A liquid chromatography-assisted assay for angiotensin-converting enzyme (peptidyl dipeptidase) in serum. I describe modification of the spectrophotometric assay described by Cushman and Cheung [biochem. Pharmacol. 20, 1637 (1971)] for serum angiotensin-converting enzyme, with use of "high-pressure" liquid chromatography to measure the hippuric acid end product. After reaction of 10 microL of untreated serum with the angiotensin-converting enzyme substrate analog hippuryl-L-histidyl-L-leucine, the hippuric acid produced is extracted into ethyl acetate and quantitated, relative to an added internal standard, by liquid chromatography. Total chromatographic running time is 3 min per sample, with a within-run CV of 4.3% and a day-to-day CV of 6.6%, for aliquots of plasma supplemented with 1 mmol of hippuric acid per liter. The measurements are linear for hippuric acid in amounts up to 20 nmol per assay. The presence of large quantities of lipid in the serum did not affect the accuracy of the determination.


Human corticotropin (ACTH) radioimmunoassay with synthetic 1--24 ACTH. A corticotropin antiserum was obtained from rabbits immunized with synthetic 1--24 corticotropin conjugated with bovine serum albumin. The antiserum did not cross react with synthetic alpha-melanotropin or with synthetic beta-endorphin and had a cross reactivity of 0.23% with human beta-lipotropin. We developed a radioimmunoassay with the antiserum obtained, in which we used polyethylene glycol in conjunction with a second precipitating antibody for fast (15-min) separation of antibody-bound and free corticotropin. The assay had a sensitivity of 16 ng/L and was validated on patients with various pituitary and adrenal diseases. From 103 normal subjects, the median value for corticotropin in specimens collected during the morning was 34 ng/L of plasma; the upper 95% confidence limit of the normal range was 98 ng/L.


Evaluation of commercial heparin preparations for use in the heparin-Mn2+ method for measuring cholesterol in high-sensity lipoprotein. Commercial heparin preparations (18 lots) from seven manufacturers were compared in the heparin-Mn2+ procedure for high-density-lipoprotein cholesterol quantitation. With normotriglyceridemic samples, 16 heparin lots, isolated from porcine intestinal mucosa, gave mean values for supernatant cholesterol that did not differ statistically; all were within 7 mg/L. Two heparin preparations from bovine lung gave results that were slightly (16 mg/L, average) but significantly (p less than 0.005) lower. With hypertriglyceridemic samples, we observed greater variation in supernatant cholesterol among the heparin preparations, which was ascribable to variable sedimentation by centrifugation of very-low-density and low-density lipoproteins precipitated by heparin-Mn2+ treatment. If the precipitated lipoproteins were completely removed by an ultrafiltration procedure, we saw no significant difference among the heparin preparations for results with hypertriglyceridemic samples.


Radioimmunoassay of total urinary estrogens. We describe a radioimmunoassay for total estrogens in urine. Estrogens are extracted by adsorption on XAD-2 resin, eluted with methanol, and acid-hydrolyzed. Estrogens are then determined in a diluted aliquot of the hydrolysate. Within-run coefficients of variation for estriol values between 6.0 and 100 micrograms/L were less than 5%; between-run CV was 19.6% at an estriol concentration of 16.8 microgram/L. A correlation coefficient of 0.96 was found on comparison of the radioimmunoassay (y) with a standard fluorometric assay (x); the regression equation is y = 2.18 x - 6.70 micrograms/L. Determination on pregnancy and non-pregnancy urine can be done within 4 h.


Four electrophoretic methods compared for diagnosis of type III hyperlipoproteinemia. Blood drawn from 192 probands and 1129 first-degree relatives who were participants in a collaborative family study of hyperlipoproteinemia at nine Lipid Research Clinics was used to prepare aliquots of whole plasma and top (d less than 1.006 g/mL) and bottom (d greater than 1.006 g/mL) ultracentrifugal fractions. Each aliquot was analyzed at a central laboratory by electrophoresis on paper, agarose, and polyacrylamide gel, and by a combined electrophoretic precipitation technique. The electrophoretograms were evaluated for the presence or absence of a "floating-beta" lipoprotein band. All four methods agreed completely for 92.3% of the samples. An additional 2.0% of the samples were in agreement for three electrophoretic methods, but the paper electrophoretic results were not interpretable. Another 1.9% were considered to be "floating-beta" positive by paper electrophoresis but negative by the other three electrophoretic methods.


Improved method for simultaneous determination of cholesterol in high- and low-density lipoproteins. A previously described procedure for simultaneous determination of cholesterol in high- and low-density lipoproteins (HDL and LDL) [Clin. Chem. 24, 1504--1508 (1978)] has been improved by using centrifugation instead of ultrafiltration. Addition of NICl2 (2.0 mmol/L) to the reagent produced a good separation of HDL from the complex of low- and very-low-density lipoprotein-heparin-Ca2+ on centrifugation at 3000 rpm for 15 min. Replicate analyses for high-density lipoprotein cholesterol by the present method demonstrated the following intra-assay precision: mean = 389 mg/L, SD = 11 mg/L, CV = 2.8%. The present (y) and original (x) methods gave results that agreed reasonably well (n = 50, r = 0.960, y = 1.0x + 0.9). The enzymic method for HDL- or (HDL + LDL)-cholesterol after the separation of their fractions gave erroneous results, in particular in the cases of hyperbilirubinemic sera and in (HDL + LDL) fractions.


Triglyceride-poor very low density lipoprotein in human serum. The chemical and physical properties of very low density lipoproteins, isolated from the pool of the sera of 60 persons with high pre-beta and normal triglyceride and cholesterol concentrations, have been studied. These very low density lipoproteins, a designated as triglyceride-poor very low density lipoproteins, consist of 20.5% phospholipids, 30.8% free cholesterol, 15% cholesterol esters and 33.7% triglycerides. Their protein content consists of 54.5% apo B, 26% apo A, 11.5% apo E and only 8% apo C, so they differ from any serum lipoprotein described until now. Triglyceride-poor very low density lipoproteins consist of spherical particles 300-450 A in diameter as revealed by electron microscopy.


Urinary high density lipoprotein in minimal change glomerular disease and chronic glomerulopathies. Serum lipids and lipoproteins and urinary apolipoprotein A (Apo A) were determined in two groups of patients. One group consisted of 11 children (ages ranging from 4 to 14 years) with minimal change glomerular disease. The other group consisted of 13 patients, eight less than 19 years old five adults, with different types of chronic glomerulopathy. Elimination of urinary lysozyme was a feature of chronic glomerulopathies, and creatinine clearances were also significantly lower in this group. Patients with chronic glomerulopathies had significantly lower HDL cholesterol and Apo A concentrations in their sera. In contrast, urinary Apo A concentrations were significantly higher in patients with chronic glomerulopathies, who also showed significantly lower urinary protein selectivities. Lipoprotein electrophoresis of urines containing Apo A showed distinct high-density lipoprotein (HDL) fractions, suggesting that HDL is eliminated in the urine as a result of increased glomerular permeability. This is also supported by a correlation coefficient of 0.77 between the selectivity indices and the ratio of urinary Apo A to total proteinuria. The determination of urinary Apo A appears to give valuable diagnostic information in patients with glomerular disease. According to our results the absence of urinary Apo A is very suggestive of minimal change glomerular disease.


The lipoprotein composition of amniotic fluid. The lipoprotein composition of amniotic fluid (AF) has been studied and it was found that it consists of high density lipoprotein (HDL) only. At about the 26th week of pregnancy the HDL content reaches a maximum and decreases thereafter. On crossed immunoelectrophoresis the HDL of AF was separated into two components if the agar layer contained an antiserum against apolipoprotein A-I. The component with the higher electrophoretic mobility (but not the slower moving component) could also be visualized with an antiserum against HDL. When maternal or umbilical cord serum (UCS) was analysed by crossed immunoelectrophoresis using the anti-apoA-I antiserum, we found that they only contained the faster moving lipoprotein component. If UCS was treated with phospholipase A2 and then subjected to crossed immunoelectrophoresis, the slower moving component appeared at the expense of the faster one. Likewise when UCS was incubated with AF from the 15th week of pregnancy or later, the lipoprotein component of UCS with the high mobility was partially converted to the component with the slower mobility. It is concluded that AF probably contains a phospholipase, an enzyme which may promote the onset of labour.


The effect of fasting on the interactions between human low density lipoproteins and calcium, heparin and chondroitin sulfate. Fasting modified the in vitro interactions between low density lipoproteins (LDL) and glycosaminoglycans. Calcium had a potentiating effect on these interactions. LDL-calcium-heparin interaction was dependent on the presence of sialic acid whereas LDL-calcium-chondroitin sulfate interaction was inversely correlated with cholesterol content.


Polymorphonuclear leucocyte fluorescence and cryoglobulin phagocytosis in systemic lupus erythematosus. Peripheral blood polymorphonuclear leucocytes (PMN) and cryoglobulins were isolated from patients with systemic lupus erythematosus (SLE). Fluorescent inclusions were found in PMN. Normal donor PMN were incubated with the sera and cryoglobulins from SLE patients. In most cases inclusions were observed after incubation. The high incidence of anti-IgG activity in phagocytosed cryoglobulins confirms the importance of the rheumatoid factor in phagocytosis of immune complexes. It is concluded that phagocytosis of cryoglobulins supports the suggestion that cryoglobulins are a subpopulation of immune complexes.


Induction of plaque-forming cells in human blood lymphocytes cultured in the presence of antigen and Epstein-Barr virus: a study with normal donors and infectious mononucleosis patients. Normal human peripheral blood lymphocytes, stimulated in vitro with SRBC in the presence of Epstein-Barr virus (EBV), gave rise to plaque-forming cells (PFC) specific for the antigen. PFC levels were very low before day 4 and increased thereafter, reaching a maximum around day 8. However, the kinetics of the response varied considerably from donor to donor and from experiment to experiment. In some instances a second peak of PFC was obtained beyond day 10. Large differences in the magnitude of the response were observed among different normal donors, the overall responsiveness range covering four orders of magnitude. Peripheral blood lymphocytes from infectious mononucleosis patients in the acute stage of the disease, when a high titre of heterophil and anti-EBV antibodies were present, did not give rise to PFC. A return to normal responses was observed during recovery from the disease.


Opsomyoclonus and neuroblastoma. Response to ACTH. Children with neuroblastoma whose opsomyoclonus recurs or persists after initial surgery should be carefully evaluated for tumor. If no tumor is detectable, therapy with ACTH should be considered in patients whose symptomatology is severe enough to compromise normal development.


