10 days before grafting led to curtailed survival of syngrafts without altering the normal rejection time of allografts. Similar sensitization, combined with local injection of streptococci into the graft site at the time of grafting, was followed by rapid rejection of both syngrafts and allografts. The time interval between exposure and grafting was critical. Injection with streptococci 5 days before grafting led to a prolonged survival of allografts and no demonstrable effect on syngrafts. In contrast, injection of streptococci 15 days before grafting did not alter survival of either type of graft. The data indicate heart grafts implanted in the ear may serve as a useful model for the study of the host responses to streptococcal antigens.


Acute and chronic infection of human lymphoblastoid cell lines with measles virus. Several human continuous lymphoblastoid cell lines (LCL) having T or B characteristics were infected with low and high passage strains of measles virus. All of the cell lines were susceptible to one or the other or to both strains of measles virus with the production of typical syncytial giant cells and released cell-free infectious virus into the supernatant medium. There was no consistent pattern of susceptibility of LCL with either T or B characteristics to infection by measles virus. Viral induced cytolysis of the lymphoblastoid cells in many of the lines was marked, but in the LCL that could be maintained over longer periods of time, a state of chronic, less cytolytic and persistent infection could be established. The infection was characterized by the production of moderate amounts of cell-free infectious virus for up to 4 1/2 months after initial infection with little change in the number of viable cells in culture. Long-term low multiplicity of infection (MOI) experiments demonstrated that the cell-free infectious virus was being produced only by a small number of cells, but the majority of cells in culture contained measles antigen that was in a cell-restricted, noninfectious, or defective form. Electron microscopic examination of the chronically infected cells demonstrated that many of them contained aggregates of hollow tubular intranuclear nucleocapsids whose "stripped" appearance was in marked contrast to the larger granular intracytoplasmic nucleocapsids found during earlier stages of infection. It is theorized that the persistent infection of LCL may serve as a model in understanding the immune mechanisms which permit latent and chronic measles infection in man.


A microplate culture method for assay of guinea pig mitogenic factor. An improved method for assay of guinea pig mitogenic factor (MF) is described. The technique is based on the use of thymocytes in microplate cultures and yields significant savings in time, labour and materials. The quantitative potential of the assay is examined using antigen- and concanavalin A-indiced MF, and its application to assessing in vitro immune responsiveness of individual guinea pigs is explored.


Antibodies ot double-stranded RNA: specificity and serum nucleases. An antiserum was prepared in rabbits to the synthetic double-stranded ribonucleic acid (ds RNA) poly rI:rC. Using a liquid-phase radioimmunoassay, the antiserum cross-reacted with a natural ds RNA isolated from the cytoplasmic polyhedrosis virus of the silkworm, binding 95% of the RNA at a 1 : 20 serum dilution. Preliminary tests of the specificity of the antiserum showed that it did not bind single-stranded RNA (ss RNA) or deoxyribonucleic acid (DNA), but also revealed that the serum contained an enzyme activity which degraded ss RNA into acid-insoluble fragments. It was therefore possible that the failure to bind ss RNA resulted from the degradation of the antigen rather than from an absence of cross-reacting antibodies. However, when the serum ribonuclease activity was inhibited by macaloid, the antiserum still did not bind the ss RNA antigen. This demonstrated that the antibodies to ds RNA did not cross-react with ss RNA. The existence of serum enzymes capable of degrading nucleic acid antigens emphasizes the need for caution in assessing the specificity of such antisera.


Dilatometry, a neglected method in immunological studies. Dilatometry has become a useful method for the study of proteins owing to its simplicity and accuracy. However, it has seldom been used in the study of immunoglobulins. Therefore possible applications of the method for that study are being discussed. A description is also given of the most common experimental set-up and procedure for dilatometric experiments. Finally, several papers describing the application of dilatometry to the study of immunoglobulins are reviewed.


Reproducibility of the immunofluorescent test for antimelanoma antibodies. The reproducibility of the indirect immunofluorescent test for antibodies to human malignant melanoma cytoplasmic antigens was investigated by testing panels of melanoma and normal sera against cells of each of several different melanomas. Tests were performed in replicate, read blindly by two observers, and repeated on different days. Different observers agreed in the interpretation of replicate assays in approximately 80 to 90% of cases. Variability increased considerably when assays were repeated on different days or when different in as few as 21% of tests. Thus, the results of the indirect immunofluorescent test for melanoma cytoplasmic antibodies must, at the present time, be interpreted with great caution.


Experimental cutaneous candidiasis in rodents. Epicutaneous application of viable Candida albicans or Candida stellatoidea under an occlusive dressing resulted in subcorneal microabscesses in newborn rats and mice. Neither heat-killed C. albicans nor occlusion alone resulted in epidermal micropustules. These results were dependent upon the number of organisms applied and the duration of application. Four additional species of Candida tested did not produce epidermal micropustules. The microscopic pathology of the abscesses revealed penetration of the epidermis by Candida pseudohyphae; the tips of the hyphal processes were seen within the pustules when sections were stained by the periodic acid-Schiff method. The resident flora of the newborn mouse or rat was not substantially altered by occlusion with or without C. albicans during the interval tested. The animal model of cutaneous candidiasis with permit greater manipulation and control in investigations of cutaneous candidiasis and the factors influencing neutrophil accumulation in the epidermis.


Epidermal cytoplasmic antibodies: incidence and type in normal persons and patients with melanoma. Antibodies to epidermal cytoplasmic antigens were present in 25 to 34% of 32 normal persons and in 34 to 50% of 53 patients with melanoma. There were at least two different types of cytoplasmic antibodies. The most common reacted to cytoplasmic antigens present only in the upper layers of the epidermis. This type of antibody occurred with equal frequency in patients with melanoma and in normal persons. The increased incidence of cytoplasmic antibodies in melanoma was due to antibodies to cytoplasmic antigens present throughout the epidermis. These antibodies were 2 1/2 times more common in patients with melanoma than in normal persons. The presence of different cytoplasmic antigens in distinct strata of the epidermis suggests they result from epidermal cell differentiation. The epidermal cytoplasmic antigens present throughout the epidermis appear to partially cross-react with cytoplasmic antigens in melanoma cells.


Pharmacokinetics of ticarcillin in patients with abnormal renal function. The pharmacokinetics of ticarcillin were determined in patients with abnormal renal function. In individuals with rates of creatinine clearance of greater than 60 ml per min, the half-life (+/- standard deviation) of ticarcillin was 71 +/- 6 min after intravenous administration. In patients with rates of creatinine clearance of 30-60 ml per min, 10-30 ml per min, and less than 10 ml per min, ticarcillin had a half-life of 3.0 +/- 0.6 hr, 8.5 +/- 2.1 hr, and 14.8 +/- 3.7 hr, respectively. Urinary concentrations of ticarcillin after intravenous administration were adequate at all levels of renal function. Ticarcillin was removed by hemodialysis with a reduction in half-life to 3.4 +/- 0.8 hr, but peritoneal dialysis was minimally effective in removing the drug. A program for the use of ticarcillin patients with renal insufficiency was outlined.


