The sensitivity of gel electrophoresis as a detector of genetic variation. Three experiments based on an idea of YOUDERIAN have been performed to determine the proportions and kinds of amino acid substitutions that are detected by gel electrophoresis when applied to surveys of protein variation in populations. The experiments involved applying the sequential method of electrophoresis under several conditions of pH and gel concentration to a large sample of human hemoglobins with known amino acid substitutions. In the first experiment, a random sample of 20 different hemoglobin variants was studied, and these were separated into 17 distinct electrophoretic classes by three sequential gel conditions, thus giving a detectability of 85%. A single pass under standard conditions detected eight classes. The second experiment compared groups of substitutions that were chemically identical, but in different positions in the alpha and beta chains, while the third experiment compared pairs of substitutions that were charge equivalent, but chemically different at the same chain position. The sequential method distinguished 90% of all chemically identical substitutions when they were at different chain locations, and four out of five charge equivalent but chemically different substitutions at the same site. Examination of the location of each substitution in the three-dimensional structure of hemoglobins showed that interior substitutions usually are less different from Hb A than are surface substitutions and that local interactions with chain and spatial neighbors are sufficient to distinguish substitutions in very similar positions on the outside of the molecule. The "charge ladder" model of electrophoretic classes is clearly incorrect, and it appears that sequential gel electrophoresis as practiced in our Drosophila surveys has detected a substantial fraction of amino acid substitutions if hemoglobin is regarded as a model. This estimate may be modified as other molecules beside hemoglobin are subjected to similar calibration experiments.


Polymorphisms, linkage and mapping of four enzyme loci in the fish genus Xiphophorus (Poeciliidae). Electrophoretic variants at four additional enzyme loci--two esterases (Est-2, Est-3), retinal lactate dehydrogenase (LDH-1) and mannose phosphate isomerase (MPI)--among three species and four subspecies of fish of the genus Xiphophorus were observed. Electrophoretic patterns in F1 hybrid heterozygotes confirmed the monomeric structures of MPI and the esterase and the tetrametric structure of LDH in these fishes. Variant alleles of all four loci displayed normal Mendelian segregation in backcross and F2 hybrids. Recombination data from backcross hybrids mapped with Haldane's mapping function indicate the four loci to be linked as Est-2--0.43--Est3--0.26--LDH-1--0.19--MPI. Significant interference was detected and apparently concentrated in the Est-3 to MPI region. No significant sex-specific differences in recombination were observed. This group (designated linkage group II) was shown to assort independently from the three loci of linkage group I (adenosine deaminase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase) and from glyceraldehyde-3-phosphate dehydrogenase and two isocitrate dehydrogenase loci. Evidence for conservation of the linkage group, at least in part, in other vertebrate species is presented.


Adult triploids in a rainbow trout family. Six triploid individuals were found in a full-sib family of 11 adult rainbow trout (Salmo gairdneri) from a domesticated hatchery stock. The triploid individuals were normal in size and external appearance, had underdeveloped gonads, and showed no evidence of 3n/2n chimerism or mosaicism. XXY triploids were males, suggesting that the Y chromosome is male determining in trout. Because they may avoid production losses associated with sexual maturation in normal fish, triploid trout and salmon could potentially be useful in fish culture.


Genic heterogeneity within electrophoretic "alleles" and the pattern of variation among loci in Drosophila pseudoobscura. An investigation, similar to our previously reported xanthine dehydrogenase study, was undertaken to examine the extent of hidden genic variation at nine loci (five larval proteins, three esterases and one aldehyde oxidase) by sequential application of various electrophoretic criteria employing pH, gel concentration and buffer variation. Polymorphic loci appear to fall into two distinct groups: weakly polymorphic, including larval protein 6, 7, 8, 10 and 13 and esterase-1 and -6; and highly polymorphic, including esterase-5, Xdh and possibly Ao. Monomorphic loci may belong to a third group different from all polymorphic loci. Bogota, a geographical isolate that is reproductively isolated from the mainland population, was found to be genetically distinct at four of the ten loci examined in detail so far, including Xdh, whereas previously it was found to be genetically distinct at none. These results are discussed in the light of balancing selection, neutral and mutation-selection hypotheses of genic variation in natural populations.


Geographical disproportions of multiple sclerosis in Czechoslovakia. In the sanatorium in Vráz near Pisek in 1970-1978 3310 patients with multiple sclerosis received spa treatment. By analysis of their birth place, domicile at age of 15 years and present domicile we tried to assess the geographical distribution of the disease in Czechoslovakia. The area of the suspected highest frequency of MS is the northwestern part of Bohemia with the maximum near Teplice and Most. The lowest frequency was recorded in the lowland areas of southern Moravia and southern Slovakia including Bratislava. The frequency gradient from the north-west towards the south-east, the demarcation line formed by the 49th northern parallel and some ecological relations are very striking and deserve further research and confirmation.


Organochlorinated pesticides in the Argentine Antarctic sector and Atlantic coastline waters. The presence of organochlorinated pesticides in water samples drawn in the Argentine Antarctic Sector and Atlantic coastline has been proved. In general, these samples showed quantities that varied between a few hundredths of a ppmm to slightly more than 1 ppmm, although in some cases and for certain pesticides, depending on the locations, the levels found were definitely higher. With the exception of Dieldrin, which appeared in only one sample/in what would seem to be its course few hundredths of a ppm, to slightly more than 1 ppmm, although in some cases and for certain pesticides found were the same as those reported in previous investigations. The isolated cases of high pesticide contents in water samples drawn at wharves and of snow in the vicinity of Almirante Brown Base show up clearly the influence of human activity on the contamination of the environment.


Researches for the prevention of dangers of pesticides used in agriculture. The authors surveyed pesticide use during a 7-year period in Szabolcs-Szatmár county. Evaluating their research data they introduced a new concept on the basis that they assured the possibility for stating the different pesticide load of different regions.


Effects of the m gene for muscular hypertrophy on birth weight and growth to one year of age in beef cattle. 182 calves, mostly Angus but also of Hereford and Shorthorn breeding, born in the University herd, were studied, including 55, born during Spring 1970, genotypically distributed as 8 mm, 33 m+ and 14 ++. Eighteen of the 24 homozygotes (mm) had the typical double-muscled conformation at birth (mmY); in the remainder, the typical bulging muscles usually manifested themselves within one month after birth. Averaged over sexes, mmY calves were 4.4 kg heavier (P less than 0.05) than the Angus (++) breed average of 26.6 kg. Compared within-sires, mmY calves were 4.0 kg heavier than m+. Heterozygotes (m+) were 0.4 kg heavier than the normal (++) average. Estimating differences between mm, m+ and ++ for post-natal traits was confined to the 1970 calves because experimental conditions were controlled. Double-muscled (mm) calves had a higher (+7%) 180-day weight compared with normal (++) calves. After weaning at approximately 180 days, until yearling age, bulls and heifers received diets with roughage:concentrate ratios of 75:25 and 70:30, respectively. The post-weaning growth rate of mm calves was depressed (-17%) due to a reduced (-11%) feed intake compared with non-mm controls. Homozygotes had a lower (-4%) liveweight at 365 days, compared with ++ animals. Least squares estimates of the difference (m+ - ++) at birth, 180 days and 365 days were -1.0 +/- 1.0 kg, +7.3 +/- 4.5 kg and +7.3 +/- 6.3 kg, respectively.


Histones and histone-DNA ratios in diploid and polyploid cottons. Histones are nuclear proteins which repress gene transcription and modify chromosome structure. They are remarkably conservative in structure throughout a wide evolutionary array of plants and animals; however, quantitative histone differences have been detected by cytological means in species having extra chromosomes. The purpose of this study was to: 1) isolate and characterize the histones of several Gossypium species and 2) relate differences to known differences in vigor, ploidy level, and genome constitution or size. Histones extracted from isolated nuclei of leaf tissues were characterized by electrophoresis on polyacrylamide gels into 14 subfractions of the five major histone classes. The subfractions were identified by various means including co-electrophoresis with known histone standards. Densitometric analysis revealed only slight quantitative differences in subfraction ratios between species. Histone-DNA ratios were significantly higher in the pentaploid species. This observed increase is considered a result of genome imbalance. These data support the premise that histones may function as generalized gene deactivators in plant species having multiple genomes.


Height increments in prepubertal children. The heights of 51 normal boys and girls were measured at regular intervals throughout a year. The data were analyzed to determine in the normally growing child, the shortest time interval regardless of the time of year during which an increase in height should be expected to be recognized in every child. That interval is 16 weeks.


A new nonparametric technique for constructing percentiles and normal ranges for growth curves determined from longitudinal data. A new nonparametric method is proposed for the construction of percentile curves and normal ranges which can be used to classify an individual's growth, velocity, and acceleration of growth dynamically over a time interval selected for its biological importance. Although the procedure requires longitudinal data, it is not necessary that all subjects be measured at identical times. Unlike classic static methods it does not provide percentile curves that are typical of no one. The median curve is the actual curve of the most "central" individual. The method is applicable to growth curves of any form with a general computer program already available for many forms. The technique is demonstrated for growth in weight curves smoothed by high degree polynomials.


Effect of maternal ethanol administration on physical growth of the offspring in rats. To study the effects of maternal alcohol consumption on the postnatal growth and physical development of the offspring, female Sprague-Dawley rats (200-220 g) were assigned to one of 3 groups. Group I (alcohol) received alcohol in drinking water (up to 20% v/v) for at least 4 weeks prior to mating, and 30% (v/v) throughout gestation. Purina Lab Chow was ad libitum. Group II (pair-fed) received the same amount of chow as was consumed by alcohol-fed animals, and an amount of corn starch calorically equivalent to the amount of alcohol consumed. Group III (ad libitum) were given chow and water ad libitum. Postnatally all animals were given chow and water ad libitum until day 51 post conception (PC). During pregnancy alcohol provided about 28% of the calories in group I, and the total calorie intakes of the alcohol and pair-fed groups were approximately 60% of that of the ad libitum controls. Weights at birth of offspring of pair-fed and ad libitum control mothers are not significantly different, but the offspring of animals given alcohol show a weight deficit of 28%, compared to the ad libitum controls. During the ensuing four weeks weight shows no indication of catching up to the controls. Total length shows the same pattern as body weight. Skeletal and muscle measurements are significantly less (p less than 0.01) in young of alcohol treated mothers than in those of the ad libitum control mothers. Skeletal maturity in the alcohol group lags behind (p less than 0.01) that of the pair-fed and ad libitum control groups and catch-up is not evident to day 51 PC. It is concluded that young born to animals given alcohol prior to and throughout gestation are physically and developmentally retarded and fail to catch up to the controls during the first four weeks after birth, although not exposed to alcohol postnatally.


