[The enzymes of the enterocyte. Prospects for a functional exploration]. The authors recall the enzymatic activity and transport activities which were localised in the glycocalyx, the microvillous membrane, the intracellular compartment and the basolateral membrane of the enterocyte. The relationships between the processes of digestion and absorption, on the one hand, active transport, energy metabolism and sodium and potassium dependent ATPase, on the other hand are considered. The application of these data to functional investigation of the enterocyte is discussed, emphasizing the complementary character of the biological tests used currently and the importance of sodium and potassium dependent ATPase in the phenomena of active transport.


Elevated serum angiotensin I converting enzyme in sarcoidosis. Serum angiotensin I converting enzyme was found to be elevated in 56 patients with sarcoidosis (52.7 +/- 25.4 nmole per min per ml) compared to 84 normal control subjects (28.2 +/- 11.3 nmole per min per ml), 22 patients with tuberculosis (26.4 +/- 10.9 nmole per min per ml), and 20 patients with lymphomas (31.8 +/ 13.9 nmole per min per ml). Forty-eight per cent of the patients with sarcoidosis had values of enzymatic activity that were higher than 2 SD above the mean value of the control population; the false-positive rate in all nonsarcoidosis subjects combined was 5.5 per cent. Patients with parenchymal disease with or without hilar adenopathy had values somewhat higher than those with hilar adenopathy alone, but the difference was not significant (P less than 0.1). Patients with a known duration of disease greater than 2 years also had somewhat higher values than those with known disease less than 2 years, but the difference was not significant (P less than 0.1). There was no significant difference in values for patients receiving or not receiving steroids.


[Enzymopathic congenital hyperlactacidemia]. Congenital enzymopathic hyperlactacidemia results from a defect of utilisation of pyruvate either at the level of the pyruvate junction (pyruvate-carboxylase, pyruvate-dehydrogenase and Kreb's cycle), or at the level of the unidirectional enzymes on neo-glucogenesis and of neo-glycogenogenesis, e.g. glucose-6-phosphatase, phosphoenol-pyruvate-carboxykinase and glycogen synthetase. The enzymopathies which affect neoglucogenesis associate hyper-lactacidemia and fasting hypoglycemia and more or less marked hepatomegaly. Type I glycogenesis (von Gierke's disease) is the best known example. Enzymopathies which affect the pyruvate junction and the Krebs cycle, may be manifested in addition by: --either chronic neuropathies, e.g. Leigh's disease, recurrent ataxia, and moderate hyperalactacidemia,--or, as in congenital lactic acidoses, which have a rapid and severe prognosis with major hyperlactacidemia. Functional investigation, in particular, loading tests are of great value in orientation and justify the practice of tissue biopsy which permits the enzyme diagnosis. Recent, still unconfirmed knowledge of the pathogenesis of these diseases emphasizes the considerable importance of estimation of blood lactic acid in the investigation of metabolic acidoses of hereditary origin.


The biological effects of mineral fibres, especially asbestos, as seen from in vitro and in vivo studies. Two in vitro models have been extensively used to compare the biological action of different types of asbestos fibres: the haemolytic effect and the cytotoxic one on macrophages grown in cell culture. The use of both techniques as led towards a better understanding of the chemical reactions which occur between fibres and the biological membranes of cells or intracellular organelles. Studies on the prevention of haemolysis and cytotoxicity have also been of use in explaining how asbestos acts a the cellular and molecular levels. Regarding in vivo studies, useful comparisons have been made of the fibrogenic and carcinogenic effects of different types of fibres in man and experimental animals. Both the in vitro and the in vivo aspects of the problem are discussed in some detail and an attemps is made to provide a reasonably unified concept for both.


The fine structure of lung macrophages from rhesus and squirrel monkeys, with special reference to the large numbers of mitochondria. Alveolar macrophages from 2 species of nonhuman primates, rhesus and squirrel monkeys, were studied by electron microscopy. These macrophages seemed to differ from their counterparts in other animal species with respect to their large numbers of mitochondria. Monkey alveolar macrophages often contained crystalline material and a variety of inclusions in their cytoplasm, which did not seem to interfere with their phagocytic ability. The significance of these findings as they might relate to pulmonary infestation with mites and nematodes is discussed.