Delayed hypersensitivity to fungal antigens in mice. I. Use of the intradermal skin and footpad swelling tests as assays of active and passive sensitization. Mice were sensitized to Coccidioides immitis and Candida albicans antigens and tested for sensitivity by the intradermal and footpad swelling methods. In mice actively sensitized with killed antigen, antigen-specific intradermal and footpad induration responses occurred 24 and 48 hr after sensitization. Antigen-specific intradermal and footpad responses were transferred to normal mice with spleen cells from immune animals. Such responses were also transferred with normal spleen cells that had been incubated in vitro with immune RNA preparations. Histologic studies of intradermal reactions showed a mixed response of neutrophilic and mononuclear leukocytes, with slight vascular involvement compatible with delayed hypersensitivity. No intradermal or footpad responses were observed 4, 24, or 48 hr after injection in recipients of serum from actively sensitized mice. Histologic examination of skin sites in these mice revealed only a polymorphonuclear response. It is concluded that these intradermal and footpad responses are the result of delayed hypersensitivity and can be used as assays for this type of immunity in mice.


Genetics of resistance to infection with Salmonella typhimurium in mice. Eight strains of inbred mice fell into two sharply defined groups. Four strains (CBA, A/JAX, C3H/He, and DBA/2) were resistant (LD50, greater than 10(5)) to Salmonella typhimurium C5 given subcutaneously. The other four strains (Balb/c, C57BL, B10.D2 [new line], and DBA/1) were susceptible (LD50, less than 10). No intermediate resistance was seen. Examination of the F1, F2, and parental backcross generations bred from matings of CBA and Balb/c mice showed that resistance behaved as a simple Mendelian dominant. Resistance was not linked to H-2 genes, and no useful marker has yet been found. However, as previously demonstrated in the parent strains, resistance in the hybrids was related to the ability to produce a good delayed-type hypersensitivity reaction to an extract of S. typhimurium.


Enteropathogenic Escherichia coli: lack of correlation of serotype with pathogenicity. Forty-eight strains of Escherichia coli isolated from children with diarrhea were classified according to nine enteropathogenic serotypes. The strains were examined for production of enterotoxin and for invasiveness by study of bacteria or bacteria-free filtrates in conventional animal and tissue culture models. Filtrates of only three strains (6%) consistently dilated rabbit ileal loops, while all 48 strains yielded negative results in suckling mice, adrenal cells, and guinea pig eyes. When filtrates of the three strains that dilated the rabbit ileum were heated at 60 C for 30 min, the reaction in rabbit ileal loops was negative; this finding indicated the production of a heat-labile enterotoxin. This study shows the lack of correlation between classical enteropathogenic serotypes of E. coli and presently known virulence properties in animal models. The results raise doubts about the value of serotyping E. coli isolates from sporadic cases of diarrhea. When it is suspected that an E. coli isolate is enteropathogenic, it may be important to perform more than one laboratory assay.


Immunofluorescent localization of factor VIII-related antigen, fibrinogen, and several other plasma proteins in hemostatic plugs in humans. Factor VIII-related antigen and its low ionic strength subunits were demonstrated in endothelium and hemostatic plugs obtained from punch biopsies of bleeding time wounds according to Mielke. No IgG, IgA, IgM, C4-C3, albumin, or prothrombin was demonstrated in the cryostat sections of hemostatic plugs. Antifibrinogen stained the hemostatic plug and fibrin fibers along the edges of the skin wound. Antiplatelet actomyosin stained the hemostatic plug and the endothelium and pericytes of blood vessels. These results suggest that factor VIII is present in the hemostatic plug not by trapping of plasma but by close association with or presence within the blood platelets. The positive staining of a hemostatic plug in which most platelets have undergone the release reaction indicates that antifactor VIII may be useful for the detection of platelet thrombi in tissue of patients with suspected diffuse intravascular coagulation.


Genetic typing of alpha1-antitrypsin by immunofixation electrophoresis, identification of subtypes of Pi M. Prolonged electrophoresis in alkaline agarose gels, followed by immunofixation, is a valuable addition to acid starch gel electrophoresis and isoelectric focusing for the genetic phenotyping of alpha1-antitrypsin. This technique is helpful in clarifying certain variants and in ascertaining types in serum and amniotic fluid samples with secondary changes. In addition, heterogeneity may be detected within the variants found at pH 4.95, analogous to hemoglobin polymorphism. Two "new" variants, PiMLamb and PiMBaldwin, have been detected by a combination of immunofixation electrophoresis and acid starch gel electrophoresis.


Biological properties of a granuloma glycoprotein that inhibits macrophage phagocytosis. The biological activity of a purified glycoprotein inhibitor isolated from mature (42-day) polyvinyl sponge granulomas on macrophage phagocytosis was examined under a variety of in vitro conditions. Its activity was compared with partially purified inhibitor isolated from young (14-day) sponge granulomas. Experiments were conducted to define the mechanism of action of the inhibitor protein, and to differentiate it from other substances known to affect macrophage function. Inhibitor activity was demonstrated in neutral salt-soluble extracts from open wound granulation tissue, guinea pig and NZB/NZW mouse spleen, and acute-phase guinea pig serum. Fresh guinea pig serum and extracts of normal guinea pig tissues did not contain inhibitor material. Immunofluorescent studies demonstrated that the inhibitor was localized in or on a subpopulation of mononuclear cells within the sponge granuloma.


Host resistance to Serratia marcescens infection: serum bactericidal activity and phagocytosis by normal blood leukocytes. Serratia marcescens strains isolated from clinical specimens can be divided into those which are sensitive or resistant to the bactericidal activity of normal human serum. Serum bactericidal activity is heat labile, cation dependent, and is absorbable by whole, serum-sensitive Serratia or ethanol-insoluble extracts of these organisms. Bacteremic Serratia infection is invariably caused by the serum-resistant strains. Serum resistant Serratia are ingested and killed by normal human leukocytes and fresh normal serum. Heating or preabsorption of serum with whole, heat-killed, or ethanol-insoluble antigen extracts of the serum-resistant Serratia diminishes opsonization and phagocytosis. Serratia opsonins in the serum of healthy individuals are type-specific IgM globulins which combine with the organism and activate complement by the alternate pathway.


