Identification of human male meiotic chromosomes. The study is based on examinations of surgical testicular biopsies performed in eleven men aged from 19-79 years (300 cells at leptotent stage, 19 at zygotene, 300 cells at pachytene, 490 spermatocytes at diakinesis/first metaphase and 23 cells at the second metaphase of meiotic division), as well as post-mortem necropsies taken from six men aged from 19-51 years (6,000 cells at the first meiotic prophage). Identification of chromosomes at leptotene and zygotene stages is limited to the determination of X and Y chromosomes enclosed in the sex vesicle. At the pachytene stage, identification of chromosomes can make use of the differences in their length and number of chromomeres, but is feasible only in figures with good chromosome spreading. Identification of chromosomes at diakinesis/first metaphase in preparations stained by classical methods rests on the size and shape of the bivalents. Application of centromeric heterochromatin staining technique enables us to differentiate among bivalents Nos. 1, 2, and 3, to recognize bivalents belonging to the B group, to identify bivalents Nos. 9, 16, 17-18, and to distinguish between bivalent No. 21 and 22. It further permits the modality of pairing of the X and Y chromosomes to be determined by their short arms. Chromosomes of secondary spermatocytes at metaphase show typical morphological characteristics essential for karyotyping, so that it is possible to arrange them into the haploid karyotype, analogous to the karyotype of somatic cells. Male germinal cells undergo very rapid autolytic changes. No spermatocytes at diakinesis/first metaphase stage could be detected in specimens taken as early as 2 hours after death. The morphology of chromosomes of cells at earlier stages of the first meiotic prophase was markedly altered. Post-mortem testicular material was found unsuitable for an analysis of meiotic chromosomes.


Onset and progression of diabetic glomerulosclerosis; a prospective study based on serial renal biopsies. Clinical factors related to the development and progression of renal lesions were studied in twenty-three diabetics by the use of serial renal biopsies or autopsy. The results were as follows: Most of the juvenile and intermediate type diabetics were poorly controlled, with the glomerular lesion progressing rather rapidly. In contrast, many cases of the adult type were able to be maintained under good control and the renal lesion neither developed nor progressed. Two of the adult type diabetics with poor control showed slowly and slightly progressing renal lesions. The progression of glomerular lesions was significantly related to the control of blood glucose, type of diabetes, age at onset, type of treatment, and degree of obesity, but not to the duration of diabetes or the length of the follow-up period. There was a significant correlation between the type of diabetes and the control of blood glucose over the years. Arteriolar lesions developed concurrently with the progression of the glomerular lesion. Retinopathy also had a tendency to develop in proportion to the progress of glomerular lesions although it was not statistically significant. We have discussed the clinical factors responsible for the progression of diabetic glomerulosclerosis and have suggested that the type of diabetes rather than the degree of control of blood glucose might be more important in determining the development and progression of diabetic glomerulosclerosis. Nevertheless, the possibility remains that successful control of blood glucose may prevent or retard the development of diabetic glomerulosclerosis.


Physiologic mechanisms in the development of starvation ketosis in man. The present study was undertaken to determine whether alterations in ketone body utilization and hepatic production, independent of the FFA load, were also involved in the development of fasting ketosis. Plasma Beta-OH butyric acid (Beta-OHB) increased to 2.5-4.5 mM and plasma FFA to 1,000-1,400 muEq/L. in normal weight individuals after five to seven days' starvation and in obese subjects after ten to fourteen days' fasting. Acute elevations fo the plasma FFA greater than 1,500 muEq/L. for sixty minutes in fed normal weight and obese subjects with a fat meal-heparin regimen resulted in peak elevations of plasma Beta-OHB (0.25-0.45mM), only 10 percent of that seen during fasting. When plasma FFA were lowered acutely during fasting with the antilipolytic agent Pyrazole to control levels (400-600 muEq/L.), plasma Beta-OHB decreased 35 plus or minus 5 per cent. Comparable lowering of plasma FFA in normal weight or obese starved subjects given dexamethasone to maintain elevated fasting plasma insulin levels resulted in an 87 plus or minus 3 per cent decrease in plasma Beta-OHB. Similar studies in obese fasted subjects pretreated with an intravenous infusion of insulin (1.0 U/hr. for eight hours) before receiving Pyrazole resulted in a 65 plus or minus 5 per cent decrease in plasma Beta-OHB. Plasma Beta-OHB half-life, determined after injections of 12 gm. Beta-OHB, increased significantly during fasting (110 plus or minus 15 minutes) and was decreased when the fasting subjects were maintained on dexamethasone (65 plus or minus 7 minutes). These studies indicate that accelerated hepatic ketogenesis during starvation is a result of both enhanced activity of the enzymatic system(s) involved in ketone body production as well as an increased FFA load. The increase in plasma Beta-OHB during fasting reflects not only an accelerated rate of hepatic ketogenesis but also an impairment of peripheral utilization, both processes apparently being sensitive to insulin. Diabetes 24:10-16, January, 1975.


Confirmation of impaired early insulin response to glycemic stimulus in nonobese mild diabetics. The initial insulin responses of nonobese normal subjects and mild diabetics were analyzed during and after five-minute and two-minute infusions of 0.5 gm. per kilogram of glucose by vein. It was found that the two-minute injection elicited faster and significantly higher absolute hormonal output in both normals and diabetics, and that on both tests the normal subjects secreted significantly more insulin than did the mild diabetics. Comparison of respective "insulinogenic indexes" (net insulin output per unit of glycemic stimulus) showed that the corrected early insulin responses were in fact the same on both the five-minute and two-minute tests in normal subjects, and also in mild diabetics; but that the corrected insulin output was still twice as great in control subjects on both tests. It was also found that obese mild diabetics had significantly greater absolute insulin responses to both the five-minute and two-minute glucose injections than did their nonobese counterparts. These findings reconfirm that the earliest clinically recognizable state of diabetes mellitus is characterized by an impaired initial insulin secretory response to glycemic stimulus. They also indicate that valid interpretations of the influence of mild diabetes per se on the early insulin response can only be drawn from data obtained in nonobese individuals. Diabetes 24:17-24, January, 1975.


Insulin therapy in phenformin-associated lactic acidosis; a case report, biochemical considerations and review of the literature. A patient with phenformin-associated lactic acidosis was treated with insulin and showed marked improvement coincident with the expected onset of action of the insulin administered. Relative insulin deficiency was demonstrated although several phenomena characteristic of phenformin-associated lactic acidosis obscured its reflection in the usual indices. From data presented and a review of the literature the following pathogenesis is proposed for the observed metabolic derangement. A background of relative insulin deficiency would permit enhanced pyruvate (and hence lactate) formation from protein sources. Insulin deficiency would also lead to inhibition of pyruvate dehydrogenase which slows pyruvate removal. Phenformin accumulation (cf impaired renal function) further reduces pyruvate removal by decreasing its conversion to glucose, but in addition alters the redox state. For the lactic acidosis which results, insulin administration may thus constitute specific therapy. Diabetes 24:28-35, January, 1975.


Cell-mediated immunity in diabetes mellitus; lymphocyte transformation by insulin and insulin fragments in insulin-treated and newly-diagnosed diabetes. Using a radioisotope labeling technic, the ability of bovine and porcine insulin antigens to induce lymphocyte transformation was tested with cells from the peripheral blood of thirty nondiabetic controls, fifty established insulin-dependent diabetics with no evidence of insulin allergy, and ten newly diagnosed diabetics (five untreated, five insulin-treated for less than three weeks). Lymphocytes from twenty-six (42 per cent) of the diabetics showed significant blastogenesis to bovine or porcine insulin, as compared with two (7 per cent) of controls; the phenomenon was shown by both established and newly diagnosed patients including four who had never recieved insulin. The results indicate that cellular hypersensitivity to insulin, as judged by an in vitro test, is relatively common in insulin-treated diabetics without in vivo evidence of allergy, and suggest that hypersensitivity may also be present in untreated diabetics. Lymphocytes from twenty-one of the twenty-six diabetics who responded to intact insulin were further tested using bovine and porcine insulin A chain bovine B chain as antigens. The A chain of either insulin induced significant blastogenesis in only one diabetic but bovine B chain induced significant blastogenesis in fourteen (67 per cent) of the patients tested. These results suggest that B chain is the major antigenic site determining cellular hypersensitivity to insulin. Diabetes 24:36-43, January, 1975.


An automated ECG-system in a large hospital: coding, storage and retrieval of tracings. This paper describes an automated ECG-system as it is used in the 1000-bed University Hospital Utrecht, The Netherlands. The system involves a "hybride" approach, combining computer analysis of the ECG by means of the Pipberger program with the reading by a cardiologist via a specially developed coding system. Up until now (since January 1, 1972) 35,000 ECGs have been handled systematically at a rate of approximately 100 ECGs per working day. All the ECGs, together with the ECG-diagnoses and other relevant data of the patient are stored and can be retrieved whenever wanted. The system enables comparison of computer analysis and cardiologist's reading of the ECG. The boundary between reliable computer analysis and the necessity of human reading and verification lies with the normal ECGs. This apparently meagre result of the computer ECG-analysis for hospital use is, however, a great achievement for its use in epidemiological studies.


[Automatic teleprocessing of ECG problems and prospectives of a service center (author's transl)]. The different alternative for an ECG's computer interpretative service are described and analyzed. The important factors which can be used as measures of effectiveness for each alternative are identified as: 1) waiting time before sending ECG; 2) waiting time before receiving the report; 3) capability of satisfying emergency calls; 4) cost per ECG. Three major alternatives are compared on the basis of these measures of effectiveness. The utility of a computer center serving more hospitals is evaluated against the utility of having an in-house computer in the hospital, on the basis of cost and convenience of service considerations.


[Multivalvular replacement: short and long term results in 367 cases (author's transl)]. Early and late results of 273 mitro-aortic, 66 mitro-tricuspid, 28 mitro-aortic-tricuspid valvular replacements performed between 1964-1973, were evaluated. The hospital mortality rate was 17, 5 percent for mitro-aortic, 30, 5 percent for mitro-tricuspid, 10, 7 percent for mitro-aortic-tricuspid replacement. The best results obtained in patients with mitro-aortic replacement, the worse one in patients with mitro-tricuspid replacement, that present also a poor functional rehabilitation. The actuarial curves were calculated: 9 years after operation the probability of survival was of 67 percent for patients operated of mitro-aortic valvular replacement and 47 percent for patients operated of mitro-tricuspid replacement.