Mannose-contaminating agglutinin for Actinomyces viscosus and Actinomyces naeslundii. Rapid agglutination of Actinomyces viscosus and Actinomyces naeslundii cells by D-mannose solutions was observed during studies of their attachment to mammalian cells in vitro. The specificity of the agglutination reaction was studied by slide agglutination tests and by measuring the rate of decrease in optical density of bacterial phosphate buffer suspensions caused by the setting of bacterial aggregates. Actinomyces cells were agglutinated by protein-containing mannose solutions of several chemical suppliers. Solutions of sugars other than D-mannose and solutions of mannitol and mannan all failed to agglutinate A. viscsus and A. naeslundii. "Mannose-enhanced" agglutination was impaired by boiling or autoclaving the mannose but was not affected by heating the bacteria, the presence of chloramphenicol, running the assay in the cold, or incorporating any of several commercially purchased sugars in the reaction mixture. During these hapten inhibition experiments, only 6-deoxy-L-talcose-containing extracts of an A. viscosus strain retarded the rate of mannose-enhanced agglutination. Protein-containing fractions of D-mannose mother liquors also agglutinated cells of A. viscosus and A. naeslundii. Other species of oral gram-positive rods were not agglutinated by mannose solutions. Together the data indicate that plant seed-derived D-mannose contains a protein-associated agglutinin for A. viscosus and A. naeslundii which may function via a "lectin-like" selective affinity for the unique cell wall sugar 6-deoxy-L-talose.


Intoxication of cells from different-aged embryos by diphtheria toxin. Different methods were used to assay diphtheria toxin sensitivity of fibroblast and heart cell cultures from chicken embryos of various ages. As defined by inhibition of protein synthesis, fibroblasts from 18-day and younger embryos respond more rapidly to toxin than fibroblasts from older embryos. The response of heart cells cultures is independent of the age of the embryos and is similar to the response of fibroblasts from 18-day embryos. Since the EF-2 content is 10-fold less in fibroblasts from 21-day-old embryos, the different responses of protein synthesis to intoxication appear to reside at the membrane level. Cytotoxicity assays in cell culture and in vivo toxin sensitivity assays show that cells  from both young and old embryos, as well as whole embryos, are equally sensitive to toxin. Thus, short-term (5-h) measurements of inhibition of protein synthesis are insufficient for determining the relative sensitivity of cells to intoxication as defined by cell death.


Regression of a murine fibrosarcoma after intralesional injection of a synthetic C39 glycolipid related to cord factor. Intratumoral injection of ultrasonically prepared emulsions of the synthetic glycolipid methly 6-O-(2-tetradecyl-3-hydroxyoctadecanoyl)-alpha-D-glucopyranoside (designated C39) induced complete regression of transplants of a syngeneic murine fibrosarcoma in most of the treated animals as did 6,6'-di-O(2-tetradecyl-3-hydroxyoctadecanoyl)-alpha,alpha,-trehalose (designated C76) in a previous study. The C76 compound, about twice the molecular weight of C39, was more effective therapeutically than the smaller molecule. Ultrasonically prepared emulsions of C39 and C76 were not toxic when given intravenously. Intravenously administered emulsions of C39 prepared by mechanical grinding were more toxic, but less granulomagenic, than those containing C76. Squalane and squalene, but not peanut oil, were effective substitutes for mineral oil as carriers of C39 in the treatment of the tumor.


Kinetics of phagocytosis of Staphylococcus aureus by alveolar and peritoneal macrophages. The rate of uptake of radiolabeled Staphylococcus aureus by macrophages in vitro was studied by use of Lineweaver-Burk analysis. It was found that competition for ingestion by excess unlabeled particles, either staphylococci or unrelated particles, resulted in diminished uptake of the labeled particles and that opsonization of particles with specific antiserum enhanced that uptake solely by altering the maximum velocity of uptake (Vmax). Uptake of radiolabeled staphylococci opsonized with specific antiserum was not inhibited by excess numbers of unopsonized organisms; the ingestion was inhibited by excess numbers of opsonized unlabeled organisms, and that inhibition was characterized by depression of Vmax. Inhibition of phagocytosis by indoacetate and cytochalasin B resulted from depression in both Vmax and Michaelis constate (Km). In addition, the phagocytic function of macrophages improved during in vitro culture, a phenomenon which was particularly striking for alveolar macrophages. That enhancement of activity resulted from improvements in both Vmax and Km. Addition of opsonizing antibody at any stage of in vitro maturation resulted in further increases in phagocytic uptake, increases which affected only Vmax. The in vitro maturation of phagocytic function by alveolar macrophages could be inhibited by both 2-deoxy-D-glucose and cycloheximide, but not by culture in hypoxia. The data indicate that the terms of Lineweaver-Burk analysis cna be correlated with functional aspects of phagocytosis and that Vmax represents the avidity of the macrophage surface for the particle, whereas Km is an index of the capacity of the cell for ingestion.


Synergistic protection against experimental cholera by immunization with cholera toxoid and vaccine. Rabbits were immunized with two parenteral injections of Wellcome toxoid PX389A, Wyeth toxoid 20101, or Merck bivalent vaccine. Other groups of rabbits were immunized with combinations of the Merck vaccine and each of the two toxoids. Antitoxin responses were monitored in each group of rabbits before livecell challenge of each animal by the ligated intestinal loop assay. Inaba and Ogawa strains of Vibrio cholerae were used for challenge experiments. Basically, the data indicate that the toxoids were equivalent in antigenic potency and antitoxin responses were unaffected by combination of the toxoids with the whole-cell vaccine. The 50 microgram doses of each toxoid as well as the 4 X 10(9) cells of the bivalent vaccine provided the same magnitude of protection against live-cell challenge with either Inaba or Ogawa vibrios. Immunization with either toxoid in combination with the bivalent vaccine resulted in a synergistic protective response against live-cell challenge of intestinal loops with V. cholerae. Synergistic protection was observed when toxoid and vaccine were administered together by the oral and parenteral routes. Maximum protection was obtained when rabbits were immunized with the combined toxoid-whole-cell vaccine administered by both oral and parenteral routes.


Polymorphonuclear leukocyte adherence to nylon: effect of oral corticosteroids. The effect of orally administered glucocorticoids on polymorphonuclear leukocyte adherence was studied by using the nylon fiber adherence assay. Inhibition of adherence 4 h after administration of the agent was confirmed, and in addition, augmentation of adherence was noted 24 h after ingestion of prednisone. Crossover studies revealed inhibition and augmentation to be cell associated as well as plasma mediated. Suspension of washed, dextran-sedimented polymorphonuclear leukocytes, harvested 4 h after prednisone ingestion in base-line or 24-h plasma failed to reverse inhibition of adherence. Adherence of 24-h polymorphonuclear leukocytes was augmented when suspended in all test plasmas. Plasma-mediated effects were demonstrated by inhibition of base-line adherence of polymorphonuclear leukocytes suspended in 4-h plasma and augmentation of adherence of cells in 24-h plasma. Plasma-mediated effects were reversible by washing.


Suppression of in vitro lymphocyte transformation during an experimental dermatophyte infection. During primary Trichophyton mentagrophytes infection of strain 2 guinea pigs, the colony-forming units (CFU) of fungi present within the lesion peaked between days 7 and 14, whereas the severity of the lesion itself peaked between days 11 and 16. Concomitant with the latter peak, a pronounced depression in the in vitro mitogenic activity of spleen cells (SPC) and lymph node cells (LNC) was observed. Only after resolution of the primary infection (day 21) did LNC show increased deoxyribonucleic acid (DNA) synthesis in the presence of fungal antigens. During cutaneous reinfection, there was no distinct peak fungal load and CFU appeared to decrease steadily during the accelerated course of a reinfection disease. LNC from guinea pigs with severe, ulcerated reinfection lesions generally exhibited a heightened response to fungal antigen in vitro. LNC from guinea pigs with mild reinfection dermatophytosis had depressed in vitro reactivity to mitogens and dermatophyte antigen. The suppression of blastogenic activity during dermatophyte infection appeared to be associated with autologous serum components, since increased DNA synthesis resulted when SPC or LNC were cultured with fetal calf serum. The depressed in vitro DNA synthesis of lymphocytes (cultured with dermatophyte antigens) that were harvested during reinfection was not accompanied by an impaired ability of infected guinea pigs to respond with a delayed-type hypersensitivity skin test in vivo. These results support the hypothesis that experimental T. mentagrophytes dermatophytosis is a cell-mediated hypersensitivity disease that can be modified by immunosuppressive control mechanisms elaborated or induced by the fungus.


Humoral immunity in the resolution of genital infection in female guinea pigs infected with the agent of guinea pig inclusion conjunctivitis. Female guinea pigs infected in the genital tract with the chlamydial agent of guinea pig inclusion conjunctivitis were selectively immunosuppressed by varying regimens of cyclophosphamide (Cy) treatment. Temporary suppression of both humoral and cell-mediated immunity by daily treatment of Cy (25 mg/kg) for 13 days resulted in a prolonged infection, whereas daily treatment for the duration of the experiment totally prevented the development of humoral and cell-mediated responses and produced an intense and prolonged infection which did not resolve. When humoral immunity alone was suppressed by treatment with Cy (250 and 150 mg/kg) at 9-day intervals, the infection again did not resolve. Treatment with 100 mg of Cy per kg at 9-day intervals resulted in an extended infection which resolved concomitantly with the development of antibody to guinea pig inclusion conjunctivitis. These data indicate that humoral immunity is essential for the recovery of female guinea pigs from guinea pig inclusion conjunctivitis genital infection. A market weight loss was observed which could not be attributed to Cy treatment alone. Edematous and ulcerative changes of the external genitalia were also noted.