Ovarian tumours in children--a review of 40 cases. The records of 40 children treated for ovarian tumours in Manchester since 1940 are reviewed. The series includes 12 dysgerminomas, nine endodermal sinus tumours, six malignant teratomas, three granulosa theca cell tumours, one Sertoli-Leydig cell tumour, one mixed chorioncarcinoma/dysgerminoma and eight cases of non-germ cell or uncertain origin. Presenting features and treatment are discussed. Survival is generally poor apart from the dysgerminomas, granulosa theca and Sertoli-Leydig cell tumours, all but one of whom are alive and disease free. The need for routine post-operative radiotherapy in cases in dysgerminoma confined to one ovary following total resection is questioned. Combination chemotherapy may improve survival in the future, particularly of children with endodermal sinus tumour. The only surviving child other than those mentioned above has had combination chemotherapy for an endodermal sinus tumour.


Newer knowledge of vitamin D and its metabolites in health and disease. Renewed interest in vitamin D, over the past several years, has resulted in increased knowledge of (1) the metabolic pathways which result in production of an active metabolite, (2) the role of its various metabolities at target tissues, and (3) its interaction with other control systems in the maintenance of calcium/phosphorus homeostasis. Presently, a role for 1, 25-(OH)2 D3 can be identified in the regulation of (1) calcium and phosphorus absorption from the intestine, (2) PTH production, and (3) calcium mobilization from bone (in conjunction with PTH). Several other actions at the kidney, muscle and other organs have been suggested but not proven. In contrast, the actual process of bone mineralization may be dependent, in part, on 25-(OH) D3. Despite these major advances in our knowledge, many gaps remain and controversial data continues unresolved. However, application of this new information to analysis of a wide variety of disease states has resulted in remarkable progress in appreciation of the pathogenesis and appropriate treatment for these diseases. Nonetheless, continued research promises further advances and more precise definition of disease states as well as delineation of the therapeutic role of Vitamin D metabolites in their treatment.


Clostridium perfringens septic arthritis. Report of a case and review of the literature. This is a report of Clostridium perfringens septic arthritis in a 5-year-old boy and a review of the literature on 6 previously reported cases. The treatment is the same as for other organisms producing septic arthritis.


Some effects of short-term methylprednisolone therapy in normal cats. Four normal cats were treated weekly for 4 weeks with repositol methylprednisolone. Hemograms, serum chemistry panels, urinalyses, 24 hour water consumption, physical and constitutional parameters, and blood cortisol responses to ACTH stimulation were monitored before and during a day were done prior to steroid therapy on each cat. Clinicopathologic abnormalities induced by steroid therapy were, on the whole, inconsistent and minor. The most important findings were: (1) marked steroid-induced suppression of blood cortisol responses to ACTH, and (2) a circadian rhythm of blood cortisol production that was opposite to that of humans and dogs.


Bovine abortion associated with mixed Movar 33/63 type herpesvirus and bovine viral diarrhea virus infection. A herpesvirus and cytopathogenic bovine viral diarrhea virus were isolated from a bovine fetus aborted in the 6th month of gestation. The herpesvirus was serologically indistinguishable from bovine herpesviruses DN-599 and Movar 33/63.


Cytochemistry of esterases.

Cytochemistry of esterases. Esterases are enzymes that cleave aliphatic and aromatic ester bonds. With the use of synthetic substrates, esterases can be demonstrated in hematopoietic cells. Using alpha naphthyl acetate or alpha naphthyl butyrate, nonspecific esterase activity can be demonstrated. Intense activity of nonspecific esterase that is inhibited by fluoride is characteristic of cells of monocytic or reticulum cell origin. Using naphthol ASD-chloroacetate, specific esterase activity can be detected. Specific esterase is a lysosomal enzyme and is a useful marker for cells of granulocytic origin. Distinctive patterns of specific and nonspecific esterase activities are found in marrow cells from patients with various types of hematologic disorders. With the use of electrophoretic techniques, isoenzymatic analysis of esterases can be achieved.


Relative importance of alpha and beta adrenergic receptors during resuscitation. Successful resuscitation from cardiac arrest in the asphyxiated dog model has been ascribed to the use of artificial ventilation, closed chest cardiac massage, and administration of a vasopressor. Controversy remains over whether the most commonly employed vasopressor, epinephrine, exerts its effects primarily by elevating diastolic pressure and reestablishing coronary flow, or by exciting cardiac pacemaker cells and enhancing myocardial contractility. To observe pure alpha and beta adrenergic receptor influences during resuscitation, three groups (alpha-blocked, beta-blocked, unblocked) of dogs were studied. beta-blocked dogs resuscitated with phenylephrine and unblocked dogs resuscitated with epinephrine experienced 100% successful resumption of spontaneous circulation after 5 min of asphyxia-induced arrest. Only 27% of alpha-blocked animals resuscitated with isoproterenol were successfully revived. The appearance of the ECG during cardiac arrest and resuscitation could in no way be used to predict the outcome of resuscitation attempts. Results suggest that, initially, alpha receptor stimulation with concomitant diastolic pressure elevation is more important to the success of resuscitation than beta receptor stimulation.


Influence of hydrochlorothiazide on the plasma levels of triglycerides, total cholesterol and HDL-cholesterol in patients with essential hypertension. The influence of hydrochlorothiazide (HCT) treatment on the plasma levels of triglycerides, total cholesterol and high density lipoprotein cholesterol (HDL-cholesterol) was studied in 10 patients with essential hypertension. After a placebo period of 4 weeks, 50 mg HCT twice daily was given for a period of 9 months, followed by a second placebo period of 4 weeks. Triglycerides, total cholesterol and HDL-cholesterol were determined at the end of both placebo periods and after 1, 3, 6 and 9 months of HCT. For the whole group, there were no significant changes in triglycerides or HDL-cholesterol, whereas total cholesterol significantly increased during HCT. In 6 patients, plasma triglycerides were higher during HCT as compared to both placebo periods. In only 4 patients did HDL-cholesterol increase during HCT. Changes in triglycerides, total cholesterol and HDL-cholesterol were not related and no correlation was found with changes in blood pressure, body weight or serum potassium. In conclusion, this study confirms a possible adverse effect of diuretic treatment on plasma lipids, which should be considered when determining therapeutic regimens for hypertension.


[Autotransplantation of pancreas fragments in dogs with total pancreatectomy without separation of exo and endocrine tissue]. By total pancreatectomy 41 dogs were made diabetic. Of these, 15 dogs remained untreated, served as diabetic controls, and died within x = 7 +/- 1 days postoperatively. In the experimental groups the best results were obtained (as judged by K values of iv glucose tolerance test and iv Tolbutamide test curves) after 20 min of incubation with collagenase and immediate transplantation into the splenic pulp. After 15 min and 25 min of incubation, normoglycemia regularly occurred, but in the 20 min collagenase group the K values resembled most closely those of normal dogs. The animals stayed normoglycemic and were observed for up to 6 months. Splenectomy led to reocurrence of the diabetic state and death within 4.8 +/- 1.5 (S.E.) days.


[Experimental studies of enzyme activity in the large intestine]. 1. A kallikrein is isolated from the human colon. 2. A collagenolytic enzyme is detectable in this tissue. 3. On day 3 after surgery on the colon of the rat, an enhanced collagenolytic activity is demonstrated.


[Methylprednisolone-induced inhibition of the sympathetic vasomotor tonus in hemorrhagic shock]. In anesthetized cats massive doses of methylprednisolon (40 mg/kg body wt.) induce a blood pressure decrease with bradycardia, which is not transmitted through vagal efferents. These effects are due to an inhibition of sympathetic outflow to heart and vessels.


Studies on the source of cyclic AMP in canine gastric juice. The possibility that cyclic AMP in gastric juice is derived from plasma by simple clearance was evaluated. The effect of exogenous cyclic AMP administration (1 mg/min) on radioimmunoassayable cyclic AMP in plasma and gastric juice was studied in dog stomach during histamine (8--16 micrograms/kg/hr) infusion. Experiments were performed in vagally denervated fundic (Heidenhain) and in innervated gastric pouches. During infusion of cyclic AMP with histamine, plasma cyclic AMP concentration rapidly increased 500-fold. Simultaneously, gastric juice cyclic AMP levels decreased almost 50%. In contrast, dibutyryl cyclic AMP infusion increased gastric juice cAMP concentration 5-fold, suggesting that the gastric mucosa is more permeable to dibutyryl cyclic AMP. In Heidenhain pouch experiments the clearance of dibutyryl cyclic AMP into gastric juice was only 1.5% of the clearance of aminopyrine. It appears that plasma clearance of cyclic AMP fails to account for most of the cyclic AMP present in gastric juice.


Effects of endogenous hyperglucagonemia on lower esophageal sphincter pressure and gastric acid secretion. Lower esophageal sphincter function and gastric acid secretion were studied in a patient with endogenous hyperglucagonemia due to a functioning islet cell carcinoma. Complete resection of the tumor resulted in a fall of the serum concentration of immunoreactive glucagon to a normal level. Pre- and postoperative resting lower esophageal sphincter pressures and lower esophageal sphincter pressure responses to administration of pentagastrin, edrophonium, and bethanechol were unchanged. After surgery, preoperative immunoreactive glucagon concentrations were reproduced by intravenous infusion or intramuscular injection of exogenous glucagon. Lower esophageal sphincter resting pressures and responses to agonists were unchanged. In contrast, glucagon administered at 36 micrograms/kg/hr, which produced a serum concentration of immunoreactive glucagon (32,000 pg/ml) much greater than observed preoperatively (1200 pg/ml), diminished resting lower esophageal sphincter pressure and sphincter responses to pentagastrin, edrophonium, and bethanechol. Similarly, pentagastrin-stimulated gastric acid secretion was unaffected by tumor resection or low-dose glucagon infusion but was decreased at a glucagon infusion rate of 36 micrograms/kg/hr. This series of observations supports the thesis that endogenous glucagon plays no physiological role in the regulation of lower esophageal sphincter pressure or gastric acid secretion.


Perforation of the colon associated with cytomegalovirus infection. Cytomegalovirus (CMV) inclusions were found at colonic perforation sites in three patients with clinical settings suggesting a compromised immunologic status. This could be interpreted as: (1) CMV was an etiologic agent in these perforations; or (2) CMV was an opportunistic superinfection in areas of preexisting inflammation. To determine which interpretation was more likely, we examined material from these three and ten similar patients with colonic perforations, identifying any potential causative factors present. Eleven of the 13 patients had an identifiable cause of perforation, either tumor, diverticulitis, arteritis, or pancreatic pseudocyst, while two remained unexplained. If the presence of CMV merely represented an opportunistic superinfection, then all 13 should have been at equal risk of infection. However, CMV was present in only one of the 11 cases with another identifiable cause of perforation but was present in both of the cases without another cause. This is consistent with the hypothesis that CMV was in fact an etiologic or contributing factor in those cases where it was present. Other cases of CMV infection of the gastrointestinal tract were studied to determine the mechanism by which this infection could lead to perforation of a viscus. In our three cases with perforation, four additional cases of CMV infection of the colon which we have studied, and 30 other cases in the literature, CMV inclusions were found only in areas of ulceration or perforation, never in undamaged mucosa. Thus there is as yet no evidence that CMV can be a primary cause of colonic mucosal injury. It remains likely, however, that a mucosal injury due to another cause may be followed by CMV infection of the granulation tissue which then may lead to further injury and perforation.