[The vectorcardiogram in A-V canal in the first three years of life: qualitative and quantitative analysis (author's transl)]. The electrocardiograms and the vectocardiograms of 26 childrens, up to 3 years of age, affected by A-V canal, were been analyzed. The pattern of QRS loop in the 3 planes, the voltage of right maximum spatial vector (RMSV) and left maximum spatial vector (LMSV), their projection onto the horizontal (azimuth) and frontal plane (elevation) were related to right ventricular systolic pressure. The qualitative and quantitative evaluation of VCG Showed a better correlation with the haemodynamic data than the electrocardiogram. In all but 3 cases, the high frequency of superior orientation and counterclockwise rotation of QRS loop in the frontal plane was found; moreover the good correlation of right ventricular systolic pressure to the rotation of QRS loop on the horizontal plane was also confirmed. In fact, the latter was counterclockwise in the cases with a low pressure, whereas it became clockwise in those with higher pressure. Moreover a good direct correlation of the right ventricular pressure with forward orientation of LMSV on the horizontal plane was found. In differential diagnoses with other congenital heart diseases with superior orientation and anticlockwise rotation on the frontal plane, useful results were obtained by using the calculation of elevation time (the interval between point 0 and intersection of the QRS loop with the axis) which is significantly lower in the A-V canal. Terminal forces directed posteriorly and to the right and with a delay no longer than 0,03 inches do not warrant the diagnosis of left anterior hemiblock with a right bundle branch block associated. On the contrary, on the basis of anatomical and electrophysiologic studies we believe that in this disease there is an asinchrony in the activation i.e. the postero-inferior region, then the left lateral wall and finally the tree high part of right ventricle wall are sequentially activated.


[Diagnostic difficulties of isolated left posterior hemiblock(author's transl)]. Out of 4000 vectorcardiograms, 23 of them showed the most tipical pattern of left posterior hemiblock (BFP). According to the vectorial characteristics, they were divided into 5 groups; only two groups (for a total of 8 cases) showed, according to the Authors' opinion, the characteristics of the isolated BFP. The diagnostic difficulties of such a conduction disturbance are discussed since, it simulates, the vectorial pattern of the vertical heart. Moroever there is an underlining possibility that conduction disturbances in the right heart coexist in many cases of supposed isolated BFP with marked axis rotation to the right. The Authors agree with the hypothesis already expressed by Polu on the vectorial progress of the isolated BFP which should have an AQRS of about 70 degrees; an exact electrocardiographic diagnosis might be therefore impossible.


[Junctional bradycardia and junctional non-paroxysmal tachycardia. A study with atrial and His electrogram and influence of neurovegetative system (author's transl)]. Two cases of unusual A-V nodal rythms, very slow junctional bradycardia and junctional non paroxysmal tachycardia, are described. The Authors emphasize the study of the His bundle electrograms, the atrial electrograms and the time of the intrinsecoid deflection for correct diagnosis of these dysrrhythmias. From the results of the clinical and electrofisiological study and neurovegetative responses some conclusions about the mechanism and clinical significance of these arrhythmias are drawn.


[Transitory transesophageal atrial electric stimulation. Preliminary report on 19 cases and considerations on the method, indications and results (author's transl)]. Literature provides sufficient evidence that transitory electric stimulation via esophagus (SATE) - after the first positive experimental attempts on dogs - can be applied to man with a simple, rapid and harmless method. The study covers 19 patients subjected to high frequency transesophageal atrial stimulation by way of a bipolar electrode inserted through a nasogastric tube and connected to an external generator capable of producing tension impulses. Said impulses are variable up to 150 volts, lasting 2.5 microsec. with a frequency of up to 450/min. The 19 patients can be divided into 2 groups. The first including 15 patients on which SATE was effected for diagnostic purposes: in coronary deficiency (8 patients), in the disease of sinus node (3 patients), and lastly in the research for the A-V-block latent in 4 patients with acute post-infarctual A-V-block which regressed during the immediate clinical course of the illness. The other group includes 4 patients in which the atrial stimulation indication was the treatment of rapid, paroxysmic atrial rhythms, inaffected by drugs. By using impulses of 25-30 volts, the AA. have obtained a stable stimulation.


[Clinical trial by perexiline maleate in treatment of angina pectoris (author's transl)]. A controlled double blind clinical trial has been conducted in 16 patients with "angina pectoris" in order to investigate the effect of Perexiline maleate as compared with prenilamine. Perexiline at the dose of 400 mg/die and prenilamine at the dose of 120 mg/die have been administered over a period of 4 weeks each. Between these periods placebo has been administered for two weeks. The number of attacks of angina and the number of tablets of nitroglycerine used per week by the patient during each period has been used for the evaluation. Furthermore ECG at rest and after exercise has been performed every two weeks. Our results statistically evaluated show a definite antianginal effect of Perexiline. According to our experience Perexiline should be considered the drug of choise in the treatment of angina complicated by bradicardia, left ventricular failure, bronchospasm, and in angina unresponsive to other drugs.


Experimental bases for diagnosis of left bifascicular and trifascicular block. It seems to be justified to establish the diagnosis of fascicular blocks, in clinical tracings, on the basis of experimental findings. For this, we produced left bifascicular block as well as trifascicular block in the heart of 20 mongrel dogs. Because of left bifascicular block, the activation process is unequally delayed in high lateral and in posterior aspects of the free left ventricular wall. Nevertheless, it begins at normal time in middle left septal mass via intermediate strands of the left bundle. Consequently, left bifascicular block which resembles intermediate degree left truncular block, is recognizable by unequally delayed onset of intrinsicoid deflection in aVL, aVF and V6 and often by the manifestation of the first septal vector. Vectorcardiographic curves are diphasic and show initial and terminal slurrings. Trifascicular block delays the activation process in both ventricles. For this, the asynchronism between the electromotive forces of ventricles is lessened. The said conduction disturbance can be diagnosed by electrocardiographic and vectorcardiographic features suggesting intermediate degree RBBB as well as left bifascicular block.


[The automated ECG laboratory: equipment and operational problems (author's transl)]. In the light of recent advances in technology, the basic equipment of an automated ECG laboratory is described. The main features of data acquisition terminals, data receiver/controller units, A/D converters, computers, visual displays and systems for storage and retrieval of tracings, are briefly discussed. Three major alternatives are open for computer-aided ECG interpretation today: 1) complete, dedicated system in the hospital; 2) ECG data collection system with offline analysis by hospital business computer; 3) ECG service center outside of the hospital. Advantages and possible limitations of these methods are discussed. At the Ospedale Civile Regionale of Udine we have choosen the first method. An HP 1530 ECG interpretative system and the 12-lead ECG analysis program developed by Caceres-USPHS are used. Analog tracing and interpretative printout are available in the laboratory and/or at the patient location in about one minute. Our system has been working for less than one year. At present, 150-200 ECG are processed daily. Such an ECG processing system has proven to yield considerable savings in time and manpower. Some operational problems related to shifting from manual to computer work have been gradually overcome and will be discussed.


Neural organization of esophageal peristalsis: role of vagus nerve. The purpose of this investigation was 2-fold: first, to determine the velocity of peristalsis in the smooth muscle area of opossum esophagus before and after administration of atropine; second, to evaluate the role of the vagus nerves in the control of the propagative nature of esophageal peristalsis. Intraluminal pressures were measured through a pressure transducer recorder system attached to continuously perfused catheters. The velocity of peristalsis in the lower third of the esophagus progressively decreased from 3.25 plus or minus 0.20 (SE) cm per sec at the 70 to 80% level to 2.17 plus or minus 0.14 (SE) at the 80 to 90%level to 1.83 plus or minus 0.10 (SE) at the 90 to 100% level. After administration of intraperitoneal atropine (100 mug per kg), the velocities were 3.1 plus or minus 0.26 (SE) cm per sec, 2.38 plus or minus 0.22 (SE), and 1.74 plus or minus 0.10 (SE), respectively, at the 70 to 80%, 80 to 90%, and 90 to 100% levels. The changes were not statistically significant. Electrical stimulation of the distal cut end of the vagus nerve induced peristaltic contractions. The velocities of peristalsis after electrical stimulation of the vagus nerve were 3.24 plus or minus 0.72 (SE) cm per sec, 2.81 plus or minus 0.64 (SE), and 1.84 plus or minus 0.34 (SE), respectively, at the 70 to 80%, 80 to 90%, and 90 to 100% levels. Results of this study indicate that the velocity of peristalsis in the smooth muscle area of the opossum esophagus has a caudally decreasing gradient. Bilateral cervical vagotomy and stimulation of the distal cut end initiates peristaltic contraction indicating that the propagative nature of peristalsis in the smooth musurrent, does not alter mucosal cyclic AMP. Dibutyryl cyclic AMP decreased net sodium absorption and increased short circuit current; findings which were qualitatively identical to those produced by taurochenodeoxycholic acid. These studies support the proposal that bile salts stimulate colonic electrolyte secretion by increasing mucosal cyclic AMP.


Biological characterization of Fusobacterium necrophorum.  Cell fractions in preparation for toxin and immunization studies. Fusobacterium necrophorum isolated from bovine liver abscesses was grown in bulk at 37 C for 24 h under a strict anaerobic atmosphere. Harvested washed cells were disrupted ultrasonically and fractionated by differential centrifugation into the intracellular (cytoplasm) and cell wall fractions. Both intact cells and cell fractions induced generalized cytopathic effect on primary pig kidney cultures and caused a variety of signs of illness and/or death of intraperitoneally injected mice. The intact cells, disrupted cells, and cell walls produced necrotic lesions and erythema on intradermally injected guinea pigs and rabbits, whereas the cytoplasm mainly erythema. By contrast, the used culture medium (culture filtrate) of F. necrophorum did not show any detectable toxicity. The toxic component of the cytoplasm appears to be associated with nondialyzable, hemolytic, high-molecular-weight proteins and its toxicity is reduced by trypsin and pronase. Heating at 60 C for 10 min decreased markedly its erythemal and cytotoxic ability, wheras the toxicity of the cell walls appeared to be only slightly affected even when heated at 100 C for 1 h. These results suggest that at leasttwo distinct cell-bound toxic factors are present in F. necrophorum cells.