Protection against experimental cholera by oral or parenteral immunization. Comparisons were made between the antigenic potency and protective capacity of several cholera toxin derivatives. Rabbits were immunized parenterally with 50 microgram of cholera toxin, A subunit, B subunit, procholeragenoid, or Wyeth glutaraldehyde toxoid 20101. Examination of the antibody response curves revealed that cholera toxin elicited serum antitoxin responses that rose more quickly than in the subunit-immunized animals; however, antitoxin levels were of the same magnitude after 10 weeks. Parenteral immunization with procholeragenoid evoked antibody titers that were similar to the toxin, whereas Wyeth toxoid yielded only one-tenth the level of antitoxin. Oral immunization with procholeragenoid as well as Wyeth toxoid resulted in lower serum antitoxin titers than that achieved with parenteral immunization, despite the oral administration of 10 times the parenteral dose. Analysis of protection against live-cell challenge revealed that parenteral administration of procholeragenoid provided the best protection against fluid accumulation. Oral immunization with procholeragenoid also was very effective, whereas oral immunization with B subunit or Wyeth toxoid resulted in minimal protection. Also, the A subunit provided surprisingly more protection than did cholera toxin.


Generalized infection and lack of delayed hypersensitivity in BALB/c mice infected with Leishmania tropica major. The susceptibility of a few strains of mice to a subcutaneous injection of Leishmania tropica major, the causative agent of cutaneous leishmaniasis in humans, was studied. The infection in six strains (CBA, AKR/J, AKR/cu, C57BL/6, A/J, and C3H) remained cutaneous, and the animals recovered within 3 to 4 months. In contast, the infection in BALB/c became generalized and killed 1005 of infected animals. Intraperitoneal injection of infected liver of BALB/c to A/J and syngeneic mice produced a lethal disease in BALB/c but no infection in A/J mice. Lower doses of the parasite produced a lethal infection in BALB/c but no apparent disease in A/J. Hence, the host rather than the parasite is responsible for the outcome of the disease. The peak antibody titer of BALB/c mice was not significantly higher than that of A/J mice. However, BALB/c failed to show any delayed hypersensitivity to leishmania tested by footpad reaction, whereas A/J mice showed a strong response.


Effect of influenza infection on the phagocytic and bactericidal activities of pulmonary macrophages. The effect of mouse-adapted influenza A/PR/8/34 virus on pulmonary macrophage function was evaluated by using an in vitro system which allowed direct virus interaction with macrophages and then separate analysis of the steps required for bacterial clearance by macrophages. Infection of macrophages with this virus resulted in the appearance of a hemagglutinating activity on the macrophage surface; expression of this activity was inhibited by amantadine, 2-deoxyglucose, and cycloheximide and by pretreatment of the virus inoculum with ultraviolet light and specific antiserum. Since there was no release of extracellular virus, this growth cycle appeared to be incomplete (abortive). After influenza infection, net ingestion of viable Staphylococcus aureus by macrophage monolayers was unaltered and there was no change in the fraction of the monolayer which ingested cocci over a wide range of bacterial inputs. Influenza-infected macrophages also inactivated intracellular S. aureus at a rate indistinguishable from controls. Therefore, these in vitro studies do not support the hypothesis that the defect in pulmonary antibacterial mechanisms associated with influenza infections results from a direct effect of virus infection on either the phagocytic or bactericidal activity of resident pulmonary macrophages.


Synergistic effect of polyriboinosinic acid:polyribocytidylic acid and either bacterial peptidoglycans or synthetic N-acetylmuramyl peptides on production of adjuvant-induced arthritis in rats. Lewis rats developed polyarthritis after a single injection of a water-in-oil emulsion containing various peptidoglycans (PGs) derived from Lactobacillus plantarum. A copolymer of polyriboinosinic acid and polyribocytidylic acid markedly potentiated the arthritogenicity of these PGs. The synthetic adjuvants N-acetylmuramyl-L-alanyl-D-isoglutamine (MurNAc-L-Ala-D-isoGln) and MurNAc-L-Ala-D-Gln were non-arthritogenic, but they did produce severe arthritis when mixed in a water-in-oil emulsion with a copolymer of polyriboinosinic acid and polyribocytidylic acid. Substitution of either L-isoGln or D-isoAsn for the D-isoGln in the MurNAc-L-Ala-D-isoGln markedly reduced its capacity to induce the disease. Taken together with the results of skin testing against various PGs and MurNAc-L-Ala-D-isoGln in the diseased rats, the present results suggest that (i) a minimal essential structure required for development of polyarthritis is related to a larger molecule than either MurNAc-L-Ala-D-isoGln or a monomer of PG, probably to a dimer of PG, and (ii) an antigenic determinant(s) for the delayed-type skin hypersensitivity to PGs exists on a common structure shared among these PGs, possibly somewhere on a monomer of PG not on N-acetylmuramyl peptides.


Respiratory syncytial virus infection in inbred mice. Respiratory syncytial virus infected the nose and lungs of each of 20 strains of inbred mice, with viral titers varying 100-fold from least permissive to most permissive strains. Viral titers appeared to be under genetic control, but did not correlate with the H-2 haplotype.


Plague antibody in large African mammals. Plague hemagglutinating antibodies to a titer of 1:1,024 were demonstrated in 6.6% of buffalo and 0.3% of elephant sera tested 1 year after a plague epidemic in the same area.


Enhanced hydrogen peroxide release from macrophages stimulated with streptococcal preparation OK-432. Wheat germ lectin was found to be a potent triggering agent for hydrogen peroxide release from mouse peritoneal macrophages. Macrophages stimulated by intraperitoneal injection of OK-432, a lyophilized attenuated streptococcal preparation, were highly responsive to wheat germ lectin.


Phosphoenolpyruvate-dependent sucrose phosphotransferase activity in five serotypes of Streptococcus mutans. An inducible phosphoenolpyruvate-dependent sucrose phosphotransferase system has been demonstrated in decryptified cell suspensions of the various common serotypes of the cariogenic microorganism Streptococcus mutans.


Investigation into the occurrence of toxoplasma in pharyngeal and palatine tonsils by inoculation of tonsillar tissues into mice. Among randomly chosen persons in the age group one to seven years the generally accepted proportion of positive dye tests for Toxoplasma is about eight per cent. The 124 children in this age group studied by us represent a selected group in which a higher percentage is to be expected. The group included 63 boys and 61 girls, from whom no blood samples were available for serological investigation. An arbitrary percentage of twice the eight per cent would result in 20 cases with a positive dye test. In the four successful attempts to isolate Toxoplasma all four cases were boys. Among 20 persons aged 14 to 21 years in whom a positive dye test could actually be established, one attempt to isolate the organism in a girl of 16 years of age was successful. No generalized lymph node enlargements were mentioned in the history of the five successful cases, only relapsing laryngo-pharyngeal symptoms. In the five successful isolations the Toxoplasma organisms present in the tonsillar organs appeared not to be virulent for the mice used; the organism was recovered in the cystic form from the brains of the mice. In subsequent animal passages the virulence could be influenced by the number of cysts per inoculum.


Radioimmunoassay in the detection of the hepatitis B e antigen/antibody system in asymptomatic carriers of hepatitis B surface antigen. Correlation with serum Dane particle associated DNA polymerase activity. A radioimmunoassay for hepatitis e antigen (HBeAg) and antibody to e (anti-HBe) was developed and sera of 71 asymptomatic chronic carriers of hepatitis B surface antigen (HBsAg), in 44 of whom liver biopsy was obtained, were tested. In addition, testing for Dane particle associated DNA polymerase activity was performed in all sera. HBeAg was detected in 14 subjects (19.7%) and anti-HBe in 46 (64.8%). The highest proportion of HBeAg positivity (40%) was found among carriers with histological evidence of chronic hepatitis, whereas anti-HBe was present in 80% of carriers with normal liver histology, in 58% of carriers with non-specific reactive hepatitis and in 60% of carriers with chronic liver lesions. DNA polymerase activity was present in 92.8% of sera positive for HBeAg, in 13% of sera positive for anti-HBe, and in 9% of sera negative for both markers. Our results demonstrate that not all HBsAg carriers reactive to HBeAg show evidence of chronic hepatitis nor, conversely, that anti-HBe is invariably associated with the healthy carrier state of HBsAg. Finally, circulating Dane particles, as revealed by the presence of serum specific DNA polymerase activity, may also be present in anti-HBe positive sera other than those of some HBsAg carriers lacking both HBeAg and anti-HBe.


Rapid high-pressure liquid chromatographic analysis of verapamil in blood and plasma. A high-pressure liquid chromatographic assay procedure has been developed for verapamil in blood or plasma. A paired-ion solvent system with a reversed-phase column is employed. The procedure is specific for verapamil and the retention times of the major metabolites are identified. This procedure is sensitive to a lower blood concentration of 1 ng/ml and standard curves were found to be linear up to the highest concentration tested, 500 ng/ml. Several drugs were tested for interference with the assay, but none were found to cause any problems. The procedure is simple, rapid and permits the analysis of up to 25 samples per day.


Protein-protein interactions studied by counter-current distribution. I. Theoretical computations. Many biological macromolecules are known to interact either with themselves, with other macromolecules or with small compounds. A simple equilibrium method for detecting and quantifying these interactions is to study the mutual influence of the molecules on their respective counter-current distribution in liquid-liquid biphasic systems. The theoretical counter-current distribution patterns for the components in an interacting system, A + B in equilibrium AB, have been calculated for two models in order to establish the boundary conditions and to optimize the experimental procedure. The patterns have been calculated for a range of association constants, partition coefficients and initial concentrations of the two reactants.


Determination of residues of methomyl and oxamyl and their oximes in crops by gas-liquid chromatography of oxime trimethylsilyl ethers. The gas-liquid chromatographic behavior of methomyl (methyl N-[(methyl-carbamoyl)oxy]thioacetimidate), oxamyl (methyl N',N'-dimethyl-N-[(methylcarbamoyl)oxyl]-1-thiooxamimidate), their respective oxime hydrolysis products and the trimethylsilyl (TMS) ethers of the oximes on 5% OV-1 was studied under isothermal conditions using a flame-photometric detector in the sulfur-selective mode. In contrast to the behavior of the parent carbamates and underivatized oximes, the oxime-TMS ethers readily produced symmetrical peaks of consistent size. Quantities of derivative equivalent to at least 0.25 ng of oxime were easily measurable. Derivative formation was reproducible for standards over the range 10.0 to 0.25 microgram/ml in benzene and at 10.0 and 0.50 microgram/ml in the presence of extractives from tomato, carrot and celery at concentrations equivalent to 10 g/ml of crop. Derivative yields from crop extract fotifications were 89% or better in most cases. Both the carbamates and oximes were simply and consistently recovered in high yield from crops fortified at 1.00 and 0.05 ppm using the procedures described. The inclusion of a second analytical step provided separate analysis for oximes and carbamates. The application of these observations to the analyses of residues in crops is discussed.