Benign liver tumors: what is their relationship to oral contraceptives? A series of eight cases of benign hepatocellular tumors from the New Orleans area is presented. Clinical and histological features of these unusual tumors are discussed. Also, a series of 56 similar cases reported in the literature is reviewed. We conclude that these tumors have shown definite evidence of hormonal sensitivity and there is strong circumstantial evidence that oral contraceptive medications have contributed to their recent increased incidence. Although the risk of this problem in the overall population of women taking oral contraceptives seems to be quite small, further clinical and experimental research on this problem is clearly indicated.


The common hyperlipoproteinemias: an understanding of disease mechanisms and their control. Our review focuses on low density lipoprotein (LD lipoprotein) and very low density lipoprotein (VLD lipoprotein) in their roles as transporters of cholesterol and triglyceride and as factors contributing to premature arteriovascular disease. We describe the clinical manifestations of the common, primary hyperlipoproteinemias--that is, hyper-beta-lipoproteinemia, combined hyperlipoproteinemia, hyper-pre-beta-lipoproteinemia, and sporadic hyperlipoproteinemia--and discuss the variations in lipoprotein structure and metabolism that occur in these diseases. Based on an understanding of the physiologic control of lipoprotein metabolism, it is possible for the physician to alter the concentrations of LD lipoprotein and VLD lipoprotein by selecting a course of therapy appropriate to the specific disease. We describe the effects of obesity, diet, insulin, ethanol, estrogens, and the drugs clofibrate, nicotinic acid, and cholestyramine.


[The mechanism of releasing and bio-behavioral significance of the hypothalamo-hypophyso-adrenal gland reaction to the environment (alarm reaction)]. Laboratory experimentation has demonstrated the large increase in the rate of circulating norepinephrin (N.E.), in a parallel with an improvement in cardiovascular dynamism of suprarenalectomized rabbits under the action of injection IV of hydrocortisone. As from this ascertainment, the author attempts to make a synthesis of known experimental facts demonstrating that next to a regulation in constancy (with negative retroaction) of the C.R.F. leads to A.C.T.H. leads to cortisol system, there exists a servomechanism (control external to the system) as from the activity of the nervous systems as compared with environment. Hydrocortisone favouring passive avoidance, and extinction facilitating the activity of the cholinergic inhibiting system of action (I.S.A.). A.C.T.H. facilitates active avoidance and the catecholaminergic activating system of action (A.S.A.). Now, it has already been demonstrated in the same laboratory that stimulation of I.S.A. or inhibition of the A.S.A. freed N.E. from the peripherous nerves of a adrenalectomized animal. It, therefore, seems that the early freeing of A.C.T.H. following aggression is capable of favouring locomotive activity (flight or fight). Should the latter be efficient, there is a return to the prior behavioural and endocrinal balance. On the other hand, should they be inefficient, there arises a vicious circle (regulation with tendency, positive retroaction) because secondary secretion of glucocorticoids will maintain inhibition of action and the secondary freing of the mineralocorticoids are discussed as well as the central mechanism of illness so called "psychosomatic".


Effect of disinfectants on variola virus in cell culture. Twenty kinds of disinfectants were examined for ability to inactivate variola virus. Cytopathic effect and plaque formation on monolayer cultures of an established monkey kidney cell line were used as indicators of virus inactivation. A micromethod using microplate cultures, and not requiring a CO2 incubator, was adopted. The procedures were straightforward, showing good reproducibility. Among the compounds tested, several were found to be superior because of the minimum concentrations required for complete inactivation of virus. The purified viruses were shown to be more sensitive to the compounds than were the crude samples. The virus inactivation kinetics curves were determined by plaque counting. The usefulness of this method for quantitative analysis of disinfecting effect is suggested.


Laboratory diagnosis of foodborne diseases. Many bacterial species are responsible for sporadic cases and outbreaks of foodborne intoxication and infection. The foodborne diseases are classified on the basis of the pathogenetic mechanisms involved into four categories: performed toxin, enterotoxin formed in the colonized small intestine, mucosal invasion (enterocolitis) and mucosal invasion with bacteremia. Invasive and toxigenic strains of enteropathogenic Escherichia coli are discussed. In vivo test systems for the identification of enterotoxigenic organisms and tissue culture assays for the heat-labile enterotoxin of E. coli are described. Current laboratory methods for the diagnosis of foodborne diseases of major public health interest are reviewed - botulism, staphylococcal intoxication, Clostridium perfringens enteritis, salmonellosis, enteropathogenic E. coli infection, Vibrio parahaemolyticus infection and Bacillus cereus enteritis. The role of the laboratory in the epidemiologic surveillance and investigation of foodborne diseases is emphasized.