Interaction of cultured mammalian cells with [125I] diphtheria toxin. The characteristics of cell adsorption and pinocytotic uptake of diphtheria toxin by several mammalian cell types were studied. Purified toxin iodinated by a solid-state lactoperoxidase method provided preparations of high specific activity and unaltered biological activity. Dephtheria toxin-sensitive HEp-2 cells and guinea pig macrophage cultures were compared with resistant mouse L-929 cells. At 37 C the resistant cells in monolayer adsorbed and internalized [125I] toxin to a greater extent than did the HEp-2 cell cultures; no significant differences were observed at 5 C. Ammonium chloride protection levels did not alter uptake of toxin by either L-929 OR HEp-2 cells. Biological activity of the iodinated toxin, however, was negated provided the presence of ammonium chloride was maintained. The ammonium salt appears to maintain toxin in a state amenable to antitoxin neutralization. Guinea pig macrophages internalized iodinated toxin to a level 10 times greater than the established cell lines. In spite of the increased uptake of toxin by the endocytic cells, ammonium chloride prevented expression of toxicity. In an artificial system, toxin adsorbed to polystyrene latex spheres and internalized by guinea pig macrophages during phagocytosis did express biological activity. Ammonium chloride afforded some but not total protection against toxin present in the phagocytic vacuoles. The data suggest that two mechanisms of toxin uptake by susceptible cells may be operative. Toxin taken into the cell by a pinocytotic process probably is not ordinarily of physiological significance since it is usually degraded by lysosomal enzymes before it can reach cytoplasmic constituents on which it acts. When large quantities of toxin are pinocytized, toxicity may be expressed before enzymatic degradation is complete. A more specific uptake involving direct passage of the toxin through the plasma membrane may be the mechanism leading to cell death in the majority of instances.


Alterations in the metabolism of hamster tracheas in organ culture after infection by virulent Mycoplasma pneumoniae. Exposure of hamster tracheal rings in organ culture to virulent Mycoplasma pneumoniae organisms leads to alterations in macromolecular biosynthesis and metabolic activity of the respiratory epithelial cells. Avirulent organisms derived from the same parent strain do not produce these effects. During the course of infection by virulent mycoplasmas, tracheal rings show an initial increase in [14C]galactose uptake followed by a significant decline as infection progresses which is also accompanied by abnormal processing of galactose as evidenced by amounts of 14CO2 released. Parallel decreases in the rate of [3H]orotic acid and [3H]amino acid uptake are observed. Within 24 h after infection of tracheal rings by virulent mycoplasmas, inhibition of host cell ribonucleic acid and protien synthesis is evident. Ribonucleic acid synthesis in infected cells, analyzed by gel electrophoresis, is reduced by 80% at 48 h and is negligible by 96 h. The course of mycoplasma infection can be interrupted or reversed by erythromycin after the initial mycoplasma-host cell interaction since addition of erythromycin 24 h or earlier after infection prevents the onset of abnormal orotic acid uptake. However, 48 h after infection, rescue of host cells by erythromycin cannot occur and cytopathology becomes evident. These data suggest that mediation of host cell injury requires continued protein synthesis by attached mycoplasmas, and the primary effect of mycoplasma infection on tracheal organ culture may be at a transcriptional or translational level.


Prevalance of antibody to hepatitis B surface antigen in various populations. Sera from individuals with different degrees of exposure to the agent of hepatitis B were tested for antibodies to hepatitis B surface antigen (anti-HB-s) by passive hemagglutination and for hepatitis B surface antigen (HB-s Ag) by radioimmunoassay and immunoelectroosmophoresis. In a plasma fractionation plant, anti-HB-s was detected in 82 percent of workers processing human plasma and 3.3 percent were healthy carriers of the antigen. Fifty-six percent of the workers having only casual contact with plasma processing exhibited anti-HB-s and 24 percent of workers with no contact had anti-HB-s, yet HB-s Ag was not found in either of these two groups of workers. A similar correlation was shown in hospital personnel; 31 percent of employees with direct contact to serum specimens and only 8 percent without direct contact had anti-HB-s. The frequency of HB-s Ag (0.8 percent in patients with disorders not involving the liver; 49.8 percent in patients tentatively diagnosed as viral hepatitis) and anti-HB-s (14.5 percent to 28.5 percent, respectively) in selected groups of hospitalized patients varied greatly. In 508 paid blood donors, anti-HB-s was present in 19.9 percent, whereas it was present in only 6.6 percent of 1,146 volunteer donors. These data demonstrate a correlation between frequency of exposure to human blood or blood products and the prevalence of anti-HB-s.


Multiple sclerosis-induced reduction in the yield of a mouse cell line. Cultures of a mouse cell line (PAM) were treated with 71 multiple sclerosis (MS) and 45 non-MS samples. Of the cultures treated with MS material, 80 percent (58) showed a reduction in cell yield (compared to untreated controls) of at least 20 percent by the third passage after inoculation. The MS samples were from 40 MS cases, and a total of 36 cases yielded at least one positive sample. The agent responsible for the decrease was not limited to nervous tissue, but was also found in serum, cerebrospinal fluid, spleen, kidney, and lymph node of MS patients. Positive samples were present at every stage of the disease. None of the non-MS samples yielded cell counts significantly different from untreated controls. The non-MS category included 12 samples from healthy individuals, 13 assorted non-central nervous system disease samples, and the following central nervous system disease samples: six subacute sclerosing panencephalitis, three Huntington's chorea, two Parkinsonism, six amyotrophic lateral sclerosis, one stroke, one encephalopathy, and one epilepsy. Brain homogenates from mice inoculated with MS tissues elicited the decrease, whereas brain homogenates from mice inoculated with non-MS samples did not.


Lymphocyte responses to rubella antigen and phytohemagglutinin after administration of the RA 27/3 strain of live attenuated rubella vaccine. Lymphocyte phytohemagglutinin (PHA) responsiveness was found suppressed in both rubella sero-negative and sero-positive recipients of RA 27/3 strain of live attenuated rubella vaccine; the suppression was readily demonstrable only when a suboptimal dose of PHA was applied in the test. Lymphocytes from sero-negative vaccinees, which initially showed little or no in vitro response to concentrated rubella virus, became responsive after vaccination by day 21, when the highest sensitization to rubella antigen was seen. In the sero-positive vaccinees. lymphocytes responded to rubella antigen in vitro before vaccination, and in most cases vaccination did not result in significant changes in lymphocyte response. These results suggest that rubella vaccination leads to temporarily increased lymphocyte reactivity to rubella antigen, and the increased lymphocyte response to specific antigen may occur at the time of mild suppression of PHA response.


Preparation and chemical composition of the cell walls of Streptococcus mutans. Purified cell walls from Streptococcus mutans strain BHT were prepared without the use of proteolytic enzymes in order to retain all cell wall constituents for chemical analysis. Of four methods employed, the Ribi cell fractionator produced disrupted cell suspensions which could be most thoroughly purified on sucrose gradients. Results of chemical analyses on purified cell walls prepared in this 8.9% glycerol teichoic acid, 33.6% non-peptidoglycan polysaccharide, and 49.9% peptidoglycan.


Interactions between Toxoplasma gondii and its host cells: function of the penetration-enhancing factor of toxoplasma. A protein with a molecular weight of 70,000 to 150,000 which was extracted from merozoites of Toxoplasma gondii enhanced the host cell penetration of the merozoites. The optimal pH and temperature for penetration of merozoites coincided with those favoring the action of the penetration-enhancing protein. In addition, a dependence on Ca and Mg existed for penetration of merozoites, either in the presence or absence of this protein. No evidence was found that indicated that the enhancing effect on penetration elicited by the protein was due to increased phagocytic capacity of host cells (HeLa) or improved motility of the merozoites. Electron microscopy demonstrated that the protein, in high concentration, caused disruption of cytoplasmic membranes. In a 100-fold-lower concentration, which still caused a marked enhancement of penetration, no such effect was observed. However, the vacuoles surrounding the penetrated parasites seemed smaller than for merozoites penetrating in cultures to which no penetration-enhancing factor was given, and the membranes limiting the vacuoles demonstrated discontinuities more often. The penetration-enchancing effect of some known enzymes was studies. However, none of these enzymes seemed to correspond to the penetration-enhancing protein of toxoplasma. The mode of entry of toxoplasma merozoites into host cells is discussed. It is concluded that phagocytosis must play a less important role and that merozoites actively penetrate the cytoplasmic membranes of the host cells. The penetration is proposed to be a result of combined mechanical and chemical actions. It is suggested that an enzymatic function of the penetration-enhancing factor released by the merozoites is of importance. The membrane limiting the vacuole of a penetrated merozoite seems to be newly formed in the cell after penetration is completed.


Immunogenicity of aryl esters of salicylic or acetylsalicylic acid in guinea pigs. A variety of derivatives of acetylsalicylic and salicylic acid have been investigated for their immunogenic properties in guinea pigs including salicylsalicylic acid (SSA), acetylsalicylsalicylic acid (ASSA), disalicylide (DI), trisalicylide (TRI), acetylsalicylic acid paracetamol ester (ASPE) and acetylsalicylic acid guajacol ester (ASGE). Contact sensitivity could be elicited by the sensitizing agent, however, with acetylsalicylic acid anhydride (ASAN) a more pronounced contact reaction could consistently be observed. Systemic anaphylactic reactions elicited by intravenous injection of N-salicyloyl bovine serum albumin could only be induced by ASAN, DI, TRI and ASSA, whereas SSA, ASPE and ASGE did not induce an anaphylactic state at a comparable dose level. From these results it is anticipated that all aryl esters of acetylsalicylic or salicylic acid are immunogenic when applied intradermally, leading to a N-salicyloyl specific immune response.


Effect of substitution on reactivity of B 512 dextran fractions with anti-B 512 dextran in heterologous passive cutaneous anaphylaxis. The influence of type and degree of substitution of B 512 dextran fractions on reactivity with rabbit antibodies against unmodified B 512 dextran was studied on heterologous passive cutaneous anaphylaxis in guinea pigs. Low degrees of substitution, i.e. 1 substituent group per 7-30 glucose residues, affected reactivity with anti-dextran only slightly or not at all. This was shown for the following substituents: sulphate, carboxymethyl, phosphate, diethylaminoethyl, hydroxypropyl, butyryl, caprylyl, stearoyl, and fluoresceinoyl groups. With high degress of substitution, i.e. 1-3 substituents per 2 glucose residues, reactivity with anti-B-512-dextran is completely abolished for some substituents, and a new immunological identity conferred on the substituted dextran. Stongly charged groups like sulphate, carboxymethyl and diethylaminoethyl abolish reactivity with anti-B-512-dextran at relatively lower degrees of substitution than more neutral groups like methyl and acetyl. The anti-B-512-dextran represents a specific reagent for alpha-1,6-linked polyglucose, as evidenced by complete cross-reactivity with synthetic linear dextran; its specificity is emphasized by non-reactivity with alpha-1,6-linked synthetic manna, the monomeric residues of the two polymers differing only in position of the C-2 hydroxyl groups.