Gas chromatographic determination of methylcyclopentadienylmanganesetricarbonyl in biological tissues and fluids. The procedure described for quantitating methylcyclopentadienylmanganese-tricarbonyl (MMT, a fuel additive) in small samples of biological fluids and tissues is based on extracting the MMT into hexane containing biphenyl as internal standard, followed by gas chromatographic analysis. With flame ionisation detection, as little as 1-2 ppm of MMT in tissue can be determined relatively easily. The method is also applicable to in vitro investigations of MMT metabolism, and it has been used to show that the enzymic oxidation of MMT by rat-liver microsomes is a cytochrome P-450-dependent process.


Chromatographic study of local anaesthetics--basic esters of substituted carbanilic acids. II. The relationship between chromatographic values, other physico-chemical parameters and activity. RM values and the increments delta RM(CH2) have been calculated from the chromatographic values RF of a group of anaesthetics comprising an homologous series of basic esters of substituted carbanilic acids. A linear relationship is demonstrated between RM values from partition chromatography and the partition coefficient (log P) or the substitution constant mu. The chromatographic parameter RM is also correlated with the pharmacological characteristic log U, the logarithm of the surface anaesthetic activity.


Use of native fluorescence measurements and stopped-flow scanning technique in the high-performance liquid chromatographic analysis of catecholamines and related compounds. Simultaneous separation of catecholamines and tryptophan metabolites has been carried out using a reversed-phase partition mode of high-performance liquid chromatography (HPLC). Compounds are detected and measured via their native fluorescence emitted with an excitation wavelength of 285 nm and the emission cut-off filter of 340 nm. Sample preparation is minimized and the assay is selective and well-suited for routine analyses. The sensitivity of the method is in the nanogram range. The identity of chromatographic peaks is confirmed by their excitation spectra, obtained by the stopped-flow fluorescence scanning method.


Atenolol determination by high-performance liquid chromatography and fluorescence detection. A high-performance liquid chromatographic procedure using a fluorescence detector for the analysis of atenolol in plasma and whole blood is described. It employs a simple and rapid method of preparation. Atenolol and metoprolol as the internal standard are chromatographed as ion pairs with heptanesulfonic acid. The method is sensitive and reproducible with accurate detection at concentrations as low as 2 ng/ml in whole blood and plasma, and a coefficient of variation of 4.7% over the range 2 ng/ml to 1000 ng/ml.


Quantitative determination of plasma oxyphenbutazone by gas-liquid chromatography with selective nitrogen detection. A sensitive and specific gas chromatographic method, using the nitrogen-phosphorus detector for the detection and determination of oxyphenbutazone extracted from plasma is described. The method involves extraction and back-extraction steps followed by derivatization of both oxyphenbutazone and the internal standard with trifluoroacetic anhydride. The procedure permits the rapid and specific routine determination of oxyphenbutazone in plasma with a detection limit of 0.5 microgram/ml. The procedure is linear over the range of concentrations encountered after administration of a single oral therapeutic dose. No interference from the biological matrix is apparent. The suitability of the method for the analysis of biological samples was tested by studying the variation with time of oxyphenbutazone plasma concentrations in normal human volunteers over a period of several biological half-lives.


Charge-state and charge-continuum models in electrophoresis and isoelectric focusing of genetic variants. A model which combines the charge-state and charge-continuum models is considered in terms of the pH at which the electrophoretic analysis is made. "Unit" charge changes are obtained when the mutated charged group is at least 2 pH units apart from the pI of the protein. "Fractional" charge changes are found when the differences between the pK of the mutated charged group and the pI of the protein becomes progressively smaller, or when the pK of the ionizable group lies on the "wrong" side of the protein pI. It is possible to measure the pK of a modified group in a protein when the delta pI per protonic unit in a given family of proteins (e.g., haemoglobin mutants) is less than unity.


Affinity elution from a phosphonic acid-Sepharose derivative in the purification of human liver alkaline phosphatase. The compound p-aminobenzylphosphonic acid has been coupled via an azo linkage to tyraminyl-Sepharose 4B. This derivative at pH 6.0 bound most of the protein and all of the alkaline phosphatase in a crude preparation from human liver. The phosphatase was selectively eluted with the substrate 2-naphthylphosphate and a purification of 400-fold obtained. This step, when incorporated into a procedure for the purification of human liver alkaline phosphatase, yielded essentially pure enzyme.


Analysis of nicotine as a trichloroethyl carbamate by gas chromatography with electron-capture detection. Nicotine was subjected to reaction at 90 degrees with trichloroethyl chloroformate in the presence of pyridine to form a carbamate in which the pyrrolidine ring was opened. Upon heat treatment, this carbamate partially formed the corresponding olefin. About 10 pg could be detected with an electron-capture detector and 60 pg with an alkali flame-ionization detector. The extraction was studied with 14C-labelled nicotine. Methylene chloride was suitable for extraction from diluted plasma, whereas toluene containing 5% of heptafluorobutanol was used in a re-extraction step and also as the chloroformate reaction medium. Due to a nicotine blank the limit for quantitative determinations was 10 ng/ml in plasma (sample volume 1 ml). N-n-Propylnornicotine was used as an internal standard. The precision at the 30 ng/ml level was +/- 8.8% (n = 7).


Determination of (E)-2,3-dichloro-4-methoxyphenyl)-2-furanylmethanone-O-[2-(diethylamino) ethyl]oxime methanesulphonate (ANP-4364) in plasma using gas chromatography with electron-capture detection. A gas chromatographic method has been developed that permits the accurate and specific determination of the antianginal agent ANP-4364 in plasma. ANP-4364 is extracted with n-hexane containing ethyl chloroformate and, after a clean-up procedure, derivatized to the trichloroethyl carbamate, which is assayed on a gas chromatograph equipped with an electron-capture detector. Accurate determinations are possible over a concentration range from 1 to 50 ng/ml of ANP-4364 in plasma with a relative standard deviation of 7.5%. The minimum detectable concentration is 0.5 ng/ml. Plasma levels of ANP-4364 in dogs receiving oral (10 mg/kg) or intravenous (0.1 mg/kg) dosing have been determined.


High-performance liquid chromatographic analysis of rosoxacin and its N-oxide metabolite in plasma and urine. A high-pressure liquid chromatographic method for the analysis of rosoxacin and its pyridyl N-oxide metabolite in plasma and urine extracts is described. A statistical evaluation of the assay data has shown acceptable accuracy and precision for 0.5 to 25 microgram of rosoxacin or the metabolite per ml of plasma and for 2.5 to 60 microgram/ml of either compound in urine. The minimum quantifiable level for rosoxacin was 0.13 microgram/ml in plasma and 0.64 microgram/ml in urine; for the metabolite in plasma and urine, the corresponding values were 0.21 and 0.60 microgram/ml, respectively. The method was applied to plasma and urine from three dogs medicated orally with 5 mg/kg of rosoxacin. The pharmacokinetic parameters calculated for rosoxacin were: plasma halflife, 1.9 h; plasma clearance, 65 ml/min; volume of distribution, 11.31. The average total urinary excretion of rosoxacin as free and conjugated rosoxacin and its free N-oxide was 7.7 +/- 0.2% over the 48-h collection period.


Determination of cefatrizine in serum and urine by reversed-phase high-performance liquid chromatography. A fast and simple high-performance liquid chromatographic procedure for the determination of cefatrizine, an orally active cephalosporin, in serum and urine is proposed. Reversed-phase liquid chromatography on the octoadecylsilane chemically bonded microparticulate packing, using methanol in 0.03 M sodium phosphate buffer (pH 5) as eluent, was used to separate and quantitate the antibiotic. The samples were analysed after deproteinization with trichloroacetic acid and injection of the clear supernatant. The accuracy and reproducibility of the procedure were investigated by determination of the cefatrizine content in spiked serum and urine samples, using cephradine as the internal standard.


Celite column chromatography followed by reversed-phase high-performance liquid chromatography: a simple, two-step method for separating 14 testicular steroids. The separation of 14 major testicular steroids has been effected by a simple, two-step procedure involving Celite column chromatography followed by reversed-phase high-performance liquid chromatography. The technique does not alter the resolved steroids, which are then available for subsequent analysis.


Classification of fungi by means of pyrolysis-gas chromatography-pattern recognition. Repetitive samples of three strains of the mould Penicillium were subjected to pyrolysis-gas chromatography (Py-GC). From the chromatograms, 26 peak heights were used in a subsequent SIMCA pattern recognition analysis. This data analysis gives a marked improvement in the classification of the samples (100% correct, 85% unique) in comparison with the traditional analysis based on the average chromatogram of each class (92% correct, 45% unique). The data analytical method is described in detail using the Py-GC data as an illustration.


Rapid and simple clean-up and derivatization procedure for the gas chromatographic determination of acidic drugs in plasma. A rapid and simple clean-up and derivatization procedure that can be generally applied to the gas chromatographic (GC) determination of acidic drugs of various chemical and therapeutic classes is described. The drugs are extracted from acidified plasma with chloroform containing 5% of isopropanol, which is then evaporated. The residue is dissolved in toluene, then the drugs are back-extracted into a small volume of a methanolic tetramethylammonium hydroxide solution. The solution obtained is added to N,N-dimethylacetamide and the drugs are treated with n-butyl iodide and chromatographed as their n-butyl esters. Retention times are given for three different columns; the results show that in all but three instances one derivative is formed. The recoveries in the extraction steps were determined by GC and UV spectrophotometry. The recovery of the back-extraction and of the extraction from plasma with chloroform-isopropanol is fairly good. For some drugs toluene can be used as the extraction solvent, which has the advantage that no evaporation of the extract is necessary, and it can be extracted directly with the tetramethylammonium hydroxide solution. Chromatograms of blank plasma treated according to the described procedure do not show interfering peaks from endogenous compounds. The advantages of the proposed method are as follows. It is a standard procedure that can be used for different types of drugs, using one standard column, with no interference from endogenous compounds. Separation is achieved between drugs that differ only in the number or position of the methyl groups and between a drug and its demethylated metabolite(s). No decomposition of the products occurs during the alkylation process and injection of the resulting neutral solution is not detrimental to the column material.


Reproducibility of pyrolysis-gas chromatographic analyses of the mould Penicillium brevi-compactum. The reproducibility of multivariate measurements is discussed. Reproducibility of a second kind is defined, in which part of the variability between samples is described by a principal components model. The use of this generalized reproducibility is shown to give an improved precision in the pyrolysis-gas chromatography of a Penicillium species.