Prevalence of gallbladder disease in hyperlipoproteinemia. An analysis of the occurrence of gallbladder disease (ie, cholelithiasis, cholecystitis, cholecystectomy) in 210 consecutive patients with primary hyperlipoproteinemia showed that the prevalence of gallbladder disease was 8%, 18%, and 42% in males with type IIa, IIb, and IV hyperlipoproteinemia, and 22%, 48%, and 72% in the corresponding groups of females. The 40-59-year-old patients were compared to three necropsy series from Malmö, Sweden. The occurrence of gallbladder disease was within normal limits in type IIa and abnormally high in type IV hyperlipoproteinemia. There were no differences with regard to age, body weight, glucose intolerance, or ischemic heart disease between type IV patients with and without GBD. It is suggested that certain forms of disturbances of lipoprotein metabolism are associated with an increased risk for development of gallbladder disease.


Structure and function of photoreaction-centre chlorophyll. Evidence from electron paramagnetic resonance (e.p.r.) studies suggests that the unpaired spin in oxidized P700+. or P865+. is shared by two special chlorophyll a (Chl a) or bacteriochlorophyll a (Bchl a) molecules respectively. Three classes of models have been proposed for special pair reaction centre chlorophyll: asymmetric, in which one Chl a (or Bchl a) acts as electron donor to a second acting as acceptor; models with translational symmetry only; and models with C2 symmetry. Models with C2 symmetry have been synthesized in vitro with two chlorophyll macrocycles tied together by a covalent link. The singlet and triplet states of the in vitro models have been characterized by e.p.r., nuclear magnetic resonance, and optical studies involving absorption, emission, and lasting behaviour. The fact that lasting occurs only from the folded configuration of the linked dimers suggests the availability of a highly effective nonradiative decay path from the S1 state of the excited open dimer. A radical-pair mechanism that accounts for the unusual spin polarization of the special pair triplet is proposed for the primary photochemistry in the reaction centre.


Pharmacology of bromocriptine in health and disease. Bromocriptine, a lysergic acid derivative with a bromine atom at position 2, has been found to have unique effects on the dopamine receptors in the pituitary and central nervous system and peripherally. It is rapidly and completely absorbed from the gut and is mainly excreted in the bile and faeces. It seems to have a particular specificity for the pituitary prolactinotrophe although it does have other effects in different diseases states. In spite of the fact that it is an ergot derivative, it is remarkably free of ergot vascular side effects in the doses needed for therapeutic benefit. The most common adverse effect are nausea, vomiting and postural symptoms. These can be overcome by starting at low doses and increasing the therapeutic levels. Its major use is in the suppression of prolactin in states where this hormone is elevated irrespective of cause. It has also been used in the treatment of acromegaly and is under investigation for use in other disease states probably linked with prolactin system or dopaminergic receptors.


The influence of normal human IgG and of thyroid-stimulating antibody (TSAb) on the binding of thyrotropin to thyroid plasma membranes. The influence of serum IgG from normal and Graves' disease subjects on the binding of 125I-thyrotropin to isolated thyroid membranes was studied. IgG from either source inhibited binding in a concentration -dependent fashion. Human membranes were more sensitive to the human IgG than were bovine thyroid membranes and, when membranes were purified by discontinuous sucrose gradient centrifugation, they were more sensitive than were those less-purified, obtained by differential centrifugation. Using a Dixon plot, inhibition by normal IgG showed non-competitive kinetics. Fab fragments were more effective, on an equimolar basis, than was intact normal IgG, but were less potent when Graves' disease IgG was the parent molecule; this difference implies distinct modes of inhibition of thyrotropin-binding. The degree of inhibition by normal IgG was variable so that multiple control preparations are required to assess the additional effect characteristic of IgG from the subject with Graves' disease.


Inhibition of adenosine 3',5'-monophosphate accumulation and parathyroid hormone release by sodium nitroprusside. Sodium nitroprusside effected a significant reduction in intracellular cAMP accumulation and parathyroid hormone release in dispersed bovine parathyroid cells. The inhibition was apparent at 3 x 10-4 M and maximal at 10-2 M nitroprusside. The effect was rapid and reversible and could be demonstrated in both the presence and absence of stimulating agonists [i.e. (-)isoproterenol, dopamine, and cholera toxin]. The inhibition was additive with that previously described for alpha-adrenergic agonists and prostaglandin F2 alpha and was not affected by phentolamine, suggesting that nitroprusside does not act through the inhibitory receptors previously described in this system. The nitroprusside effect on cAMP accumulation and parathyroid hormone release was present at virtually all concentrations of extracellular calcium tested; 2mM EGTA failed to prevent the inhibition. While extracellular calcium may play some role in this inhibition, it is not required for demonstration of the effect.


The role of the carbohydrate in the stabilization, processing, and packaging of the glycosylated adrenocorticotropin-endorphin common precursor in toad pituitaries. The neurointermediate lobes of dark adapted toads, Xenopus laevis, were incubated for 30 min in [3H]arginine, [3H]arginine plus [14C]glucosamine, or [3H]glucosamine and then chased for various time periods ranging from 1--3 h. The labeled polypeptides synthesized and secreted by the lobes were analyzed by acid-urea polyacrylamide gel electrophoresis. A glycosylated ACTH-endorphin precursor (32,000 mol wt) was synthesized during the pulse and identified by immunoprecipitation by ACTH-(11--24) antiserum. During the chase, this precursor was processed to various glycopeptides and peptides, including ACTH, beta-lipotropin, and alpha-MSH, which were subsequently secreted into the medium. An immunoprecipitable ACTH-related glycoprotein (approximately 150,000 mol wt) and other nonimmunoprecipitable glycoproteins (approximately 80,000--100,000 mol wt) were also synthesized and secreted by the neurointermediate lobe. The secretion of these glycoproteins and peptides was inhibited by dopamine. The significance of glycosylation of the precursor for the biosynthesis, processing, and secretion of the ACTH, beta-lipotropin-, and MSH-related peptides was examined by using a specific inhibitor of glycosylation, tunicamycin. Tunicamycin treatment did not affect the synthesis of the 32,000 mol wt ACTH-endorphin precursor but did prevent its glycosylation. The absence of carbohydrate on the precursor resulted in its rapid intracellular degradation. Precursors that escaped degradation were processed incompletely, leading to the formation and secretion of an unglycosylated intermediate and various other abnormal peptides. The data indicate that glycosylation of the ACTH-endorphin precursor may not be involved in the processes of intracellular transport, packaging, and secretion per se but, rather, may provide specific conformational stability to the precursor as a signal for directed limited proteolysis.


Anticonvulsant drug actions on in vitro and in vivo levels of cyclic AMP in the mouse brain. Injections of pentylenetetrazol to mice produced a rise in steady state levels of cyclic AMP in the cerebrum and cerebellum subsequent to sacrifice by focused microwave irradiation. One hour pretreatment with anticonvulsant drugs--phenytoin, phenobarbital, carbamazepine, clonazepam, and diazepam--prevented this pentylenetetrazol-induced rise in cyclic AMP. Anticonvulsant agents which alone depressed steady state levels of cyclic AMP were phenytoin and carbamazepine in the cerebral cortex and carbamazepine in the cerebellum while elevations in the cyclic nucleotide were evoked by clonazepam in the cerebrum and cerebellum or diazepam and phenytoin in the cerebellum. Under in vitro conditions, in which incubated tissue slices of mouse cerebral cortex were used, the stimulation of cyclic AMP by norepinephrine was blocked by carbamazepine greater than phenobarbital greater than phenytoin. Inhibition of adenosine-induced accumulation of cyclic AMP was observed with phenobarbital greater than carbamazepine greater than phenytoin. Clonazepam enhanced this response to adenosine. Ouabain stimulation of cyclic AMP was prevented by carbamazepine, phenytoin greater than clonazepam greater than phenobarbital greater than diazepam. Only the highest concentrations (0.5 mM) of phenytoin, phenobarbital, and carbamazepine reduced the action of KCl on cyclic AMP elevation. Clonazepam and diazepam elevated the basal levels of the cyclic nucleotide.


Binding of adenosine 5'-monophosphate to bovine liver fructose 1,6-bisphosphatase. Bovine liver fructose 1,6-bisphosphatase bound 4 mol of its allosteric inhibitor AMP per mole of enzyme with half-saturation at 17 mumol/l AMP. The presence of a mixture of positive and negative cooperativity in the binding of AMP to the enzyme was suggested by several procedures for analyzing binding data. In particular, calculation of the intrinsic binding constants for AMP yielded the relationships: K1' less than K2' greater than K3' less than K4', indicating mixed cooperativity.


The effect of dephosphorylation on the properties of a helix-destabilizing protein from meiotic cells and its partial reversal by a protein kinase. Properties of the helix-destabilizing protein from Lilium meiotic cells, 'R-protein', have been examined after treating it either with alkaline phosphatase or with two types of protein kinase. Dephosphorylation with the phosphatase increases binding capacity for single-strand DNA, but abolishes specificity of binding. Dephosphorylated R-protein binds equally to single and double-strand DNA. The capacity to facilitate denaturation or renaturation of DNA is also abolished by the treatment, but cooperativity characteristics are unaffected. The consequences of protein kinase treatment of native or dephosphorylated R-protein depend upon the origin of the kinase. Heterologous cyclic-AMP-dependent protein kinase cannot reverse the effects of dephosphorylation. However, it abolishes the binding affinity of either native or dephosphorylated R-protein for DNA. A protein kinase isolated from meiotic cells has no effect on the native protein, but it does restore all native properties tested to the dephosphorylated form after phosphorylating approximately two residues/molecule of protein.