Fluorescamine as a terminating agent in solid phase peptide synthesis. Fluorescamine was shown to be an excellent terminating agent for blocking unreacted amino groups during solid phase peptide synthesis. A comparison of the termination efficiency of fluorescamine versus that of acetylation revealed that the former method gave superior products as assessed by peptide analysis, dansyl-amino end group determination and biological assay. In addition, fluorescamine terminated fragments were converted to non-fluorescent spirolactones during the deprotection stage. These spirolactones were stable to subsequent solid phase reaction conditions and were readily removed from the target peptide.


Conformational analysis of circular dichroism spectra of insulin, proinsulin and c-peptides by non-linear regression. A method of resolving CD spectra in alpha-helix, beta-structure and random coil conformations is described. The residue ellipticites for alpha-helix and beta-structure given by Greenfield & Fasman or by Chen,, Yang & Martinez are used together with CD spectra from at least two similar peptides to determine, by an iterative least-squares method, the number of amino acids in the three reference conformations as well as a set of residue ellipticities characteristic of the random coils of the family of peptides in question, but not necessarily of other peptides. The fits between computed and experimental spectra improve significantly and systematic deviations disappear by allowing the random coil coefficients to vary from one family of proteins to another, a liberty justified by the different types of random coils that have been encountered. The method of analysis showed that 5 M urea did not change the conformations of C-peptides of proinsulin from ox, pig and duck, all being mainly in the random coil conformation and all having 3-4 amino acids in beta-structure. Bovine insulin and proinsulin showed a transfer of amino acids from alpha-helix to beta-structure with increasing concentrations of urea, the latter at a higher concentration, indicating a stabilizing effect of the connecting peptide. The numbers of amino acids found in the alpha-helical conformation in insulin and proinsulin were equal and in agreement with the X-ray crystallographic data for insulin when the Greenfield & Fasman coefficients for alpha-helix and beta-structure were employed, whereas the Chen, Yang & Martinez coefficients yielded too few amino acids in alpha-helix in proinsulin. Both sets of coefficients estimate more beta-structure in proinsulin than in insulin.


A general method of purification of adenosine deaminase by affinity chromatography. Affinity chromatography has been used to purify adenosine deaminase from various sources: calf spleen, calf intestinal mucosa, chicken duodena and human erythrocytes. For this purpose a specific inhibitor, 9-(p-aminobenzyl) adenine, was synthesized and covalently joined to agarose. Adenosine deaminase is selectively retained by such an inhibitor-resin when highly impure solutions are chromatographed through it. After elution from the resin with guanylurea, a competitive inhibitor, the enzyme is homogeneous and can be recovered in yields of 80 percent or more and the same number of multiple forms of the enzyme is present in the purified preparation and in the crude extract.


Renal stone formation. The inhibitory effect of urine on calcium oxalate precipitation. The mechanism of renal stone formation is not fully understood. This report describes an investigation into the effect of urine on the precipitation of calcium oxalate from dilute solution. Over 33 min a 5 per cent solution of urine caused considerable inhibition of precipitation. The degree of this inhibition was related to the concentration of the original urine. An "artificial urine" composed of 12 solutes present in normal urine had some inhibitory effect, but less than that of equiosmolar true urines. Inhibition was also related to the percentage of urine present. This inhibitory effect may be important in the prevention of renal stone formation in normal people by delaying crystallization until urine reaches the lower urinary tract.


Effect of shape on contraction of abdominal stoma. We compared the contraction of round, square, and triangular abdominal stomas of isolated ileal segments. Twelve stomas of each of these three different shapes were observed in six dogs for periods ranging from 1 to 4 months. Our observations indicate that the square shaped enterocutaneous stoma is preferable to the more conventional circular one. The square shape provides the longest perimeter for a given surface area with minimum contraction, whereas the circle has the shortest perimeter and results in maximum contraction.


Epsilon-aminocaproic acid in traumatic renal hematuria in the rabbit. Epsilon-aminocaproic acid (EACA) was intravenously administered to rabbits subjected to bilateral renal crushing trauma. Posttraumatic hematuria in 10 control rabbits lasted a mean of 5.5 days (range 5 to 7 days). Rabbits treated with 1250 mg 5 min before renal trauma and with repeat EACA injections 1 hr (500 mg) and 24 hr (500 mg) after the injury had significantly less hematuria. Only eight of 20 EACA-treated rabbits had any hematuria, and this bleeding terminated by the 2nd posttrauma day in all. EACA may have an important role in the management of trauma-induced renal bleeding.


Effects of pressoreceptor stimulation on micturition relfex and urethral pressure profile in rabbits. The influence of pressoreceptor stimulation on the micturition reflex and urethral pressure profile was studied. Stimulation of pressoreceptors of the aortic arch and carotid sinus causes an increase of parasympathetic tone and a decrease of sympathetic tone. Striated muscular tone was also decreased. Pressoreceptor stimulation was simulated by electrical stimulation of the depressor nerve in the rabbit. Our studies show that with pressoreceptor stimulation micturition occurred with 24 per cent less bladder filling and 32 per cent less intravesical pressure than before stimulation, and urethral pressure profile showed a 17 per cent decrease of the maximum and mean urethral pressure. The interpretation of the action of parasympathicomimetic and sympathicomimetic drugs on bladder and bladder neck must take into consideration the cardiovascular effects of these drugs and therefore the effect on the pressoreceptors.


Theory of hydrokinetic clearance of bacteria from the urinary bladder. I. Effect of variations in bacterial growth rate. If the bladder is regularly emptied in appropriate circumstances the concentration of bacteria in successively voided samples progressively falls. By making a number of assumptions about conditions of bacterial growth in the bladder the way in which this washout of bacteria will occur can be predicted. Such predictions give a form of washout curve which differs significantly from that commonly encountered in patients. The shape of the predicted washout curve is affected by the form of the bacterial growth curve but this influence is not sufficient to account for the observed difference between patients and predictions.


Theory of hydrokinetic clearance of bacteria from the urinary bladder. II. Effects of "bound" organisms and diuresis. When patients with urinary infection drink large volumes of water and empty the bladder at frequent regular intervals, the concentration of bacteria in the voided samples commonly falls rapidly at first and then remains almost constant. The present theoretical analysis leads to the conclusion that the rapid initial fall in concentration is a function of diuresis and that the subsequent constant output originates from bacteria bound to multiplication sites on the uroepithelium from which their progeny are shed into the urine.


Incorporation of 3H-thymidine into rat ventral prostate in organ culture. Influence of hormone-cytostatic complexes. The effects of hormones, hormone-cytostatic complexes, and their derivatives on 3H-thymidine incorporation in explants of rat ventral prostate and rat uterus have been studied. The histologic appearance has also been used as a parameter of investigation and the results were in good agreement with 3H-thymidine incorporation. The results showed that when a nitrogen mustard was linked in position 3 to estradiol-17beta-phosphate (Estracyt), this complex had a profound inhibitory effect on growth of rat ventral prostate explants, far more pronounced than that seen by estradiol-17beta. The active nor-nitrogen mustard (LEO72a) alone also had a profound inhibitory effect, while a complex consisting of the nitrogen mustard linked in position 17 to dihydrotestosterone (DHT-17cyt) showed no inhibition. The possible intracellular mechanisms of action of the hormone-cytostatic complexes are discussed.


The physical properties of polyglycolic acid sutures (Dexon) in sterile and infected urine. In a series of in vitro tests the time-dependent alteration of the resistance to breaking and of the extensibility of catgut and of polyglycolic acid sutures (Dexon) was examined. The threads were incubated in sterile and infected urine up to 10 days at 36 C. In the sterile urine environment Dexon dissolved on the 6th day and in the infected urine on the 3rd day. Catgut did not dissolve at all in the sterile urine and in the infected urine it dissolved only on the 8th day. In view of these results the use of Dexon in the urine environment may cause some concern, especially in an infection exists.


The response of smooth muscle cells in the rabbit urinary bladder to outflow obstruction. The morphologic response of smooth muscle cells in the urinary bladder to a standardized bladder neck obstruction and the cell changes subsequent to release of that obstruction were studied in both growing and adult rabbits. The bladder reacted first by gross dilation and then by concentric thickening of the wall at the expense of the lumen. Microscopically, thickening of the bladder wall was due to temporary submucosal edema, then to the increased fibrocollagenous connective tissue, and finally to the increased muscle mass which in part replaced the connective tissue. Hypertrophy of the smooth muscle cells and their nuclei was apparent and this preceded cellular hyperplasia, which did not occur until 30 days after the obstruction was induced in the adult rabbits. In contrast, there was early hyperplasia of smooth muscle cells in the growing rabbits, followed by hypertrophy of the cells and their nuclei. In no instance could mitosis of smooth muscle cells be found in either young or adult rabbits. Quantitation showed that the degree of cellular hypertrophy was proportional to the duration of the obstruction. In the adult animal, the muscular thickening was mainly due to an initial 5 fold increase of the muscle cell volume together with a later 3 fold increase of muscle cell number. In the growing rabbits, the muscle cells first doubled in number and then after 8 weeks a 6 fold enlargement of cell volume contributed to the total muscular thickening. Upon release of obstruction in the adult rabbits, both enlarged cell and nuclear sizes regressed to normal values and the cell numbers gradually decreased to near normal values over periods up to 4 months after release.


A quantitative study of smooth muscle cells in reflux, obstructed, and triad bladders. A preliminary report;. This quantitative study has shown that the average size of the vesical smooth muscle cells of humans increases with age, whereas the average nuclear size remains constant. In the bladders of six babies with proven outflow obstruction, the cell size was signigicantly increased. In 23 of 25 bladders of infants and children with vesicoureteral reflux there was no apparent hypertrophy, but in two the cells were larger than normal and the micturition cystourethrograms exhibited features which may signify impairment of outflow. In two autopsy bladder specimens of two newborn, triad syndrome babies, there was no hypertrophy of muscle cells, indicating absence of obstruction in fetal life. Three giant bladders associated with rectovesical fistula deformities (two newborn specimens) and dyskinesia of the small and large bowel showed no hypertrophy of muscle cells, indicating that the enlargement was unlikely to be obstructive and appeared to be developmental in origin. Hypertrophy of muscle cells of the bladder indicated obstruction to the outflow, even when the degree of obstruction was mild. Muscle cell size can be measured on full thickness small surgical biopsy specimens prepared as paraffin sections and hence the method can be useful in clinical practice.