Stability of human enteroviruses in estuarine and marine waters. Studies of the effects of temperature and salinity on the survival of three enteric viruses (poliomyelitis type 1, echovirus-6, and coxsackievirus B-5) under controlled laboratory conditions and in situ indicate that temperature rather than salinity is the critical factor affecting their stability, in that the higher the temperature the more rapid was the loss of viral infectivity. In the laboratory studies, all three viruses were quite stable at 4 degrees C, with infectious virus still detectable after 46 weeks of incubation. In situ studies on virus survival in free-flowing estuarine or marine waters showed that, although the viruses were more labile in natural waters than in the laboratory studies, they persisted for several months, in some cases during the winter months. At all temperatures and salinities, coxsackievirus B-5 was the most stable, echovirus-6 was intermediate, and poliovirus 1 was the least stable of the viruses tested.


[Demonstration, by means of electron microscopy, of the penetration of somitic cells into the mesoblast of the limb buds of reptile embryos (Anguis fragilis, Lacerta viridis)]. An electron microscopic study of the components of anterior limb buds of the slow-worm (Anguis fragilis) and of the green lizard (Lacerta viridis) (embryos of Anguis whose allantoic bud reach 0,7 to 4 mm of length; embryos of Lacerta 2 to 7 days old) provides data on the cytological characteristics of the components of the limb bud at these early stages. 1. The cells of the distal extremity of the somitic processes extending in the limb bud of Anguis and Lacerta, are elongated cells with ovoid nuclei containing large nucleolus; they possess mitochondria always thin and with dense matrix; they are rich in lipid droplets; they possess cilia; they are devoid of myofilaments; endoplasmic reticulum, free ribosomes and polyribosomes are abundant. Golgi networks display signs of activity. These characteristics are also observed in the cells of the "dermatome" layer of the dermo-myotome; and so, it appears probable that the cells of the "dermatome". Furthermore, in Anguis embryos, the cells of the distal extremities of the somitic processes possess numerous lysosomes and a certain number of cells among them, degenerate early. 2. The somatopleural mesoblastic cells of the limb bud of Anguis and Lacerta embryos keep the characters of the cells of the mesodermic layer of lateral plate from which they originate; they have rounded nuclei, cilia, and their mitochondria are always larger and more transparent to electrons, than the ones of cells of the somitic processes and of cells of the epiblastic apical crest. Golgi networks are well developped, endoplasmic reticulum is abundant, lipid droplets are rare. 3. The processes of somites which extend in the dorsal part of the limb bud of Anguis embryos are cords of cells with thin lumina; at the stage of the allantoic bud of 0,6 to 0,8 mm long, the distal extremity of these processes dislocate in group of cells which afterwards dissociate, releasing individual somitic cells which are integrated among the mesoblastic somatopleural cells. In young lizard embryos (2 to 4 days old) the distal extremity of the somitic processes enlarges into a vesicle from which cells are released and penetrate in the mesoblast of the limb bud. 4. The somitic cells released from the somitic processes of Anguis and Lacerta keep--at least at early stages--the cytological characteristics they displayed when they were still in situ in the somitic processes: grounded on the presence or absence of lipid droplets, on the width and density of the mitochondria, the distinction, at these stages, between the somitic and mesoblastic somatoplerual cells is possible; and it is also possible to observe the integration of the somitic cells into the mesoblast. This study brings the demonstration of the cellular contribution of the somites to the formation of the limb bud in Reptiles. 5...


Cytology of rheumatoid synovial cells in culture. IV. Further investigations of cell lines cocultivated with rheumatoid synovial cells. A previous report described a cell isolate presumed to have arisen by accidental cocultivation (contamination) of the Chang 'liver' cell line and rheumatoid synovial cells. This cell isolate had the same glucose-6-phosphate dehydrogenase isoenzyme as the Chang cell and also some shared antigens. It clearly differed in its karyotype, its ability to grow in semisolid agar, and in the possession of bleb-like projections of the cytoplasmic membrane filled with collections of beaded or granular material. In addition, it had a novel antigen(s) not present in the Chang cell. As these properties might have been acquired from the synovial cells and because the bleb structures resembled those seen in some cell lines transformed by leucovirus the cell isolate has been further studied. Cytochemical methods at the light and electron microscope level showed that the granular material was polysaccharide in nature, probably glycogen. No evidence was found of the presence of a virus or a viral genome using a variety of techniques including attempted induction followed by 3H-uridine labelling of the cultures, and assay of the supernatant fluid from the culture for viral RNA-dependent DNA polymerase. In addition, cell extracts were not found to contain viral RNA-dependent DNA polymerase or RNA-dependent RNA polymerase. No rubella virus or leucovirus interspecies antigens were detected on the cell membranes.


