Couples group therapy as an adjunct to lithium maintenance of the manic patient. Use of couples therapy groups in conjunction with lithium in the long-term management of married manic-depressive patients is described. Among bipolar patients on lithium, those in couples group therapy had more benign posthospital course than those given minimal support beyond medication. The structure and benefits of the couples group are discussed.


Evidence for glomerular actions of ADH and dibutyryl cyclic AMP in the rat. Experiments were performed on 54 chronically water diuretic Munich-Wistar rats to investigate the effects of various antidiuretic peptides on the determinants of glomerular ultrafiltration. Transition from water diuresis to antidiuresis, induced either by intravenous infusion of 1) exogenous peptides (Pitressin, synthetic arginine vasopressin, or synthetic [1-deamino,4-valine]-8-D-arginine vasopressin) or 2) dibutyryl cyclic AMP, or by stimulation of endogenous ADH release by acute, mild arterial hemorrhage, was associated with near-constant or decreased values for single nephron (SN) and total kidney GFR. Nevertheless, the glomerular transcapillary hydraulic pressure difference (deltaP) uniformly increased with antidiuresis, due to consistent reductions in Bowman's space hydraulic pressure rather than to increases in glomerular capillary hydraulic pressure, the former a consequence of the fall in urine flow rate. In all antidiuretic states, the rats were uniformly observed to be at filtration pressure disequilibrium, permitting calculation of unique values of the glomerular ultrafiltration coefficient (Kf). These values of Kf in antidiuresis were invariably lower than the values obtained during water diuresis. Whether these effects of ADH and DBcAMP on deltaP and Kf represent physiological influences in the control of GFR remains uncertain; their offsetting effects in the present studies usually failed to alter GFR appreciably.


Renin release and pressor response to renal arterial hypotension: effect of dietary sodium. Changes in plasma renin activity (PRA) and mean arterial pressure (MAP) produced by renal arterial hypotension were studied in conscious, adrenalectomized dogs maintained on low-, normal-, or high-Na diet during constant steroid replacement therapy. In animals maintained on a low-Na diet, reduction of renal perfusion pressure to 50 mmHg for 45 min increased MAP 40 +/- 3 (SE) mmHg, while PRA rose rapidly by 36.5 +/- 6 ng ml-1 h-1. Similar renal hypotension in dogs maintained on a normal-Na diet increased MAP only 21 +/- 3 mmHg while PRA rose 5.5 +/- 0.9 ng ml-1 h-1; dogs on high-Na intake had a 6 +/- 1 mmHg pressure rise without a significant change in PRA. The rise in MAP correlated well with the log deltaPRA. Calculated open-loop gain was -1.2, -0.7, and -0.1 in dogs on low-, normal-, and high-Na diets, respectively. Nonpeptide angiotensin I converting enzyme inhibitor (CEI) reversed the elevated MAP observed during reduction of renal perfusion pressure in dogs on low- and normal-Na diets, but had little effect in dogs on high-Na intake. These observations suggest that the renin-angiotensin system becomes quantitatively more important in the regulation of blood pressure as Na intake is reduced.


Involvement of cAMP-dependent protein kinase in the regulation of heart contractile force. The effects of perfusate epinephrine, 1-methyl-3-isobutylxanthine, calcium, and filling pressure were investigated in the perfused working rat heart. Epinephrine produced a rapid increase in cAMP, in the protein kinase activity ratio, and in active phosphorylase. These effects preceded the increase in contractile force produced by the hormone. There was good correlation between protein kinase activation and the increase in force. Epinephrine and the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine were synergistic in their stimulatory effects on cAMP, protein kinase activity, active phosphorylase, and contractile force. When an increase in the force of contraction was produced either by increasing the filling pressure of the heart or by increasing the perfusate Ca2+ concentration, there was no change in either cAMP levels or protein kinase activity. These data suggest that the effect of beta-adrenergic catecholamines on contractile force is due, at least in part, to cAMP-dependent protein kinase activation. The increase in contractile force produced either by increasing the filling pressure (Frank-Starling phenomenon) or by increasing the perfusate Ca2+ concentration is apparently not mediated by cAMP or the protein kinase.


The pseudocapsule of pleomorphic adenomas (benign mixed tumors): the argument against enucleation. The "well encapsulated" pleomorphic adenoma has at best a pseudocapsule which allows for bits of satellite tumor to be left behind at ""enucleation" surgery as well as for easy "spillage" of tumor by the overconfident surgeon. Recurrent pleomorphic adenomas then become difficult tumors to eradicate and place vital structures, such as the facial nerve, in jeopardy.


Multivariate analysis of visual evoked response. The visual potentials evoked by flash are analyzed in healthy subjects in cases of retinal detachment, of optic nerve lesions, and of amblyopia by means of different statistical methods: 1. Analysis of the various features of the response, evaluated separately 2. Analysis of the interdependence between the measures of the various features The first method supplies information on the differences in 'size' of the curves in the various samples, the second method reveals differences of 'shape'. The second method has proved to be able to distinguish both normal conditions from pathologic impairment of optic structures and the different sites of the lesions.


[The hypnotic effect of fentanyl and sulfentanil. An electro-encephalographic comparison (author's transl)]. 1. Electroencephalographic activity was registered in healthy subjects before, during and 90 minutes after intravenous injections of sufentanil and fentanyl. 2. With a dosage of ratio of 1:10, i.e., 0.025 mg/70 kg body weight sufentanil: 0.25 mg/70 kg body weight fentanyl as well as 0.015 mg/70 kg body weight sufentanil: 0.15 mg/70 kg body weight fentanyl, sufentanil was hypnotically more potent from fentanyl. 3. Sufentanil acts more powerfully on the respiratory regulation than does fentanyl. Periods of apnoea occur more frequently but can be interrupted by external psychophysiological stimulation. 4. The evaluation of indices of certain EEG-waves, characterizing well defined electroencephalographic stages corresponding to the waking-sleeping-behaviour confirmed the visual over-all analysis of the EEG-stages. 5. Physiological observations on heartrate, systemic blood pressure, rapid eye movements during wakefulness, and slow eye movements during sleepiness showed no reliable differences between sufentanil and fentanil. The systematically documented symptoms and vegetative signs of subjects also showed neither differences nor side-effects.


Histochemical studies on the spinal cord of the Indian buffalo (Bubalus bubalis). The distribution pattern of glycogen, alkaline phosphatase and acid phosphatase has been studied and recorded in normal and operated spinal cord of the Indian buffalo (Bubalus bubalis). Based on the observations, the following conclusions have been arrived at i) Glycogen is present in the cytoplasm and on the surface of the spinal neurons. Following injury, it is depleted from these sites. ii) Alkaline phosphatase is located in the nucleus and on the neuronal surface. It is not traceable at these sites following spinal cord injury. iii) Acid phosphatase is mostly confined to the neuronal cytoplasm and tends to increase in injured neurons.


Difficult lesions of the carotid arteries and their surgical management. We have discussed five lesions that are encounted less commonly than the usual occlusive carotid lesion that occurs at the carotid bifurcation, namely, high lesions, ulcerative plaques, tortuous carotids, carotid aneurysms, and carotid body tumors. Illustrative cases of these lesions were presented. The diagnostic difficulties related to some of them and the technicalaspects of their surgical management were discussed. New techniques for resecting internal carotid aneurysms and carotid body tumors by using autogenous saphenous vein grafts and a shunt were described and illustrated.


Immunodiffusion test for ovine progressive pneumonia. An agar gel immunodiffusion test was developed to detect precipitating antibody against ovine progressive pneumonia (OPP) virus. The test was conducted in plastic petri dishes containing 6 ml of 1% purified agar in tris buffer and 8% sodium chloride. Wells for serum and antigen were 8 mm in diameter and were cut in a hexagonal pattern 3 cm from a central well. Tests were read at 24 and 48 hours. Soluble antigen for the test consisted of concentrated nutrient medium removed every 2 weeks from a cell culture persistently infected with isolate WLC 1 of OPP virus. Specificity of results was verified by testing serums from experimentally exposed sheep and appropriate controls. Two lines of precipitate formed with some serums from experimentally inoculated sheep. Serums taken soon after exposure of sheep to the virus and those taken 3 to 4 years after exposure frequently formed only 1 line of precipitate. Of 37 lambs inoculated with OPP virus, 25% of those tested were positive by postinoculation (PI) month 1, 79% of those tested were positive by PI month 3, and all of those tested were positive by PI month 6. The test appears adequate to detect exposure of sheep to OPP virus.


Broad antigenicity exhibited by some isolates of avian adenovirus. Ten of 16 isolates of avian adenovirus could be readily classified as members of serotypes 2, 5, or 7. However, 6 exhibited broad neutralization reactions which created typing problems. Detailed studies of these interactions revealed that our prototype for serotype 5 (i.e., T-8; subsequently found to be an "intermediate" strain) contributed to this broad cross-reactivity because it shared antigens with members of serotypes 5 and 7. As a result of this study, TR-59 or 58-1 is recommended as the suitable prototype for the serotype 5 viruses. The 6 broadly reactive isolates were classified as serologic "variants" of serotypes 5 and 7.


Recovery of reovirus type 2 from an immature dog with respiratory tract disease. Two similar cytopathic agents were recovered from the throat and rectal swab specimens of an immature dog with upper respiratory tract disease. The reference isolate, 14-72R, was shown to be a member of the reovirus group by its physicochemical properties, cytopathic effects in cell cultures, and appearance when examined in the electron microscope. Both isolates hemagglutinated human type O erythrocytes and antigenically were closely related to reovirus type 2. The affected pup had an increase in antibody titer to reovirus types 2 and 3. The latter findings provide evidence for possible heterotypic antibody responses in dogs to reovirus infection.


Cutaneous abscesses. Anaerobic and aerobic bacteriology and outpatient management. Specimens from 135 cutaneous abscesses in outpatients were cultured anaerobically and aerobically. Of these, 4% were sterile and 29% yielded pure cultures, predominantly of Staphylococcus aureus. Aerobic species were isolated from all anatomic areas. Anaerobes were found with a frequency comparable to aerobes in all nonperineal areas except the hand. In contrast, abscesses in the perineal region contained a greater variety and frequency of anaerobes. Only two patients were febrile. All abscesses were treated with incision and drainage, and all healed without complication, including those 74% that were treated without adjunctive antibiotics. Primary management of cutaneous abscesses should be incision and drainage. In general, routine culture and antibiotic therapy are not indicated for localized abscesses in patients with presumably normal host defenses.


Enzymatic oxidation of diethyldithiocarbamate by xanthine oxidase and its colorimetric assay. Diethyldithiocarbamate is oxidized in vitro by purified cream xanthine oxidase in the presence of nitro blue tetrazolium and phenazine methosulfate. Disulfiram is formed during the enzymatic or nonenzymatic oxidation. When diethyldithiocarbamate is mixed nonenzymatically with nitro blue tetrazolium, the dye is rapidly reduced, but only in the presence of certain organic solvents. This reaction can be used as a convenient colorimetric assay for diethyldithiocarbamate, which gives a stable color over a wide concentration range.