Salt-induced hypertension in rats with hereditary hydronephrosis: the effect of renomedullary transplantation. The antihypertensive action of renomedullary autotransplantation was investigated in rats with unilateral hereditary hydronephrosis showing extensive destruction of the medulla of the affected kidney. All rats were divided into three groups and unilaterally nephrectomized. The first group consisted of rats with a normal kidney remaining. The second and third groups had the hydronephrotic kidney remaining and received renomedullary and renocortical autotransplants, respectively. After completion of baseline studies, all rats were given 1 per cent saline solution instead of drinking water, and relevant parameters were re-examined 14 days later. Significant increase in blood pressure (greater than 150 mm. Hg), extracellular fluid volume, and plasma volume were found in group 3 while no changes were detected in groups 1 and 2. After administration of aspirin (and presumed blockade of prostaglandin synthesis) significant increases in plasma and extracellular fluid volumes were detected in groups 1 and 2, but no change in blood pressure was found. The results show that renomedullary transplantation protects against salt-induced hypertension and further indicate that the renomedullary prostaglandins are not a likely mediator of the antihypertensive action of the renal medulla.


Autoantigens in human sweat: purification and characterization of the D-group antigens. We have found several heteropolysaccharides in human thermal sweat which are both autoantigenic and isoantigenic for a majority of healthy adults. They are present in trace amounts and are difficult to isolate. We have facilitated extraction through the use of a DEAE-Sephadex batch technique. One of the major antigenic fractions, the D group, has been purified and characterized. It contains 88.5 per cent carbohydrate as uronic acid, hexosamine, methyl pentose, and glucose. The protein content was 12.5 per cent.


Direct quantitation of cholestanol in plasma by gas-liquid chromatography. A gas-liquid chromatographic (GLC) method for quantitation of cholestanol in plasma was developed using SP-1000 without the need for argentation thin-layer chromatography and silylation. Cholestanol was resolved well before cholesterol and the phytosterols. This method was sensitive, specific for cholestanol, and reproducible.


Systems analysis of computerized EKG processing center. A functional description of a computerized system for analysis of electrocardiograms (EKGs) is presented. Although it is recognized that the diagnostic accuracy of the program is the critical parameter in determining system acceptance, other system features such as quality control, turn around time and cost are shown to be of high importance. A digital simulation of alternative configurations for on-line data acquisition is presented. The simulation predicts the system throughout and wait times in different phases of a telephone EKG transmission for combinations of interfaces, telephone lines and acquisition procedures. These results should be helpful in selecting the appropriate configuration for each special situation.


Endocrine changes in male sexual deviants after treatment with anti-androgens, oestrogens or tranquillizers. The endocrine effects of drugs on two groups of 12 male sexual offenders in a special hospital were studied. In the first study benperidol, chlorpromazine and placebo were compared and in the second ethynyl oestradiol and cyproterone acetate were compared with no treatment. In the first study there was no difference between the three drugs in their effects on plasma testosterone or luteinizing hormone (LH). In the second study cyproterone acetate produced a reduction in plasma testosterone, LH and follicle-stimulating hormone (FSH). Ethynyl oestradiol produced a rise in plasma testosterone and LH, and no change in FSH. Neither drug changed total plasma oestrogen levels. The unexpected effects of ethynyl oestradiol were attributed to an increase in sex hormone-binding globulin (SHBG) leading to a rise in bound, inactive testosterone. Direct measurement showed a two- to threefold increase in SHBG with ethynyl oestradiol treatment and no change in SHBG with cyproterone acetate treatment. In spite of these contrasting endocrine effects, ethynyl oestradiol, cyproterone acetate and benperidol produced similar behavioural changes.


Effects of progesterone and synthetic luteinizing hormone releasing hormone on the release of luteinizing hormone during sexual maturation in the hen (Gallus domesticus). Single intramuscular injections of 0-5 mg progesterone/kg resulted in increased LH secretion in laying hens but not in pullets with completely undeveloped sexual organs. Injections of the steroid were first able to stimulate LH release 8-10 weeks before the onset of lay when the comb, ovary and oviduct had started to grow and basal plasma LH concentrations were beginning to rise. At this time, injecitons of 10 mug synthetic LH-RH/kg resulted in an incremental change in plasma LH levels of around 26 ng/ml. A similar incremental change was observed after giving the same dose of LH-RH to pullets with no signs of sexual development. Three to four weeks before the first eggs were laid, basal plasma LH levles started to fall, the pituitary became progressively more insensitive to synthetic LH-RH and injections of 0-5 mg progesterone/kg resulted in a reduced LH response. Ten mug LH-RH/kg caused incremental changes in blood levels of LH of less than 5 ng/ml. The final stage of sexual maturation occurred during the week before the onset of lay and was characterized by a rapid growth of large yolky ovarian follicles and a further fall in the sensitivity of the pituitary to synthetic LH-RH. However, injections of 0-5 mg progesterone/kg resulted in a prolonged release of LH. These observations are discussed in relation to the maturatio of the positive feedback mechanism by which progesterone stimulates the secretion of LH.


Potentiation by urethane and inhibition by pentobarbitone of oxytocin release in vitro. Isolated rat neural lobes were incubated in vitro in Locke's solution containing anaesthetic quantities of urethane, pentobarbitone or tribromoethanol. The oxytocin content of the incubation medium was estimated before, during and after stimulation of the tissue by raising the potassium chloride concentration from 5-6 to 56 mmol/l. Urethane (25 mmol/l) significantly potentiated oxytocin release (P less than 0-01) whereas tribromoethanol (0-5 mmol/l) had no obvious effect and pentobarbitone (0-4 mmol/l) significantly (P less than 0-01) inhibited its release. Reduction of the sodium chloride concentration in the medium potentiated the release of oxytocin in each case but did not alter its pattern. Urethane which increased secretion of oxytocin also increased calcium ion uptake by the neural lobes and pentobarbitone which decreased oxytocin release decreased calcium ion uptake. The results may explain why the blood concentration of the neurohypophysial hormones tends to be higher in rats anaesthetized with urethane than with tribromoethanol. Inhibition of hormone release by pentobarbitone suggests that this anaesthetic is unsuitable for use in studies of neurohypophysial hormone release. A partial explanation of the anaesthetic properties of urethane and pentobarbitone may also have been found if the release of neurotransmitter substances is influenced in a similar manner.


Genetical and biochemical aspects of resistance to p-fluorophenylalanine in Saccharomyces cerevisiae. Growth of haploid yeast strains was inhibited by the phenylalanine (PA) analogue DL-p-fluorophenylalanine (FPA) in yeast extract media containing 0-2 mg PA/ml. Most strains had a maximum FPA tolerance of about 0-25 mg/ml when glycerol was the carbon source and 0-5 mg/ml in in glucose medium. Spontaneous FPA-resistant mutants isolated on glucose medium showed little or no increase in FPA tolerance over that of the parent when metabolizing glycerol. Resistance was controlled by a different nuclear gene in each of four mutants analysed. In a proportion of the mutants the amount of FPA incorporated into cellular proteins in competition with PA was less than into the proteins of sensitive parental cells, whether glucose or glycerol was used as carbon source. This suggests that the mutational change allowed the cytoplasmic system to descriminate against the analogue without affecting its incorporation into mitochondrially-synthesized proteins. Although attempts to measure the latter were not made, the observed decrease in respiratory activity of cells grown in the presence of FPA suggests such incorporation. In other mutants showing resistance to FPA in glucose medium, the amount of FPA incorporated into cellular proteins varied with the carbon source, less analogue being incorporated in glucose medium than in glycerol medium.