A kinetic analysis of the uptake of cytosine-beta-D-arabinoside by rat-B77 cells. Differentiation between transport and phosphorylation. We present here a differentiation by kinetic methods of the tandem processes of transport and metabolic during uptake of cytosine-beta-D-arabinoside by intact rat fibroblasts. Transport across the cell membrane occurs by a carrier-mediated mechanism displaying a Km of approximately 500 microM and a V of approximately pmol x min-1 x (10(6) cells)-1. The subsequent metabolic trapping (phosphorylation) has a Km of approximately 15 microM and V of approximately 0.25 pmol x min-1 x (10(6) cells)-1. In this system, transport is rate-limiting for the first phase of the uptake process whereas phosphorylation becomes rate-limiting when internal concentration of radioactive labeled substrate exceeds that in the extracellular medium. The duration of the first phase depends on the substrate concentration.


Effects of cytosol on transcription in isolated simian-virus-40-infected nuclei. The transcription in vitro of nuclei isolated from monkey kidney cells infected with simian virus 40 was stimulated by a cytosol fraction from the same uninfected cells. Transcription in nuclei was inhibited 60--80% by 0.1 microgram/ml of alpha-amanitin, in the presence or in the absence of the cytosol preparation. Treatment of nuclei after transcription in vitro with 0.02 M EDTA preferentially solubilized viral-specific RNA partially bound to the viral chromatin. The increase in incorporation into total RNA seen in the presence of cytosol was not paralleled by any detectable increase in transcription of viral sequences as shown by RNA . DNA hybridizations.


Calf spleen NAD glycohydrolase. Comparison of the catalytic properties of the membrane-bound and the hydrosoluble forms of the enzyme. The catalytic properties of membrane-bound calf spleen NAD glycohydrolase were studied in comparison with previous data obtained with a solubilized hydrosoluble form of the enzyme. When the hydrolysis of NAD catalyzed by membrane-bound NAD glycohydrolase was studied at pH values below 7.5, only insignificant interference by other NAD-hydrolyzing enzymes was detected, and no proton-diffusional inhibition was observed. The kinetics could, therefore, be followed using a titrimetric assay for NAD glycohydrolase activity. The effect of pH, ionic strength on the kinetic parameters, and shifts in binding constants for several ligands of the membrane-bound enzyme indicate that the NAD glycohydrolase activity is influenced by an electrostatic potential due to negative charges (polyelectrolyte effect). No significant changes in kinetic mechanism could be found between both NAD glycohydrolase forms. The association of the enzyme with the membrane results in a remarkably increased thermal stability, in changes in binding properties of the active site and in the emergence of new inhibitor binding sites; e.g. adenosine 3':5'-monophosphate (cyclic AMP) and adenosine, which do not inhibit the hydrosoluble form of NAD glycohydrolase, are good inhibitors (respectively competitive and mixed) of the membrane-bound enzyme. These data (i.e. allotopic changes) probably can be ascribed to enzyme conformational changes induced and stabilized by interaction with membrane constituents.


The dolichol pathway of protein glycosylation in rat liver. Stimulation by GTP of the incorporation of N-acetylglucosamine in endogenous lipids and proteins of rough microsomes treated with pyrophosphate. Incorporation of N-acetylglucosamine into endogenous lipid and protein acceptors was investigated on heavy microsomes from rat liver, incubated with UDP-N-acetyl[14C]glucosamine and GDP-mannose in the absence of detergent. This subcellular preparation derived for 95% or more from the rough endoplasmic reticulum and was devoid of Golgi components which contain the enzyme that adds the peripheral N-acetylglucosamine units to glycoproteins. The label was found almost exclusively in dolichyl diphosphate N-acetylglucosamine, except when the subcellular preparation was treated with pyrophosphate and subsequently incubated with the nucleotide sugars in the presence of GTP. Then, the incorporation of N-acetylglucosamine was considerably enhanced, and the additional label was associated with dolichyl diphosphate N,N'-diacetylchitobiose, with dolichyl diphosphate oligosaccharides and with proteins. The time-course of N-acetylglucosamine incorporation in these products was compatible with the pathway of dolichyl diphosphate glycoconjugates for the biosynthesis of the core portion of saccharide chains linked to asparagine residues of glycoproteins. The addition of GDP-mannose to the incubation medium was required to produce labeled dolichyl diphosphate oligosaccharides, but not to incorporate N-acetylglucosamine in protein. It is concluded that rough microsomes are capable of assembling dolichol-linked oligosaccharides from exogenous nucleotide precursors and of transferring N,N'-diacetylchitobiose, or its mannosylated derivatives, from the lipid intermediate to endogenous proteins. However, these metabolic activities are hindered in the original subcellular preparation, and in the absence of GTP. Although the earliest perceptible effect produced jointly by the treatment with pyrophosphate and by GTP was the synthesis of dolichyl diphosphate N,N'-diacetylchitobiose, the primary action of these factors remains uncertain. They may stimulate directly the reaction forming dolichyl diphosphate N,N'-diacetylchitobiose from dolichyl diphosphate N-acetylglucosamine, or activate the synthesis of this latter intermediate from a particular pool of dolichyl monophosphate which is readily converted afterwards into disaccharide and oligosaccharide derivatives and glycosylates protein. The requirement for GTP might have a functional meaning, for GTP acted maximally at a concentration distinctly lower than its actual concentration in liver. The detachment of ribosomes from rough vesicles was the major alteration induced by treatment with pyrophosphate. It is suggested that the removal of ribosomes unmasks the membrane sites where GTP acts.


Computer simulation of the fructose bisphosphatase/phosphofructokinase couple in rat liver. Recycling of fructose 6-phosphate and fructose 1,6-bisphosphate in the rat liver under gluconeogenic and glycolytic conditions was investigated with a computer model containing representations of the kinetic properties of phosphofructokinase and fructose 1,6-bisphosphatase under realistic physiological conditions. The two enzyme submodels were constructed from data for the isolated enzymes in vitro by formal optimization. Tissue metabolite concentrations were corrected for cytosolic/mitochondrial compartmentation and effects of chelation and protonation equilibria. This model, which mostly considers the behavior of livers from starved rats, predicts negligible recycling under physiologically realistic conditions. Metabolic regulation of fructose 6-phosphate, the magnesium ion concentration and the distribution of adenine nucleotides appear to prevent operation of a 'futile cycle' in vivo. Rate-limiting chemical species were identified by sensitivity analysis.


Methionyl-tRNA synthetase from Escherichia coli. Inactivation and labeling by periodate-treated initiator tRNA. Both the aminoacylation and isotopic ATP-PPi exchange activities of native and trypsin-modified methionyl-tRNA synthetases from Escherichia coli are specifically inactivated by incubation in the presence of periodate-treated initiator tRNA Met. The inactivation proceeds through the formation of a reversible Schiff's base between the epsilon-amino group of a lysine within the catalytic center of the enzyme and the 2',3'-aldehyde groups created at the 3'-terminal ribose of tRNA. The Schiff's base may be stabilized by reduction with sodium borohydride. Intact tRNA Met f competes with the inactivation by its dialdehyde. It has been verified in the case of the modified enzyme that the protection is afforded according to an equilibrium constant identical to that for tRNA Met f binding at the active site of the enzyme. Finally it is shown that the incorporation of one molecule of the dialdehyde of [14C]tRNA completely destroys the activity of the monomeric trypsin-modified methionyl-tRNA synthetase.


Benign cystic nephroblastoma.

Benign cystic nephroblastoma. A benign cystic nephroblastoma in a 6-month-old boy is presented. Erroneous interpretation as malignant nephroblastoma led to unnecessarily aggressive therapy. There is no evidence of recurrence or metastatic disease 12 years after the initial resection. As shown in our case and in the others previously reported in the literature, benign cystic nephroblastoma represents a distinct clinicopathological entity which does not show malignant behavior.


Adenylate kinase activity and glutathione concentration of cerebrospinal fluid in different neurological disorders. Adenylate kinase activity and glutathione concentration were measured in cerebrospinal fluid (CSF) of 64 consecutive patients admitted for various neurological disorders. These two analyses were performed in addition to conventional examination of the CSF. Neurological symptoms most probably connected in some cases with no and in others with only subtle changes in the central nervous system were linked to no or only moderate activities of adenylate kinase together with no glutathione. 1 patient with meningioma had no adenylate kinase activity at all while 3 patients with malignant brain tumours showed clear activities similar to 3 patients with well established diagnoses of multiple sclerosis. On the contrary, glutathione was absent in CSF of the patients with brain tumours and multiple sclerosis. Various cerebrovascular diseases involving larger areas of the brain tissue resulted in clear adenylate kinase activities in CSF either alone as in 11 of the patients with cerebral infarction or in combination with the appearance of also glutathione as in the remaining 7 patients with cerebral infarction as well as in the 14 patients studied with hemorrhages of the brain.


Reversible abnormalities of low density lipoprotein composition in familial hypercholesterolaemia. Low density lipoprotein (LDL) composition was analysed in twenty-one patients with familial hypercholesterolaemia, including six homozygotes. By comparison with nineteen controls the patients' LDL had an increased ratio of cholesterol:phospholipid and a decreased ratio of lecithin:sphingomyelin; these changes were more marked in homozygotes than in heterozygotes. Treatment of sevel patients with plasma exchange temporarily resulted in near-normalization of the composition of their LDL. These results suggest that abnormalities of LDL composition in familial hypercholesterolaemia are secondary to hypocatabolism of LDL, which prolongs the half-life of LDL and thus increases the mean age of the population of particles circulating in plasma.


Mechanism of PGE inhibition of catecholamine release from adrenal medulla. Catecholamine (CA) secretion from the adrenal medulla was induced in vitro by acetylcholine (10(-4)M) (ACh), by incubation in potassium-free medium, by addition of ouabain (10(-3)M), by theophylline (10(-2)M) or by salbutamol (10(-6) and 6 x 10(-6) M). Theophylline and salbutamol, but not ACh, released CA in a calcium-free medium supplemented with 2mM EGTA. PGE2 significantly inhibited both CA secretion evoked by ACh and that evoked by salbutamol, i.e. both secretion dependent on, and independent of, extracellular calcium, PGE2 counteracted the increase of cAMP levels caused by ACh or salbutamol in adrenal medullary slices. PGE2 also diminished the salbutamol-induced activation of adenylate cyclase in an adrenal medullary membrane preparation, PGE2 reduced the rate of 45Ca efflux from slices of adrenal medulla preloaded with 45CaCl2. It is suggested that PGE2 inhibits CA secretion through the following sequence: inhibition of adenylate cyclase, a fall of cellular cAMP resulting in reduced release of calcium from intracellular binding sites and reduced free cytoplasmic calcium.