Characterization of native multicomponent protein mixtures by one- and two-dimensional gradient electrophoresis. An arrangement of one- and two-dimensional polyacrylamide gradients has been applied to the analysis of multicomponent mixtures of soluble proteins: (1) two-dimensional 3-25% slabs; (2) 3-40% cylinders; and (3) 20-50% cylinders. The run in the first dimension is performed in non-restrictive agarose gel to ascertain a "pure" charge abscissa. Gradient electrophoresis is performed over prolonged periods in order to reach or closely approach the exclusion (pore) limit, which is taken as an operational parameter for the hydrodynamic size of globular molecules irrespective of their frictional ratios. For each of the three gradients one to four reference mixtures were formulated that allow size evaluations of non-denatured proteins. Human serum again proved its usefulness as a universal reference mixture for newly developed techniques. The number of stainable components is in accordance with expectation based on the broad knowledge of serum proteins. Two-dimensional agarose/gradient electrophoresis seems to fulfil the one band-one protein strategy of high-resolution techniques.


Determination of food preservatives and saccharin by high-performance liquid chromatography. The quantitative analysis of benzoic and sorbic acid, methyl, ethyl and propyl esters of p-hydroxybenzoic acid and saccharin in foodstuffs is described. These compounds are quantitatively extracted with disposable clean-up columns packed with Extrelut and simultaneously determined by high-performance liquid chromatography on reversed-phase columns. Complicated matrices such as cheese, cake, ketchup and chocolate were tested and recoveries were generally better than 95% in the concentration ranges normally used in the food industry.


Measurement of testosterone with a high-performance liquid chromatograph equipped with a flow-through  ultraviolet spectrophotometer. A technique is described for measuring nanogram amounts of testosterone using high-performance liquid chromatography with detection by a flow-through spectrophotometer. The addition of a non-radioisotopic internal standard (4-androsten-11 beta-ol-3,17-dione) to the biological specimen automatically corrects for testosterone losses due to extraction and non-quantitative sample injection into the high-performance liquid chromatograph. This new method, which can be performed by inexperienced personnel, is shown to be rapid, precise, accurate and specific for testosterone.


Affinity chromatography on concanavalin A-Sepharose of antigenic fractions of human seminal plasma. Elution diagrams obtained on affinity chromatography show that the antigenic fractions of human seminal plasma studied, namely (a) the non-dialyzable components of human seminal plasma and (b) its trichloroacetic acid-soluble fraction, (c) the trichloroacetic acid-soluble fraction of whole human seminal plasma and (d) the pronase digested human seminal plasma, are complex mixtures of glycoproteins with minor amounts of polysaccharides. Some of these glycoproteins contain significant percentages of carbohydrates while others contain only trace amounts. Most of the glycoproteins carry non-reducing end-chain groups comprising alpha-D-glucopyranosyl, alpha-D-mannopyranosyl or sterically related residues.


Fractionation of histones on a metal ion equilibrated cation exchanger. I. Chromatographic profiles on an Amberlite IR-120 (A1 3+) column. Amberlite IR-120, a polystyrene sulphonate type of cation exchanger, equilibrated with A1 3+ ions, has been employed for the fractionation of whole histone. This adsorbent permits the quantitative and reproducible recovery of whole histone in six fractions.


Enzymatic reactions on thin-layer chromatographic plates. II. Phospholipase A2 hydrolysis of phosphatidylcholine and separation of the products on a single plate. A procedure for the phospholipase A2 hydrolysis of phosphatidylcholine on a thin-layer chromatographic plate and subsequent separation of the products on the same plate is described. A 0.2-0.8-mg amount of Russell's viper venom (phospholipase A2) in 0.2 ml of 0.005 M calcium chloride solution was applied on a 0.5-mm silica gel G plate as a band over which 2-5 mg of egg phosphatidylcholine in 0.2 ml of diethyl ether containing 5% of methanol was evenly applied. After the reaction had proceeded for 15-20 min in a diethyl ether-saturated chamber at 25 degrees, the plate was developed with chloroform-methanol-water (65:25:4). The bands were identified and their contents extracted. The extent of hydrolysis under different reaction conditions was evaluated from the amount of lysophosphatidylcholine formed. Approximately 74.6% (maximum) conversion was obtained within 15 min at 25 degrees using a substrate to enzyme ratio of 4:1. The acyl group distributions in the 1- and 2-positions of hen egg phosphatidylcholine obtained from the gas-liquid chromatographic analysis of the methyl ester corresponding to the lyso and free fatty acid band agreed with those obtained by the method of Wells and Hanahan. The method is also applicable to phosphatidylethanolamine.


[Sensitive method for continuous detection of peptides and proteins using the biuret reaction and a copper-Sephadex reactor (author's transl)]. We describe a detection method relying both on the copper displacement from a Sephadex gel by peptides and proteins, and on the subsequent colorimetric determination of the complexed copper. The system described is fully automated and it permits a continuous analysis of column effluents. The choice of cuprizone as a detecting reagent for copper, enables one to bring the detection limit down to 200 ng for albumin and 60 ng for alanylglycylglycin. The specificity of the method is the same as the biuret reaction. Some examples of the possible applications are given.


High-performance liquid chromatographic assay for acetaminophen and phenacetin in the presence of their metabolites in biological fluids. We propose a method in which tracer amounts of a radiolabeled compound are used as the internal standard for the same unlabeled compound in high-performance liquid chromatography. The approach is valuable when a response from the internal standard becomes undesirable due to the presence of interference by the metabolites. We tested our approach with phenacetin and its metabolites, acetaminophen, 2-hydroxyphenacetin, N-hydroxyphenacetin, phenetidine, acetaminophen sulfate conjugate and acetaminophen glucuronide conjugate in biological fluids with the use of [14C]phenacetin and [3H]acetaminophen as the internal standards, and were able to quantitate both phenacetin and acetaminophen simultaneously. We also tested the alternative approach in which the unlabeled drug was used as internal standard for tracer amounts of the same radiolabeled compound, with phenacetin and acetaminophen as the internal standards for tracer amounts of [14C]phenacetin and [3H]acetaminophen. Again, we were able to quantitate the two tracer radiolabeled compounds simultaneously.


Gas-liquid chromatography of some alkyl derivatives of 5-fluorouracil. Various procedures for converting 5-fluorouracil into its methyl, butyl and hexyl derivatives are described. Structures were established as the N,N'-dialkyl derivatives using mass spectrometry or combined gas-liquid chromatography-mass spectrometry. The reaction conditions, i.e., the amount of derivatization reagents and reaction time, were optimized. Gas-liquid chromatographic characteristics of the derivatives were investigated on different stationary liquid phases, and 2% or 3% SP-2250, 5% XE-60 and 5% OV-1 were found to be superior. With 5-chlorouracil as the internal standard a linear response for the various derivatives was observed in the microgram range. The applicability of the different dialkyl derivatives in the measurement of 5-fluorouracil in biological materials is discussed.


Determination of bromocriptine in plasma: comparison of gas chromatography, mass fragmentography and liquid chromatography. Gas chromatographic, mass fragmentographic and liquid chromatographic techniques for the determinations of bromocriptine (2-bromo-alpha-ergocriptine; Parlodel) in human plasma are described. These methods were found to be suitable for determining concentrations of bromocriptine down to 0.5, 1.0 and 10.0 microgram/l, respectively. Accuracy, specificity and analytical capacity were satisfactory for all three methods. Gas chromatography was compared with liquid chromatography, and the two methods were demonstrated to give identical results in patients treated with bromocriptine for Parkinson's disease. Gas chromatography was also compared with mass fragmentography, and the results from these two assays were also in agreement.


Temperature effects in affinity chromatography of alanine aminotransferase. The effects of temperature on the elution parameters of alanine aminotransferase and albumin were studied on substituted agaroses designated for the affinity chromatography of the enzyme. The elution volume of alanine aminotransferase depended logarithmically and and the elution volume of albumin linearly on temperature. Both decreased when the temperature increased. It was concluded that the observed elution volume of alanine aminotransferase was due to two types of retardation mechanisms: specific (the logarithmic mode) and non-specific (the linear mode), both of which were additive. Thermodynamic parameters were estimated for the specific mode and the calculation resulted in delta Ho and delta So values of ca. -40 kJ/mole and -140 J/mole . degrees K, respectively.


High-performance liquid chromatography of 2,6- and 2,4-diaminotoluene, and its application to the determination of 2,4-diaminotoluene in urine and plasma. 2,4-Diaminotoluene is used for the production of industrial dyes, and along with the 2,6-isomer, as an intermediate in the production of polyurethane foams. 2,6- and 2,4-diaminotoluene were resolved as sharp peaks by normal-phase high-performance liquid chromatography in 3 min by an acetonitrile-water-saturated chloroform elution solvent (8:2, v/v) with detection by ultraviolet absorbance at 250 nm. The relationship between peak height and amount injected was linear over a range of 0.025-2 microgram for both compounds. Retention times and peak heights were highly reproducible. Detection was very sensitive, allowing quantitation of 1-2 ng of either compound. Quantitative recovery of 2,4-diaminotoluene from spiked urine and plasma samples was obtained by extraction with methylene chloride.


Separation of peptides by high-pressure liquid chromatography for the identification of a hemoglobin variant. High-pressure liquid chromatography on a reversed-phase column has been used to separate the tryptic peptides of a human hemoglobin variant which was then identified as hemoglobin E.


High-performance liquid chromatographic separation of cobalamins. Physiological cobalamins were separated by means of high-performance liquid chromatography (HPLC). Optimal conditions for elution of methylcobalamin, adenosylcobalamin, hydroxycobalamin and cyanocobalamin were determined. Excellent separation and resolution of these physiological cobalamins by HPLC were achieved. In addition, several cobalamin analogues were also studied and shown to be separable from the physiological forms. HPLC provides a rapid, sensitive, reproducible means of characterizing physiological cobalamins.