A mucocutaneous disease in the dog, resembling pemphigus vulgaris in man. A chronic mucocutaneous disease was diagnosed in 3 dogs. Clinically, the disease was characterized by erosions and ulcerations of the oral mucosa, various mucocutaneous junctions, and the skin. Histologically, there was acantholysis or a separation of the epithelial cells from one another, resulting in the formation of clefts and bullae. Based on clinical and laboratory findings, the disease seemed to be the canine equivalent of pemphigus vulgaris (PV) in man.


Hydronephrosis secondary to focal papillary hyperplasia of the urinary bladder of cattle. Focal papillary hyperplasia in the trigone of the urinary bladder caused partial urethral and ureteral obstruction in 4 Holstein-Friesian cows. Secondary chronic hydronephrosis resulted in clinical and laboratory evidence of chronic renal failure.


Left displacement of the abomasum in a clinically normal cow. A cow with left displacement of the abomasum (lda) was found to be clinically normal for 1 1/2 years. During this period, the cow calved twice and ate and produced milk normally. Auscultation and percussion at approximately weekly intervals revealed the continuing abomasal displacement, which was confirmed at necropsy.


Experiences with a veterinary preceptorial program. An off-campus, practitioner-supervised training (preceptorial) program for veterinary students at Michigan State University was developed and introduced in the fall of 1972. The program was designed to meet the needs of students concentrating on food animal medicine. It has enabled senior students to receive degeee credit for on-the-job experience with practitioners. Students opting for the program gain experience and knowledge in segments of clinical veterinary medicine that are most available with off-campus practitioners. Exposure of students to the clinical abilities of private practitioners, the case method of study, and diagnostics in a realistic environment are the goals of the program. The program has afforded faculty members and private practitioners meaningful insights into the changing face of veterinary education. Indeed, the consensus of the faculty in the Department of Large Animal Surgery and Medicine at Michigan State University is that an off-campus curriculum. Participating students and practitioners report that the program has been successful and is worthy of expansion and continuation. Both groups rate the program favorably and believe it has been a worthwhile teaching-learning experience.


Experimentally induced vitamin E-selenium deficiency in the growing dog. Eight 5-to 8-week-old Beagle pups were allotted to 4 groups of 2 dogs each. For 55 to 70 days, they were fed either a semisynthetic basal diet (BD) deficient in vitamin E and selenium (Se) (group 1) or the BD supplemented with either 30 IU alpha-tocopherol/kg (group 2), 0.5 ppm Se as selenite (group 3), or 1.0 ppm Se as selenite (group 4). In the dogs fed the BD, clinical signs of vitamin E-Se deficiency developed after 40 to 60 days. These signs were accompanied by increased plasma activity of creatine phosphokinase (CPK) and glutamic oxalacetic transaminase (GOT). The dogs were euthanatized after 10 to 15 days of progressive clinical signs, including muscular weakness, subcutaneous edema, anorexia, depression, dyspnea, and eventual coma. Gross lesions seen at necropsy included ventral subcutaneous edema, generalized skeletal muscular pallor and edema with scattered white longitudinal streaking, prominent brownish yellow discoloration of the intestinal musculature, and a layer of white chalky material at the renal corticomedullary junction. Microscopically, there was evidence of extensive skeletal muscular degeneration and regeneration, focal subendocardial necrosis in the ventricular myocardium, intestinal lipofuscinosis, and renal mineralization. Mean hepatic Se content in the dogs fed the BD was 0.10 ppm (wet weight basis) at necropsy. In the dogs fed the 3 supplemented diets, clinical signs of deficiency did not develop. At necropsy, mild skeletal myopathy was evident histologically in the dogs fed BD and 0.5 ppm Se (group 3) but not in the dogs fed the other supplemented diets. Intestinal lipofuscinosis was found in the dogs fed the 3 supplemented diets but was less severe in the dogs fed the diet supplemented with vitamin E than in those fed diets supplemented with Se.


Cervical abscess and pharyngeal fistula in a horse. A weanling Quarter Horse filly developed ventral swelling of the lower cervical area after difficult passage of a stomach tube for deworming. Intermittent bilateral nasal discharge developed. Radiography revealed fluid and gas density dorsal to the trachea and esophagus. Surgical incision with drainage and debridement of the abscess and fistulous tract, facilitated by use of drains, led to complete recovery. Contrast medium injected after surgery demonstrated a communication between the abscess and the pharyngeal region.


Coagulation studies during experimental hemoglobinemia in humans. Infusion of autologous hemolyzed blood in humans has served as a model for various experimental investigations for many years. Numerous studies have shown this model to be unattended by any adverse clinical reactions. In this study evidence of subclinical disseminated intravascular coagulation (DIC) was sought in normal humans infused with autologous hemolyzed blood. Hemoglobinemia was induced in 10 experiments by a single injection of frozen-thawed blood and in 4 experiments by such an injection of hemolysate followed by a 5-h maintenance infusion. Mean peak plasma hemoglobin following single dose injections was 540 mg/100 ml, while levels during continuous infusion averaged 240 mg/100 ml. The induction of hemoglobinemia was asymptomatic. Coagulation studies showed no significant alteration in prothrombin time, partial thromboplastin time, thrombin time, clottable fibrinogen, or WBC. Fibrin degradation products were not found. Platelet counts fell slightly in the 5-min postinfusion sample but returned to preinfusion levels within 30 min, suggesting a temporary sequestration of platelets rather than consumption. The induction of moderate brief experimental hemoglobinemia in normal subjects did not result in the development of demonstrable DIC.


Flow and age dependence of airway closure and dynamic compliance. The influence of expiratory flow rate and age on the results of measurement of closing volume (CV) of the lung have been studied by a nitrogen single-breath method in 66 asymptomatic lifetime nonsmoking normal subjects between 20 and 82 yr of age. Normal was defined as having values for spirometric measurements within a 95% tolerance interval of reported predicted normal mean values. For the CV determination, inspiratory flow rate was held constant at 0.51/s and studies were carried out at expiratory flow rates of 0.25, 0.5, 1.0, and 1.5 1/s. Our results show that CV expressed as a percentage of vital capacity (VC) and the slope of the alveolar plateau increases with increasing flow rate and age. Dynamic compliance (Cdyn) at frequencies corresponding to peak flow rates of 0.5 and 1.5 1/s was also measured and correlated well with the CV results. Frequency dependence of compliance with aging was demonstrated. Nine smokers with normal spirometric measurements and abnormal CV %VC were also studied. Since the results of Cdyn measurement differentiated only two-thirds of the smokers from the normal population, we suggest that the CV method is probably more sensitive than the Cdyn method for the detection of small airway obstruction.


Effect of physical training on the metabolism of thyroid hormones in man. The effect of a 6-wk program of physical training (track running) on the peripheral metabolism of thyroxine (T4) and triiodothyronine (T3) was evaluated in a group of 11 men. Measurements were made of hormone turnover, urinay and fecal clearances, plasma hormone concentrations, and hormone binding by plasma proteins in all subjects before and after training, After training, metabolic clearance of T3 was increased 8.5% above the pretraining level due to an increased deiodinative clearance of this hormone. No significant change was observed in plasma T3 concentration. The absolute degradation of T3 increased 10.3% after training. In contrast, no significant change in the metabolic clearance of T4 was detected. Significantly decreased plasma concentration of total T4 after 4 and 6 wk of training was apparently not due to decreased hormone binding by plasma protein since no significant alteration in the dialyzable fraction of T4 was detected. The absolute degradation rate of T4 was decreased 8.8% after training. Possible implications of the observed differential impact of training on the degradation of T4 and T3 regarding thyroid hormone economy are discussed.


Assessment of the degradation of thyroid hormones in man during bed rest. The effect of bed rest on the absolute turnover rates of thyroid hormones, thyroxine (T4) and triiodothyronine (T3), was evaluated in man. Bed rest resulted in physical deconditioning, measured by a decreased rate of maximal oxygen consumption; physical deconditioning was associated with no significant alterations in the metabolic clearance of T4 and T3. Plasma concentrations of these iodothyronines were not changed as a result of bed rest. Absolute hormone turnover rates were similar in control and bed-rest subjects. The data suggest that the degradation of thyroid hormones is not influenced by physical deconditioning or hypodynamia.


Inflection point on transpulmonary pressure-volume curves and closing volume. In 20 healthy subjects and 18 patients with bronchial obstruction, closing volume (CV) on single-breath nitrogen washout curves and inflection point (IP) on transpulmonary pressure-volume curves were recorded simultaneously during slow expiratory vital capacity maneuvers. IP and CV did not occur at identical lung volumes, IP being systematically larger than CV for small CV values. This discrepancy could not be attributed to an esophageal or mediastinal artifact. It is suggested that, though CV and IP both express "airway closure," their sensitivity to closure may differ: CV underestimates closure because of a dead space effect; the latter may vary individually. On the other hand, IP may not reflect the true beginning of closure, particularly when it occurs at higher lung volumes.


Glucose metabolism in thyroidectomized and normal dogs during rest and acute cold exposure. Uniformly labeled (14C)glucose was infused iv at a constant rate into unanesthetized surgically thyroidectomized (THY) and normal dogs during the basal state at 22 degrees C and during cold exposure at 4-5 degrees C. 02 uptake, CO2 output, and plasma glucose levels were determined; from these rates of glucose turnover and oxidation were calculated. Comparison of THY with normal dogs shows that at a mean plasma glucose level of 103 mg/100 ml, a significantly lower glucose turnover rate was observed in THY dogs on the basis of a kg body weight, body surface area (m-2), but not per cal BMR. The same percentage of glucose turnover was immediately oxidized in THY and normal dogs, accounting for 16 and 15% of the total caloric expenditure, respectively. Acute cold exposure increased energy expenditure in both THY and normal dogs without a change in glucose level or turnover rate. However, the percentage of glucose turnover which was immediately oxidized as well as the amount of glucose oxidized increased significantly and proportionally to the increase in energy expenditure so that the percent of calories derived from plasma glucose oxidation remained the same as that observed during the control period.


A test of time invariance in models of ferrokinetics. Application of a particular mathematical property of time-invariant systems to experimental data obtained from injection and infusion of radioactive tracer iron in normal subjects is used to show that the generally applied assumption of time invariance to ferrokinetic systems is unjustified and may lead to substantial errors in analysis.