Studies on the effects of pyrogallol and the structurally related dopa decarboxylase inhibitor RO4-4602 on acetaldehyde metabolism. Microanalytic procedures for the determination of AcH in whole blood from EtOH-intoxicated animals given PG or related drugs should utilize a hemolysis step in 0.5 N PCA in order to inhibit PG-dependent AcH production in vitro. Thiourea may also be included as an added protective measure. RO4-4602, a clinically important drug that contains a PG ring structure, is a moderate in vitro inhibitor of AldDH activity, comparable in potency to PG, chloral hydrate, or diethyldithiocarbamate.


Pattern of muscle and nerve pathology in alcoholism. One hundred and ninety-one inpatients undergoing detoxification from alcoholism were studied electrodiagnostically. Of them, 115 (57%) exhibited muscle pathology; only 11 (5%) showed no electrodiagnostic evidence of pathology. A common occurrence in the more severely toxic patients was a phenomenon called "burst suppression," which revealed an inability of the patient to sustain a normal consistent firing of motor units upon muscle contration; the significance of this phenomenon is not thoroughly understood. Electrodiagnosis is a helpful tool in the diagnosis, prognosis, treatment, and follow-up of patients with alcoholism.


[Derivatographic analysis of 6-beta-[(hexahydro-1H-azepin-1-yl) methylenamino]penicillanic acid]. Derivatographic analysis of 5 samples of 6-beta-(hexahydro-IH-azepin-I-yl)methylenamino penicillanic acid was performed. In addition to the antibiotic the samples had water and acetone in their composition. No effects associated with changes in the physical and chemical state of the substance were observed on the derivatogramme of the samples containing 0.2 and 0.8 per cent of water up 140 degrees C. With a further increase in the temperature an exothermic effect was observed with maximum at 152--153 degrees C connected with melting and chemical degradation of the substance. The derivatogrammes of the samples containing 89--96.4 per cent of the antibiotic were characterized by an endothermic effect with minimum at 65 degrees C caused by evaporation of acetone and partially water from them and by an exothermic effect with maximum at 120 degrees C practically not accompanied by any change in the weight resulting from chemical interaction of the antibiotic with water. The study showed sensitivity of 6-beta-(hexahydro-IH-azepin-I-yl)methylenaminopenicillanic acid to the presence of even insignificant amounts of water in it, maximum elimination of which from the antibiotic is an important factor of increasing its stability.


[Mechanism of penicillinase inhibition by alkyl sulfates in the presence of synthetic polyeletrolytes]. Competing inhibition of Bacillus licheniformis 749/C penicillinase by alkylsulfates CnH2n+ 1OSO3N1 where n was 8--16 was studied. The values of the inhibition constants Ki of individual homologues were estimated. It was shown that stability of complex "enzyme-inhibitor" increased with lengthening of the hydrocarbon radical which was probably due to increased hydrophobic interaction of the alkyl radical with the lipophilic areas of the penicillinase active center. Inhibition in the presence of sopolymers of vinylpirrolidone with N-alkylvinylamine was studied with the aim of modeling the process of penicillinase inhibitions by alkylsulfates in the presence of the blood serum. It was shown that polyelectrolytes posessing hydrophobic substituents had an ability of reducing the activity of inhibited penicillinase, cooperative transfer of alkylsulfate molecules from the enzyme to polyelectrolyte being observed. The maximum effect was registered in the polyelectrolytes with substituents C12H25 and C16H33 which was confirmed in the experiments with the penicillinase-producing strains of staphylococci.


Antiviral activity of 3-deazaguanine, 3-deazaguanosine, and 3-deazaguanylic acid. 3-Deazaguanine (ICN 4221), 3-deazaguanosine (ICN 4793), and 3-deazaguanylic acid (ICN 5412) represent a new class of synthetic guanine analogs having antiviral activity. In vitro, nine ribonucleic acid and seven deoxyribonucleic acid viruses were inhibited, including influenza, parainfluenza, rhino-, vesicular stomatitis, adeno-, herpes-, cytomegalo-, vaccinia, pseudorabies, and myxoma viruses. They were effective orally against influenza types A and B and parainfluenza type 1 (Sendai) virus infections in mice, with a therapeutic index of 16 against the latter two viruses. The course of herpes encephalitis was altered only when the drugs were applied directly into the brain. In addition, these drugs were effective inhibitors of Friend leukemia virus-induced splenomegaly in mice; treatment also produced extensions of life in these animals.


Uptake of glucose and maltose by Bacillus popillae. Results of experiments with glucose and its analog, methyl alpha-D-glucopyranoside, indicated that when glucose was present at low concentrations, it was transported into Bacillus popilliae NRRL B-2309MC cells as glucose 6-phosphate by a phosphoenolpyruvate:sugar phosphotransferase system. An additional mode(s) of entry may be operative at higher glucose concentrations. Maltose appeared to enter the cells by a nonphosphorylative process and was hydrolyzed intracellularly to glucose. No phosphoryl donor was necessary for this hydrolysis.


Aflatoxin in corn: ammonia inactivation and bioassay with rainbow trout. Four samples of corn were compared with respect to their hepatocarcinogenicity in rainbow trout. One corn sample was found by chemical analysis to contain no detectable aflatoxin. A second sample was contaminated with aflatoxins at a level of 180 microgram/kg. Each of the above-mentioned samples was divided, and one-half of each was ammoniated. These four samples were added to a semipurified basal diet and fed to a sensitive strain of rainbow trout. It was found that ammoniation inactivated the aflatoxins and reduced the carcinogenicity of the contaminated corn to a level that was not significantly different from that with the basal control diet. It was also found that the ammoniation process did not reduce the nutritive value of the corn.


Inheritance and longevity of infectious pancreatic necrosis virus in the zebra fish, Brachydanio rerio (Hamilton-Buchanan). Zebra fish (Brachydanio rerio) were injected with infectious pancreatic necrosis virus (IPNV) and then spawned to determine whether the virus was passed on to the eggs, and if it was, how long it remained in the free-swimming F1. The mating variations included parents receiving one or two injections of virus, and within these categories, matings in which both parents were treated or only one parent was treated. The results showed that transmission of IPNV to the egg did occur, and that this transmission was via the female alone. However, if the female was allowed to produce antibodies to the virus, as when she received two injections of IPNV, she transmitted the virus to the eggs for only a short period of time. In addition, when the virus was transmitted to the egg, it remained in the free-swimming F1 for a period of at least 5 months.


Eczema herpeticum caused by herpesvirus type 2. A case in a patient with Darier disease. This, to our knowledge, is the first documented report of eczema herpeticum due to infection with herpesvirus hominis type 2. The virus was isolated from a patient with known Darier disease who developed Kaposi varicelliform eruption. Recent increases in the incidence of infection with type 2 herpes simplex as well as the high frequency of recurrence with this virus make this infection of potential significance in patients with preexisting cutaneous disease.


Sporotrichoid leishmaniasis.

Sporotrichoid leishmaniasis. American leishmaniasis that is acquired in Panama may appear clinically as a sporotrichoid eruption. When leisons reminiscent of sporotrichosis are encountered, a careful history of the patient's travels should be made, as well as a search for the organism of leischmaniasis in tissue smears, histopathological sections, and cultured media. We report the case of an American soldier stationed in Panama who had developed an ulcer on the dorsum of his right wrist, and nodules on his right forearm that were arranged in a linear pattern. The initial clinical impression was that of sporotrichosis, but on careful study of the patient's history, and after other appropriate investigations were made, it was discovered that the patient had leishmaniasis.


The hepatic ultrastructure of chlordene-fed pigeons. Adult male pigeons were fed 500 ppm of nontoxic chlordene per day for 6 weeks. The ultrastructure of hepatic cells of treated pigeons was examined and compared with that of the control birds. Chlordene feeding induced cytological changes in the structure of liver cells. Thinning of the microvilli of bile canaliculi and clear marking of fibrous long spacing and non-fibrous segment long spacing on collagen fibers were noticed. Increase in the number as well as the vacuolation of lipid bodies in certain portions of the cytoplasm, presence of myeloid bodies in mitochondria, and an increase in the smooth-surfaced endoplasmic reticulum were observed in the liver cells of pigeons given chlordene.


Location of three key enzymes of gluconeogenesis in baker's yeast. The subcellular location of hexose diphosphatase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase in baker's yeast (Saccharomyces cerevisiae) was investigated by density gradient centrifugation of spheroplast lysates obtained by osmotic shock treatment of spheroplasts and centrifugation for 10000 g x min. On the evidence obtained from zonal separations these three enzymes of gluconeogenesis are most probably located in the soluble cytosol.


[Cerebral tumors of primitive germinal origin]. The authors attempt to justify the term cerebral tumour of primitive germinal origin from four of their own cases and a review of the literature. They emphasise the specific features. The tumours are more common in boys and involve the pineal, the walls of the third ventricle, the hypothalamus and the posterior pituitary. Several histological types may be distinguished by the degree of differentiation but the stromal reaction, which is partly responsible for the symptoms, is always present. The clinical course of the illness is biphasic. The first is manifest by endocrine disorders and is of relatively long duration. Water homeostasis is always affected and may be associated with other hypothalamic disorders. In the second phase, neurological symptoms and raised intracranial pressure appear. Surgical removal is not always possible, but radiotherapy improves the outlook.


[Severe viral pneumonia. A radiological classification with prognostic value]. In one year eighteen children were admitted to an intensive care unit with severe viral pneumonia. Four groups were identified by the appearance of the chest X-ray. Bronchiolitis was seen in eight patients, alveolitis in four, interstitial pneumonia in two and combined bronchiolitis and alveolitis in four. Three of the four children with alveolitis died and six of the twelve with bronchiolitis, alone or with alveolitis, had residual bronchial obstruction.


[Bone maturation in the fetus and newborn infant]. On the basis of a study of X-ray films of the lower limbs of 994 babies (included 245 hypotrophics) a "bone age" scale of the fetus between 28 and 42 weeks of gestation has been established. The Acheson's method has been used and the bone age determined by addition of notes given to different evolutive phases of the first five ossification centers of the ankle and the knee. To more practical purpose, the results of boys and girls have been joined, although the bone age is in advance among the girls and a theorical scale established with the 10th, 50th and 90th percentiles curves. The comparison with the curves established among 245 hypotrophic newborns, seems to prove that the bone age is a more resistant criteria to malnutrition than the weight and even the length. This new criteria to determine the fetal age does not escape to the criticisms given to the other existing criteria, but radiograph is an objective and fixed document. Its repetition allows to follow the growth of the child and probably to study the role of various factors, mainly nutritional, on this growth. The measure of the length of the tibia and of its increament on the same X-ray films is an other possible method of great interest for the same purpose.