Changes in brain levels of cyclic nucleotides and gamma-aminobutyric acid in barbiturate dependence and withdrawal. Rats exposed to chronic intake of sodium barbital maintained high circulating levels of barbital in blood and brain and exhibited increased sensitivity ot audiogenic convulsions during the withdrawal period. Levels of gamma-aminobutyric acid (GABA), glutamate, guanosine 3',5'-monophosphate (cyclic GMP) and adenosine 3',5'-monophosphate (cyclic AMP) were measured in selected brain regions after sacrifice with high power microwave inactivation. Cyclic GMP during chronic barbital administration was significantly lower than controls in most brain regions, especially the hindbrain. During the withdrawal period cyclic GMP in the cerebellum was significantly increased, while returning at least to control levels in all other regions. GABA throughout the brain tended to be reduced during barbital dependence, while cyclic AMP and glutamate levels remained unchanged in all groups. These results indicate a possible role for cyclic GMP in the mediation of the central nervous system response during barbiturate dependence and withdrawal.


Modification by SQ 14225 of blood pressure and adrenal catecholamine response to hemorrhage. SQ 14225 infusion to cats exposed to hemorrhage suppressed adrenomedullary catecholamine release. The immediate compensatory blood pressure response was unaffected, while the later compensatory response was of a higher magnitude in intact cats that in SQ 14225-treated animals. Adrenal gland blood flow was better preserved in SQ 14225-treated hemorrhaged cats than in cats exposed to hemorrhage only. These  data emphasize the role of angiotensin II in adrenal catecholamine and blood pressure responses to hemorrhage.


Effects of monovalent cations on (Na+ + K+)-ATPase in rat brain slices. The influence of monovalent cations on membrane (Na + K+)-ATPase was estimated in vitro in intact cells from the oxygen consumption of rat brain cortical slices. High concentrations of K+, Rb+ or Cs+ stimulated the respiration in the presence of Na+. This stimulation was antagonized by ouabain in a concentration- and time-dependent manner. Additionally, only combinations of monovalent cations, that stimulate (Na+ + K+)-ATPase, increased oxygen consumption, indicating that the stimulated portion of respiration is realted to the (Na+ + K+)-ATPase activity. Low concentrations of Rb+ and Cs+, however, failed to affect oxygen consumption. Li+ slightly and transiently stimulated oxygen uptake at low concentrations and inhibited it at higher concentrations. Low concentrations of Tl+ also stimulated respiration in a K+-free medium. However, the inhibitory effects of Tl+ were predominant at higher concentrations or in the presence of K+. Thus, monovalent cations can alter (Na+ + K+)-ATPase activity. While Rb+ and Li+ produce opposite effects on this enzyme system under certain conditions, these actions do not seem to be related to the antidepressant action of Rb+ and the antimanic action of Li+.


Decrease of cyclic GMP in cerebellar cortex by intrastriatal (--)-sulpiride. Intraperitoneal administration of (--)-sulpiride (12 to 50 mg/kg) produced a dose-related decrease in cGMP content in cerebellar cortex, while the (+)-sulpiride (10 microgram), but not after intracerebellar injection. These findings support the contention that (--)-sulpiride modifies cerebellar cGMP levels through and action on striatal dopamine receptors.


An intracerebral, physiological role for angiotensin: effects of central blockade. Although exogenous angiotensin II (AII) exerts a multitude of effects on the central nervous system, there is little evidence supporting a physiological role for the endogenously produced peptide. Some investigators have tested the hypothesis that AII is physiologically active in the brain with intracerebral infusions of blockers of the renin-angiotensin system. If blocker infusions produce effects that are opposite to exogenous AII infusions, it is evidence supporting a physiological role for endogenously generated angiotensin. Previous work has demonstrated that intraventricular infusion of AII elicits thirst and stimulates antidiuretic hormone and ACTH release. Intracerebral administration of AII also suppresses aldosterone secretion. Experiments that employed the blockers saralasin, a competitive inhibitor of AII, and SQ 20881, a converting enzyme blocker, are presented; results suggest that endogenous AII is involved in the control of thirst and peripheral hormone levels. Infusion of the blockers in the ventricular system led to changes in peripheral hormone concentrations opposite to that observed following infusions of AII.


Evolutionary aspects of some neuropeptides. The study of biologically active peptides is a study of structural homology and functional diversity. Some fundamental peptide structures used as chemical messengers by simple organisms were evidently maintained throughout evolution with little change. In higher organisms these structures may still be used for functions related to the original use as well as for very different purposes. Examples are given of the multifunctional roles of common peptides and of the mechanisms by means of which these functions are separated and regulated. The active core of ACTH appears to be used as a signal in regulation of reproduction throughout the range of eukaryotes and also appears in a baffling range of other important proteins. The ubiquitous nature of this peptide sequence suggests that it plays a universal role in recognition or activation of specific receptors. Peptides used in lower species as hormones or chemical defense substances play an important role in research as presagers of the discovery of corresponding mammalian peptides.


Localization and possible function of peptidergic neurons and their interactions with central catecholamine neurons, and the central actions of gut hormones. The localization of various neuropeptides is described in the gut and in the hypothalamus in the rat. Evidence is given for the presence of material resembling corticotropin-like intermediate peptide in arcuate and periarcuate neurons, projecting to various hypothalamic nuclei, limbic areas and the thalamus. beta-Endorphin and glucagon decrease dopamine turnover in the median eminence, while secretin increases dopamine turnover and vasoactive intestinal polypeptide (VIP) has no effect. beta-Endorphin, VIP, secretin, and glucagon all produce discrete changes in norepinephrine turnover in various hypothalamic nuclei. Mainly increases of norepinephrine turnover were observed. These catecholamine turnover changes appear to cause changes in the secretion of prolactin and growth hormone. The results therefore indicate that gut hormones and opioid peptides may act directly on the hypothalamus on specific types of receptors to participate in the control of hypothalamic functions such as control of hormone secretion from the anterior pituitary and of food intake. It seems possible that gastrointestinal peptides released from the gastrointestinal tract into the circulation under certain circumstances could reach the hypothalamus and modulate its activity via the above-mentioned mechanisms. It may therefore be speculated that disturbances in gastrointestinal functions could lead to pathological changes in food intake via modulation of hypothalamic activity.


[Heart rate in cats during sleep after coagulation of the locus coeruleus]. The relationship between heart rate (HR) modulation and sleep mechanisms were analysed in 14 unrestrained cats with implanted electrodes and additional tripolar electrodes in the lateral geniculate nucleus and the abducens nucleus for recording the ponto--geniculo--occipital spikes. Anterior lesion of the locus coeruleus (LC) reduced the heart rate by about 18%, and a severe arrhythmia with augmented dispersion of R--R intervals occurred during paradoxical sleep (PS). Cats with posterior lesion had the heart rate increased by 20% and the spontaneous fluctuations of the R--R interval totally disappeared in them. The obtained data show that anterior and posterior LC exert different influences upon the heart rate fluctuations in sleep.


[Effect of parathyroid hormone on the myocardium in the presence of ouabain-induced reduced membrane ATPase activity]. The effect of parathyroid hormone (PTH) on the action potential of myocardium cells under conditions of the oubain--reduced activity of transport ATPase tells in reduced duration of the plateau and in increased amplitude of evoked potentials. The form of the AP alters on administration of the hormone against the background of expressed activity of the cardiac glycoside. The increase in the AP amplitude and the appearance of the "peak"preseding the plateau phase due to the hormone administration seem to be entailed by an action of the hormone upon the transport of sodium ions and activity of the membrane Na, K--dependent ATPase. The effect of PTH upon the contractile activity of the myocardium cells under the oubain influence becomes inverse: instead of a decrease in the contractions amplitude the hormone administration entails an elongation of the "therapeutic" phase of oubain action. The elongation also occurs after a preliminary work of the myocardium strip in the PTH solution. The effect seems to follow the increase of amount of calcium in the cell under the hormone effect.


[Mechanism of dilatation of the thoracic duct upon stimulation of somatic nerves]. In acute experiments on 31 dogs, dilatation of the thoracic lymphatic duct on moderate (5--8 V) stimulation of afferents of the sciatic nerve and i. v. administration of noradrenaline (3--5 mcg/kg) was shown to be due to specific effects of excitation of the duct alpha-adrenoreceptors mainly. The alpha-adrenoreceptors seem to be represented in the thoracic duct in a much greater amount that the beta-adrenoreceptors. Stimulation of both types of the duct's adrenoreceptors leads to an increase in its capacity.


[Adrenergic and cholinergic mechanisms of the contractile reactions of major blood vessels]. Epinephrine, norepinephrine, and isoproterenol induced circular contraction of isolated segments of canine intracerebral internal carotid artery. The alpha--and beta-antagonists inhibited the effects of catecholamines. Acetylcholine showed dual effects involving both dilation (5.10(-12)--5.10(-9) g/ml) and contraction 5.10(-8) g/ml and higher). Atropine inhibited both effects. The K+ depolarization abolished the contractile effects of norepinephrine and acetylcholine. The blockade of calcium permeability by Mn++ abolished the contractile effects of sympathetic and parasympathetic mediators.


[Effect of creatine phosphate on the slow inward calcium current, action potentials and the strength of myocardial contraction]. The effect of creatine phosphate on the frog heart muscle involved maintenance of its contraction at high level even after inhibition of mitochondrial oxidative phosphorylation by sodium cyanide. The effect of creatine phosphate on the contractile force and action potential is similar for the frog heart ventricle and atrium. The voltage--clamp technique showed that creatine phosphate controlled the slow inward calcium current through the surface membrane of the frog atrium cells.


[Changes in the morpho-functional properties of gastric parietal cells during activation of secretory activity]. Electron--microscope and biochemical studies in rats and dogs showed that secretory membranes of parietal cells were in dynamic equilibrium with plasmalemma in activated secretion. During secretion, a transposition of secretory membranes occurs: 53.2% of tubulovesicular membranes transform into membranes of intracellular canaliculi and apical surface. K+--ATPace and carbonic anhydrase activities increased (55.5% and 62.9%, resp.). The system of secretory membranes and enzyme systems of parietal cells seem to take part synchronously in the process of gastric acid secretion.


[Effect of etimizol, ethyrasol, and caffeine on the electrical activity of mollusk neurons]. In experiments on Limnae stagnalis neurons, ethymisol increased the AP duration while reducing the trace hyperpolarization and the rate of development of the AP descending phase. Ethymisol (10 mM/1) induced either hyperpolarization of neurons with an increase in membrane resistance or their depolarization (20 mM/1) with a decrease in membrane resistance. A drop in the medium temperature by 2-4 degrees C prevented the hyperpolarization. The ethymisol-induced hyperpolarization seems to be connected with a decrease in membrane permeability for sodium ions in resting conditions and with activation of electrogenic ion transport. The increase of extraneuronal potassium up to 4 mM/1 and depolarization induced by currents of 1-3 nA intensified the ethymisol effects. Ethymisol decreased efflux and influx of ions through membrane thus affecting the AP parameters. The effects of ethymisol also involved an increase of neurons excitability and intensification of synaptic activity. Neither ethyrasol nor caffein exerted these effects.