Nature of induction of tryptophan pyrrolase in cold exposure. The activity of hepatic tryptophan pyrrolase in rats exposed to cold increased rapidly and reached a maximum of three-fold at 8 h. On continued exposure up to 48 h stress, the activity partly decreased but remained at a level higher than the initial. Withdrawal from the cold stress reversed the change. Adrenalectomy or treatment with inhibitors of protein synthesis abolished the increase in the enzyme activity during cold stress indicating a possible involvement of corticosteroids and de novo protein synthesis. Treatment with drugs known to block autonomic nervous system failed to inhibit the cold-mediated increase in enzyme activity. The results suggest that the increase in enzyme activity obtained on cold exposure is mediated by corticosteroids and not by either indoleaklylamines or autonomic nervous system. The changes in the enzyme obtained under cold stress with respect to the overshoot phenomenon, relationship to the degree of stress and reversibility on withdrawal from the stress indicate the "adaptate" nature of the response.


Sleep metabolism and age.

Sleep metabolism and age. Continuous recordings were made of oxygen consumption (VO2) and EEG, EMG, and EOG during two nights of sleep for 20 men who were between 19 and 63 yr. old. There was a linear decrease in overnight oxygen consumption with age, even though the older men slept less and woke more often than the younger ones. On the first night, VO2 was higher, and sleep stages less like normal than on the second. In the second night data, individual oxygen consumptions averaged over the whole night ranged from 138 to 482 ml/min (61-247 ml/m2timesmin). No relationship appeared between stage of sleep and VO2 level, but there was usually a decline in VO2 in the first hour of sleep, and a slow rise in the 2-3 h before awaking. Periodic breathing was observed in the men older than 45 yr.


Longitudinal studies of aging in 37 men. The physiological adjustments of the men to both aerobic (5.6 km/h, up 9% grade), and maximal treadmill work were first determined when they were healthy college students, ages 18-22 yr. They were restudied at ages 40-44, and again at ages 49-53 yr. In the aerobic walk VO2 increased in proportion to weight gain, but efficiency did not change with age. The men who had gained most weight showed the greatest elevations of heart rate, blood lactate and "ventilatory equivalent," and reductions of the "O2 pulse" in the walk. At age 50 mean "O2 pulse" in exhausting work had declined 13%, even though mean maximal heart rate had declined 15 beats/min from values observed in youth. At ages 40-44 yr mean VO2 max had declined 25% to values 12% lower than mean values reported in 1938 for men of this age. Eight of the men imporved an average of 11% in VO2 max between ages 40-44 and 49-53 yr; mean VO2 max of the others continued to decline with age. The five men who improved most had increased their participation in vigorous activities (tennis, squash, skiing, jogging, etc.) and had quit or reduced their smoking.


Brain temperature during reversible upper respiratory bypass. Seven male New Zealand white rabbits were trained to run in an exercise wheel. At least 5 days prior to experimentation, each rabbit had a reversible tracheal bypass canula and a hypothalamic thermocouple guide tube chronically implanted. The reversible tracheal bypass enabled the rabbit to breathe normally (through its upper respiratory pathways) or to be placed on bypass (breathe through its neck). Prior to and following exercise, hypothalamic temperature in rabbits breathing normally was lower than rectal temperature. This difference was greatest following a mild heat stress induced by exercise. When the rabbits breathed through the bypass, there were no differences between hypothalamic and rectal temperature. These data suggest that even in a species such as the rabbit, which lacks a carotid rate, hypothalamic temperature is influenced by upper respiratory cooking of venous blood and that the ensuing transfer of heat from the warmer  internal carotid artery to the cooler venous sinuses can effectively cool the brain.


Cardiac acceleration in man elicited by a muscle-heart reflex. The shortening of the R-R interval in response to voluntary and electrically induced isometric muscle contractions of short duration was investigated in 15 volunteers. In some of those experiments the effect of vagal blockade was also studied. The results show: 1) a lag time between the start of the contraction and the following decrease in R-R interval duration of 550 milliseconds; 2) a similar R-R interval response due to voluntary and electrically induced contractions of the same force; 3) no shortening of the R-R interval when the skin is stimulated without ensuing muscular contraction; 4) a complete disappearance of the response to isometric contractions during vagal blockade. A difference in lag time between the onset of arm contraction and cardiac acceleration could not be demonstrated. Most of the results give strong evidence to the existence of a muscle-heart reflex in man, involved in the instantaneous cardiac acceleration at the onset of exercise, that has its origin in the muscles and the vagal nerves as its efferent pathway.


Relative susceptibility of altitude-acclimatized mice to acute oxygen toxicity. The influence of hypoxic acclimatization at altitudes of 0, 5,000, or 15,000 ft on the relative susceptibility to acute oxygen poisoning was determined in 288 adult female mice. After acclimatization periods of 1, 2, 4, or 8 wk, the mice were exposed to oxygen at high pressures (OHP) of 4, 6, or 9 ATA and the times to convulsion and death recorded. A factorial analysis of variance indicated that altitude and OHP level had inverse, log-linear effects on both parameters. The duration of acclimatization progressively decreased the time to death. The onset of convulsions and death was independent of body weight. There were significant interactions on the measured parameters between various combinations of altitude, OHP level, and duration of acclimatization. While alterations in the metabolism of gamma-aminobutyric acid and high-energy compounds are common to both hypoxia and hyperoxia, the most plausible explanation of the results relates to the decrease in buffer base induced by hypoxic acclimatization which might have caused CO2 potentiation of OHP symptoms.


Excretion of retained fluoride in man.

Excretion of retained fluoride in man. In fluoride studies performed in man the excess fluoride excretions in urine and stool over and above the base-line excretions were determined following the discontinuation of fluoride supplements. The fluoride supplements contained either 9.1 mg fluoride as sodium fluoride, given daily for 32 days, or 7.6 mg fluoride as fish protein concentrate (FPC), given daily for 26 days. Following discontinuation of these fluoride supplements the excess fluoride excretions were low and of short duration, in most cases for 6-12 days. Since the fluoride supplements were given for a known period of time, the fluoride retention could be calculated from the fluoride balances, and the excess fluoride excretion corresponded to 9.1% of the retained fluoride given as NaF and to 14.1% of the retained fluoride given as FPC. The excess fluoride was predominantly excreted via the kidney, i.e., 87% of the total excess fluoride excretion was passed in urine and only 13% in stool.


Changes of thermal balance induced by passive heating in resting man. Heat acclimatization has been induced in 12 resting healthy men by 90-min exposure to 45C dry bulb and 24% relative humidity for 9 successive days. The most significant results ovserved were 1) increased sensitivity of sweating with marked quickening of sweat measured, 2) decreased rate of body heat storage associated with a lower rectal temperature at end of exposure, as follows: 14.07 plus or minus 1.58 Wtimeshtimeskg-1 before and 9.39 plus or minus 1.69 afterward for body heat storage; 37.55 plus or minus 0.15C before and 36.99 plus or minus 0.24C afterward for rectal temperature. In contrast, no significant changes were observed in the final sweat rates, mean skin temperatures, or the heat conductance between the body interior and skin surface. The quickness of the heat dissipation process caused by both increased sensitivity of sweating and lower internal body temperature is the major factor in achieving a thermal balance and a decreased body heat content after acclimatization.


Maximal aerobic capacity at different levels of carboxyhemoglobin. Previous studies had indicated that maximum aerobic power (VO2 max) would be seriously impaired when HbCO levels were above 7% but was not altered if HbCO was around 2.7%. The present studies indicated that the critical level at which HbCO influenced VO2 max was approximately 4.3%. This was accompanied as in the above-noted studied with a reduction in total work time to the attainment of VO2 max. Two procedures to raise HbCO to appropriate levels were employed, i.e., a buildup wherin HbCO was incrementally increased by breathing ambient air containing 75 or 100 ppm CO and a bolus plus maintenance procedure. In the latter, HbCO was raised to the level attained in the buildup test by giving a "bolus" of CO followed by the continued inhalation of CO at a level to just maintain this level of HbCO regardless of the magnitude of the ventilation. Regardless of the mode of presentation, the decrement in VO2 max occurred at the same level of HbCO. These observations are of considerable significance, since it indicated that even low ambient levels of CO (23.7 ppm) would result in lowering maximum aerobic power if the individual had been previously exposed to CO such that the level was raised to this critical point.


Pulmonary venous waterfall and perivenous pressure in the living dog. Evidence has been obtained that a waterfall effect occurs in the pulmonary veins of the living dog. Anesthetized open-chest dogs were used. Small catheters were passed from the left atrium retrogradely up the pulmonary veins and venous tributaries until they finally pierced the venous walls. They were then pulled out through the surface of the lung until their bellshaped inner ends wedged in small side branches of the vein. They were used to measure upstream venous pressures via the occluded tributary without occlusion of the vein. The pulmonary lymphatics and bronchial circulation were untouched. Alveolar pressure (transpulmonary pressure, Ptp) was held constant during the periods of measurement. We found that intrapulmonary (upstream) venous pressures did not rise with elevation of left atrial (downstream) pressures at any Ptp until a pressure of at least 7 cmH2O above the base of the lung was exceeded. This left atrial pressure (below which a venous waterfall or flow-limiting segment effect was present) increased with Ptp when Ptp exceeded about 15 cmH2O. The waterfall occurs in the larger veins at the lung surface. Its presence and location suggest that intrapulmonary veins in the living animal are held open by the surrounding lung tissue and that neither the flow through them nor their contained volume is influenced by left atrial pressure when this is low.


A new method for the measurement of percent oxyhemoglobin. A new method is presented for the spectrophotometric determination of percent oxyhemoglobin (percent saturation) in whole blood. The method is based on a theory of light absorption and scattering by particulate suspensions and requires the measurement of optical densities (D) of blood at three closely spaced wavelengths. The contribution of scattering to the optical density at each wavelength is determined from optical density values at two isosbestic wavelengths (546 and 520 nm) and the optical density at the third wavelength (555 nm) is related to the extent of oxygenation of the hemoglobin. The wavelength independence of the scattering contribution (B) induced by red cells was established from 510 and 575 nm. The optical density of red blood cell suspensions was measured with a spectrophotometer and a linear relationship was found between percent saturation and the corrected optical density ratio, (D555  B)/(D546  B). For a given saturation, this ratio was independent of optical path length (0.1-2 mm) and hematocrit (3-50%).