The inhibitory action of fatty acids on the growth of Escherichia coli. The effect of fatty acids on Escherichia coli K12 was dependent on the source of the inoculum, the growth phase and the washing of the bacteria. The effects of saturated fatty acids from C4 to C16 and oleic acid at two concentrations (0-I and 0-4%, w/v) were determined on E. coli K12/154 growing exponentially in five different culture media. Depending on the media, 0-I % fatty acids increased the doubling times of the cultures by up to 96 %. Fatty acids of medium chain length (C6 to CII) at 0-4 % produced a decrease in cell concentration, nonanoic and decanoic acids being the most effective. A correlation was found between the decrease in cell concentration and the loss of viability of the culutres after addition of 0-4 % decanoic acid, with stationary-phase bacteria being affected more than those from exponential-phase cultures. Experiments carried out with E. coli B and C gave results similar to those obtained with E. coli K12/154.


Hybridization and selection for increased penicillin titre in wild-type isolates of Aspergillus nidulans. Repeated hybridization and selection among wild-type isolates produced strains of Aspergillus nidulans with increased penicillin titre. Four independent selection lines were established, each originating from a sexual cross between two different heterokaryon-incompatible wild-type isolates. In each generation, two selected high-titre sister strains were crossed to produce the next generation. An initial increase in titre was obtained in each line, but after four or five generations of selection the genetic variation was considerably reduced and the rate of response to selection had decreased. From a base population of wild-type isolates with a mean titre of 8-6 units/ml the progeny mean titre was raised to between 16 and 20 units/ml in each line. The gradual nature of the response suggests that a number of genes determine penicillin titre in the wild-type isolates used. The gene action throughout the selection programme was predominantly additive.


Hybridization and selection for increased penicillin titre in wile-type isolates of Aspergillus nidulans. Repeated hybridization and selection among wild-type isolates produced strains of Aspergillus nidulans with increased penicillin titre. Four independent selection lines were established, each originating from a sexual cross between two different heterokaryon-incompatible wild-type isolates. In each generation, two selected high-titre sister strains were crossed to produce the next generation. An initial increase in titre was obtained in each line, but after four or five generations of selection the genetic variation was considerably reduced and the rate of response to selection had decreased. From a base population of wild-type isolates with a mean titre of 8-6 units/ml the progeny mean titre was raised to between 16 and 20 units/ml in each line. The gradual nature of the response suggests that a number of genes determine penicillin titre in the wild-type isolates used. The gene action throughout the selection programme was predominantly additive.


The inheritance of penicillin titre in crosses between lines of Aspergillus nidulans selected for increased productivity. Selection for increased penicillin production among the progeny of pairwise crosses between wild-type isolates of Aspergillus nidulans resulted in the production of high-titre strains. Three crosses were made between strains with increased titre derived from independent selection lines. Significant genetic variation was found among the progeny of each cross, indicating that different genes for increased titre had been selected in each line. Gene action was additive in each cross. Renewed selection from the progeny of one 'between-line' cross resulted in further increases in titre, over three successive generations.


A timing control of cell division in Escherichia coli. The effect of heat treatment at 42degreeC on a thermosensitive division-defective strain of Escherichia coli K12, MAC1, has been studied under conditions which support a generation time of about 50 min. Synchronous cells gained simultaneously the ability to divide at 42degreeC and to divide in the presence of nalidixic acid or chloramphenicol, 20 min before physical separation of daughter cells. When synchronous cells of different ages (between 0 to 20 min after elution from an absorbent membrane) were subjected to a heat shock, division always took place 55 to 60 min after the shock. A similar treatment of an exponential culture resulted in synchronous cell division after a lag of 55 to 60 min during which no division occurred. Division is probably controlled for 40 to 45 min by the gene mutated in MAC1. Thus MAC1 cells of different ages appear to return to the same point of their division cycle when they are heated at 42degreeC. We propose that the gene mutated in MAC1 has a role in the timing control of E. coli cell division. Progress to division appears to require a fixed period in which the function controlled by the gene is performed: this period ends, under physiological conditions, when division does not require further protein or DNA synthesis.


Mutagenic DNA repair in Escherichia coli: conditions for error-free filling of daughter strand gaps. Two situations have been observed in which daughter strand gaps in DNA synthesized after exposure of excision-deficient Escherichia coli to ultraviolet light are filled but in which no mutations are formed as judged by loss of photoreversibility: (i) during the first 20 min of growth after u.v. irradiation, and (ii) when repair is allowed to occur in buffer. We suggest as an explanation that the majority of daughter strand gap-filling is error free and that mutations arise through a minor error-prone repair pathway which is inoperative under these conditions.


The molecular relatedness of R factors in enterobacteria of human and animal origin. The molecular length and DNA homology of R factors isolated from enterobacteria of human and animal origin have been examined. DNA from plasmids of the same compatibility group, whether of human or animal origin is indistinguishable, after allowance has been made for the regions coding for different antibiotic resistances. These results indicate that there is a common pool of R factors in man and ainmals.


Mutations in Escherichia coli that relieve catabolite repression of tryptophanase synthesis. Tryptophanase promoter-like mutations. From a strain lacking adenyl cyclase and the catabolite-sensitive gene activator protein, two mutants were isolated that can synthesize tryptophanase. Each mutation is extremely closely linked to the tryptophanase structural gene. The mutations differ from one another in the rate of synthesis of tryptophanase that they permit in the genetic background in which they were isolated; they differ from one another and also from the wild type in the maximum rate of synthesis of tryptophanase that they permit in a genetic background with intact adenyl cyclase and catabolite-sensitive gene activator protein. Both mutations appear to lie in the tryptophanase promoter.


Genetical and physiological studies on a thermosensitive mutant of Escherichia coli defective in cell division. A new temperature-sensitive mutant of E. coli, defective in cell division, was isolated after selection for tolerance to colicin E2. The mutant strain, ASHI24, growing in either minimal or complex medium, commences filament formation immediately upon shift to high temperature. High densities of bacteria or the presence of 0-44 M-sucrose prevents filament formation at 42 degrees C and division continues. Filament formation in the mutant is reversible and upon return to 29 degrees C the multinucleate filaments divide up into normal-sized bacteria by a series of rapid but sequential divisions. In the presence of chloramphenicol at 29 degrees C, 25% of these division sites are still expressed. A genetic locus designated ftsH, apparently controlling both temperature sensitivity and filament formation, was provisionally mapped at minute 80 on the E. coli K12 map.