Alteration of cell surfaces with increased tumorigenicity. A comparison of four mouse tumorigenic cell lines derived from transformed peritoneal macrophages is reported. Tumorigenicity of various cell concentration inocula was compared with their saturation densities, agglutinability by Con A, and their surface morphologies as revealed by SEM. It is shown that increase in tumorigenicity after repeated passage through syngeneic mice is associated with changes in their growth characteristics, Con A agglutinability, and correlates with surface changes observed by SEM. The results show that after repeated transfer of transformed cells in syngeneic mice the cells become more tumorigenic, have a higher degree of agglutinability, and show progressive changes of their surface morphology.


Introductory studies on metabolism of free glucose in aqueous extracts from smooth muscles of the stomach of geese and ducks. Aqueous extracts from smooth muscles of the stomachs of geese and ducks contain enzyme systems which not only oxidize free D-glucose, but also determine its further metabolism. The successive metabolites are probably 1,5-D-gluconolactone and 2-ketogluconic acid, and the final product is either pyruvic acid of alpha-ketoglutaric acid. Better understanding of this metabolism will require further studies.


Acute radiation nephritis. Light and electron microscopic observations. Light and electron microscopy were used to observe acute radiation nephritis. By light microscopy the changes were of fibrinoid necrosis of the arteries and atrerioles with segmental necrosis of the glomerular tufts. By electron microscopy the endocapillary cells reacted by hypertrophy and hyperplasia with increase in cytoplasmic organelles. In addition, disruption of endothelial and epithelial cells from the basement membranes were seen. It is concluded that the electron microscopic changes were unique and may be helpful in differentiating the necrotizing glomerulitis seen in other conditions, especially malignant hypertension.


Further studies on human enteric coronaviruses. Comparisons were made between human enteric coronaviruses and the enteric coronaviruses of pigs and calves by negative staining. Examination of human intestinal organ culture fluids at various time intervals after inoculation with the human enteric coronavirus showed increasing numbers of particles in the fluids. Thin sections of the columnar epithelial cells of these explants showed a number of features consistent with the replication of known human and animal coronaviruses. Virus particles found in thin sections had a mean diameter of 68 nm. In addition, a structure was found in thin sections which has not been described previously. This structure may represent the viral nucleocapsid.


Type specificity and use of human sera for rapid typing of herpes simplex virus isolates by an indirect peroxidase-labeled antibody technique: a comparison with three other methods. Human sera from patients showing seroconversion following a primary herpes simplex virus type 1 (HSV-1) or HSV type 2 (HSV-2) infection, as well as 19S and 7S fractions obtained from the same sera, have been used for typing 191 HSV isolates by an indirect peroxidase-labeled antibody (PLA) method. Typing has also been performed on all of the isolates using a microneutralization (MN) and the indirect hemagglutination (IHA) inhibition test. Furthermore, 30 isolates have been typed by a plaque method. Results obtained with the PLA method was in complete agreement with those of the other two procedures. The PLA method is rapid and simple, offers easy interpretation and a permanent record of results.


Differences in multiplication of virulent and vaccine strains of poliovirus type I, II, and III in laboratory animals. Multiplication of virulent and vaccine strains of poliovirus type I, II and III in laboratory animals of different species was studied comparatively. The main criterion of virus reproduction was the production of the photoresistant virus progeny after inoculation of the animals with proflavin-photosensitized virus strains. On the whole, virulent poliovirus strains were characterized by replication in a wide range of hosts (monkeys, cotton rats, white mice, guinea pigs, rabbits, chickens, chick embryos), a low infective dose, production of the photoresistant progeny to a high titre, clinically overt disease in some animal species. The vaccine strains multiplied in a norrower range of hosts, had a high infective dose, a low titre of virus progeny, and caused no clinical symptoms of infection. These differences may serve as a marker for differentiation between virulent and attenuated strains in vivo. Administration of guanidine before inoculation of newborn cotton rats completely prevented or delayed by several days the production of photoresistant virus progeny. This fact confirms the stability of the proflavin-poliovirus complex under conditions ruling out virus replication.


Isolation of Marek's disease virus DNA from infected cells by electrophoresis on polyacrylamide gels. Marek's disease virus DNA isolated from the nuclear fraction of infected chicken embryo fibroblasts and sucrose-purified particles was electrophoresed on 3 per cent polyacrylamide gels and was compared in its electrophoretical behaviour with isolated pseudorabies and herpes simplex DNA, strain HF. The DNA molecules eluted from the gel were identified by their sedimentation coefficient (53--55S) and buoyant density (1.707 g/ml) to be of viral origin. MDV DNA molecules were electrophoretically also detected and identified in DNA preparations of the lymphoblastoid Marek's disease tumour cell line MSB-1 which therefore has to be considered as a producer line. The electrophoresis of DNA preparations from Marek's disease virus-infected cells on polyacrylamide gels provides a semipreparative method for the isolation of MDV DNA.


Prevalence of type-specific antibody against type 1 and type 2 herpes simplex virus in women with abnormal cervical cytology: evidence towards pre-pubertal vaccination of sero-negative female subjects. Patients with abnormal cervical cytology demonstrated a higher prevalence of type-specific complement-fixing antibody to type 2 herpes simplex virus than patients with negative cervical cytology and patients with carcinoma of other body sites. Case-control differences were apparent irrespective of age, socio-economic class and marital status. By contrast, case groups demonstrated a lower prevalence of subjects with type 1 specific antibody. This raises the possibility that pre-adolescent exposure to type 1 herpes simplex virus may offer some measure of protection against pre-malignant and malignant cervical pathology.


Improvement of arbovirus HA antigens by treatment with a colloidal silica gel and sonication. A remarkable increase in HA titers for weakly haemagglutinating Norwegian arbovirus strains, Uukuniemi and Runde viruses, was achieved by including treatment with the colloidal silica gel Aerosil in the antigen preparation scheme. By combining this procedure with sonication, the titers of sucrose-aceton extracted, infected suckling mouse brains could be increased several hundred times. Good antigens also were obtained from virus grown in BHK21/c 13 cell cultures and concentrated by polyethylene glycol 6000/NaCl. Rubella virus HA antigen and HBsAg were adsorbed to the gel, and excluded from a preparation by treatment with Aerosil. This indicates a limitation to the universal use of the method, presumably related to the particle size.


Analysis of the structure of human papilloma virus DNA. Human papilloma virus DNA has been examined for heterogeneity by electron microscopy, reassociation kinetics, and restriction endonuclease digestion. No heterogeneity in contour length was detected. The reassociation rate of papilloma DNA isolated from pooled warts was consistent with that expected for a homogeneous, unique DNA of molecular weight 5 X 10(6). Restriction endonuclease digestions of five papilloma DNA preparations isolated from pooled warts yielded predominantly fragments of the expected sizes, but significant quantities of fragments atypical in size were also commonly produced.


Blood-to-cerebrospinal fluid barrier for cyclic adenosine monophosphate in man. A definite blood to lumbar CSF barrier for cyclic adenosine monophosphate (cAMP) exists in man under physiologic conditions. Lumbar CSF cAMP level remained unchanged, while the CSF glucose level rose significantly after a glucagon hydrochloride infusion that caused a 40-fold increase in the plasma cAMP level.


Anterior interosseous nerve syndromes. Diagnostic methods and alternative treatments. Electromyographic and nerve conduction studies on 46 normal controls (12 men and 34 women, aged 9 to 67 years) are compared with findings in seven patients (three men and four women, aged 9 to 60 years) with the anterior interosseous nerve (AIN) syndromes, including two patients with bilateral involvement (the first such reported cases, to our knowledge). Routine motor and sensory nerve conduction studies of the radial, median, and ulnar nerves show no difference between controls and patients. In 46 normal controls (84 different AIN studies), the latency and duration of the evoked action potential from elbow to pronator quadratus was 5.1 msec +/- 0.9 (95% confidence interval) and 3.6 msec +/- 1.1 (95% confidence interval), respectively. Five of the seven patients had abnormal latencies from elbow to pronator quadratus while all showed prolonged duration of the action potential. Electromyographic abnormalities of the pronator quadratus, flexor pollicis longus, and flexor digitorum profundi, as well as delayed motor latencies and prolonged duration of the evoked potential of the AIN from elbow to pronator quadratus provide sufficient information to diagnose the AIN syndrome. Surgical exploration may not be necessary unless there is evidence of a penetrating injury, significant entrapment, or certain fractures.


A method for characterizing subgroups and related survivals in cancer patients. Application to brain tumors. It has long been known that if the probability of dying of a given cancer remains constant over time, the logarithmic plot of survival of a group of patients with that cancer will be a straight line. More frequently, however, the probability of dying varies with time and the logarithmic survival plot is a curve. We believe that in some cases a population of patients with one type of neoplasm may be composed of several subgroups, each of which has a simple predictable course with a rectilinear survival plot. This article describes a method for partitioning such survival plots and identifying and characterizing the subgroups and applies the method to a group of patients with brain tumors. The value of being able to identify these subgroups and their relative proportions, the observations that can be made with this analysis regarding the nature of the various subgroups of brain tumors, and the implications of changes in the half-lives as well as the proportions of the subgroups with time are discussed.


Abrupt memory loss associated with thalamic tumor. We report a case in which glioma with apparent major involvement  of the right thalamus was manifested initially solely by abrupt onset of severe impairment of both recent and moderately remote memory. Distracting stimuli plays a role in prevention of consolidation of memory.


Perinatal plasma lipid concentrations.

Perinatal plasma lipid concentrations. The effect of the maternal environment on umbilical cord plasma lipid concentrations was studied in a group of mothers and babies. Changes in neonatal plasma concentrations during the first week of life were followed in a small group of infants. The lipoprotein distribution of the cholesterol and triglyceride content of umbilical cord plasma was similar to that seen in maternal plasma at delivery. Neonatal plasma cholesterol was significantly correlated with the cholesterol content of maternal low density lipoprotein. Neonatal plasma triglyceride concentration was related to maternal parity, duration of labour and the existence of maternal hypertension and to the occurrence of foetal distress and dysmaturity.


Arousal from sleep: the physiological and subjective effects of a 15 dB(A) reduction in aircraft flyover noise. The present research was concerned with whether or not a 15 dB(A) reduction in overall noise level would lessen the sleep disturbing properties of jet aircraft flyover noise and, if less disturbing, whether this would be subjectively appreciated by the sleeping individual. The results indicate that a reduction of 15 dB(A) does result in less sleep disruption but only during sleep characterized by fast-wave electroencephalographic activity. During sleep characterized by slow-wave electroencephalographic activity, such a reduction in the sleep-disturbing properties of jet aircraft noise has little effect. Moreover, even when effective during fast-wave sleep, the decreased arousal produced by the lower noise levels is not subjectively appreciated by the individual in terms of his estimate of the quality of his night's sleep. Thus, reducing the overall noise level of jet aircraft flyovers by some 15 dB(A), is, at best, minimally beneficial to sleep.