Measurement of percent oxyhemoglobin in the microvasculature. A new method is applied to the determination of percent oxyhemoglobin (percent saturation) in whole blood. The method is based on a theoretical treatment of light absorption and scattering by particulate suspensions and requires the measurement of optical densities (D) of blood at three closely spaced wavelengths. The optical density of red blood cell (RBC) suspensions was measured with a video microdensitometer and a linear relationship was found between percent saturation and the corrected optical density ratio, (D555  B)/(D546  B). For a given saturation, this ratio was independent of optical path length (12 mum-2 mm), hematocrit (3-50%), and RBC velocity (1.5-17 mm/s). The applicability to microvascular measurements has been assessed through the use of TV microdensitometry on micropipettes with flowing RBCs and on microvessels in the hamster cheek pouch.


Moment analysis of multibreath lung washout. A moment analysis of multibreath nitrogen washout has been developed to provide a sensitive, quantitative measure that characterizes the inhomogeneity of pulmonary ventilation. To test the analysis scheme, we studied 5 normal subjects and 16 subjects with obstructive lung disease who performed each washout test at constant tidal volume and frequency. Subjects executed the wahout test 3-4 times at different tidal volumes (0.5-1.5 liters) and frequencies (10-30/min). Plotting washout data as dimensionless end-tidal nitrogen concentration versus the cumulative expired volume normalized by the functional residual capacity (CEV/FRC) renders the washout curves of each individual almost superposab le despite changes in breathing frequency and tidal volume from test to test. The dimensionless washout curve is treated as a distribution from which the normalized first (M1/Mo) and second (M2/Mo) moments are obtained. These parameters clearly display diagnostic clustering for various disease states. With respect to the normal subjects, the magnitude of M1/Mo was 26% greater for asthmatics, 38% greater for bronchitics, and 52% greater for emphysematics. This moment analysis provides an objective, quantitative assessment of the extent of ventilation inhomogeneities without specification of a lung model.


A method for the assessment of phasic vagal influence on tidal volume. Vagal influence related to lung volume changes results in reduction in tidal volume during spontaneous breathing due primarily to premature termination of inspiration. The strength of this vagal influence was traditionally assessed by the duration of apnea following lung inflation, a method recently shown to be inadequate and potentially misleading. An alternate method is described utilizing analysis of the volume tracing of spontaneous breaths and the tracheal pressure tracing during the first breath following airway occlusion at FRC. A formula was devised which, on the basis of previous observations, should predict the tidal volume to be obtained in the absence of phasic vagal influence. The formula was tested in four pentobarbital-anesthetized rabbits using a technique of vagal cooling which rapidly eliminated the vagal influence under study. It was found that the tidal volume obtained following vagal block could be accurately predicted provided allowances were made for the vagally mediated terminal inhibition during spontaneous breathing and the relative stiffness of the respiratory system at high lung volumes.


A microindentation technique to measure rheological properties of the vascular intima. A microindentor was developed to measure the depth of indentation of the intimal surface of arterial tissue loaded by flat-ended, cylindrical probes. The depth of indentation depended on the initial stretch of the tissue which required a rigid support (plaster of Paris) beneath the adventitial surface. Probe tips used ranged from 550-mum down to 65-mum diameter while loads ranged from 800 to 15 mg. The depth of indentation was markedly time dependent; that obtained 30 s after loading (variation of 30) was reproducible and served as a useful parameter of viscoelasticity of the aortic intima and supporting tissues of dog and man. The mean variation of 30 (0.19-mm diam tip, 120-mg load), obtained from longitudinal series of indentations of nine dog aortas, ranged from 40.8 to 68.8 mum while coefficient of variation in these series ranged from 4.8 to 15.9%. Intimal pads were found to have greater resistance to identation than adjacent tissue; likewise the tissue on the dorsal, intimal surface of the aorta had lower variation of 30 values compared with the rest of the intima. Lipid-filled intimal regions were about twice as complaint as macroscopically spared areas. The technique should prove useful in understanding the microrheological response of the blood vascular interface to hydrodynamic stresses.


A simple relation between performance in running and maximal aerobic power. As the cost of running amounts to about 1 cal per m covered and per kg of body weight, when the aerobic power (VO2, ml/min kg) is known and the energy from glycolysis assumed to be about equal to the energy from oxidations in 1 min, the time employed to cover a given distance can be predicted: a nomogram is given to facilitate the calculation. Conversely from the time employed to cover a given distance the aerobic power in VO2, ml/kg times min can be calculated.


Apparatus for intracellular electrophysiological measurements at 200 ATA. An apparatus has been constructed and tested at pressures to 200 ATA which meets the basic requirements for intracellular microelectrode work. Standard microelectrodes, unaffected by pressure in this range, were used with lobster axon and frog sartorius fibers and action potentials have been recorded at pressure up to 151 ATA. The chamber itself has a simple roll-in door and a modular design that recommends it as a highly convenient multipurpose vessel for work at moderately high pressures.


A system to regulate aortic pressure in the rat. A servo-controlled aortic clamp has been designed and used for the first time in the anesthetized rat to control femoral arterial pressure. An electrical schematic is given and details of the mechanical clamp used to compress the abdominal aorta are shown. The system has a long-term (30 min) mean pressure control of plus or minus 2 mmHg although some 1-2 s transients of plus or minus 3 mm may occur. It responds to a desired step change in pressure in less than 2 s. The system allows accurate and convenient control of aortic pressure and minimizes the technical difficulties encountered when one attempts similar control by manual means. It therefore facilitates investigation of the effect of transient or steady-state changes in arterial pressure on organ function, e.g., autoregulation of renal blood flow and glomerular filtration rate.


Permanent cannulation of the hepatic portal vein in rats. A technique is described for chronic cannulation of the hepatic portal vein in rats. The T-shaped cannula is made in the laboratory from polyethylene tubing. Implantation of the cannula allows injection or blood withdrawal in a small laboratory animal which is unanesthetized and unrestrained. Simultaneous implantation of intracardotid and intraportal cannulas permits comparisons of blood composition in order to study the role of the liver or intestinal absorption.


A bidirectional respiratory flowmeter using the hot-wire principle. We have devised a bidirectional respiratory flowmeter using the hot-wire principle. The flow-direction sensor consists of a pair of tungsten wires strung parallel to the platinum hot-wire one on each side of the platinum wire. When the gas stream passes through the transducer, the upstream wire is cooled and the downstream wire is heated by the gas stream producing a temperature difference between the two tungsten wires. The difference in resistance thus produced between them is detected and amplified by a differential amplifier whose output serves as a triggering signal of flow inversion. The switching times of the flow inversion of the present instrument are 3 ms during panting and 10 ms during quiet breathing, when the distances from the platinum wire to the tungsten wires are 1.6 mm. Artifacts produced by the delay in switching are practically negligible. The flowmeter can be adapted for many kinds of respiratory flow measurement, except under the condition when inflammable gases are used.


Technique for accurately producing desired carboxyhemoglobin levels during rest and exercise. A method is described for accurately adjusting a subject's blood carboxyhemoglobin (HbCO) level and then maintaining this level during either rest or exercise. The data presented describe the constancy with which HbCO levels (between 3 and 5% HbCO)were maintained despite varying the inspired ventilatory volumes from 5 to 120 1/min (BTPS). After an initial bolus inhalation of CO to bring the blood HbCO to a desired level, the concentration of CO in the inspired air to maintain this level of HbCO can be predicted from the following equation: COI (ppm) = desired %HbCO divided by 0.180.


Nonrebreathing valve for respiratory measurements in unsedated small mammals. A nonrebreathing valve for measuring the respiratory volumes and gas exchange of unsedated, trained small mammals is described. The valve was easily fabricated from Plexiglas and latex flaps cut from a surgical glove. It has a low dead space and airflow restance and can be scaled to fit a variety of small mammal species. Ten hamsters were trained to breathe through the valve while expirate was collected. Respiratory frequency, tidal volume, minute volume, O2 uptake, CO2 output, respiratory exchange ratio, and ventilatory equivalent were measured.


Guanosine triphosphate binding sites in solubilized myocardium. Relation to adenylate cyclase activity. Binding of (3H)GTP to solubilized preparations of myocardial adenylate cyclase, partially purified by DEAE-cellulose chromatography, as been studied in an attempt to gain further insight into the mechanisms by which guanine nucleotides regulate adenylate cyclase activity. Although several peaks of (3H)GTP-binding activity were present in crude preparations of solubilized myocardium, one peak was associated with the adenylate cyclase peak. Binding of (3H)GTP to this material was rapid (equilibrium within 3 min at 37 degrees) and reversible and not associated with nucleotide hydrolysis. Scatchard analysis revealed a single class of (3H)GTP binding sites with KA = 3 x 10-6 M-1 and total binding capacity of 50 pmol per mg of protein. The GTP analog Gpp(NH)p competed for the sites with an affinity somewhat lower than GTP, although its ability to activate the adenylate cyclase was far greater. GTP and other guanine nucleotides activated the soluble cyclase only weakly, although they antagonized competitively enzyme stimulation by Gpp(NH)p. Ability of GTP and other nucleotides to compete with (3H)GTP for binding sites and to antagonize competitively adenylate cyclase activation by Gpp(NH)p were directly parallel. The potency series was GTP = GDP = dGTP greater than GMP greater than ITP greater than UTP, CTP. Dissociation constants of nucleotides for the sites determined by inhibition of (3H)GTP binding and inhibition of Gpp(NH)p activation of cyclase agreed closely. Gpp(NH)p dose-response curves for activation of adenylate cyclase and inhibition of (3H)GTP binding were superimposable.


Enzyme deletions and essential fatty acid metabolism in cultured cells. Conversion of 14C-labeled linoleic acid to dihomo-gamma-linolenic and arachidonic acids, precursors of the PG1 and PG2 series of prostaglandins, was examined in a number of mammalian cell lines in tissue culture. Diploid cells converted linoleate to arachidonate in good yield. Heteroploid or transformed cells did not desaturate linoleic acid but readily converted dihomo-gamma-[14C]linolenate to arachidonate. These observations demonstrate that separate delta6 and delta5 desaturases are involved in the conversion of linoleic to arachidonic acid in mammals. The results indicate that loss or modification of the delta6 desaturase system is a frequent occurrence in transformed cells.