Free mycolic acids as criteria in the classification of Nocardia and the 'rhodochrous' complex. The methyl esters of free mycolic acids from representative strains of Nocardia asteroides, N. brasiliensis, N. caviae and the 'rhodochrous' complex were subjected to detailed mass spectral analysis. The anhydromycolic esters of the Nocardia strains consisted of homologous series containing from zero to three double bonds, with the main components of the parent mycolic acids centred on C52 to C54 (range C46 to C58). The anhydromycolates from one rhodochrous strain, Nocardia opaca, had a molecular weight range similar to the nocardiae (C46 to C57) but the remaining rhodochrous strains gave an homologous series of anhydromycolates containing from zero to two double bonds, with the main components of the parent mycolic acids centred on C38, C42, C44 or C46 (total range from C34 to C50). The mycolic acids from the rhodochrous strains with chain lengths centred around C40 form a group intermediate in size between corynomycolic acids (centred around C32) and nocardomycolic acids (centred around C50). These data weaken the case for retaining the 'rhodochrous' complex in the genus Mycobacterium, and also show that many rhodochrous strains can be distinguished from true nocardiae and corynebacteria. These results confirm the value of lipid characters in the classification of these organisms.


On the nature and formation of the fibrillar nets produced by protoplasts of Saccharomyces cerevisiae in liquid media: an electronmicroscopic, X-ray diffraction and chemical study. The nets produced by protoplasts of Saccharomyces cerevisiae in liquid culture media consisted of microfibrils about 20 nm wide, forming flat, fairly straight bundles of variable width and length, up to about 500 nm wide and 4 mum long. Ends of microfibrils were seldom found. They were not attacked by chitinase or dilute acids, but the net structure disappeared in 3% (w/v) NaOH, leaving about 60% dry wt of the nets as partly microfibrillar clusters. The X-ray powder pattern from the nets, in contrast to that from normal walls, exhibited a set of well-defined rings which identified two micro-crystalline constituents: chitin and unbranched chains of beta-(1 leads to 3)-linked D-glucose residues. These latter were the alkali-soluble fraction. The X-ray diagram of the glucan, corresponding to that of paramylon, indicated an in vivo crystal modification. Up to 15% dry wt was chitin which was found de novo by the protoplasts. A fine net structure of microfibrils about 7-5 to 10 nm thick with meshes about 20 to 60 nm wide was demonstrated in normal walls, forming the entire inner layer and consisting mainly of yeast glucan. This glucan and chitin were only slightly crystalline in these walls. The features of the glucan and chitin of the protoplast nets indicate that enzymes active in normal wall formation were differentially removed or inactivated by the liquid medium.


On the nature and formation of the fibrillar nets produced by protoplasts of Saccharomyces cerevisiae in liquid media: an electronmicroscopic, x-ray diffraction and chemical study. The nets produced by protoplasts of Saccharomyces cerevisiae in liquid culture media consisted of microfibrils about 20 nm wide, forming flat, fairly straight bundles of variable width and length, up to about 500 nm wide and 4 mum long. Ends of microfibrils were seldom found. They were not attacked by chitinase or dilute acids, but the net structure disappeared in 3% (w/v) NaOH, leaving about 60% dry wt of the nets as partly microfibrillar clusters. The X-ray powder pattern from the nets, in contrast to that from normal walls, exhibited a set of well-defined rings which identified two micro-crystalline constituents: chitin and unbranched chains of beta-(I leads to 3)-linked D-glucose residues. These latter were the alkali-soluble fraction. The X-ray diagram of the glucan, corresponding to that of paramylon, indicated an in vivo crystal modification. Up to 15% dry wt was chitin which was formed de novo by the protoplasts. A fine net structure of microfibrils about 7-5 to 10 nm thick with meshes about 20 to 60 nm wide was demonstrated in normal walls, forming the entire inner layer and consisting mainly of yeast glucan. This glucan and chitin were only slightly crystalline in these walls. The features of the glucan and chitin of the protoplast nets indicate that enzymes active in normal wall formation were differentially removed or inactivated by the liquid medium.


The dispersal of an initial concentration of motile bacteria. The dispersal of an initial concentration of identical Brownian particles is accurately described by the solution of the conventional diffusion equation, and a diffusion coefficient can be assigned to the assembly of particles. However, the dispersal of an initial concentration of motile bacteria is not well described by the same solution, in spite of the similarity between the random motion of a bacterium and a Brownian particle. Reasons for the failure of the Gaussian solution of the diffusion equation to describe the dispersal of Escherichia coli are discussed. An equation is formulated which gives the concentration of dispersing organisms as a function of space and time if the speed distribution function of the assembly of organism is known and reproduction is suppressed. For three assumed speed distributions the results are compared with concentrations measured by previous authors.


The elimination of urease activity in Streptococcus faecium as evidence for plasmid-coded urease. A strain of Streptococcus faecium from the sheep rumen showed spontaneous loss of urease activity when subcultured at the normal rumen temperature of 38 degrees C, although in mixed cultures in vivo or in vitro loss of urease was not apparent. The rate of loss of urease in pure cultures was increased at incubation temperatures above 38 degrees C, but loss was never complete. However, at temperatures below 38 degrees C loss was greater, and at 22 or 18 degrees C the urease was completely eliminated. Incubation with sodium dodecyl sulphate (0-002%) or ethidium bromide (2-5 X 10(-5)M) caused complete loss of urease activity. The urease activity was also eliminated when the streptococcus was grown aerobically, and this loss of activity was irreversible. It is suggested that the urease activity is controlled by a plasmid gene and that aeration, low growth temperature and chemical agents 'cure' the streptococcus of the plasmid. Attempts to demonstrate the presence of covalently closed circular extrachromosomal DNA by caesium chloride-ethidium bromide equilibrium density-gradient centrifugation were unsuccessful.


The stability of toxigenicity in Clostridium botulinum types C and D. Several type C and D strains of Clostridium botulinum, which had been converted to the toxgenic state by phages, were serially transferred through cooked meat medium with and without specific anti-phage serum. Most of the converted strains lost their toxigenicity even during transfer without antiserum, and the non-toxigenic variants that appeared were resistant to lysis and conversion by the original phage. However, in some combinations of phage and host bacteria toxigenicity was stable after ten transfers, though it showed a transient decrease, and the non-toxigenic variants that arose remained sensitive to lysis and conversion. When converted strains were transferred in medium containing anti-phage serum, toxigenicity was lost more rapidly than in the absence of serum and the non-toxigenic variants that appeared remained sensitive to lysis and conversion by the parent phage. Filtrates of the supernatants of culture fluids of strains transferred without anti-phase serum converted non-toxigenic strains to toxigenicity at varying rates.