Ion-exchange separation of nucleic acid constituents by high-performance liquid chromatography. The high-performance liquid chromatographic separation of a large variety of nucleic acid constituents on a silica-based, weak-anion exchange column was accomplished. Using this technique it was possible to achieve some relatively difficult separations, such as the separation of 2'-, 3'-, and 5'-AMP, and the separation of a mixture of ribo- and deoxyribo-nucleosides and -nucleotides. A number of other separations are demonstrated by isocratic or gradient elution. These include the separation of a mixture of nucleoside monophosphates, the separation of a mixture of nucleoside mono-, di-, and triphosphates, the separation of a mixture of nucleosides and bases, and the separation of a mixture of nucleotide oligomers. These chromatographic separations were accomplished using relatively simple experimental procedures at ambient temperatures and involved relatively short analysis times. Excellent separations were obtained, in most cases, by adjustment of buffer concentration and pH, or by addition of an organic modifier. In some cases, it was necessary to use gradient elution to achieve optimum resolution.


[Quantitative determination of drugs by in situ spectrophotometry of chromatograms for pharmacokinetic studies. I. Sulpiride and other benzamides, vincamine, naftazone (author's transl)]. Assays are proposed for sulpiride and other benzamides, vincamine and naftazone in plasma (or blood) and urine with direct UV reflectance spectrophotometry on this are applied directly on TLC along with a calibration curve on each plate. Plasma (or total blood) samples are extracted, and an internal standard is added before aplication; slopes of the obtained calibration curves do not change significantly from plate to plate, thus allowing several determinations on the same plate. The sensitivity is 2 microgram in a 1-ml sample (amount applied 30 ng) for sulpiride and related compounds and about the same for vincamine. Naftazone is determined in plasma with simultaneous reflectance and transmittance spectrophotometric measurements at 520 nm on chromatoplates sprayed with lead acetate, the sensitivity reached is 10 ng in a 1-ml sample (amount applied 0.5 ng). For all drugs studied, the proposed techniques are specific, reliable and sensitive enough and can be used to perform pharmacokinetic studies in human or in animal after administration of doses in the therapeutic range.


Analysis of natural corticosteroids in adrenal extracts and in biological fluids by high-performance liquid chromatography. A liquid chromatographic procedure is described for the analysis of the principal natural corticosteroids in extracts of adrenal glands. Microparticulate silicic acid columns and gradients of methanol in chloroform are used: conditions are described for the quantitative analysis of the single principal steroidal components of adrenal extracts for pharmaceutical use and of adrenal extracts of rats. In the last case, the use of a 5-micron silica column with the appropriate gradient allows the determination of corticosterone and of 18-hydroxydeoxycorticosterone, which were identified by means of mass spectrometry on their eluates. A single analysis can be performed on the extract of 15 mg of rat adrenal tissue. For the last type of analysis, isocratic conditions on a 10-micron LiChrosorb Diol column are also described. The application of the gradient elution procedure to the analysis of steroidal compounds in human plasma is also described.


Normal-phase high-performance liquid chromatographic determination of neomycin sulfate derivatized with 1-fluoro-2,4-dinitrobenzene. A high-performance liquid chromatographic (HPLC) assay method has been developed for quantifying neomycin sulfate powders and ointments containing neomycin. A dinitrobenzene derivative of neomycin is formed and then chromatographed isocratically on a normal-phase system. Neomycin B and C and be separated and quantified with a relative standard deviation of approximately 1%. When monitored at 350 nm, the method is sensitive to about 1 ng of neomycin base per column injected. Gradient elution and isocratic HPLC methods for the analysis of derivatized neamine have also been developed. The relative standard deviation is less than 1% for the isocratic method. The HPLC method is applicable to the analysis of other aminoglucoside antibiotics.


Effect of transition metals on recovery from plasma of the growth-modulating tripeptide glycylhistidyllysine. Isolation and purification of growth-modulating peptides from biological sources is often accompanied by excessive losses of bioactive material. During the isolation of a growth-modulating tripeptide glycylhistidyllysine (GHL) from human plasma, copper and iron were found to co-isolate with the peptide. Studies with [3H]GHL demonstrated that these metals interfere at several steps of the procedure for the isolation of GHL from plasma (gel filtration chromatography, high-pressure silica-gel). Removal of these metals with an insoluble chelating resin (Cellex 100) enhanced recovery of [3H]GHL from plasma 8-fold. These results suggest that removal of transition metals may aid in the recovery of peptides which are difficult to isolate from biological sources.


Use of reversed-phase high-performance liquid chromatographic analysis for the determination of provitamin A carotenes in tomatoes. The usual methods for provitamin A evaluation of foods convert the total pigment amount, determined spectrophotometrically, into vitamin A units. Since the totally inactive lycopene is the major carotenoid in the tomato, such readings result in erroneously high provitamin A values. In view of the recent development of chemically bonded, reversed-phase, microparticulate packings and their use in high-performance liquid chromatography which combines highly accurate and reproducible resolution with the speed and ease of operation, a new method using such a system was developed to isolate carotenoid pigments from tomato samples. A 15-min column separation was thus achieved, dramatically decreasing the analysis time of the classical open column chromatographic procedures, which often result in unresolved and altered fractions due to long-term exposure to oxygen, light, solvents and sometimes adsorbent. beta-Carotene and lycopene were determined and quantitated in six tomato samples. beta-Carotene, 100% vitamin A-active, was expressed in International Units of vitamin A. The newly developed method gives a more reliable evaluation of the fruit potency in vitamin A than the methods of the Association of Official Analytical Chemists currently used for food composition tables.


Quantitation of polymethoxylated flavones in orange juice by high-performance liquid chromatography. A quantitative high-performance liquid chromatographic (HPLC) procedure for the determination of the five major polymethoxylated flavones (PMFs) in orange juice has been developed. It employs a unique ternary solvent system with coupled UV-fluorescence detection. The dual detectors were employed to determine the presence of interfering substances and served as a cross check on quantitation. Stop flow UV and fluorescence scanning was used to identify peaks and determine the presence of impurities. Although all five citrus PMFs fluoresce, some HPLC fluorescence peaks were too small to be of much practical use. All five citrus PMFs could be quantitated satisfactorily with the fixed wavelength UV (313 nm) detector. The HPLC procedure has been used to evaluate each step in the preparation. The optimum extracting solvent was selected and one time consuming step was eliminated, as it was found to be unnecessary. HPLC values for nobiletin and sinensetin are in good agreement with the thin-layer chromatographic (TLC) values in the literature. HPLC values for the other three flavones were considerably lower than those reported in the literature. The HPLC procedure is considerably faster than the TLC procedure with equal or superior precision and accuracy.


Thin-layer chromatographic determination of aldehydes encountered in foods as dimethones, octahydroxantheines and barbiturates. Aldehydes in foods can conveniently be characterized and determined by thin-layer chromatography (TLC) after converting them into their dimethones. For further confirmation the dimethones can be cyclized on the TLC plates or in solution to give octahydroxanthenes. Aldehydes also can be determined as their barbiturates. The aldehydes could be determined as dimethones and octahydroxanthenes in amounts up to 5 micrograms and as barbiturates in amounts up to 7.5 micrograms at their UV maxima.


Large-bore coated columns for sampling and concentration of organic volatiles in air, headspace and water analysis. Large-bore coated (LBC) columns were used as sampling and concentrating traps in analyses for traces of organic volatiles in air and water. This simple technique utilizes long metal columns thinly coated with SE-30 for direct trapping of the organics. The sample is simply passed through the LBC column; the trapped organics are then thermally desorbed onto a conventional porous polymer pre-column or onto a second LBC column. If desired, this can be shorter or narrower bore than the initial LBC sampling column. The sample is finally desorbed onto the gas chromatographic column for analysis. Multiple transfers between LBC columns are possible, with increased concentration at each transfer, resulting in a "concentration pump" effect. The technique offers the advantages of great simplicity, efficiency and ease of sample transfer. Samples are obtained with low back-pressure and minimal interfering artifacts. The system shows almost complete imperturbability to moisture. Indifference to moisture and the low back-pressure enable direct sampling of very large volumes of air and even breath. Direct sampling of aqueous systems was also possible. The latter area was not fully investigated but offers potential for water pollution analysis and in direct examination of biological fluids and aqueous flavor extracts where heat sensitivity is a problem. With LBC columns the sampling and concentration sequence exposes the substances sought to no more drastic conditions than those they will be subjected to in the process of gas chromatographic analysis.


Identification of ergot alkaloids with a photochemical reaction detector in liquid chromatography. An on-line photochemical reaction detector is described for the identification of ergot alkaloids by high-performance liquid chromatography. The fluorescence signal of alkaloids decreases within about 20 sec of irradiation and disappears selectively from complex chromatograms. The application of this principle to urine samples is described.


Use of 2,6-dinitro-4-trifluoromethylbenzenesulfonic acid as a novel derivatizing reagent for the analysis of catecholamines, histamines and related amines by gas chromatography with electron-capture detection. We have found that 2,6-dinitro-4-trifluoromethylbenzenesulfonic acid reacts rapidly and specifically with primary amines at room temperature. We have used this reagent for derivatizing phenylethylamines, including catecholamines, and histamine and 1-methylhistamine. After the reaction, hydroxyl groups were derivatized to form the corresponding trimethylsilyl ethers, and the final derivatives were analyzed by gas chromatography with electron-capture detection. These derivatives are stable, possess excellent gas chromatographic properties and are detected with high sensitivity. We have applied this method to the analysis of histamine and 1-methylhistamine in human urine.


Convenient method for the gas chromatographic analysis of hexosamines in the presence of neutral monosaccharides and uronic acids. A convenient gas chromatographic method has been devised for the analysis of hexosamines in the presence of neutral and acidic sugars, which involves sequential derivatization reactions of nitrous acid deamination, mercaptalation, and trimethylsilylation. This method allows rapid, simultaneous determination of 0.1-1 micromole samples of hexosamines with coefficients of variation of less than 3%.


Determination of unchanged hydralazine in plasma by gas-liquid chromatography using nitrogen-specific detection. A gas chromatographic method is described for the specific determination of unchanged hydralazine in plasma. On treatment with 2,4-pentanedione, hydralazine is converted into 1-(3,5-dimethyl-1-pyrazolyl)phthalazine, a stable compound which is easily extracted from biological material and determined quantitatively by gas chromatography with nitrogen-specific detection. 4-Methylhydralazine is used as an internal standard. The sensitivity of the method is ca. 10 ng/ml. Hydrazones of hydralazine do not interfere with the assay.