Preferred surgical treatment for alveolar cell carcinoma. An analysis of our experience with 48 patients having bronchiolar or alveolar cell carcinoma is reported. The remarkable biological variability of this peripheral tumor has important surgical implications. Basically, two dominant clinical presentations occur. In the less common diffuse or multinodular form, prolonged survival is infrequent regardless of the therapeutic approach. Often these patients die from respiratory compromise due to the tumor itself. In the more common localized or solitary form the prognosis for cure is good, approximating 47% or higher. Based on the material presented, lobectomy is the preferred method of surgical treatment. In patients manifesting multinodular disease, surgical resection rarely seems warranted. The concept of preserving pulmonary tissue is stressed.


Granular cell myoblastoma of the bronchus: report of 2 cases and review of the literature. Granular cell myoblastoma of the bronchus is rare. Two patients are described, bringing the total reported to 44. Review of the literature shows that cough, chest pain, hemoptysis, and wheezing are frequent presenting symptoms and that distal atelectasis and recurrent or persistent pneumonitis are common roentgenographic findings. Though the histogenesis of this tumor remains controversial, most pathologists now believe that the cells have a neurogenic origin. Adequate open surgical resection is the treatment of choice.


Nephrogenous cyclic AMP levels in primary hyperparathyroidism. The clinical utility of urinary cyclic adenosine-3',5'-monophosphate (cAMP) determinations has been limited by the overlap between hyperparathyroid and normal patients. We evaluated the potential of the parathyroid hormone (PTH)-dependent, nephrogenous cAMP in the diagnosis of hyperparathyroidism. Twenty-three patients with primary hyperparathyroidism and 19 control subjects had two-hour urine collections and blood sampling at midpoint. Nephrogenous cAMP level was calculated as total urinary cAMP excretion minus the amount filtered. The total urinary cAMP excretion (micromols per gram of creatinine) was higher in hyperparathyroid patients (6.8 +/- .5 SE), but overlapped with values obtained in controls (2.9 +/- .15). The level of nephrogenous cAMP (percent of total) was also higher in hyperparathyroid patients (72.5 +/- 1.8) than controls (26.3 +/- 4.1) and clearly separated the groups. Determination of nephrogenous cAMP levels may be useful in the diagnosis of hyperparathyroidism.


[Presence of cytomegalovirus in maternal milk]. The AA. make in evidence the role of the milk as carrier of Cytomegalovirus. The virus was isolated in the 23.5% of the 17 specimens of milk examinated within the first 48 hours from the delivery. The contemporary observation of the urins of the womans was negative.


[200 valvular disorders seen in Abidjan]. The authors have made a retrospective study of the case notes of 200 patients with valvular disorders seen over the course of 35 months (excluding those due to constrictive endocardial fibrosis). The cases represent the frequency of cardiac failure, especially in the mitral group. The authors found 61% to have mitral valve disease, 28.5% aortic valve disease, and 9.5% disease of more than one valve. Mitral valve lesions were found most frequently in young females (average age 26 years); aortic valve lesions were found more commonly in an older age group of men (average age 47 years). The authors discuss the aetiology. Rheumatic fever was responsible in 60% of cases, and caused particularly mitral valve disease. Atherosclerosis and syphilis were the commonest causes in the aortic group. Indications for surgery were found in 74 cases (37%), and 28% of these were closed heart techniques. The course of the disease is best appreciated during the first hospital admission. The mortality rate was 5.5%, and most of the patients (65%) were stabilised on treatment.


[Pseudohypothyroidism. Complementary results apropos of 4 familial cases]. In a sibship, 4 out of 5 children featured pseudo-hypoparathyroidism. The following points were verified: hypocalcaemia; increase of the serum level of PTH; normal response of bone and gut effectors to endegenous PTH; contrast between normal bone X-rays and lesions detected by histological studies.


[Tumor hyperacidulation through glucose infusion enhanced by nicotinamide adenine dinucleotide (author's transl)]. Tumor peracidity in Walker carcinosarcoma 256-bearing Wistar rats attained by glucose (Gk) infusion was significantly (t-Test; p = 3.49) increased by a following infusion of 200 mg NAD in 7 ml 40 per cent. Gk-solution. The mean pH values obtained on six animals amounted 6.88 (start), 5.21 (after Gk) and 5.79 (after Gk + NAD). Three animals were cured through the hyperacidification process.