[Bioelectrical activity and ultrastructure of medicinal leech Retzius neurons during chronic direct and synaptic activation]. The threshold direct or synaptic 1/sec stimulation of the Retzius cells increases the firing rate of the neuron in the leach. The firing rate becomes synchronized with the stimulation applied. The membrane potential does not change. After 10 min of such stimulation changes occurring in the ultrastructure of neuron are similar to those in a neuron with no stimulation: these changes reveal a decrease in the synthetic processes whereas the bioelectrical activity remains the same which suggests a certain independence of the neuron electrophysiological parameters of its ultrastructure.


[Neural regulation of coronary circulation]. In anesthetized dogs, with the aid of catheterization and perfusion of the coronary arteries roith heporinesed blood, catheterization of the heart cavities, and synchronous recording of the hemodynamics in unopened thorax, the mechanisms of neural regulation of the coronary circulation were analysed. Tonic effects of the nervous system on the coronary vessels are insignificant, coronary resistance being maintained by the high basal tonus. The cholinergic and adrenergic mechaniseins interact with the more powerful metabolic mechanisms. The concrete interrelationships between the cholinergic and the adrenergic mechanisms in the heart adrenergic responses as well as their probable physiological significance, are discussed.


[Regulation of Na, K-ATPase activity in nerves during propagation of rhythmic excitation]. The changes of Na, K-ATPase activity of the isolated squid, crab, frog, and rat nerves depended on the stimulation frequency: at the frequency specific for each nerve the maximum deviation from initial resting level occurred. During a certain stimulation the maximum deviation of SH-group content and Na/K ratio as well as of Na, K-ATPase activity developed while the level of acetylcholine esterase activity (AEA) remained at its minimum. The data obtained suggest the mechanism of Na, K-ATPase activity in nerve under rhythmic propagation of excitation. In the resting nerve the minimum level of the acetylcholine concentration is kept because of the AEA. Under stimulation the membrane depolarization induces changes of the protein conformation suppressing the AEA. This results in an increase of the acetylcholine concentration in the nerve which leads to an increase of Na+ influx and K+ efflux. The changes of Na/K ratio in the nerve activate the transport ATPase.


[Effect of adaptation to high-altitude hypoxia on the adrenoreactivity of the heart and the state of the adenyl cyclase and phosphodiesterase systems of the myocardium]. Experiments in white rats suggested that changes in activity of certain intracellular systems played a significant role in the mechanism of increasing adrenoreactivity of the heart in adaptation to high-altitude hypoxia.


Mechanisms of inactivation of hypothalamic regulatory hormones. The mechanisms of enzymic inactivation of thyrotropin-releasing hormone, luteinizing hormone-releasing hormone and somatostatin, the three fully-characterized hypothalamic regulatory hormones, and the possible physiological significance of the peptidases in neuroendocrine control has been reviewed. Application of the criteria of enzyme location (at the sites of biosynthesis, release, action, elimination and excretion), appropriate biochemical characteristics of the enzymes and changes in enzyme activity in physiological circumstances all suggest that the peptidases can contribute to the mechanisms controlling the hypothalamic hormones' release and actions. Besides their physiological function, the enzymes may also be directly involved in certain pathological conditions. There is evidence to indicate that the enzymes degrading the regulatory hormones may participate in the process of hormone activation as well as inactivation. A continuing investigation of the peptidases may lead to a better understanding of the established endocrine and other putative functions of these hypothalamic polypeptide hormones.


Effects of carbamylcholine and ionophore A-23187 on cyclic 3',5'-AMP and cyclic 3',5'-GMP accumulation in dog-thyroid slices. Carbamylcholine and acetylcholine through a muscarinic type of receptor, KCl, ionophore A-23187 and NaF increased cyclic GMP accumulation in dog-thyroid slices. These effects were abolished in calcium-depleted slices, which findings confirm that Ca2+ is required for cyclic GMP accumulation. All these agents depressed the accumulation of cyclic AMP in TSH-stimulated slices. KCl and NaF depressed cyclic AMP accumulation in TSH-treated slices even when they had been depleted of Ca2+. This suggests a cyclic GMP- and Ca2+-independent mechanism. The absence of inhibition of the effects of the ionophore, NaF and KCl in the presence of atropine suggests that these drugs do not act by inducing the release of acetylcholine in the slices. The effects of carbamylcholine and ionophore A-23187 on cyclic GMP accumulation and protein iodination were reversible; the inhibitions of TSH-induced cyclic AMP accumulation and secretion were non-competitive and were not accompanied by a depression of ATP levels. All these effects were greatly decreased in the absence of extracellular Ca2+. These data suggest that carbamylcholine and ionophore A-23187 act mainly by increasing the influx of extracellular Ca2+ in thyroid cells. However, the persistence of some carbamylcholine effect in the absence of Ca2+ in the medium suggests that this agent may also trigger the release of Ca2+ from an intrafollicular pool. The kinetics of action of carbamylcholine are compatible with a role of cyclic GMP in the inhibition of cyclic AMP accumulation. However, with the ionophore, the depression of cyclic AMP accumulation was much longer than the rise of cyclic GMP, which suggests a mechanism independent of cyclic GMP.


Prolactin receptors in primary cultures of carcinogen-induced rat-mammary tumors. 7,12-Dimethylbenz[alpha]anthracene-induced rat mammary tumors were dissociated with collagenase and hyaluronidase and placed into primary culture. In most cultures, specific binding of 125I-labeled ovine prolactin was (i) lower than that for the original tumors unless bovine prolactin (1 microgram/ml) had been added to the dissociation medium, and (ii) varied with the type of growth medium used. The level of prolactin binding in cultured cells was relatively constant for the first 7-10 days. Prolactin binding in cultured cell homogenates was maximal at pH 7.0, proportional to cell protein, specific for prolactin, and reached a steady state by 12 h at 22 degrees C. The half-maximum inhibition of 125I-labeled prolactin binding by unlabeled prolactin was 100 ng/ml for cells grown in 5-1000 ng of prolactin/ml. After prolactin was removed from the growth medium, the level of available binding sites progressively increased, reached a maximum at 48 h and then declined. At 48 h, the dissociation constant for prolactin binding (Kd approximately 1 x 10(-10) M) was comparable to that in tumors. In some cultured tumors, a 48-h treatment with 0.5 or 1.0 ng of prolactin/ml caused an apparent increase in the level of prolactin binding. Prolactin increased DNA synthesis and its removal caused a reduction in [3H]estradiol and [3H]-R5020 binding to cultured cell cytosols.


Calcific pancreatitis in a patient with type 5 hyperlipoproteinemia. The history of a 27-year-old woman with 10 years of episodic abdominal pain and the development of calcific pancreatitis secondary to hyperlipoproteinemia is presented. The relationship between familial and alcoholic hyperlipidemia and pancreatitis is discussed as well as the mechanism of injury to the pancreas.


Histocompatibility antigens in patients with hepatocellular carcinoma and their relationship to chronic hepatitis B virus infection in these patients. Although hepatocellular carcinoma is probably caused by one or more environmental carcinogens, a genetically determined susceptibility to the development of the tumor has not been excluded. In looking for such a predisposition, we have compared the histocompatibility antigens (HLA) of 102 southern African blacks with histologically proved HCC with those of 208 healthy blacks. The standard two-stage lymphocyte microcytotoxicity method was used to test for 40 antigens: 17 in the A locus, 20 in the B locus, and 3 in the C locus. None of the HLA antigens had a frequency that was significantly different in the patients and the controls. A close association undoubtedly exists between chronic hepatitis B virus infection and hepatocellular carcinoma. If this virus is proved to be oncogenic with respect to hepatocellular carcinoma, a genetic predisposition to the hepatitis B virus carrier state may have an indirect bearing on the etiology of the tumor. Sera from the hepatocellular carcinoma patients were therefore tested for hepatitis B virus markers (HBV surface antigen and antibody against HBV core antigen), and these were related to the patients' histocompatibility antigens. None of the HLA antigen frequencies was significantly different in the surface antigen-positive and the surface antigen-negative patients. As 88% of the patients were anticore positive, no meaningful correlation could be carried out with this marker. Analysis of histocompatibility antigens thus failed to show evidence of a genetic predisposition either to hepatocellular carcinoma or to chronic hepatitis B surface antigenemia in patients with this tumor.


A genetically distinct form of cyclic AMP phosphodiesterase associated with chromomere 3D4 in Drosophila melanogaster. Two cyclic AMP phosphodiesterase enzymes (E.C.3.1.4.17) are present in homogenates of adult Drosophila melanogaster. The two enzymes differ from one another in heat stability, affinity for Mg++, Ca++ activation and molecular weight. They do not differ markedly in their affinities for cyclic AMP, and both exhibit anomalous Michaelis-Menten kinetics. The more heat-labile enzyme is controlled in a dosage-dependent manner by chromomere 3D4 of the X chromosome and is absent in flies that are deficient for chromomere 3D4. Chromomere 3D4 is also necessary for the maintenance of normal cAMP levels, for male fertility, and for normal female fertility and oogenesis. The structural gene(s) for the more heat-stable enzyme is located outside of chromomeres 3C12-3D4. Whether 3D4 contains a structural gene, or a regulatory gene necessary for the presence of the labile enzyme, remains to be determined.


Effects of dihydroergotoxine (Redergine) on the sleep-wakefulness cycle in the cat. Investigations with dihydroergotoxine (DHE 1.0; 3.0 and 5.0 mg/kg i.p.) indicate that DHE exerts effect on the sleep-wakefulness cycle of the cat. In doses of 3.0 and 5.0 mg/kg i.p. DHE has a suppressant effect on sleep, and in particular on REM sleep, while wakefulness was increased. With a dose of 1.0 mg/kg, wakefulness and REM sleep were decreased, while slow-wave sleep was increased.


Platelet aggreation malondialdehyde formation in type IIA hypercholesterolemic patients. Platelet aggregation induced by threshold concentrations of ADP, adrenaline, collagen and Thrombofax was evaluated in 25 type IIA hypercholesterolemic patients in comparison with 15 control subjects. Malondialdehyde (MDA) formation induced by collagen and thrombin was also studied in a subgroup of 8 patients. In the patient group, irreversible platelet aggregation was induced by significantly lower concentrations of both adrenaline (p is less than 0.05) and Thrombofax (p is less than 0.01). MDA formation induced by both aggregating agents was markedly increased (p is less than 0.01) in type IIA hypercholesterolemic patients.