The effect of sublethal doses of rifampin on the sporulation of Clostridium botulinum. Sublethal doses of rifampin (0-005 mug/ml), added to vegetatively growing cultures of a sporogenic mutant of Clostridium botulinum at inoculation time or after 4 h, resulted in a decrease of growth and in blockage of spore formation. But when rifampin was added 6 to 24 h after inoculation, normal growth and sporulation occurred, indicating that the time of addition was critical and that rifampin was most effective on rapidly dividing, exponential-phase cells. Ultrastructural studies showed that when rifampin was added at the time of inoculation, endospore development was blocked at stage III. During subsequent incubation (greater than 10 h) the cells lost their rigidity, and lysis of the mother cell was followed by that of the forespore. When the cultures were treated with rifampin at 4 h, about 40% of the cells were blocked at stage III and about 60% reached stages IV and V. Some showed excessive elongation and contained developing spores at each pole. They appeared to be derived from two daughter cells unable to form a division septum because of a specific inhibitory effect of rifampin on division. It would seem, therefore, that two daughter cells which are genetically coded to form endospores will do so irrespective of the development of a division septum, and the spores are formed at the 'old' polar regions.


Arthritis in myasthenia gravis.

Arthritis in myasthenia gravis. Seven patients with myasthenia gravis developed clinical signs of arthropathy. In two patients, the symptoms were due to a deforming rheumatoid arthritis and the myasthenic symptoms appeared as a transitory phase during the course of the disease. Muscle antibodies of IgG class were demonstrated with sera from both patients. Autoreactivity between muscle antibodies and rheumatoid factor was detected in one patient. Both patients died from sudden cardiac failure. Necropsy was performed in one and revealed a spotty myocardial necrosis. One patient had juvenile rheumatoid arthritis. Two patients had mild articular symptoms with indices of multivisceral disease and serological findings indicating a systemic lupus erythematous. One patient had classical ankylosing spondylitis, and one, unspecified arthropathy.


Marked suppression of thyroid function in rats with gram-negative septicemia. Gram-negative septicemia was induced in rats by two daily injections of fecal mixture into the thigh, after which the thyroid function was markedly suppressed for 2 days. Iodine metabolism was studied by organ radioassay and by imaging with a multiwire proportional chamber (MWPC) at various time intervals after intravenous injection of 125I. Plasma T3, T4, and TSH, measured by radioimmunoassays, were suppressed, as were the T3-resin uptakes. Fractional blood supply to the thyroid glands of the infected rats, studied by the 81Rb uptake method, was also found to be markedly reduced. Sections of the thyroid glands showed little structural change during the period of marked thyroid suppression. There was no biochemical evidence of renal failure in the septicemic rats.


Radionuclide determination of cardiac chamber flow/volume characteristics. The flow/volume characteristics of an externally monitored radionuclide dilution curve may contain valuable information about specific cardiac chamber abnormalities. a simplified method of obtaining this flow/volume ratio on a standard scintillation camera is presented. Preliminary clinical application to right-sided cardiac lesions has been undertaken. The implications of flow/volume variation with time are discussed.


Initial assessment of a simple functional image of ventilation. Several methods for quantitatively measuring regional ventilation using an inert gas such as Xe133 have been described. One such method provides a functional image of washout. A particularly simple functional image may be derived from the mean transit time for clearance. Regional ventilation may thus be represented as a single image in which the spatial distribution of washout from multiple small regions of the lung is displayed. In 100 patients for whom such a functional image of ventilation was obtained, the functional image was found to be generally more useful and easier to interpret than sequential images taken during the period of washout. Distributions of half-time (t1/2) for washout and the mean transit time (t) in the same population were similar in shape, but the t distribution peaked about 15 sec later than the t1/2 distribution. The mathematical simplicity of the functional image described here should permit its implementation on virtually any nuclear medicine computer system.


Use of direct bonding materials for fixation and stabilization in the correction of facial deformities. Comparatively little time is required for placement of orthodontic brackets with a direct-bonding technique. No anesthesia is required and no discomfort is caused to the patient. Fixation is esthetically acceptable and periodontal health is preserved during fixation. The procedure has been used successfully for immobilization after anterior segmental osteotomies and for maxillomandibular fixation after complete mandibular and maxillary osteotomies.


Alloplastic augmentation of middle-third facial deformities. In reconstruction of deformities of the middle third of the face, an advantage in the use of an alloplastic implant rather than tissue transplantation is that only one surgical procedure is needed. A more accurate fit and better esthetic results are obtained with custom-made implants of silicone rubber. Carved implants require less fabrication time and offer adequate results although they are not always shaped as accurately.


The adverse effect of anticonvulsant therapy on renal allograft survival. A preliminary report. Administration of anticonvulsant medication to recipients of cadaver renal allografts appears to be associated with decreased allograft survival. The one-and two-year actuarial graft survival rates for 20 index grafts was significantly lower than for 92 control grafts. Since phenobarbital and diphenylhydantoin increase the metabolism of corticosteroids, it is proposed that renal allograft recipients receiving these anticonvulsants may have ineffective immunosuppression, leading to a higher incidence of graft failure.


Iron deficiency and behavior.

Iron deficiency and behavior. The literature bearing on the behavioral correlates of sideropenia in animals and man is reviewed. There is evidence to support the contention that sideropenia, per se, is causally related to the subjective complaints of the iron-deficient and/or anemic individual. And, although important biochemical pathways involving electron transport, catecholamine catabolism, and porphyrin synthesis have been shown to be deranged in iron-deficient animals and human beings, the role of these alterations in any putative behavioral aberration is conjectural at present. Given the high prevalence of iron deficiency in the world's population, these issues should be addressed by appropriate biochemical and psychologic studies in animals and human beings.


Laron dwarfism: growth and immunoreactive insulin following treatment with human growth hormone. A 13 1/2-year-old boy with features of growth hormone deficiency had elevated fasting plasma GH levels (5.7 to 66 ng/ml). Serum somatomedin values remained low despite treatment with human growth hormone. Plasma GH values were suppressed following oral administration of glucose and increased following insulin-induced hypoglycemia, L-dopa, and arginine. Chlorpromazine suppressed GH, both fasting and during IIH. These results suggest that the neuroendocrine mechanisms mediating GH secretion seemed to be intact. Peak plasma insulin levels increased in response to glucose administration after HGH suggesting that GH has a direct effect on the pancreatic beta cell which is not mediated by Sm. Plasma testosterone values increased to adult male levels, but there was inadequate secondary sexual response. Growth was enhanced by HGH and may have been due to testosterone and/or insulin. Although Laron dwarfism may result from a receptor defect, an abnormality in GH structure is also possible.