Systems simulation: theory of monosynaptic transfer between neuron populations. This paper presents a theory for the input-output transformation involved in diffuse monosynaptic activation in a neural tissue or organ system of one population of neurons by another. Main findings are: (1) highly diffuse monosynaptic linkages act very much like filters, selectively sensitive to synchronized clusters of action potentials among the fibers of the input population; (2) partially diffuse monosynaptic linkages are capable of effecting either an amplification or diminution of the number of pulses involved in a single synchronized cluster, depending on parameters of the system; and (3) partially diffuse and spatially organized monosynaptic linkages are capable of effecting a spatial inversion of fine-grained spatial patterns. Theoretical predictions are clarified by mathematical analysis and computer simulation.


Diabetes and the control of pyruvate dehydrogenase in rat heart mitochondria by concentration ratios of adenosine triphosphate/adenosine diphosphate, of reduced/oxidized nicotinamide-adenine dinucleotide and of acetyl-coenzyme A/coenzyme A. 1. The proportion of active (dephosphorylated) pyruvate dehydrogenase in rat heart mitochondria was correlated with total concentration ratios of ATP/ADP, NADH/NAD+ and acetyl-CoA/CoA. These metabolites were measured with ATP-dependent and NADH-dependent luciferases. 2. Increase in the concentration ratio of NADH/NAD+ at constant [ATP]/[ADP] and [acetyl-CoA]/[CoA] was associated with increased phosphorylation and inactivation of pyruvate dehydrogenase. This was based on comparison between mitochondria incubated with 0.4mM- or 1mM-succinate and mitochondria incubated with 0.4mM-succinate+/-rotenone. 3. Increase in the concentration ratio acetyl-CoA/CoA at constant [ATP]/[ADP] and [NADH][NAD+] was associated with increased phosphorylation and inactivation of pyruvate dehydrogenase. This was based on comparison between incubations in 50 micrometer-palmitotoyl-L-carnitine and in 250 micrometer-2-oxoglutarate +50 micrometer-L-malate. 4. These findings are consistent with activation of the pyruvate dehydrogenase kinase reaction by high ratios of [NADH]/[NAD+] and of [acetyl-CoA]/[CoA]. 5. Comparison between mitochondria from hearts of diabetic and non-diabetic rats shows that phosphorylation and inactivation of pyruvate dehydrogenase is enhanced in alloxan-diabetes by some factor other than concentration ratios of ATP/ADP, NADH/NAD+ or acetyl-CoA/CoA.


Effect of cortisol acetate on collagen biosynthesis and on the activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase in chick-embryo tendon cells. Collagen synthesis and the activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase were studied in isolated chick-embryo tendon cells after the administration of cortisol acetate to the chick embryos. When the steroid was injected 1 day before isolation of the tendon cells, collagen synthesis was decreased, even though the enzyme activities were not changed. When cortisol acetate was given as repeated injections over a period of 4 days, both collagen synthesis and the enzyme activities decreased. The hydroxylase activities decreased even more than the two collagen glycosyltransferase activities, both in isolated cells and in whole chick embryos. The amount of prolyl hydroxylase protein diminished to the same extent as the enzyme activity, indicating that cortisol acetate inhibits enzyme synthesis. The inhibitory effect of cortisol acetate on collagen synthesis and on the enzyme activities was partially reversible in 3 days. Total protein synthesis was completely restored within this time. Only massive doses of cortisol acetate inhibited collagen synthesis in vitro. Additional experiments indicated that cortisol acetate did not decrease the rate of the enzyme reactions when added directly to the enzyme incubation mixtures. The results suggest that cortisol acetate decreases collagen synthesis both by its direct effect on collagen polypeptide-chain synthesis and by decreasing the activities of enzymes involved in post-translational modifications.


The probable site of action of thenolytrifluoracetone on the respiratory chain. 1. It is shown that the electron-transfer inhibitor thenoyltrifluoroacetone abolishes a respiratory-chain electron-paramagnetic-resonance absorbance due to spin-spin interactions of ubisemiquinones at concentrations similar to those required for inhibition of succinate oxidation. 2. A specific site of interaction of thenoyltrifluoroacetone with the respiratory chain is proposed to be on the ubisemiquinone with which succinate dehydrogenase reacts. 3. Our results further demonstrate the close association of the HiPIP (high-potential iron-sulphur) centre of succinate dehydrogenase with ubisemiquinone.


Cytochrome c-specific protein methylase III from Neurospora crassa. A protein methylase III responsible for specifically methylating the cytochrome c in Neurospora crassa was partially characterized by using unmethylated horse heart cytochrome c as a substrate. This enzyme utilizes S-adenosyl-L-methionine as the methyl donor. An analysis of the distribution of [14C]methyl groups in the peptides obtained by chymotrypsin digestion of the enzymically methylated cytochrome c showed that all of the radioactivity could be recovered within a single peak after chromatography. This indicates that the enzyme methylates a specific amino acid sequence within cytochrome c. On hydrolysis of the radioactive chymotryptic peptide, Me-14C-labelled epsilon -N-mono-methyl-lysine, epsilon-N-dimethyl-lysine and epsilon-N-trimethyl-lysine were identified. The enzyme can easily be extracted from the N. crassa mycelial pads and was purified approx. 30-fold.


The use of non-aqueous chloroform/methanol extraction for the delipidation of brain with minimal loss of enzyme activities. When freeze-dried brain was extracted at -4-0degrees C with dry chloroform/methanol (2:1 v/v), four of the five enzymes examined were recovered in the diethyl ether-washed residue without inactivation. By contrast, extraction with chloroform/methanol (2:1 v/v) in the presence of water destroyed activities of all the enzymes examined. The amounts of major lipids extracted were similar whether extraction was done in the absence or presence of water. The study was carried out with special interest in 2':3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37), which is firmly bound to the membrane structures of brain white matter.


The synthesis of tritium-labelled human corticotropin of high specific radioactivity. Human [[3,5-3H2]Tyr23]corticotropin-(1-39)-nonatriacontapeptide of specific radioactivity 25.2 Ci/mmol, identical with unlabelled human corticotropin by several criteria, was prepared via the fully protected di-iodotyrosine compound. The latter was synthesized by classical procedures.


Adenosine 3':5'-cyclic monophosphate in higher plants: Isolation and characterization of adenosine 3':5'-cyclic monophosphate from Kalanchoe and Agave. 1.3':5'-Cyclic AMP was extensively purified from Kalanchoe daigremontiana and Agave americana by neutral alumina and anion- and cation-exchange column chromatography. Inclusion of 3':5'-cyclic [8-3H]AMP from the point of tissue extraction permitted calculation of yields. The purification procedure removed contaminating material that was shown to interfere with the 3':5'-cyclic AMP estimation and characterization procedures. 2. The partially purified 3':5'-cyclic AMP was quantified by means of a radiochemical saturation assay using an ox heart 3':5'-cyclic AMP-binding protein and by an assay involving activation of a mammalian protein kinase. 3. The plant 3':5'-cyclic AMP co-migrated with 3':5'-cyclic [8-3H]AMP on cellulose chromatography, poly(ethyleneimine)-cellulose chromatography and silica-gel t.l.c. developed with several solvent systems. 4. The plant 3':5'-cyclic AMP was degraded by ox heart 3':5'-cyclic nucleotide phosphodiesterase at the same rates as authentic 3':5'-cyclic AMP. 1-Methyl-3-isobutylxanthine (1 mM), a specific inhibitor of the 3':5'-cyclic nucleotide phosphodieterase, completely inhibited such degradation. 5. The concentrations of 3':5'-cyclic AMP satisfying the above criteria in Kalanchoe and Agave were 2-6 and 1 pmol/g fresh wt. respectively. Possible bacterial contribution to these analyses was estimated to be less than 0.002pmol/g fresh wt. Evidence for the occurrence of 3':5'-cyclic AMP in plants is discussed.


[Trigeminal herpes zoster].

[Trigeminal herpes zoster]. The authors, after a brief discussion about etiopathogenesis, istopathological changes and principal symptomatologic and therapeutic elements of Herpes Zoster, present three  personal observations of Herpes Zoster facialis, two affecting only the third branch of the trigeminus nerve and one only its second branch. Finally they discuss, basing on clinical peculiarities, a possible diagnostic localization of the disease.


Tracheal cylindroma: anaesthetic management. A tracheal cylindroma in a lady aged 56 yr was excised. The problems of tracheal and carinal resection, and an anaesthetic technique which obviates the use of cardiopulmonary bypass are described.


Sulphated acid mucopolysaccharides in SV40-transformed human cells from normal and mucopolysaccharidosis patients. Lines of fibroblasts have been established from normal individuals and from patients diagnosed as suffering from one of the mucopolysaccharidoses or mucopolysaccharide-storage diseases. Transformation of these lines with SV40 virus has been found to reduce their capacity to secrete sulphated mucopolysaccharides into the growth medium. No differences were detected between the individual cell types in their secretory capacity, either before or after viral transformation. A direct relationship was found to exist between the rate of acid mucopolysaccharide production and cell-doubling time. The level of sulphated mucopolysaccharide detected within the cell was also reduced for all cell types after transformation by SV40. Transformed fibroblasts from mucopolysaccharidosis patients, however, showed a relatively greater reduction in storage capacity than those derived from normal individuals.


Bilateral spontaneous pneumothorax with pulmonary metastases from synovial cell sarcoma. The association of bilateral spontaneous pneumothroax with pulmonary metastases is rare. The clinical and radiological features of bilateral spontaneous pneumothorax with pulmonary metastases from a synovial cell sarcoma are described in a 16-year-old Punjabi student.


Lymphocyte abnormalities in myeloma.

Lymphocyte abnormalities in myeloma. Lymphopenia and monocytosis were found in one third of untreated myeloma patients but there was no correlation between the two. Half of the patients studied had an increase in medium basophilic lymphoid cells, and eight had a raised PAS score. Lymphocytes from the majority of untreated myeloma patients failed to respond to antigenic stimulation in vitro compared with age matched controls. Three patients were immunized but showed no increase in immunoblasts or rise in antibody titre. The findings strongly suggest that abnormal lymphocytes are present in the blood in myeloma. The failure of lymphocytes to respond to antigen provides an explanation for the lack of normal plasma cells and depressed antibody responses in myeloma.