High-performance liquid chromatographic analysis of emetine after oxidative activation to a fluorescent product. A clinically useful analytical method is described for monitoring plasma levels of emetine. The drug is initially extracted from plasma with dichloromethane (0.3 volumes). The extract can be analyzed directly by paired-ion reversed-phase high-performance liquid chromatography to levels of 500 ng/ml of plasma by spectrophotometric monitoring of column effluent. For analysis of emetine at lower concentrations, the dichloromethane extracts are subjected to mild mercuric acetate oxidation prior to separation, thereby converting emetine to a fluorescent product. Spectrofluorometric monitoring of the column effluent readily extends the sensitivity of the assay to 10 ng of emetine/ml of plasma. At these levels measurements can be made with a precision of +/- 4%.


High-performance liquid chromatographic assay of betamethasone 17-valerate and its degradation products. A high-performance liquid chromatographic assay of betamethasone 17-valerate is described. The procedure may be use for quantitative assay of the degradation products, betamethasone 21-valerate and betamethasone, and the application to the analysis of ointments is described. The method is also suitable for the determination of the kinetics of decomposition from one experimental run, and the determination of rate constants from a four-compartment sequential reaction is described. The procedure is also applicable to other corticosteroids, and hydrocortisone 17-butyrate, hydrocortisone 21-butyrate, and hydrocortisone may similarly be determined without modification to the method.


Use of microbore columns for the separation of substances of biological origin. A gradient system for use with microbore columns is described and the repeatability of the system determined. A simple sampling device for use with aqueous biological samples is described, and the advantages of the high mass sensitivity of microbore columns for the analysis of such samples are discussed. The sensitivity of the  system is demonstrated by the determination of acetophenone in water at a level of one part per billion. The use of the apparatus for the separation of low-molecular-weight, non-ionic compounds contained in blood is also demonstrated. It is shown that excellent separations of such components can be obtained from as little as 400 microliter of serum.


Use of hydrophobic interaction methods in the isolation of proteins from endocrine and paraendocrine tissues and cells by high-performance liquid chromatography. We have recently described the separation of a large number of polypeptide hormones, related peptides and some protein standards by hydrophobic interaction high-performance liquid chromatography (HPLC). This paper reports the practical application of these methods to the reproducible isolation and separation of components of a mixture of immunoreactive calcitonin-like proteins (less than 25 kD) synthesised and secreted by human tumour cells in vitro. Using hydrophobic interaction HPLC on ODS-silica for both preliminary bulk fractionation and subsequent analytical separation greater than 80% recoveries of small (ng) quantities of immunoreactive proteins were obtained from samples containing less than 100 mg total protein, and characteristic profiles of synthesised and secreted materials were established. Using a partially purified hypothalamic extract, containing a number of small proteins (12--25 kD), we have also examined the effects of varying chromatographic conditions in an attempt to modify the separations obtained with ODS-silica using an acid-saline-acetonitrile gradient elution system at ambient temperature, and achieve further resolution of its components. No useful selective effects were observed when temperature, organic modifier, gradient profile or hydrophobic stationary phase were altered. These techniques may not therefore be inherently capable of completely resolving all components of natural protein mixtures. They do, however, offer an adjunct to and in certain cases a substitute for conventional methods of protein separation.


High-speed steric exclusion chromatography of biopolymers. Biopolymer separations were studied on Micropak TSK type SW columns, which contain an aqueous compatible steric exclusion support. Columns of two different pore sizes, designated 2000SW and 3000SW, were compared for separation of proteins and nucleic acids covering a molecular weight range of 13,500 to 340,000 and the effect of molecular shape and denaturation upon elution volume was investigated. Use of high-speed steric exclusion chromatography as a prefractionation step prior to ion-exchange chromatography in biopolymer purification schemes is discussed.


Application of 1,1'-carbonyldiimidazole-activated agarose for the purification of proteins. II. The use of an activated matrix devoid of additional charged groups for the purification of thyroid proteins. The use of 1,1'-carbonyldiimidazole-activated agarose for biospecific affinity chromatography is described. Activation of agarose with this carbonylating reagent gives a matrix devoid of additional charged groups. Conditions for the coupling of a range of ligands and leashes have been evaluated. The efficient purification of bovine trypsin, human thyroglobulin and sheep thyroid membrane glycoproteins demonstrates the suitability of the new activated matrix for affinity chromatography.


Application of an automated extractor/concentrator to the analysis of anticonvulsant drugs in blood serum by high-performance liquid chromatography. An automated extractor/concentrator has been used to extract a variety of anticonvulsant drugs from blood serum in a concentration range from 0.075 to 80 microgram/ml. The volume of serum required varies from 0.1 to 2.0 ml, depending on the concentration of drug. The average absolute extraction recovery is 90% larger; recoveries relative to an internal standard are greater than 95%. The serum extracts were analyzed by high-performance liquid chromatography on a reversed-phase column with an acetonitrile and phosphate buffer mobile phase. Comparison of results with gas chromatographic and immunoassay techniques was excellent.


Determination of apomorphine in plasma and brain tissue by ion-pair extraction and liquid chromatography. Apomorphine is extracted from plasma or tissue homogenate with ethyl acetate. After back-extraction into hydrochloric acid, the apomorphine is extracted as an ion pair with 3,5-di-tert.-butyl-2-hydroxybenzene sulphonate into a small volume of methylene chloride and the solution is injected into the chromatographic column. Apomorphine is separated on microporous silica with a mixture of aqueous perchloric acid, methanol and methylene chloride as the mobile phase. With absorbance measurement of the eluent at 254 nm the method permits the determination of 15 pmol of apomorphine in 1 ml of plasma or in a rat brain. The coefficient of variation was 4% at the 100 pmol level.


Liquid chromatographic determination of tetracycline in plasma and urine. Tetracycline is extracted from plasma (0.50 ml) as an ion pair with tetrabutylammonium into chloroform-1-heptanol (9:1). After re-extraction into an acidic aqueous phase the separation is performed by reversed-phase liquid chromatography using LiChrosorb RP-2 as the support and acetonitrile-water-phosphoric acid as the mobile phase. The chromatographic system shows a high selectivity for the separation of tetracycline analogues. A high detection selectivity is obtained by the use of photometric detection at 357 nm. Analysis of urine is performed by direct injection of the sample into the liquid chromatograph. The precision in the determination of tetracycline in plasma was about 10% (relative standard deviation) at drug levels of 200 ng/ml and 200 microgram. Urine samples containing 20 and 200 microgram/ml of tetracycline were determined with a precision of 3%.


Reversed-phase chromatography of zimelidine and similar dibasic amines. I. Analysis in biological material. Zimelidine, a new antidepressant, and its pharmacologically active metabolite, norzimelidine, have been determined quantitatively in biological material. The compounds are extracted from alkali-treated body fluid or tissue homogenate into diethyl ether-n-hexane (8:2, v/v), and re-extracted into a small volume of acidic aqueous phase (ca. 100 microliter), which is injected onto the chromatographic column. The chromatographic support is Nucleosil C18 (5 micron) and the mobile phase is phosphate buffer (pH 2)-acetonitrile (91:9, v/v) with the addition of an aliphatic amine, N,N-dimethyl-N-octylamine (4.10(-4) M), which improves the chromatographic performance. By using glass-lined stainless-steel tubes instead of the common precision-bore stainless-steel columns, peak symmetries were improved. Detection limits in plasma are in the range 2.3--4.7 nmol/1 for extractions from 1 ml. It is demonstrated that the quality of the quantitations are improved by use of an internal standard, norzimelidine E-isomer. The precisions obtained for both compounds at the concentration levels of 300 and 1500 nmol/1 are ca. 5--6% and 2% respectively for within-run variations, and 8--9% and 5% respectively for day-to-day-variations.


Determination of 5-aminolaevulinic acid and porphobilinogen by high-performance liquid chromatography. A strong anion-exchange column coupled to a strong cation-exchange column with acetate buffer as eluent or reversed-phase ion-pair chromatography on octadecylsilica with methanol-water containing 1-heptanesulphonic acid as the mobile phase is described for the simultaneous separation of 5-aminolaevulinic acid and porphobilinogen. The separation is applied to the development of a fast and simple method for determining the activity of the enzyme aminolaevulinic acid dehydrase in human erythrocytes. 5-Aminolaevulinic acid is used as the enzyme substrate and the enzyme activity is expressed as micromoles of porphobilinogen formed per ml of erythrocytes in 1 h at 38 degrees. 5-Aminolaevulinic acid and porphobilinogen can also be separated from the urine of prophyric patients but the UV detector has insufficient sensitivity for the determination of 5-aminolaevulinic acid.


Quantitative analysis of corticosteroids in adrenal cell cultures by capillary column gas chromatography combined with mass spectrometry. A quantitative method using glass capillary column gas-liquid chromatography (GLC) has been established for the analysis of corticosteroids in small biological samples such as a few milliliters of medium from steroidogenic cultured cells. The steroid extract is separated in three main thin-layer chromatographic fractions. The steroids assayed in these fractions are: less polar fraction (C21O2) steroids), 20 alpha-dihydroprogesterone, pregnenolone and 20 alpha-dihydro-pregnenolone; intermediate fraction (C21O3 steroids), deoxycorticosterone, 11-oxo- and several hydroxy-20 alpha-dihydroprogesterones; corticosteroid fraction (C21O4 steroids), corticosterone and 18-hydroxydeoxycorticosterone. The method has been assessed by characterizing the steroid structures and checking the purity of GLC effluents through direct coupling of the capillary column to the electron-impact or chemical-ionization source of a computerized mass spectrometer. Applications to the quantitative evaluation of the endocrinological status of newborn rat adrenocortical cell cultures under various conditions of development, hormonal stimulation or specific inhibition by drugs are described.


Identification and quantitation of nucleosides, bases and other UV-absorbing compounds in serum, using reversed-phase high-performance liquid chromatography. I. Chromatographic methodology. A comprehensive investigation of the high-performance liquid chromatographic separation of nucleosides, their bases and other low-molecular-weight UV-absorbing compounds that might be found in serum is reported. A buffer-methanol gradient was used in conjunction with chemically bonded, microparticulate columns to separate many of the biologically important compounds under study in minimal time with maximal resolution. Retention data, absorbance ratios (280/254 nm) and fluorescence responses are reported for 86 nucleosides, bases, nucleotides and other UV-absorbing compounds commonly encountered in biological studies.


Identification and quantitation of nucleosides, bases and other UV-absorbing compounds in serum, using reversed-phase high-performance liquid chromatography. II. Evaluation of human sera. The reversed-phase mode of high-performance liquid chromatography was used to investigate the profiles of low-molecular-weight, UV-absorbing compounds in human serum. Identification techniques are described which allow for the identification of picomole amounts of the nucleosides, bases and other compounds in several microliters of serum ultrafiltrate. The sera from 31 normal subjects (17 males, 14 females) showed very consistent profiles. A total of 12 compounds were identified and quantified in normal serum. The analysis of sera from over 150 patients with various types of neoplasia and other diseases showed serum profiles significantly different from normal profile.