Ovarian hyperthecosis in the adolescent patient. The endocrine findings in two adolescents with hyperthecosis are compared to those in a patient with an androgenic ovarian tumor. In patients with hyperthecosis, luteinizing hormone values were elevated or in the upper normal range, and plasma testosterone and androstenedione values were increased. Following dexamethasone suppression, testosterone and androstenedione values remained elevated, but after administration of human chorionic gonadotropin, they increased further in only one patient. Baseline 17-ketosteroid values were normal, suppressed with dexamethasone, and stimulated to baseline levels following HCG. The patient with a lipoid cell ovarian tumor had low baseline LH levels, and elevated testosterone, androstenedione, and 17-ketosteroid values. Dexamethasone produced little change in urinary or plasma values, but the 17-ketosteroids increased markedly after administration of HCG. The finding of low serum LH values in patients with hirsutism and elevated androgen secretion should alert the clinician to the possibility of a tumor.


Effect of synthetic luteinizing hormone-releasing hormone on the release of gonadotropins in hypophysogonadal disorders of children and adolscents. VII. Constitutional delay of puberty in males. Serum gonadotropins (LH and FSH) were measured by radioimmunoassay before and after intravenous injection of 0.1 mg/m2 of synthetic luteinizing hormone-releasing hormone in 20 male patients, aged 15 to 18 years, with constitutional delay of puberty. Basal plasma levels of LH and FSH were in the prepubertal range. After administration of LH-RH, the increase in LH was significantly high than in prepubertal control subjects, aged 1 to 13 years; the difference between test patients and pubertal control subjects was not significant. The increase in FSH was in the prepubertal range, significantly lower than that in pubertal control subjects. This discrepancy between LH and FSH responses to LH-RH is similar to that observed in normal boys at the late prepubertal stage and suggests that an elevation of readily releasable pituitary stores of LH correlates with the first step of pubertal onset in males, even if puberty is delayed.


Anorectal Manometry as a diagnostic method in the neonatal period. Presented are the results of manometric investigation of 63 neonates. The method was correct in 92% of all cases at first investigation, and if delay was justifiable on clinical grounds, this figure rose to 98%. Most problems in diagnosis using this method were related to abnormal smooth muscle contractility, which is regarded as being the most important single factor to determine. In patients in whom contractility was abnormal, sepsis, hypoxia, and hypothermia were associated with the generally poor clinical condition. The return of normal contractility paralleled the improvement in the patient's general clinical condition. Neither the maturity nor the size of the patient was felt to be a factor influencing the success or failure of manometry as a diagnostic method.


Evaluation of cleansing devices in the maintenance of interproximal gingival health. This study consisted of 24 dental students, who 10 days prior to the start of the experimental period, were thoroughly scaled and given instructions in the use of a rubber tip stimulator, unwaxed dental floss, and a modified Bass brushing technique. On day 0, the subjects had reached a high level of interproximal gingival health as measured by intracrevicular exudate flow and löe's Gingival Index. Subjects were then randomly divided into 4 groups which were to brush; brush and floss; brush and rubber tip; and brush, floss and rubber tip. Evaluation was performed on days 0, 9, 15, and 33 using Löe's Gingival Index, Podchadley's Plaque Index, and gingival exudate flow. The results of this study indicate that interproximal gingival health can be maintained in motivated patients with initially healthy gingivae (for at least several weeks) with proper use of the modified Bass brushing technique alone.


Effects of fear appeals and physiological arousal upon emotion, attitudes, and cigarette smoking. Two experiments are reported that attempted to replicate conceptually Schachter's theory of the determinants of emotion and to test the feasibility of extending the theory to attitudes and behavior that may be mediated by the emotion of fear. A total of 279 cigarette smokers were administered either epinephrine or a placebo and then exposed to situational cues suggestive of disparate emotional states (Experiment 1) or different intensities of the same emotion (Experiment 2). Contrary to preictions based upon Schachter's theory, manipulated physiological arousal was not necessary for emotional labeling and under some conditions elicited fear. The situational cues affected emotion and attitudes. Higher levels of fear appeals strengthened intentions to quit smoking, and reassurance of the efficacy of stopping smoking reduced cigarette consumption.


Antimalarial amino alcohols II: anthraceneaminoethanols and anthraceneaminopropanols (1- and 9-substituted). The syntheses of seven anthracene amino alcohols with one, two, or three additional substituents are described. These compounds include three 1-aminoethanols, two 9-aminoethanols, and two 9-aminopropanols, prepared from substituted anthraquinones or from 10-chloro-9-anthraldehydes. The antimalarial activity of these compounds, as well as tentative structure-activity relationships, is discussed in the light of previously published work.


Synthesis of 2-pyridyl-alpha-toluenesulfonates as antimalarials. A series of substituted 2-pyridyl-alpha-toluenesulfonates was synthesized for antimalarial testing. They were prepared by treating various 2-pyridinols with alpha-toluenesulfonyl chlorides in the presence of an alkali. In tests against Plasmodium berghei in mice at 640 mg/kg, only 3,5-dichloro-2-pyridyl-alpha-toluenesulfonate was considered active, i.e., doubled the mean survival time.


Calcium and stimulus-secretion coupling in the adrenal medulla: contrasting stimulating effects of the ionophores X-537A and A23187 on catecholamine output. 1. The ionophores X-537A and A23187, which are known to transfer calcuim across cell membranes, stimulated catecholamine release from perfused cat adrenal glands. 2. These stimulant effects persisted in the presence of hexamethonium and atropine and are therefore attributable to direct actions of the ionophores on the adrenal chromaffin cells. 3. Perfusion with calcium-free Locke abolished responses to A23187 and reduced those to X-537A. 4. Responses to X-537A were consistently large and comparable with those produced by 56 mM potassium. By contrast, responses to A23177, over the wide range of concentrations tested, were variable and much smaller. 5. That the two ionophores can stimulate through calcium-dependent mechanisms is considered fresh support for the calcium hypothesis of stimulus-secretion coupling. That they differ in effectiveness may mean that factors besides calcium are important. The greater potency of the less specific ionophore, X-537A, may be attributable to its ability to depolarize as well as carry calcuim, while the relatively small effects of A23187, a generally more effective ionophore for calcuim, may indicate that inward movement of calcium without a background of membrane perturbation such as may be produced by depolarization, is insufficient to elicit strong secretory responses.