Antibody activity to type 1 and type 2 herpes simplex virus in human cervical mucus. Neutralizing antibody activity in cervical mucus to type 1 herpes virus was detected in 24 of 28 patients, and to type 2 herpes simplex virus in 18 of 24 patients. The neutralizing antibody activity resisted heat inactivation for 30 minutes at 56 degrees C, was independent of complement and followed first order kinetics. There was evidence of antibody against both virus types in immunoglobulin fractions IgG and IgA, the latter containing approximately threefold greater neutralizing antibody activity per unit of immunoglobulin concentration. Type 1 and type 2 neutralizing antibody activity showed a positive but weak correlation and type 2 neutralizing antibody activity showed a positive but weak correlation and a type-common immunoprecipitin was identified in all concentrated pooled mucus samples. However, type-specific neutralizing antibody against both virus types was identified in pooled mucus samples by heterologous absorption techniques. There was a relatively higher average type 2 neutralizing antibody activity in the mucus than in the serum and there was no correlation between serum and mucus antibody levels for either virus type. These observations support the concept of an independent local antibody system for herpes simplex virus in the uterine cervix.


Trophoblastic tumour, oral contraceptive therapy and pregnancy associated alpha2-glycoprotein (PAAG), case report. Pregnancy associated alpha2-glycoprotein (PAAG) and human chorionic gonadotrophin (HCG) levels were studied in a patient with an invasive mole who was being treated with cytotoxic drugs and oral contraceptives. Both the PAAG and HCG levels fell during cytotoxic drug treatment while oral contraceptives caused a rise in PAAG concentrations. A relation between oestrogen-induced PAAG and prolonged survival of trophoblastic tumour is proposed.


Effect of dietary protein level on some key enzymes of the tryptophan-NAD pathway. 1. Six groups of rats were given diets containing protein at three levels (50, 100 and 200 g/kg), with and without nicotinic acid. After 4 weeks on these diets some key enzymes of the tryptophan and nicotinic acid-NAD pathway, liver nicotinamide nucleotide concentration, and urinary metabolites of tryptophan and nicotinic acid were studied. 2. Liver nicotinamide nucleotide levels were lower in rats given the diet with 50 g protein/kg as compared to those in rats given diets with 100 and 200 g protein/kg. The addition of nicotinic acid to the diet resulted in a significant increase in the levels of nicotinamide nucleotides only in rats given 50 g protein/kg diet but not in those given either 100 or 200 g protein/kg diet. 3. Liver tryptophan oxygenase (EC 1.13.1.12) activity increased with increasing dietary protein level. Niconitic acid in the diet had no effect on its activity. 4. Quinolinate phosphoribosyltransferase (EC 2.4.2.a) activity in liver was inversely related to dietary protein level, and nicotinic acid in the diet had no effect on its activity. 5. Liver nicotinate phosphoribosyltransferase (EC 2.4.2.11) activity and kidney picolinate carboxylase (EC 4.1.1.45) activity were not altered either by dietary protein level or nicotinic acid in the diet. 6. The addition of nicotinic acid to the diet resulted in increased excretion of N'-methylnicotinamide at all dietary protein levels. 7. The inverse relationship between protein level in the diet and liver quinolinate phosphoribosyltransferase activity, the rate-limiting enzyme of the tryptophan-NAD pathway suggests that the efficiency of conversion of tryptophan to NAD is related to protein level in the diet, the efficiency decreasing with an increase in the level of dietary protein.


The effect of dietary calcium on the activity of 25-hydroxycholecalciferol-1-hydroxylase and Ca absorption in vitamin D-replete chicks. 1. As most of the studies on the regulation of renal 25-hydroxycholecalciferol-1-hydroxylase (25-HCC-1-hydroxylase) activity have been done in marginally-vitamin D-deficient animals and as it is known that vitamin D administration suppresses the specific activity of the 25-HCC-1-hydroxylase, it was decided to study the effect of dietary calcium on the activity of 25-HCC-1-hydroxylase and on Ca absorption in vitamin D-replete chicks. 2. Chicks, 10 d old, were given diets differing in their Ca contents (65 nmol cholecalciferol/kg diet) for 10 d and the activity of 25-HCC-1-hydroxylase in kidney homogenates, Ca absorption from the duodenum, Ca-binding protein (CaBP) activity in the duodenal mucosa and plasma Ca and phosphate concentrations were all determined. 3. The CaBP activity and the efficiency of Ca absorption both decreased with increasing dietary intake of Ca. Ca absorption and CaBP activity were significantly correlated (r 0-995, P less than 0-01). 4. The activity of 25-HCC-1-hydroxylase decreased as the dietary level of Ca increased and was significantly correlated with Ca absorption (r 0-900, P less than 0-05). The plasma Ca concentration and the activity of 25-HCC-1-hydroxylase were inversely related (r-0-940, P less than 0-01). 5. It is concluded that in the vitamin D-replete chick the efficiency of duodenal Ca absorption is regulated by the renal 25-HCC-1-hydroxylase activity via production of 1,25-dihydroxycholecalciferol and CaBP synthesis.


Nearest neighbor relationships of the polypeptides in ubiquinone cytochrome c reductase (complex III). Ubiquinone cytochrome c reductase (complex III) in detergent dispersion has been cross-linked with two reversible cross-linking agents dithiobissuccinimidylpropionate and dimethyl-3.3'-dithiobispropionimidate and the cross-linked products formed have been analyzed by two-dimensional gel electrophoresis. Under mild reaction conditions, polypeptides I and II, II and VI, I and V, and VI and VII were the most prominent subunit pairs seen. With higher levels of reagent, larger aggregates were produced until an aggregate of apparent molecular weight 310 000 was the dominant band on gels. This is the complex III monomer.


Separation and quantitation of intracellular forms of poliovirus RNA by agarose gel electrophoresis. Intracellular poliovirus-specific RNA species can be measured directly by electrophoresis of total cytoplasmic nucleic acids through 1% agarose gels, resulting in the separation of single- and double-stranded forms of poliovirus RNA from each other and from HeLa cell 28S ribosomal RNA. Single-stranded RNA molecules differing by only 15% in length are resolved in this gel system. RNA species can be visualized as fluorescen bands appearing after staining of the gels with ethidium bromide and observation under ultraviolet illumination. The total amount of RNA can be determined by densitometric quantitation of the fluorescent response. In this way, the amount of poliovirus-specific RNA within the cytoplasm of HeLa cells infected for various times has been estimated. At 170-min postinfection, there are 0.67 X 10(5) molecules of single-stranded poliovirus RNA per cell and at 230 min, the amount has increased to 3.7 X 10(5) molecules/cell. Poliovirus double-strnaded RNA reaches a maximum of 0.7 X 10(5) molecules/cell at 330 min after infection.


Linear electric field effect in electron paramagnetic resonance for two bisimidazole--heme complexes, model compounds for B and H hemichromes of hemoglobin and for cytochrome b5. Bisimidazole-ferric heme is considered to be the structure at the heme site of cytochrome b5 and two different low spin ferric hemochromes spontaneously formed from ferric hemoglobin. The addition of strong base to bisimidazole-ferric heme in organic solvents alters the optical and magnetic properties of this compound. With the use of the linear electric field effect in the electron paramagnetic resonance, we demonstrate that addition of base does not lead to the exchange of hydroxide anion for ligated imidazole and that the bisimidazole structure is retained. Analysis of optical titrations indicates that 2 equiv of base react reversibly with bisimidazole-ferric heme. It is suggested that the two hemichromes formed from hemoglobin differ in structure from one another by the state of protonation of N-1 in the bound imidazoles.


Phosphoglycerate mutase from wheat germ: studies with 18O-labeled substrate, investigations of the phosphatase and phosphoryl transfer activities, and evidence for a phosphoryl-enzyme intermediate. From studies using unlabeled phospho-D-glycerate in solutions enriched in H2(18)O, and from experiments involving [18O]phospho-D-glycerate, it is shown that the intramolecular isomerization of 2- and 3-phospho-D-glycerate that is catalyzed by the phosphoglycerate mutase from wheat germ does not involve an intermediate 2,3-cyclic phosphate. It is also shown that phosphoglycerate mutase catalyzes the hydrolysis of the substrate analogues 2-phosphoglycolate, 2-phospho-D-lactate, 3-phosphohydroxypropionate, phosphoenolpyruvate, and phosphohydroxypyruvate. The substrates 3- and 2-phospho-D-glycerate are not hydrolyzed, nor are 2,3-bisphospho-D-glycerate, 2-phospho-L-lactate, 3-phospho-L-glycerate, or sn-glycerol 3-phosphate. Although no exchange of D-[14C]glycerate into phospho-D-glycerate can be detected, the enzyme catalyzes the transfer of the phosphoryl group from "unnatural" donors such as 2-phosphoglycolate, to the "natural" acceptor, D-glycerate. It is concluded that the intramolecular phosphoryl transfer catalyzed by the wheat germ phosphoglycerate mutase follows a pathway involving a phosphoryl-enzyme intermediate.


Isolation and properties of the rabbit skeletal muscle protein inhibitor of adenosine 3',5'-monophosphate dependent protein kinases. The heat-stable protein inhibitor (Walsh, D. A., et al. (1971), J. Biol. Chem. 246, 1977--1985) of the cyclic adenosine 3',5'-monophosphate dependent protein kinase has been isolated in pure form from rabbit skeletal muscle after a 430 000-fold purification with a 47% yield. The four-step procedure involves sequentially a heat treatment, batchwise anion and cation exchange, and affinity chromatography on protein kinase catalytic subunit covalently coupled to Sepharose 4B. The inhibitor is an acidic protein (pI = 4.24) of molecular weight 11 300. It contains 98 amino acid residues none of which contains sulfur and only 2 (phenylalanine and tyrosine) are aromatic. The NH2-terminus is blocked. The muscle content is ca. 0.6 mg of inhibitor per L of intracellular water. The inhibitor is tightly bound to the catalytic subunit of protein kinase (Ki congruent to 2 X 10(-9) M) and acts competitively with respect to the protein substrates. Protein kinase recognizes a short stretch of the inhibitor sequence, in which arginyl side chains play a crucial role. A study of various competitive inhibitors of the kinase confirms the importance of guanidino groups and hydrophobic side chains in the specific interaction with the substrate binding site.


Model studies for molybdenum enzymes. Reduction of cytochrome c complexes by mu-oxo-bis[oxodihydroxo(L-cysteinato)molybdate(V)]. The reduction by mu-oxo-bis[oxodihydroxo(L-cysteinato)molybdate(V)] (I) of cytochrome c complexes in which the methionine-80 ligand has been replaced by N3-, CN-, or imidazole has been investigated. The N3- and CN-substituted species are reduced by I in a first-order reaction that appears to proceed via a slow dissociation of N3- or CN-, followed by rapid reduction of native cytochrome c. At low concentrations of I, reduction of the imidazole-cytochrome complex occurs by the same mechanism, while, at higher concentrations of I, direct reduction by I and by a monomeric Mo(V) complex in equilibrium with I appears to occur, although at much lower rates than with native cytochrome c. Potentiometric measurements of E degrees for the cytochrome c complexes with N3- and imidazole indicate the lack of reducibility, or reduction in rate relative to native cytochrome c, is not due to thermodynamic reasons. In the case of the CN- complex, E degrees may be too low for direct reduction by I. The effects on the reduction rates are attributed to a conformational change accompanying the replacement of the methionine-80 ligand, which makes the exposed heme edge less available to attack by outer sphere reductants.