Diagnosis of neural crest tumors by reversed-phase high-performance liquid chromatographic determination of urinary catecholamine metabolites. Aberrations in the metabolic pathways of catecholamines in patients with neural crest tumors result in characteristic urinary excretion patterns of their catabolites. Tumors such as pheochromocytoma, neuroblastoma and ganglioneuroma usually defy clinical diagnosis because of their rarity, small size, intraabdominal position and clinical symptoms similar to those of essential hypertension. Quantitative determination of catecholamine metabolites such as vanillylmandelic acid (VMA) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) offers possibilities for reliable confirmation of diagnosis. However, previous techniques for the assessment of catabolite levels suffered from inadequate sensitivity, reproducibility or specificity, which seriously diminished their usefulness as biochemical determinants in the prognosis of these life-threatening tumors. Reported in this paper is the analysis of urinary levels of VMA and MHPG using reversed-phase high-performance liquid chromatography with electrochemical and sectrophotometric detection. We present the excretion patterns showing these metabolites in 15 control subjects, 15 patients with pheochromocytoma and 5 patients with neuroblastoma.


High-performance liquid chromatographic assay of vitamin K in human serum. A sensitive combined adsorption and reversed-phase high-performance liquid chromatographic procedure is described that permits the determination of endogenous vitamin K1 levels in serum. The separation of vitamin K1 (cis- + trans-isomers), vitamin K1 epoxide and the menaquinones MK-2, MK-4 and MK-9 by adsorption, reversed-phase and cyano-bonded phase chromatography is discussed. The methodology was further developed for the quantitative measurement of vitamin K1 in human serum. Concentrations as low as 500 pg/ml in serum could be detected. The identity of the vitamin K1 peak was confirmed by UV absorption spectrophotometry and re-chromatography. The method has been applied to the determination of serum levels in normal healthy individuals and patients treated with vitamin K1.


Determination of Kováts retention indices with a capillary column and electron-capture detection: application to the assay of the enzymatic conversion of 3,4-epoxy-1-butene into diepoxybutane. A method is described that allows for alternate operation of a "pin and cup"-type electron-capture detector (ECD) in the normal ECD mode and in the metastable argon ionization mode. By such means, marker n-alkanes can be run without a change of the gas or temperature parameters, and measurements can be made of the Kováts retention indices of unknown compounds that are detectable in trace amounts by ECD only. As an application, tests are reported which support the probable enzymatic conversion of 3,4-epoxy-1-butene into diepoxybutane during incubation of the former with rat liver microsomes.


The brain of two mammal-like reptiles (Cynodontia - Therapsida). The endocasts of the cynodonts Massetognathus sp. and cf. Probelesodon are studied from descriptive and quantitative viewpoints. The morphology of the casts is described briefly, doing special attention to those features that import to the quantitative analysis, as for instance the general appearance of the casts and some vascular impressions. The general conclusions of this study are: 1.--Massetognathus sp. has had a brain with rather long olfactory peduncles and perhaps not greatly developed olfactory bulbs which could imply a non-macroosmatic condition, up to date not known for cynodonts. Cf. Probelesodon, on the other hand, shows a brain type similar to those of other cynodonts. 2.--Certain cynodonts at least, have had brains that filled fairly well the endocranial cavity. 3.--The cynodonts Massentognathus sp. and cf. Probelesodon, as surely other species, have acquired in Middle Triassic times a relative brain size rather closed to that of certain fossil and living mammals.


The corticofugal projections from the sensorimotor cortex to the spinal cord. A neuroanatomical and autoradiographical study in the cat with some methodological comments. An autoradiographical analysis of the corticospinal projections from the sensorimotor cortex of the cat using several 3H-labelled protein precursors showed a distribution of radioactivity on the contralateral side of the spinal cord in fairly good agreement with the findings using silver impregnation techniques. The findings on the ipsilateral side were sparse and the radioactivity was located to the transitional area of lamina VII and VIII almost as intensely as over the contralateral lamina VII. This pattern was more in line with that of the monkey and indicates a more common corticospinal terminal pattern in higher vertebrates. The corticospinal tracts were difficult to demonstrate even in horizontal sections apart from the crossed lateral tract. This was interpreted as a sign of insufficiency of the labelling considering the number of strongly labelled cortical cells. The lumbosacrally projecting neurons were rather difficult to demonstrate and most of them were situated in the more superficial areas around the cruciform sulcus. The investigation was mainly based on labelling the cortex with 3H-fucose which provided the most intense, differential and reproducable labellings. This was considered due to a lesser tendency to encounter selective neuronal uptake phenomenas. The transported amounts of radioactivity were relatively poor and unsuitable for studies of the finer somatotopics or EM-autoradiography of the synaptology.


An autoradiographical study of the projections from the feline sensorimotor cortex to the brain stem. Following an autoradiographical study on the projections from the feline sensorimotor cortex (representation of the limbs) to the brain stem, new projections to nucleus pretectalis posterior, the coerulear nuclei, nuclei corporis pontobulbares, nucleus intertrigeminalis, nucleus f, x and z of the vestibular nuclear complex, nucleus parvocellularis compactus, nucleus parasolitarius, nuclei insulae cuneati laterales, the nuclei of the raphé, nucleus reticularis lateralis and nucleus fastigii were found besides the well known projections to the tectal nuclei, the reticular formation, nucleus ruber, griseum pontis, the sensory trigeminal nuclei and the dorsal column nuclei. There was no evidence of projections to nucleus tractus solitarii. The amounts of radioactivity within the nuclei seem to be in favour of EM autoradiographical studies on the synaptology of these projections, apart from faintly labelled structures like most of the reticular nuclei, the sensory trigeminal nuclei, the coerulear nuclei and nucleus fastigii.


[Autoradiography of the neurogenesis and morphogenesis of the regio cingularis in the rat. II. Proliferation kinetics in the area praecentralis agranularis]. The proliferation of the matrix-cells in the Area praecentralis agranularis of 219 embryonic rats has been investigated by means of autoradiographs, after injection of tritiated thymidine at different stages of foetal life. The course of the labeling index indicates that matrix-cells represent a cell population in which cell multiplication is both rapid and continuous. Moreover the number of labeled cells decreases following the migration of labeled neuroblasts to another site. Because the beginning of migration retards after a division a proliferative compartment and a differentiating compartment were found in the subventricular and ventricular zone. The migration appears in waves not before 24 hours after division of cells. We have found that, between the second and the third day and betw-en the third and the fourth day after injection, migrating cells arise with 1/2 or 1/4 of the number of silver grains respectively. A comparison with the values of the Regio cingularis mesoneocorticalis (RICHTER and KRANZ 1978) indicates that no differences exist between the both regions. Cell division and migration both indicate in the brain a sequential nature; after each mitosis only one of the daughtercells retains their proliferative capacity, the other becomes differentiated into neuroblast and leaves the proliferative compartment.


The visual system of the trout Salmo irideus Gibb. A degeneration and radioautographic study. The organization of the primary visual system of the trout (Salmo irideus Gibb.) was studied by Fink-Heimer methods and radioautography. In addition to normal fiber, Nissl and Golgi staining techniques, we carried out total ablation of the retina on the right side on 16 animals which survived post-operatively at 13 degrees C for 9 to 31 days. The brains were cut at 35 micrometer and then stained by the Fink-Heimer techniques. Furthermore, we injected 20 microliter (2 microCi) of 14C proline into the right eye of 8 trouts which were then fixed between 24 and 72 hours after the injection, and then prepared for radioautography. The primary visual system projections are entirely contralateral. The optic tract is divided into three bundles which pass into tectal layers 2, 4 and 6. In the trout, a very obvious deep optic layer is present which extends throughout the length of the medial part of the tectum. In addition to the accessory optic tract and to a projection at the level of the preoptic area, the trout possesses a well-developed thalamic visual system composed of a ribboned geniculate nucleus and two medial thalamic visual centres fed by the retinothalamic pathways as well as a small retinal target located in the lateral thalamus. The pretectum receives only a few small sparse retinal projections. An interpretation of the trout visual system as the whole is discussed.


Patterns of organization of the corticotectal projection of cats studied by means of the anterograde degeneration method. By using the Fink and Heimer method, patterns of organization of the cat corticotectal projection have been studied in more detail than previously known. Collicular projections from various cortical areas show specific laminar distributions in tectal layers, while there is a considerable overlap in areas of terminations. Thus the superficial layers of the superior colliculus receive fibers almost exclusively from the visual cortex, while the deep layers from the auditory and the somatic sensory areas. In the intermediate gray layer, fibers from the visual, auditory and somatic sensory areas terminate in extensive areas throughout the colliculus. These findings are in good accord with physiological observations. Fibers from the "association", orbital, proreate and cingulate areas end in the intermediate and deep layers as well. Patch-like terminations of projection zones from the somatic sensory cortex are observed in the superior colliculus, longitudinally arranged with approximately 200-250 micrometer width and spaced at 200-500 micrometer. Similar patterns of terminations appear to exist in projections from the visual cortex. The lateral portion of the mesencephalic periaqueductal gray matter has been found to be a recipient of cortical fibers from the somatic sensory, orbital and cingulate areas. Possible neuronal interconnections within the superior colliculus are discussed in connection with the recent physiological data.


[Localization of thalamic afferents in the regio cingularis of the rat]. After lesioning the nucleus mediodorsalis and the nucleus anteromedialis thalami, where from specific afferents project to the cingulate cortex, we confirm signs of transneuronal degeneratiopn at lamina-V-pyramidal neurons of the cingulate cortex. By the GOLGI-KOPSCH-method operated animals show statistically significant decrease of dendritic spines at the apical main dendrites of lamina-V-pyramidal cells compared with the controls. The average spine-decrease along 300 micrometer length of the apical main dendrite amounts to 16%. Maximal spine-decrease is present at the central part of the apical main dendrite: 23% within a 50-micrometer-segment of this dendrite. This central dendritic part passes through the lamina III of the cingulate cortex. By the FINK-HEIMER-method degeneration particles are preferentially dispersed in laminae I, III and lower layers. The results give evidence for a more precise neuronal localization of the specific afferent terminations in this region.