The influence of secretin on the secretion of pepsin in response to acid stimulants in the anaesthetized cat. Peptic secretion was studied in fasting anaesthetized cats in which the pylorus and common bile duct had been occluded to prevent the release of duodenal hormones which might stimulate or inhibit gastric secretion. Dilute acid was instilled into the stomach at intervals to aid recovery of gastric secretion and to preserve peptic activity. 2. Caerulein, histamine and N-methyl histamine did not increase the output of pepsin when given on their own. Desulphated caerulein was a weak peptic stimulant. 3. Two C.H.R. u./kg per hour secretin initiated pancreatic secretion, the volume of which increased progressively as the dose was increased by stages to 32 C.H.R. u./kg per hour. 4. Four C.H.R. u./kg per hour secretin did not increase the output of pepsin. Peptic secretion was stimulated by 8 C.H.R. u./kg per hour. A maximal output of approximately 2000 u. pepsin/15 min was obtained when 16 C.H.R. u./kg per hour was infused. 5. When each acid stimulant was infused along with 4 C.H.R. u./kg per hour secretin the output of pepsin increased significantly. The peak output, which usually occurred between 15 and 30 min after stimulation, did not exceed 1000 u. pepsin/15 min. 6. The proposed explanation for the potentiation of the peptic response when an acid stimulant is infused along with a dose of secretin, in itself below the threshold of peptic stimulation, is that each acid stimulant increases gastric mucosal blood flow, approximately doubling the effective concentration of secretin delivered to the peptic cell.


Acrosomal enzymes: Immunochemical localization of acrosin and hyaluronidase in ram spermatozoa. Acrosin and hyaluronidase have been localized in the acrosomal region of ram spermatozoa using specific antibodies raised against the highly purified enzymes. Hyaluronidase staining was denser at the periphery of the sperm head; whereas acrosin staining was denser in the equatorial region and appeared to be bound to the inner acrosomal membrane.


Functional and structural relationships in the Graafian follicle population of the sheep ovary. The Graafian follicle population in sheep can be divided into two groups on the basis of the capacity that individual follicle have for secreting oestrogen when isolated from the remainder of the ovary (Moor, 1973). The most developed one or two non-atretic follicle from each animal generally produce large amounts of oestrogen in culture and can thus be classified as steroidogenically 'activated'. The other follicles usually produce very little oestrogen in culture and are accordingly classified as 'non-activated'. In this paper the above classification will be used and the two groups of follicles will be considered separately. In the first section of the paper the response of the non-activated follicles to gonadotrophic stimuli will be described and related to growth and atresia within this population. The steroidogenic potential of the activated follicles at different stages of the cycle will be discussed in the second part of the paper. In the third part, an attempt will be made to relate the ultrastructure of the activated follicles to the functional activity of their component tissues, namely the theca interna and membrana granulosa.


Relationship between the secretion of the corpus luteum and the length of the follicular phase of the ovarian cycle. The variation in the length of the follicular phase in many mammals may be related to the cellular origin of oestradiol secreted during the luteal phase. In all species the time taken for a small developing follicle (4-5 layers of granulosa cells) to mature to a preovulatory follicle may be the same as that which has been found experimentally in the mouse (10-17 days). In animals such as the sheep, in which there is no source of oestradiol other than the Graafian follicle, follicular development proceeds unimpaired throughout the luteal phase, and the 'follicular phse' which involves only the final stages of maturation of the Graafian follicle is relatively short. In primates, however, in which there is an extrafollicular source of oestrogen from the CL, the secretion of gonadotrophins is suppressed during each luteal phase to a level too low to initiate and maintain follicular development. At the end of each luteal phase and the beginning of the subsequent follicular phase, therefore, it is necessary to initiate the growth of a new crop of small follicles. The time taken for these follicles to develop inot preovulatory follicles determines the length of the follicular phase.


Control of microflora in stallion semen with a semen extender. Sterile equipment was used to collect ten ejaculates from each of ten normal stallions and quantitative and qualitative bacterial counts were made within 15 min after collection. The mean bacterial population in undiluted semen was found to be 573,000 +/- 374,000 organisms/ml. The bacterial content of semen diluted with two parts sterile physiological saline, or with two parts of a cream-gelatin extender, was measured within 15 min after collection and again after 2 hr at 25 degrees C. The number of bacteria was slightly increased in the saline after 2 hr, but 1 93% and 99% reduction occurred in the extended semen within 15 min and after 2 hr at room temperature.


The use of Gn-RH for controlling the oestrous cycle of the mare (preliminary report). Clinical tests with synthetic gonadotrophin-releasing hormone (Hoechst) were made during the breeding seasons of 1973 and 1974, using 128 mares injected with 1-0 to 4-0 mg of the substance intramuscularly. The mares were placed in one of five groups based on ovarian condition determined by clinical evidence. Some success was obtained in the induction of ovulation in mares with inactive and sub-normally active ovaries and in a small group having cystic ovaries. A large proportion of mares having a mature follicle responded within 48 hr, but others with atretic follicles failed to respond. The usefulness of Gn-RH for this purpose is discussed.


Duration of oestrus and time of ovulation in mares treated with synthetic Gn-RH (AY-24,031). Synthetic Gn-RH, administered during oestrus, stimulates the release of pituitary LH in the cyclic mare. Duration of oestrus was significantly reduced by 1 mg Gn-RH given on Day 2 of oestrus; the time of ovulation, measured in days from the onset of oestrus, also tended to be reduced. An injection of 2 mg Gn-RH had nor further effect, but daily injections from Day 2 until ovulation significantly shortened the duration of oestrus and the time to ovulation. The profiles of LH were found to be variable from mare to mare, but in all mares, treated and control, elevation of LH was detected close to the time of ovulation.


The contribution of the mule to scientific thought. The infertility of the mule has proved a continuing challenge to scientific thought. Since the chromosomal differences between the two parental species are so great as to render normal meiosis impossible, it is postulated that all mules and hinnies are sterile. The problem now is to explain how mules and hinnies can occasionally produce spermatozoa or ova. The appearance of the mule was sufficient to persuade the ancients that both parents, not just the male, must contribute to the make-up of the offspring. The mule has also taught us that, when the number of oocytes in the ovary is reduced, the ovary becomes the time-clock that regulates the length of the oestrous cycle. A study of gonadotrophin production in horses and donkeys bearing hybrid foals has yielded fascinating results about the immunology of pregnancy. The behaviour of the sex chromosomes of female mules and hinnies has helped to confirm the Lyon hypothesis about X-chromosome inactivation. The mule has also provided spectacular proof of the doctrine of hybrid vigour, and the marked deficiency of males at birth confirms the general truth of Haldane's Law. The mule and the hinny remain man's only successful attempt at the production of a commercially viable interspecific mammalian hybrid.


Infection of the horse fetus.

Infection of the horse fetus. Many infections of the equine placenta and fetus result from ascending spread along the cervical canal. Most abortions due to infection occur during the later stages of pregnancy and the possible effects of intrauterine infection on the developing fetus and young foal are discussed.