Purification and initial rate kinetics of acyl-phosphate-hexose phosphotransferase from Aerobacter aerogenes. The enzyme acyl-phosphate-hexose phosphotransferase from Aerobacter aerogenes was purified to electrophoretic homogeneity. The molecular weight of the enzyme as determined on Sephadex gels is 150 000. The enzyme possesses potent phosphotransferase and phosphohydrolase activities. Initial rate kinetics were used to investigate the mechanism of acyl-phosphate-hexose phosphotransferase. These studies, which involved a number of different phosphoryl donors and substrate analogues, suggest that the kinetic mechanism is of the rapid equilibrium random Bi Bi type. A number of other enzymes that exhibit both transferase and hydrolase activities involve obligatory covalent enzyme-substrate intermediates in their mechanisms of action.


Phosphatidylcholine exchange between the boundary lipid and bilayer domains in cytochrome oxidase containing membranes. A phospholipid spin label, 16-doxylphosphatidylcholine, is employed in a study of lipid--protein interactions in cytochrome oxidase containing membranes. Two methods are used to label the membranous cytochrome oxidase: dispersion in cholate with subsequent detergent removal, and fusion with vesicles of the pure phospholipid label in the absence of detergent. A fraction of the label is immobilized, which is calculated to fall in the range of 0.17--0.21 mg of phospholipid/mg of protein (0.15--0.19 after correction for lipids not extracted by chloroform--methanol). This narrow range of values is independent of methods of labeling, protein isolation, and lipid depletion within experimental error. When labeling by fusion is utilized, the patches of pure phosphatidylcholine spin label diffuse in the plane of the bilayer, become diluted, and demonstrate exchange with bound phospholipid. These observations are evidence that boundary lipid, as reflected by the partitioning of the phosphatidylcholine label, is in equilibrium with adjacent bilayer regions and that it consists of a relatively constant amount of phospholipid associated with the hydrophobic portion of the protein.


Metabolically labeled cell membrane proteins in spontaneously and in SV40 virus transformed mouse fibroblasts. A family of mouse fibroblast cell lines in exponential phase of growth were compared in protein constitution of their cell membranes. In preparations from these cells enriched in cell-surface membrane we observed one protein component (apparent molecular weight about 250 000) consistently to be reduced or absent in an SV40 virus transformed cell line, when compared with the normal cell line. No such compositional difference was observed in a spontaneously transformed tumorigenic clonal derivative cell line, or in subclones of such a derivative cell line, with or without SV40 virus infection. However, in metabolic labeling experiments with 14C-labeled mixed amino acids, a consistent decrease also was demonstrated in the biosynthesis of the same protein in the SV40 virus infected subclone, as compared to an uninfected sister subclone, during exponential growth. This specific difference in biosynthesis is apparently related to the presence and functioning of the SV40 gene, and correlates with the ability of these cells to grow in viscous medium, but not with cellular tumorigenicity.


Response of adrenal tumor cells to adrenocorticotropin: site of inhibition by cytochalasin B. The ability of cytochalasin B to inhibit the steroidogenic response of mouse adrenal tumor cells (Y-1) to adrenocorticotropin (ACTH) was examined with two aims: to consider the specificity of the inhibitor and to determine at what point(s) in the steroidogenic pathway it acts. Cytochalasin B did not inhibit protein synthesis or transport of [3H]-cholesterol into the cells nor did it alter total cell concentration of ATP. Together with previous evidence, this suggests that the effects of cytochalasin observed are relatively specific in these cells. Cytochalasin inhibits the increase in conversion of [3H]cholesterol to 20alpha-[3H]dihydroprogesterone (20alpha-hydroxypregn-4-en-3-one: a major product of the steroid pathway in Y-1 cells) produced by ACTH but does not inhibit conversion of cholesterol to pregnenolone by mitochondrial and purified enzyme preparations from Y-1 cells and bovine adrenal, respectively. Cytochalasin does not inhibit the conversion of pregnenolone to 20alpha-dihydroprogesterone but was shown to inhibit increased transport of [3H]cholesterol to mitochondria resulting from the action of ACTH. These findings indicate that cytochalasin acts after cholesterol has entered the cells and before it is subjected to side-chain cleavage in mitochondria. In view of the known action of cytochalasin on microfilaments, it is proposed that these organelles are necessary for the transport of cholesterol to the mitochondrial cleavage enzyme and that at least one effect of ACTH (and cyclic AMP) is exerted upon this transport process. The specificity of the effects of cytochalasin is considered in relation to this conclusion.


Studies on the 5' termini of Novikoff ascites hepatoma ribosomal precursor RNA. The 5' termini of 18S and 28S rRNA and their 32S, 41S, and 45S nRNA precursors were analyzed by fractionating alkaline hydrolysates of the RNAs on columns of diethylaminoethyl-Sephadex A-25 at neutral pH followed by electrophoresis on Whatman 3MM paper at pH 3.5. The results indicate that each RNA begins with a mononucleoside diphosphate of heterogeneous composition. The principal nucleoside diphosphate was pCp in 28S, 32S, 41S, and 45S RNA and pUp in 18S RNA. The 18S rRNA also contained the most homogeneous composition with approximately 70% of the molecules beginning with pUp; the 41S and 45S nRNA termini were the most heterogeneous, each containing pCp in only one-third of the molecules. Each terminal composition was reproducible and characteristic for its RNA. The composition of 28S rRNA was very similar to that of 32S nRNA, but distinctly different from 5.8S or 18S rRNA or 45S nRNA. The isomeric forms of 45S nRNA contained no appreciable amounts of a terminal 5'-triphosphate, although they had similar nucleoside diphosphate compositions. These results are discussed in relation to the transcription of ribosomal genes and the arrangement of the 5.8S rRNA sequence within these genes.


Comparative study of hen yolk phosvitin and plasma vitellogenin. Vitellogenin, the only phosphoprotein detectable in the plasma of laying hens, is present at an approximate concentration of 1 mg/mL and can be isolated by chromatography on diethylaminoethylcellulose. Vitellogenin has a molecular weight of 235 000--240 000 and contains approximately 3% phosphorus by weight. Evidence that this protein is the precursor of phosvitins includes its ability to act as an acceptor for phosphate with a phosvitin specific kinase, the generation of a peptide similar to phosvitin by trypsinization, and the presence of distinctive peptides of multiple clustered phosphoserine upon partial acid hydrolysis. This partial sequence similarity between phosvitins and vitellogenin has not been previously reported. The phosphorus content and amino acid composition of vitellogenin are consistent with a model which contains two phosvitins and one lipovitellin. The total molecular weights of these proteins (28 000 + 34 000 + 170 000 = 232 000) are close to that of vitellogenin.


Redetermination of the pressure dependence of the lipid bilayer phase transition. The effect of pressure on the phase transition temperature for the dipalmitoyllecithin bilayer was redetermined by following the volume change accompanying the transition. These measurements were carried out isothermally with the transition from the ordered to the disordered phase induced by decreasing the pressure. This contrasts with our previous measurements which were carried out at constant pressure and increasing temperature. The transition at every temperature was sharp and confirmed our previous observation that the volume change associated with the transition (0.033 mL g-1) is invariant with pressure. However, our present measurements, in contrast to our previous results, indicate that dP m/dTm at all pressures is in agreement with the 1 atm value of delta H/Tm delta V within experimental error where Tm and Pm are the temperature and pressure of the phase transition, respectively. These results, which are now in agreement with all other known pressure data, indicate that the entropy change associated with the transition is invariant with pressure.


Phospholipid-protein interactions in human low density lipoprotein detected by 31P nuclear magnetic resonance. 31P nuclear magnetic resonance (NMR) spectra of human low density lipoprotein (LDL) has been obtained and the major phospholipid components identified. Analysis of the spectra revealed two phospholipid environments: one occupied by 4/5 of the phospholipid with high resolution resonances possessing properties similar to phospholipids in vesicles, and a second occupied by 1/5 of the phospholipid with broad lines indicative of immobilization. Limited trypsin treatment of the particle cleaved all of the B peptide into smaller molecular weight peptides which remained with the particle. Trypsin-treated LDL eluted from a Sepharose CL-6B column similarly to native LDL so that the modified particle remained intact. 31P NMR spectra of trypsin-treated LDL showed little or no immobilized phospholipid. The immobilization in the native LDL particle is attributed to lipid-protein interactions between 1/5 of the phospholipid and the B peptide.


Nitrite and nitric oxide treatment of Helix pomatia hemocyanin: single and double oxidation of the active site. The reaction of nitrite and nitric oxide with Helix pomatia hemocyanin has been studied. One or both of the two copper ions in the active site can be oxidized, depending upon reaction conditions. The single oxidation of the oxygen binding site can be reversed by reduction with hydroxylamine, and the oxygen binding properties of the protein are simultaneously restored. The experiments, including electron paramagnetic resonance, indicate that nitric oxide is not a ligand of copper in the singly oxidized active site and that the oxidized copper ions is coupled to at least two nitrogen atoms of amino acid residues. The doubly oxidized protein can be reduced to a singly oxidized one with ascorbic acid or hydroxylamine; the latter reagent is again able to reduce the singly oxidized state and to restore the oxygen binding properties.


Structural studies on the sialic acid polysaccharide antigen of Escherichia coli strain Bos-12. A polysaccharide, antigenically related to group C meningococcus, has been isolated from Escherichia coli strain Bos-12 (016; K92; NM). Like groups B and C meningococcal polysaccharide, the Bos-12 antigen is a pure polymer of sialic acid. 13C NMR studies on the meningococcal group B and C polysaccharides have indicated that the former consists of sialic acid units linked 2 leads to 8- alpha, whereas the latter contains the sialic acid residues linked 2 leads to 9-alpha (Bhattacharjee, A.K., Jennings, H.J., Kenny, C.P., Martin, A., and Smith, I.C.P. (1975), J. Biol. Chem. 250, 1926). Comparison of natural abundance 13C NMR spectra of the Bos-12 polysaccharide with group B and C meningococcal polysaccharides established that Bos-12 was either (a) an equimolar mixture of 2 leads to 8-alpha linked sialic acid homopolymers or (b) a 2 leads to 8-alpha/2 leads to 9-alpha heteropolymer. These possibilities were distinguished in the following manner. The fact that Bos-12 polysaccharide precipitated with anti-group C serum but not with anti-group B serum would seem to exclude a. Further, chemical studies (periodate oxidation followed by tritiated NaBH4 reduction) gave saccharides with a radioactive-labeling pattern expected for alternating 2 leads to 8-alpha/2 leads to 9-alpha sialic acid linkages. Bos-12 is thus an 2 leads to 8/2 lead to 9-alpha heteropolymer.