Diagnostic and prognostic significance of electroencephalography with intravenous diazepam in epilepsy. Thirty cases of intractable epilepsy in children were investigated to observe the correlation between the suppression of seizure discharges to intravenous diazepam and the type of seizure or effects of subsequent therapy. Suppression of seizure discharges were bilaterally poor in 75% of the cases with infantile spasms and 89% of cases with Lennox syndrome and other generalized seizures while it was unilaterally poor in 80% of the cases with focal or unilateral seizures. ACTH or steroid therapy was clinically and electroencephalographically effective in 80% of the cases with good suppression responses and in none of cases with good suppression that was resistant to the therapy. On the other hand, clinical seizures were not controlled in 73% of the cases showing poor suppression and EEG did not reveal improvement in 82% of the cases with poor suppression. EEG with intravenous diazepam is valuable for understanding the pharmacophysiological mechanism of epilepsy and it may be possible to select cases as candidates for ACTH or steroid therapy using this technique.


[Quantitative aspects of specific binding of cardiac glycosides to membrane receptors]. Although the exact mechanism of positive inotropic action of cardiac glycosides is unknown, specific membrane bound proteins with high affinity for this group of drugs have been characterized. These "receptors" for cardiac glycosides have been measured quantitatively in cardiac tissue of humans and several species as well as in other tissues. The occupation of receptors by cardioactive steroids has been found to agree quantitatively with the drug effects in respect to inhibition of (Na+ + K+)-ATPase and in respect to positive inotropy (these experiments were performed in electrically stimulated contracting cardiac muscle). Changes in receptor concentration or receptor properties have been observed in hyperthyroidism, chronic hypokalaemia, thalassaemia or in acutely changed serum concentrations of K+, Ca++ and several drugs. These changes may be of great significance in patients treated with cardiac glycosides as their effects are not reflected by the serum concentration of cardiac glycosides. The understanding of drug-receptor-interactions on the molecular level--especially under the pathological conditions in the patient--will increase our diagnostic and therapeutic knowledge.


[Conservative treatment of peripheral nerve lesions]. Conservative treatment for cases of peripheral nerve damage may be subdivided into electrotherapy, positioning on splints and active as well as passive physiotherapy. Electrotherapy is generally carried out with exponential current which makes selective stimulation of the paralyzed muscles possible without stimulation of sensory fibres. The adjustment of the stimulation parameters, duration of stimulus and pause, gradient and stimulus intensity to the conditions of stimulation for each case is possible by determining the most favorable pulse duration. Successful electrotherapy depends upon an early beginning, the selective stimulation of the paralyzed muscles with exponential current at a sufficiently high intensity under isometric conditions and by avoiding overstretching of the muscles. Positioning of the extremities on splints in a functional position of the joints prevents overstretching of paralyzed muscles and thus contractures. Passive physiotherapy is indicated for the prevention or mobilisation of joint contractures. Active physiotherapy stimulates the regeneration of motor nerve fibres. The aim is a reintegration of the paralyzed muscles with the mobile muscles. Here the training of intact synergists, the innervation of antagonists and the activation of symmetrical healthy muscles of the contralateral extremity should be considered. The treatment of single nerve lesions with vitamin B does not appear reasonable.


[Clinical and hemodynamic studies before and after mitral valve replacement (author's transl)]. Clinical and hemodynamic studies were carried out before and after replacement of stenotic mitral valves in 17 patients, 10 of whom received Björk-Shiley (low profile mechanical) prostheses and 7 who received (porcine heterograft) bioprostheses. Mitral valve replacement led to amelioration of symptoms, improvement of phonocardiographic findings and, partially, to a lowering of pressure in the pulmonary circulation, augmentation of cardiac output, lessening of the pulmonary arteriolar and mitral valvular resistances as well as incrementation of mitral valve orifice area. Preexistent ECG changes, radiologic heart size, right ventricular filling pressure and stroke volume remained unaltered. In consideration of variables such as prosthetic size, severity of the disease and post-operative follow-up periods, the results obtained with both types of prostheses were comparable. The prostheses, however, inherently are responsible for a moderate degree of mitral stenosis which compromises exercise capacity and generally does not lead to normalization of pressure in the pulmonary circulation. Irreversible myocardial damage appears responsible for the fact that electrocardiographic abnormalities, the heart size, the right ventricular filling pressure and stroke volume remain unaltered postoperatively. Thus, patients who have undergone mitral valve replacement continue to require limitation of physical activities, a tailored medical regimen and close cardiologic follow-up.


Glycerokinase in rat and human adipose tissue: response to hormonal and dietary stimuli. Glycerokinase activity was measured in homogenates of rat and human adipocytes. Human adipocyte glycerokinase activity was not altered by various dietary and hormonal treatments. In contrast, glycerokinase activity in rat adipocytes was decreased by fasting 48 hr and returned toward normal levels after refeeding for 36 hr. This increase in enzyme activity during refeeding was blocked by prior administration of uromycin. Glycerokinase activity was also significantly increased following prolonged incubation of rat adipocytes with dexamethasone in vitro. This stimulation of glycerokinase was further augmented by the simultaneous addition of insulin. Glycerokinase activity in rat and human adipocytes was also dependent on the body weight of the respective tissue donor. Other data presented indicate that glycerokinase is not involved in the "anti-lipolytic" action of insulin. The possible metabolic significance of glycerokinase in adipose tissue is discussed.


Effects of thyrotoxicosis on mitochondrial enzymes of rat soleus. Cytochrome oxidase, glycerol-3-phosphate dehydrogenase, and succinate dehydrogenase were measured in mitochondrial fractions obtained from rat soleus muscle of control and 8 week T3 + T4 treated animals. Under these conditions of prolonged treatment, there is a five-fold increase in the specific activities of both cytochrome oxidase and glycerole-3-phosphate dehydrogenase. Significant increases in total cellular mitochondrial content and enzyme activities were observed in T3 + T4 treated animals as compared to controls. These results indicate that thyrotoxicosis can induce selective changes in mitochondrial enzymes in slow twitch red (Type I) muscle fibers.


Evidence of adrenal 18-hydroxylase inhibition by metyrapone in man. The influence of metyrapone (M) on the adrenal 18-hydroxylation was studied in two groups of healthy young men. In group I, serum concentrations of 18-OH-11-deoxycorticosterone (18-OH-DOC) fell significantly after a single oral dose of 40 mg/kg of M at 8.00 h, while those of 11-deoxycorticosterone (DOC) increased by a factor of about 500 within 4 hours after drug administration. Serum concentrations of 18-OH-DOC remained suppressed up to 14,00 h and tended to increase up to 16.00 h with a concomitant increase of plasma ACTH. In group II, serum concentrations of 18-OH-DOC and corticosterone (B) were slightly lowered eight hours after oral administration of 30 mg/kg of M at midnight in comparison with measurement of the previous day. Serum concentrations of 11-deoxycortisol (S) and DOC were markedly increased after drug administration. These findings indicate an inhibitory effect of M on adrenal 18-hydroxylation in addition to 11-hydroxylation under in vivo conditions. The slight increase of 18-OH-DOC at 16.00 h in group I and the only slight decrease of this steroid 8 hrs after drug administration in group II may be explained by declining enzyme blockade and a superimposed ACTH stimulation of the adrenal cortex at this time.


Immunohistochemical localization of prolactin-binding sites in R3327 rat prostatic cancer cells. Light microscope immunohistochemistry was used to test for and to localize prolactin-binding sites in the transplantable R3327 rat prostatic carcinoma. Fixed tissue sections of the tumor were first incubated with vehicle or varying concentrations of highly purified NIAMDD rat prolactin and then exposed to an immunoperoxidase staining sequence. Prolactin produced dose-related, immunospecific, staining in the cytoplasm of some neoplastic cells, indicative of intracellular prolactin-binding sites (IPBS). While cells with IPBS have been demonstrated in both differentiated and undifferentiated regions of this cancer, a role for prolactin in prostatic neoplasia, be it stimulatory or inhibitory, remains to be elucidated.


Cytophotometric DNA determination in human astroglial tumours. The DNA content is determined by the cytophotometric method in 16 astroglial tumours, classified according to the degree of clinical malignancy. The values are compared to those found in non-tumoural astrocytes. This method appears to be more reliable in assessment of the degree of malignancy of the tumour in biopsy material than histological criteria and the mitotic index. It is also shown to be of great value in cases where the biopsy is taken at the margin of the tumour and in which malignant histological features are missing.


COPE: a systematic approach to moving chronic patients into the community. COPE, a program designed to help chronic psychiatric patients make the transition from institutionalization to independent living in the community, emphasizes both in-hospital training the systematic aftercare as two points on a continuum of treatment. The program includes four training phases, with each phase based upon the skills acquired in the preceding one. The residents are thereby assured of having the necessary skills for progressing to more complex tasks as they proceed through the program. During the last phase, support by the mental health system is gradually replaced by help from community support systems.


Oligodactyly and multiple synostoses of the extremities: two cases in sibs. A variant of Cenani-Lenz syndactyly. Two sibs with a rare phenotype of oligodactyly with metacarpal, carpal, radioulnar, and metatarsal synostoses and shortening of the forearms were born of healthy parents. Genetic counselling with regard to future children of one of these sibs was given on the assumption of an autosomal recessive inheritance, which is highly probable, judging from the other rare familial cases found in the literature.


Organ culture of adult mouse intestine. I. Morphological results after 24 and 48 hours of culture. Explants of adult mouse intestine have been maintained in organ culture for 24 to 48 hr. The best results have been obtained with a mixture of DMEM-HEPES medium and NCTC-135 enriched with 10% fetal bovine serum. The morphology of the mucosa is well preserved at the light and electron microscopic level: absorbing cells exhibit an increase in secondary lysosomes; goblet cells and Paneth cells remain active; numerous mitoses are observed in the crypts; and vigorous re-epithelization takes place on the margin of the explants.


Effect of in vitro cultivation and Mycoplasma pneumoniae infection on intracellular cyclic AMP levels in hamster tracheal organ cultures. Exogenous cyclic AMP and dibutyryl cyclic AMP decreased the relative ciliary activity values of tracheal organ cultures. In contrast, theophylline and cholera toxin were not ciliostatic. The use of a radioimmunoassay for cyclic AMP indicated that all of the tested substances increased intracellular cyclic AMP levels to some extent (from 3-fold for cholera toxin to almost 40-fold for dibutyryl cyclic AMP). Physical inactivation of explants by either freeze-thaw or heat destroyed all ciliary activity and greatly decreased intracellular cyclic AMP levels. Cyclic AMP levels of explants remained relatively constant during in vitro cultivation. Three strains of Mycoplasma pneumoniae were found to contain extremely low amounts of cyclic AMP. Infection of tracheal explants produced a significant decrease in relative ciliary activity, but only a slight decline in organ-culture cyclic AMP levels.