Activation of pigeon erythrocyte membrane adenylate cyclase by guanylnucleotide analogues and separation of a nucleotide binding protein. Metabolically stable GTP analogues were 10 to 40 times more potent activators of DL-isoproterenol-stimulated adenylate cyclase (EC 4.6.1.1) from pigeon erythrocyte membranes that GTP. The order of effectiveness was guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) greater than guanylyl imidodiphosphate (Gpp(NH)p) greater than guanylyl methylenediphosphonate (Gpp(CH2)p greater than GTP. In contrast to activation with GTP, activation by analogues was independent of ATP concentration. The analogues seem to bind, however, to the same regulatory sites in membrane preparations to which GTP is bound but with higher affinity; Kdiss for (14C)Gpp-(nh)p and (3H)Gpp(CH2)p and membranes was 0.7 and 2.4 x 10-7 M, respectively. DL-Isoproterenol did not increase the amount of guanylnucleotide bound, it merely accelerated and potentiated activation. Bound radioactive GTP analogues were recovered unchanged from the membrane pellet. This and mutual displacement of analogues and GTP ruled out covalent attachment of the whole or of part of the nonphosphorylating GTP analogues. Treatment of the membrane preparation with Gpp(NH)p effectively (greater than 80%) protected DL-isoproterenol-activated adenylate cyclase against the action of Filipin or Lubrol PX. Activation of membrane-bound adenylate cyclase with GTP analogues resulted in a stable enzyme which could be nearly completely resolved from membranes with Lubrol PX and stripped of lipids and detergent without loss of activity. This effect was synergistically amplified by DL-isoproterenol. A protein fraction with an apparent molecular weight of 230,000, containing about 90% of (14C)Gpp(NH)p originally bound to membranes, could be solubilized and separated from adenylate cyclase activity by chromatography on Sepharose 4B. The binding protein was purified about 40- to 80-fold from activated membranes. Removal of the nucleotide binding protein was also achieved by affinity chromatography with GTP gamma S coupled to Sepharose via a spacer. When membranes which were not or only weakly and reversibly activated (with GMP) were used as source of the soluble preparation, removal of the binding protein resulted in 75% loss of Gpp(NH)p activation without change in basal and Mg2+/F-stimulated adenylate cyclase activity. It is assumed that the GTP analogues cause an unphysiological, irreversible activation of membrane-bound adenylate cyclase, because, in contrast to the natural guanylnucleotides whose action they mimic, they are metabolically inert and bound quasi-irreversibly to regulatory sites.


Alteration of human erythrocyte membrane properties by complement fixation. Erythrocyte survival studies of complement-coated radiolabeled erythrocytes have shown rapid removal of these cells from the peripheral blood with a return of these cells into the circulation within a few hours. We studied complement-coated human erythrocytes and measured surface charge and deformability,  two parameters believed to be important in erythrocyte survival. Erythrocytes were coated with complement by two in vitro techniques: the addition of (a) low ionic strength sucrose, and (b) IgM cold agglutinins. Erythrocytes obtained from three patients with cold agglutinin disease were used as a source of in vivo complement-coated cells. No difference was found in surface charge as measured by electrophoretic mobility between erythrocytes from normal subjects and complement-coated erythrocytes from any of the three sources. When deformability was measured by filtration through 3-mum polycarbonate sieves, marked decreases in deformability were found in complement-coated erythrocytes. The filtration returned toward control levels by incubating the complement-coated erythrocytes in serum for 1 h and correlated with decreases in immune adherence. Using screen filtration pressure as a measure of deformability, a positive correlation between number of C3 molecules per erythrocyte and decreased deformability was found. C3b appeared responsible for the decreased deformability of the erythrocytes, since conversion of C3b to C3d resulted in a return of deformability toward normal. The data suggested that the sequestration of complement-coated human erythrocytes in the microvasculature can be explained in part by decreased deformability and changes in immune adherence.


Hormonal characteristics of the human menstrual cycle throughout reproductive life. The changes in serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FHS), estradiol, and progesterone that occur both early and late in reproductive life were characterized and compared with findings in young, normal women and in patients with certain menstrual disorders. A total of 50 complete menstrual cycles in 37 were examined. Five distinct patterns of hormonal regulation were found, three of which are reported here: (a) A long follicular phase and delayed follicular maturation in young women with long, unpredictable intermenstrual intervals from menarche; (b) a short follicular phase with increasing age and in short cycles in perimenopausal women; and (c) true anovulatory vaginal bleeding in long cycles in perimenopausal women. The short cycles before and during the menopausal transition were found to have lower E2 levels and high FSH concentrations throughout, while LH remained in the normal range. During long cycles in perimenopausal women, concentrations of LH and FSH were in the menopausal range. However, follicular maturation was observed months after high levels of gonadotropins were attained. These studies permit the characterization of the menstrual history of the normal woman in terms of the hormonal changes that occur and provide a basis for the definition of several disorders of follicular maturation.


Evidence for physiological importance of calcitonin in the regulation of plasma calcium in rats. To determine the physiological importance of calcitonin in the regulation of plasma calcium, studies were carried out in fasting animals to (a) assess the acute effects of thyroparathyroidectomy (TPTX) and thyroidectomy (TX) on plasma and urinary calcium; (b) investigate whether the changes in plasma calcium produced by removal of the glands were dependent on the presence of the kidney; and (c) determine if the effect of TPTX on plasma calcium is affected by age. Except where otherwise indicated, all studies were carried out on fasting male Wistar rats weighing over 300 g. The following observations were made. (a) TPTX and TX caused an increase in plasma calcium in nephrectomized animals. (b) This increase was not dependent on nephrectomy since in intact animals bearing autoparathyroid transplants TX also caused a significant rise in the mean plasma calcium level (0.37 mg/100 ml at 1 1/2 h). (c) Urinary calcium increased twofold in the 3-h period immediately after TX. (d) In unnephrectomized immature (50-g) rats, TPTX caused a progressive decrease in plasma calcium in contrast to old (360-g) rats, where a significant fall observed at 6 h was preceded by an increase in plasma calcium (0.5 mg/100 ml at 1 1/2 h). From these observations we conclude that: (a) calcitonin must play an important physiological role in the regulation of plasma calcium since the termination of its basal secretion caused an immediate but transient increase in plasma calcium in old unfed rats; (b) the relative importance of calcitonin and parathyroid hormone in the acute regulation of plasma calcium is age-related; and (c) the action of parathyroid hormone on bone may be modified by changes in ambient calcitonin concentration.


Stimulation of active and passive sodium absorption by sugars in the human jejunum. The effects of glucose and fructose on water and sodium absorption in the human jejunum were compared to assess the relative contribution of active and passive sugar stimulation of sodium transport. The effect of fructose is assumed to be entirely passive, and the difference between the effects of fructose and glucose is assumed to be a measure of sugar-stimulated, active sodium absorption. Water and sodium movement with mannitol was the base line. Three sets of test solutions with differing sugar concentrations were studied. Fructose stimulated 66-100 per cent as much net sodium and water absorption as glucose. Fructose stimulated potassium absorption, whereas glucose stimulated potassium secretion. Urea absorption was stimulated by both sugars. Glucose and fructose stimulated sodium absorption when chloride was the major anion, but they had relatively little effect on net sodium movement when chloride was replaced by bicarbonate or sulfate. It is concluded that glucose stimulates passive and active sodium transport in the human jejunum. Stimulated active sodium absorption generates an electrical potential across the mucosa that causes sodium (and potassium) secretion and partly or completely nullifies the effect of active sodium transport on net sodium movement. Net sodium absorption sitmulated by glucose is mainly (66-100 per cent) the passive consequence of solvent flow. The accompanying anion determines the degree to which sugars stimulate sodium absorption (C1 greater than SO-4 greater than HCO3). The effects of bicarbonate and sugars on jejunal sodium absorption are not additive.


Urinary prostaglandins. Identification and origin. Human urine was analyzed by mass spectrometry for the presence of prostaglandins. Prostaglandin E2 and F2alpha were detected in urine from females by selected ion monitoring of the prostaglandin E2-methylester-methoxime bis-acetate and the prostaglandin F2alpha-methyl ester-Tris-trimethylsilylether derivative. Additional evidence for the presence of prostaglandin F2alpha was obtained by isolating from female urine an amount of this prostaglandin sufficient to yield a complete mass spectrum. The methods utilized permitted quantitative analysis. The origin of urinary prostaglandin was determined by stimulating renal prostaglandin synthesis by arachidonic acid or angiotensin infusion. Arachidonic acid, the precursor of prostaglandin E2, when infused into one renal artery of a dog led to a significant increase in the excretion rate of this prostaglandin. Similarly, infusion of angiotensin II amide led to a significantly increased ipsilateral excretion rate of prostaglandin E2 and F2a in spite of a simultaneous decrease in the creatinine clearance. In man, i.v. infusion of angiotensin also led to an increased urinary eliminiation of prostaglandin E. These results show that urinary prostaglandins may originate from the kidney, indicating that renally synthesized prostaglandins diffuse or are excreted into the tubule. Thus, urinary prostaglandins are a reflection of renal prostaglandin synthesis and have potential as a tool to delineate renal prostaglandin physiology and pathology.


Ozone interaction with rodent lung. III. Oxidation of reduced glutathione and formation of mixed disulfides between protein and nonprotein sulfhydryls. Nonprotein sulfhydryls (NPSH), a major source of cellular reducing substances, were examined in lung tissue after short-term exposure of rats to O3. While the NPSH level was unaffected by low-level exposures (e.g., 0.8 ppm for up to 24 h or 1.5 ppm for up to 8 h), it was significantly lowered by higher exposure regimens (e.g., 25 per cent after 2 ppm for 8 h and 49 per cent after 4 ppm for 6 h). After exposure to 4 ppm O3 for 6 h the level of reduced glutathione (GSH), which accounted for approximately 90 per cent of NPSH in the lung, decreased 40 per cent but without a rise in the level of oxidized gluathione (GSSG). Treatment of lung homogenate with borohydride led to recovery of NPSH in exposed lungs to control values, suggesting that NPSH or GSH oxidation during in vivo O3 exposure resulted in formation of mixed disulfides with other sulfhydryl (SH) groups of lung tissue. Extracts of borohydride-treated particulate and supernatant fractions of lung homogenate were analyzed for NPSH by paper chromatography. From this analysis GSH appeared to be the only NPSH bound to lung tissue proteins via mixed disulfide linkage. The formation of mixed disulfides appeared to be a transient phenomenon. Immediately after a 4-h exposure to 3 ppm O3 the level of mixed disulfides was small (15 per cent of the total NPSH) but attained a peak (equivalent to 0.6 mumol NPSH/lung) after a recovery for 24 h. However, the level diminished considerably within 48 h of recovery.