Modification of ribonucleic acid by vitamin B6. 1. Specific interaction of pyridoxal 5'-phosphate with transfer ribonucleic acid. Whole tRNA preparation obtained from a human cell line (HT-29) of colon carcinoma and purified specific Escherichia coli tRNA were reacted with pyridoxal 5'-phosphate, reduced by sodium borohydride and digested with RNase A and snake venom phosphodiesterase. Two-dimensional chromatography of the pyridoxal 5'-phosphate treated tRNA digest showed that pyridoxal 5'-phosphate binds specifically to GMP, presumably in the form of a Schiff base with the exocyclic amino group of the purine. The reaction of pyridoxal 5'-phosphate with whole tRNA was competitively inhibited by N-acetoxy-2-acetylaminofluorene. This suggests that binding occurred primarily to the G20 base residue at the unpaired region of the dihydrouridine loop (Fujimura et al., 1972). The modification of tRNA by pyridoxal 5'-phosphate resulted in the inhibition, to varying extent (10-80%), of amino acid acceptance in the aminoacyl-tRNA synthetase reaction. Defects in codon recognition by pyridoxal 5'-phosphate modified amino acid acylated tRNAs in the presence of the corresponding guanine-containing polynucleotide triplets were observed by the ribosomal binding assay.


Relationship between hydroxypyruvate and the production of oxalate in vitro. Chicken liver lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC1.1.1.27) catalyses the reversible reduction reaction of hydroxypyruvate to L-glycerate. It also catalyses the oxidation reaction of the hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form to glycolate. At pH 8, these latter two reactions are coupled. The coupled system equilibrium is attained when the NAD+/NADH ratio is greater than unity. Hydroxypyruvate binds to the enzyme at the same site as the pyruvate. When there are substances with greater affinity to this site in the reaction medium and their concentration is very high, hydroxypyruvate binds to the enzyme at the L-lactate site. In vitro and with purified preparation of lactate dehydrogenase, hydroxypyruvate stimulates the production of oxalate from glyoxylate-hydrated form and from NAD; the effect is due to the fact that hydroxypyruvate prevents the binding of non-hydrated form of glyoxylate to the lactate dehydrogenase in the pyruvate binding site. At pH 8, THE L-glycerate stimulates the production of glycolate from glyoxylate-non-hydrated form and NADH since hydroxypyruvate prevents the binding of glyoxylate-hydrated form to the enzyme


Effect of temperature on arginine incorporation by ribosomeless extracts of cells transformed by a temperature-sensitive mutant of Rous sarcoma virus. The effect of transformation of normal rat kidney cells by a temperature-sensitive mutant of the Prague strain of Rous sarcoma virus (ts LA 24 PR-A) on the post-translational addition of arginine to the NH2-terminus of preformed acceptor molecules has been studied. Cells maintained at the permissive (35 degrees C) temperature show a high arginine-incorporating activity in ribosome free extracts compared to that found in extracts of cells grown at the non-permissive (40 degrees C) temperature. Temperature shift experiments as well as studies with cells transformed by wild type Rous sarcoma virus suggest that the decreased activity in cells grown at 40 degrees C is not due to a high temperature per se. The lower arginine incorporation in the 40 degrees C cell extracts is partially due to a decrease in the activity of arginyl transferase which catalyses the transfer of arginine from arginyl tRNA to the acceptor protein. Polyacrylamide gel electrophoresis of the radioactive product shows that the acceptor molecules present in extracts of cells grown at 40 degrees C are larger and qualitatively different from those found in extracts of cells grown at 35 degrees C.


Adenosine 3',5'-monophosphate-dependent protein kinases of myocardial non-histone nuclear proteins. Myocardial acidic non-histone nuclear proteins (NHPs) contain endogenous protein kinase activity. Phosphocellulose chromatography of purified NHPs identifies nine separate peaks of protein kinases which can phosphorylate both endogenous and exogenous substrates to a variable degree; endogenous NHPs are the best substrates. Cyclic AMP-stimulated protein kinase induced phosphorylation of endogenous and exogenous substrates; the extent of this stimulation varied according to the protein kinase fraction and substrate used. Cyclic AMP also enhanced NHP-induced stimulation of RNA polymerase activity. This enhancement was dependent on protein kinase-induced phosphorylation of NHPs since it was prevented by alkaline phosphatase pretreatment. It is concluded that nuclear protein kinases regulate myocardial RNA synthesis by enhancing phosphorylation of NHPs and that this regulation is under cyclic AMP control.


Interactions of some nitro-derivatives of substituted 9-aminoacridine with DNA. The mechanism of the biological activity of the 1-nitro and 2-nitro aminoacridine derivatives containing the dimethylaminopropyl side chain was studied. RNA synthesis in the isolated rat liver nuclei was only slightly influenced by both compounds. They do not differ in their ability to form an intercalative complex with DNA. Only the 1-nitro derivative exhibited strong inhibitory effect on RNA biosynthesis and caused distinct ultrastructural changes (nucleolar segregation, chromatine margination etc.) in a living cell. The 1-nitro derivative binds covalently to DNA in vivo resulting in crosslink formation. It is concluded that the biological activity of 1-nitro acridine derivatives depends more on their crosslinking activity than on their ability to intercalate into DNA.


Yaba virus-specific DNA in the host cell nucleus. DNA-DNA hybridization studies show that Yaba virus-specific DNA is present in the host cell nucleus late in the infection cycle. The nuclear DNA appears to exist as a complete genome, not convalently linked to host cell DNA, as demonstrated by sedimentation analyses. The DNA apperas to be synthesized in the nucleus, since its level of incorporation of label is ten times the background incorporation detectable in the cytoplasm. Extraction of the nuclei by treatment with SDS and EDTA after precipitation with 1 M NaCl separates most of cellular DNA from the Yaba virus-specific DNA.


Studies on viral DNA protein complexes isolated at different times after infection of monkey kidney cells with simian virus 40. The major protein components of the DNA complex, isolated from SV40-infected monkey cells, are the major viral structural polypeptide VP1 and cellular histones. At early times (24 h) after infection, VP1 is present in large amounts relative to histones, whereas at late times (48 h), the complex contains mostly histones. The amount of VP1 in the complex can be correlated to the amount of "free" VP1 present in the cells, i.e. VP1 not yet incorporated into virus particles. At early times about 40% of VP1 is "free" VP1; at late times, most of the VP1 is incorporated into virus particles. In contrast, viral DNA is produced in huge excess and only about 13% is incorporated into virions. In agreement with the above result, we find that only about 16% of the DNA in the DNA complex can be chased into virions. There is, apparently, no turnover of newly synthesized VP1 that is associated with the DNA complex at late times after infection.


The mechanism of ADP-ribosylation of elongation factor 2 catalyzed by fragment A from diphtheria toxin. Measurements of the initial rate of ADP-ribosylation of elongation factor 2 (EF-2) catalyzed by Fragment A from diphtheria toxin support a sequential mechanism and suggest that the reaction proceeds through a central ternary complex involving Fragment A and the substrates, EF-2 and NAD. The Michaelis constants for EF-2 and NAD are 0.15 and 1.4 muM, respectively. As determined by equilibrium gel permeation, EF-2 does not bind Fragment A significantly, alone or in the presence of adenine, ADPribose, nicotinamide or NADH. Based on these and earlier results, we propose an ordered sequential mechanism for the reaction; the sequence of binding of substrates is NAD, followed by EF-2.


Dissociation of phosphohistone phosphatases from canine heart. Phosphohistone phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) of canine heart extract has been separated by DEAE-cellulose chromatography into 4 molecular forms, namely phosphatases A (Mr = 156 000), B (Mr = 161 000), C (Mr = 95 600) and U (Mr = 61 000). ATP inhibited phosphatase A, stimulated phosphatase B and did not significantly affect phosphatase C activity. Phosphatase U requires Mn2+ for activity, under which condition ATP is inhibitory. Phosphatases A, B and C, but not phosphatase U, were dissociated by ethanol into catalytic subunits that were inhibited by ATP, insensitive to Mn2+, and had a common molecular weight of 34 800 (phosphatase S). The dissociation was accompanied by an increase of enzymic activity. Chromatography of the ethanol-treated 55% (NH4)2SO4 fraction of canine heart extract on DEAE-cellulose demonstrated that the multiple forms of phosphohistone phosphatase could be reduced to two forms: phosphatase U and phosphatase S, which may represent two basic constituents of the multiple forms of phosphohistone phosphatase in canine heart.


The detection and characterisation of collagenase inhibitors from rabbit tissues in culture. As tissue cultures, rabbit bone, skin and non-gravid uterus synthesise inhibitors of collagenase (EC 3.4.24.3). An assay for the inhibitors is described and their action on collagenase from different tissue sources demonstrated. Evidence for the involvement of the tissue inhibitors of collagenase in the latency of the enzyme in culture media is presented. Latent collagenase was activated by treatment with 4-aminophenylmercuric acetate, and then reacted with the inhibitors to form inactive complexes with properties similar to the naturally occurring latent enzyme forms. The associated changes in molecular weight are detailed, and discussed in relation to the observations of other workers concerning the extracellular control of collagenase activity.


Kinetic studies of the old yellow enzyme. I. The reaction mechanism of the enzyme with reduced nicotinamide adenine dinucleotide. The reaction mechanism of old yellow enzyme (NADPH:(acceptor) oxidoreductase, EC 1.6.99.1) was kinetically investigated using NADH as substrate. The enzyme was reduced by NADH via a reaction intermediate which has a specific absorption spectrum. This intermediate decomposed to yield a reduced enzyme and NAD+ through an inrreversible first-order reaction step. The reduced enzyme was reoxidized by oxygen through a second-order reaction process. Individual values of elementary rate constants were measured and a computer simulation of the reaction process was carried out. No involvement of free radical of flavin semiquinone in the reaction process could be shown.


Regulation of low density lipoprotein receptor activity by cultured human arterial smooth muscle cells. The ability of cultured human arterial smooth muscle cells to regulate low density lipoprotein (LDL) receptor activity was tested. In contrast to human skin fibroblasts incubated with lipoprotein deficient medium under identical conditions, smooth muscle cells showed significantly reduced enhancement of 125I-labeled LDL and 125I-labeled VLDL (very low density lipoprotein) binding. Smooth muscle cells also failed to suppress LDL receptor activity during incubation with either LDL or cholesterol added to the medium, while fibroblasts shoed an active regulatory response. Thus, in comparison with the brisk LDL receptor regulation characteristic of skin fibroblasts, arterial smooth muscle cells have and attenuated capacity to regulate their LDL receptor activity. These results may be relevant to the propensity of these cells to accumulate LDL and cholesterol and form "foam cells" in the arterial wall in vivo, a process associated with atherogenesis.