Quantitative immunoelectrophoretic analysis of human antibodies against herpes simplex virus antigens. By use of crossed immunoelectrophoresis with intermediate gel, antidbody titers against six individual herpes simplex virus (HSV) glycoproteins and two nonglycosylated proteins were determined in 100 human sera. High antibody titers were found against two different HSV type-common glycoproteins designated Ag8 and Ag11 (containing glycosylated polypeptides D and B, respectively). The anti-Ag8 and -Ag11 titers correlated with HSV neutralizing antibody titers. Most of the serological cross-reactivity between HSV type 1 and type 2 was probably caused by antibodies to Ag8 and Ag11. Human antibodies against one HSV type 1-specific glycoprotein (Ag6, containing glycosylated polypeptide C) and two HSV type 2 glycoproteins (Ag4 and Ag9) were also demonstrated, and the titers correlated better with neutralizing antibody titers of the homologous than of the heterologous virus type. The data presented can be directly applied to the further development of diagnostic reagents.


Effect of tobacco smoking on the functions of polymorphonuclear leukocytes. Eight tests investigating the function of circulating polymorphonuclear leukocytes were performed in 68 subjects, half of whom smoked at least 20 cigarettes per day. Comparison of the two groups allowed determination of the in vivo effect of tobacco smoke on the nonspecific defense system of the body. Ingestion ability, oxygen consumption, and bactericidal activity were normal in smokers. Myeloperoxidase and neutrophil alkaline phosphatase activities also were unchanged. The nitroblue tetrazolium reduction and the serum lysozyme levels were slightly increased in smokers. The capillary tube random migration, though, was depressed, and intensive smoking further aggravated this change. It is suggested that tobacco smoke acts directly on one (or several) unidentified target site of polymorphonuclear leukocytes. This impairment, demonstrated in vivo, probably plays a role in the genesis of the bronchopulmonary diseases so frequent in heavy smokers.


Polymyxin B sulfate modification of bacterial endotoxin: effects on the development of endotoxin shock in dogs. The effects of endotoxin (lipopolysaccharide [LPS]) on the pathogenesis of canine endotoxin shock were compared with those of LPS which had interacted with polymyxin B sulfate prior to administration. Both LPS and polymyxin B-modified LPS caused comparable early decreases in aortic blood pressure, leukocyte and platelet numbers, and serum complement levels. However, in dogs receiving polymyxin B-modified LPS the late hypotensive phase was significantly ameliorated and lethality was significantly decreased. These data indicate that polymyxin B-modified LPS, though significantly less lethal than unmodified LPS, was capable of major interactions with several components of the humoral defense system, and support the concept that such interactions are not determinative in the pathogenesis of canine endotoxin shock.


Suppression of in vitro antibody response by spleen cells of mice infected with Friend-associated lymphatic leukemia virus. The ability of spleen cells of mice infected with oncornaviruses to depress the in vitro antibody responsiveness of normal lymphoid cells was exploited in an attempt to clarify the role played by the lymphatic leukemia virus (LLV) component in the immunodepressive properties of the Friend leukemia complex. Spleen cells of mice infected with LLV or, for comparison, with the entire complex were added to cultures of sheep erythrocyte-primed uninfected spleen cells, and the antibody-forming cells produced by the latter, after antigen restimulation, were assayed. The addition within 2 days from culture initiation of low numbers of cells infected with either virus preparation suppressed all stages of the response affecting the production of both immunoglobulin M and immunoglobulin G antibody. The activity of infected cells resisted doses of ultraviolet radiation which inhibit cell multiplication but was abolished by disrupting the cells and was prevented by the presence of anti-LLV antibodies. The LLV-infected spleen cells responsible for suppression were not removed by treatments which selectively remove or kill macrophages and exhibited surface properties of B lymphocytes. These results were interpreted as indicating that the effect is due to virus (or viral products) released by B cells. The suppressing cells in the spleens of mice in the early days of Friend leukemia complex infection presented superimposable properties, supporting the concept that their activity is also due to the LLV they release in large quantities. However, in later stages of infection, the spleens of Friend leukemia complex-infected mice also contained non-B-suppressing cells possibly derived from the proliferation of nonlymphoid LLV-producing cells caused by the neoplastic process.


Development of antibodies reactive in antibody-dependent cellular cytotoxicity in infectious mononucleosis. Serial sera from patients with infectious mononucleosis were examined for the emergence of antibodies reactive in antibody-dependent cellular cytotoxicity tests, using Epstein-Barr virus-superinfected Raji cells as targets. For this specific purpose, the antibody-dependent cellular cytotoxicity test proved to be of limited sensitivity because only relatively high serum dilutions can be tested dependably, due to prozone effects at low serum concentrations, and because antibody-dependent cellular cytotoxicity reactions at the 5% level are not always statistically significant. Under the conditions of the test, antibody-dependent cellular cytotoxicity-reactive antibodies were not measurable, or only barely measurable, in early-acute-phase sera, but they became detectable during convalescence and increased thereafter, gradually over many months to the range of titers seen in healthy persons after long-past-primary Epstein-Barr virus infections. The percentages of antibody-dependent cellular cytotoxicity ultimately attained were on the order of 20% in most patients and healthy individuals, but in others did not exceed 10%. The likely identity of the antibodies reactive in the test with antibodies to late Epstein-Barr virus-determined cell membrane antigens has been discussed.


Interaction of inflammatory cells and oral microorganisms. VIII. Detection of leukotoxic activity of a plaque-derived gram-negative microorganism. In the present study we identified a gram-negative anaerobic rod referred to as Y4 which was cytotoxic for human polymorphonuclear leukocytes. Y4 was isolated from dental plaque of a patient with juvenile periodontitis and presented most of the taxonomic characteristics of Actinobacillus species. Under experimental conditions, viable Y4 were cytotoxic for human peripheral blood polymorphonuclear leukocytes in serum-free cultures. Cytotoxicity was dependent on bacterial concentrations and was enhanced in the presence of a fresh or heat-inactivated (56 degrees C, 30 min) autologous serum. Leukotoxicity was independent of phagocytosis. Y4 leukotoxic effect was abolished when bacteria were heat treated (56 degrees C, 30 min) or when incubations were carried out at 4 degrees C instead of at 37 degrees C. The leukotoxicity was monitored by electron microscopy and biochemically by measuring lactate dehydrogenase indicator of cell viability. No cytotoxic effects of Y4 on human mononuclear cells, chicken fibroblasts, or mouse macrophages were detected under the conditions studied. Polymorphonuclear leukocytes may play an important role in the host defense against bacteria in periodontal disease. The cytotoxic effect of Y4 for polymorphonuclear leukocytes presented in this study is the first report of a direct offensive microbial vector in a plaque-derived microorganism and may prove to be relevant in the pathogenesis of juvenile periodontitis.


Ultrastructural-immunohistochemical evidence for a maturation defect of temperature-sensitive G31 vesicular stomatitis virus in murine spinal cord neurons. Ultrastructural immunoperoxidase studies were done in spinal cords of mice infected with wild type vesicular stomatitis virus or its temperature-sensitive (ts) mutant G31. Infected neurons showed subplasmalemmal staining of viral antigen and staining of viral particles budding from the neuronal membrane in wild-type vesicular stomatitis virus infection, whereas diffuse membrane and cytoplasmic staining with no budding virus was observed in ts G31 infection. Such findings suggest rapid viral assembly and release of viral particles from cells infected with wild-type virus. In contrast, maturation of ts G31 appears defective, and this would lead to accumulation of viral antigen in the cytoplasm of infected cells. These results correlate with studies in neuroblastoma cells which investigated the growth cycles of wild type, ts G31, and the spinal cord isolate of ts G31 as well as the viral protein-synthetic capacity of these viruses.


Endogenous RD-114 virus of cats: absence of antibodies to RD-114 envelope antigens in cats naturally exposed to feline leukemia virus. Healthy cats, including those exposed to feline leukemia virus in leukemia cluster households, lack evidence of humoral immunity to virion surface antigens of the endogenous RD-114 virus.


Inhibition of adenosine 3',5'-monophosphate-dependent protein kinase by staphylococcal alpha-toxin. Staphylococcal alpha-toxin inhibited the activity of cyclic adenosine 3',5'-monophosphate-dependent protein kinase by competitive inhibition, probably by its interaction with a cyclic adenosine 3',5'-monophosphate-binding site in the protein kinase molecule.


Experimental reproduction of neonatal diarrhea in young gnotobiotic hares simultaneously associated with Clostridium difficile and other Clostridium strains. Clostridium difficile, C. perfringens, and C. tertium are very often present simultaneously in the feces of conventional diarrheic young hares, whereas these three bacterial species are rarely encountered and never present simultaneously in the feces of healthy young hares. When a strain of each of the three bacterial species was monoassociated with axenic young hares, the appearance of pathological disorders was only observed in animals monoassociated with C. difficile, when the number of C. difficile exceeded 10(8) per g of fresh feces. When a strain of C. perfringens or a strain of C. tertium, or both, was associated with C. difficile, diarrhea and death occurred more rapidly than in hares monoassociated with C. difficile. C. difficile and C. perfringens became established more rapidly when disassociated than when monoassociated with axenic hares. The association of C. perfringens and C. tertium with axenic hares did not bring about any pathological disorders. It may be concluded that C. difficile is the causal agent of neonatal diarrhea in conventional and gnotobiotic young hares and that other strains of Clostridium enhance its pathogenic effect. C difficile alone or associated with C. perfringens or C. tertium does not play any pathogenic role in young rats, mice, or rabbits.


Evaluation of a ribosomal vaccine against pertussis. A crude ribosomal vaccine derived from Bordetella pertussis administered to ICR and N:NIH (SW) strains of mice protected them effectively against a standardized intracranial challenge. The dose of vaccine that protected half the mice was less for N:NIH (SW) than for ICR mice and compared favorably with a killed reference vaccine. Ribosomes prepared from bacteria ground with washed sea sand were more immunogenic than those obtained by rupture with alumina or with a Braun homogenizer. The protective effect of the crude ribosomes was not an innate part of the organelle but was due to a substance or substances that could be removed from them by a 1 M NH4Cl wash. The material in the wash was highly immunogenic and retained both the histamine-sensitizing and leukocytosis-promoting properties. It lost much of the dermonecrotic activity and was poorly pyrogenic in rabbits. The most potent pyrogen was present in the washed ribosomes, which apparently, retained the endotoxic components of the cell wall. The best vaccines permitted acceptable weight gain in the immunized mice.