The effect of portacaval shunt on plasma lipids and lipoproteins in swine. Plasma lipids and lipoprotein composition and distribution were studied in fasted miniature swine prior to and at 5 and 19 weeks following portacaval shunt surgery or a sham operation. Plasma triacylglycerol and cholesterol levels were significantly reduced in the portacaval shunt swine at 5 weeks. These reductions were accompanied by significant decreases in the plasma very low density lipoprotein (d less than 1.006), low density lipoprotein (d = 1.02-1.07) and high density lipoprotein (d = 1.09-1.21) levels. The very low density lipoprotein were shown depleted in lipids and the low density lipoprotein was a cholesterol-depleted, triacylglycerol-enriched particle. No changes in the composition of the high density lipoprotein were observed. These reductions and changes in composition were maintained until killing at 19 weeks post-surgery.


Fatty acid synthetase activity in Mycobacterium smegmatis. Characterization of the acyl carrier protein-dependent elongating system. Mycobacterium smegmatis extracts contain two fatty acyl synthetase systems (Brindley, D.N., Matsumura, S. and Bloch, K. (1966) Nature 224, 666-669). One is the extensively studied multienzyme complex, (molecular weight 1.39 - 10(6)) which produces shorter C16 and C18) and longer (C24 and higher) fatty acids in a bimodal pattern. The second synthetase is acyl carrier-protein (ACP) dependent and elongates the CoA derivatives of C12 and longer chains. In contrast to the type I synthetase which also extends long fatty acyl chains, the ACP-dependent system produces homologous fatty acids up to 30 carbon atoms long in approximately equal proportions. Other properties which distinguish the ACP-dependent system from the multienzyme complex include the resistance to high concentrations of palmitoyl-CoA and to low ionic strength and the lack of stimulation by mycobacterial polysaccharides. The possibility that the two fatty acid synthetases are complimentary in their function is discussed.


Role of lamellar bodies in the biosynthesis of phosphatidylcholine in mouse lung. 1. A lamellar body-enriched fraction was isolated from whole lung homogenates of mouse lung and its contamination with microsomes, mitochondria, and cytosol protein assessed by marker enzyme analyses. 2. By measuring the activity of cholinephosphotransferase (EC 2.7.8.2) in varying amounts of microsomes in the presence and absence of a fixed quantity of lamellar bodies, it could be demonstrated unequivocally that lamellar bodies of mouse lung lack the capacity to synthesize phosphatidylcholine de novo. 3. A similar approach allowed the conclusion that lamellar bodies of mouse lung do not contain lysophosphatidylcholine acyltransferase (EC 2.3.1.23) and lysophosphatidylcholine:lysophosphatidylcholine acyltransferase (EC 2.3.1.--), enzymes which play a putative role in the formation of pulmonary 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine. The activities of these enzymes observed in lamellar body fractions could be attributed completely to contaminating microsomes and cytosol respectively. 4. Lamellar bodies contributed to the activity of microsomal lysophosphatidylcholine acyltransferase by a cooperative effect. The possible role of this cooperation in the biosynthesis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine is discussed.


Two copper-containing proteins from white and gray matter of brain. The procedure for the isolation of two water soluble copper-containing proteins from the white and gray matter of bovine brain is described. One of the proteins, cerebrocuprein I, is superoxide dismutase; and three molecular forms of this enzyme are to be found in brain. The other protein present in gray and white matter is devoid of superoxide dismutase and amine oxidase activities. The amino acid composition, molecular weight, isoelectric point and copper content of this protein were determined. The effect of some agents, pH and thermal treatment of the optical and EPR spectra of the protein were also studied. The copper of the protein may be removed and the holoprotein reconstituted again from apoprotein and copper. The results obtained led to the conclusion that in brain a new copper protein is discovered, which is named neurocuprein.


Calcium and pancreatic beta-cell function. I. Stimulatory effects of pentobarbital on insulin release. Islets microdissected from ob/ob-mice were exposed to 3mM pentobarbital in media which were normal or deficient in Ca2+. This treatment resulted in marked decrease of the islet content of cyclic AMP recorded in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Pentobarbital had a dual effect on insulin release. In addition to being a potent inhibitor of glucose-stimulated insulin release in media containing 2.56 mM Ca2+ it increased the amounts of insulin released in high glucose media deficient in Ca2+. There was a transient stimulation with ordinary concentrations of Ca2+ and 3mM glucose whtn the media also contained 3-isobutyl-1-methylxanthine. The stimulatory effect of pentobarbital persisted after replacing part of the Ca2+ in the beta-cell membrane with lanthanum ions and it could not be mimicked by lowering the oxygen tension of the incubation medium. It is suggested that pentobarbital stimulation of insulin release is the result of a specific action of the drug on the distribution of Ca2+ within the pancreatic beta-cells.


Cyclic GMP-dependent and cyclic AMP-dependent protein kinases, protein kinase modulators and phosphodiesterases in arteries and veins of dogs. Distribution and effects of arteriovenous fistula and arterial occlusion. Possible involvement of cyclic GMP-dependent and cyclic AMP-dependent protein kinases, protein kinase modulators and cyclic nucleotide phosphodiesterases in functions of vascular tissues were investigated in the dog. All of the above activities, localized in the smooth muscle-rich inner layer of the blood vessels, were found to be higher in the arteries than in the veins. The peripheral arteries were disproportionately richer in cyclic GMP-dependent protein kinase (as indicated by high ratios of cyclic GMP-dependent to cyclic AMP-dependent protein kinase) than were the veins, with the exception of the pulmonary artery, an atypical arterial tissue exposed to low blood pressure. Interestingly, the protein kinase ratio for the aorta, an artery with no significant role in blood pressure regulation, was not higher than that for the vena cava. Creation of femoral arteriovenous fistulae in the dogs led to preferential reductions in the cyclic GMP-dependent enzyme activity both in the proximal and distal arteries, whereas it was elevated in the stressed vein distal to the anastomotic site. The cyclic GMP-dependent enzyme was preferentially reduced in the saphenous artery distal to occlusion. Changes in the cyclic GMP-dependent enzyme activity appeared to precede gross atrophy or hypertrophy of the vessels. It is suggested that the vascular cyclic GMP-dependent protein kinase may be closely related to peripheral resistance and its regulation.


Protein phosphorylation in a dopamine-sensitive homogenate of rat caudate. Effect of adenosine 3',5'-cyclic monophosphate and putative neurotransmitters. Adenosine 3',5'-cyclic monophosphate (cyclic AMP) and its 8-methylthio derivative stimulate the incorporation of 32P into proteins endogenous to a homogenate of rat caudate nucleus when 4 micrometer [gamma-32P] ATP is used as substrate. Higher concentrations of ATP reduced the effect of the cyclic nucleotide until at 400 micrometer no significant increase in protein phosphorylation was seen. Incubation of the homogenate with 400 micron ATP and 100 micron dopamine resulted in an approx. 2-fold increase in cyclic AMP but did not alter caudate protein phosphorylation suggesting that the catecholamine could not stimulate protein phosphorylation under the experimental conditions used in the present study.


Effects of calcitonin and parathyroid hormone on the metabolism of chondrocytes in culture. Rabbit articular chondrocytes in suspension culture synthesize Type II collagen [3alpha1(II)] in the absence of extracellular Ca2+ and Type I collagen [2alpha1(I) - alpha2] in the complete medium. As a result of pre-treatment in monolayer culture with calcitonin or parathyroid hormone in the complete medium, an influx of Ca2+ into the cells occurs. These cells produce mainly Type I collagen when transferred to suspension cultures in the medium devoid of CaCl2. If added directly to the suspension culture medium containing no CaCl2, calcitonin stimulates an active efflux of Ca2+ from the cells into the medium and leads the cells to synthesize Type I collagen. Under similar conditions, parathyroid hormone does not change the collagen-phenotype.


Interrelation of prostaglandin endoperoxide (prostaglandin G2) and cyclic 3',5'-adenosine monophosphate in human blood platelets. The prostaglandin endoperoxide, prostaglandin G2, in platelet-rich plasma may produce reversible platelet aggregation without secretion, irreversible aggregation with secretion of platelet constituents inhibited by indomethacin, or the latter effects despite indomethacin, depending on the concentration of the endoperoxide. Irreversible aggregation and platelet secretion induced by prostaglandin G2 apparently result from the action of ADP, since these responses are inhibited by 2-n-amylthio-5'-AMP (an inhibitor of the actions of ADP on platelets) and they do not occur in heparinized platelet-rich plasma. Prostaglandin G2 lowers the platelet level of cyclic 3',5'-AMP. Its actions are inhibited by elevation of cyclic AMP levels by prostaglandin E1 or dibutyryl cyclic AMP or adenosine. Like malondialdehyde production induced by thrombin, ADP, or arachidonic acid, prostaglandin G2-induced malondialdehyde production is reduced by dibutyryl cyclic AMP and prostaglandin E1. Platelet activation by prostaglandin G2 is enhanced by the adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)-adenine. The action of prostaglandin G2 on platelets is more complex then previously reported.


[Liberation and reassociation of the microsomal membrane glucokinase in cat liver]. The particulate glucokinase of cat liver is shown to be microsomal. The activity is readily solubilized by glucose-6-phosphate, ATP, pyrophosphate, high salt concentrations and, to a lesser extent, ribonucleoside triphosphates. The solubilization by glucose-6-phosphate is inhibited by Pi. Solubilizations by ATP and glucose-6-phosphate differ in their sensitivity to temperature changes; they are relatively specific for glucokinase as compared to solubilization by detergent (Triton X 100). The enzyme can be bound again to previously eluted microsomal membranes. Treatment of membrane with trypsin, at 0 degrees C, destroys the ability to rebind the enzyme to the membrane. It is suggested that electrostatic forces are of considerable importance for the binding of glucokinase to a possible protein binding site in the membrane.


[Absorption spectra of chlorophyll a adsorbed on aerosil]. The adsorption spectra of chlorophyll a adsorbed on aerosil surface have been studied. It has been supposed that the spectral properties of adsorbed chlorophyll a layers are mainly due to the interactions between the pigment molecules. The derivative absorption spectra of adsorbed chlorophyll a have been obtained. It has been determined that descrete absorption maxima of adsorbed chlorophyll a aggregates are characteristic of the native chlorophyll a. It has been supposed that the fundamental spectral forms of chlorophyll a in vivo are reproduced in adsorbed layers of the pigment.


Effect of cyclic nucleotides in vitro assays of rat liver galactosyltransferase. Experiments with rat liver microsomal galactosyltransferase has been developed to test the effect of cyclic nucleotides on the transfer activity. An overall stimulation is observed when cAMP or cGMP (concentration higher that 10(-6) M) are added to the incubation medium. However, more detailed experiments show that the cyclic nucleotides do not act as direct effectors of the enzyme but present the precursors degradation by the glycosylnucleotide pyrophosphatases.