Scanning electron microscopic observation on intracellular structures of ion-etched materials. Ion-etching technique on the cracked surface of biological material may give a plastic visualization of intracellular structures under the scanning electron microscope, because membraneous structures in the cell are generally etch-resistant and the cytoplasmic matrix is easily ion-etched. Mild ion-etching using low voltage was applied to the cells of the pancreas. Nuclear pores were clearly disclosed but were enlarged slightly in the process of etching. Endoplasmic reticulum with ribosomes, Golgi apparatus, mitochondria, some filamentous structures and crystalline inclusions (B cell granules) were also effectively disclosed by ion-etching technique. It is necessary, however, to compare the etched specimens carefully with non-etched ones to determine whether given structures observed are intrinsic ones or artifacts caused by etching.


Nerve conduction velocity measurements: improved accuracy using superimposed response waves. A new procedure of serial motor nerve conduction velocity (NCV) measurements with the use of "superimposed response waves" technique (or double stimulus technique) was performed on 29 normal subjects. Six peripheral nerves were tested once a week for four to six weeks. A total of 760 NCV measurements were thus obtained to try to assess the magnitude of error in serial NCV testings. With the double stimulus technique employed, a significant reduction in variations of serial NCV measurements was found. The overall standard deviation of four to six consecutive NCV measurements in the 34 subjects was 1.3 meters per second with a coefficient of variation of 2.4%. These findings obtained with the double stimulus technique have proven to be approximately three times more accurate than results obtained by investigators who studied nerve conduction velocity measurement variation with single stimulus standard NCV testing techniques.


Intraductal "noninfiltrating" carcinoma of the breast. We are reviewing 40 patients with noninvasive intraductal carcients; 10% developed oppostie breast cancer of the invasive variety. Twenty-one patients underwent radical mastectomy in which one patient was found to have a positive axillary node. The 19 remaining patients had total mastectomy alone. No recurrence of death attributed to intraductal carcinoma was found in 39 patients available for follow-up.


[Interrelationship between the quality of dietary protein and the function of the adrenal cortex. 3. Reactivity of the adrenal cortex and ACTH content of the pituitary gland of rats fed with proteins of various quality]. Trials were performed investigating the reactivity of the adrenals and the ACTH content of the pituitary gland in rats fed proteins of varying quality. A positive correlation was found to exist between the relative weight of the adrenals and the rate of body weight increases as well as between the weight of the pituitary gland and the rate of body weight gains. In either case this process was found to be primarily dependent on the quality of the dietary protein. It was shown that the quality of the dietary protein did not affect the in vitro reactivity of the adrenal glands. The ACTH content of the pituitary gland( determined on the basis of 100 mg of pituitary tissue) did not change considerably.


[Acid phosphatase activity in breast dysplasias and in carcinomas of the breast]. The histochemical reactions of acid phosphatase were studied in tissues deriving from normal breasts, from mammary dysplasia, and from breast carcinoma. Acid phosphatase was demonstrated in all cases with more intense activity in breast carcinoma where activity was particularly accentuated in the stroma. In benign tumours and fibroadenosis, phosphatase was prevalently localized in the periductal cells and within the ducts. Exceptions were the cases of fibroadenosis with acute or chronic collateral inflammation where phosphatase granules were located in the stroma also, even though they were always quantitatively inferior to the corresponding localization of cases of breast carcinomas.


Antibody class and complement requirement of neutralizing antibodies in the primary and secondary antibody response of cattle to infectious bovine rhinotracheitis virus vaccine. Calves responded to a single intramuscular injection of an attenuated strain of infectious bovine rhinotracheitis virus by producing IgM followed by IgG antibody. Both IgM and IgG antibody produced during the first month were primarily complement-requiring neutralizing antibody (CRNAb), especially IgM antibody. After a month, IgG had replaced IgM as the predominant immunoglobulin, and titers with and without complement (C') decreased in both IgG and IgM fractions. The largest decrease was in the IgM CRNAb fraction. Seven days after a second injection given on day 196, calves responded with an anamnestic IgG response in which CRNAb titers were 1 or 2 two-fold dilutions higher than non-CRNAb titers. One calf developed an IgM response similar to its primary response, whereas inhibition of the IgM response occurred in the other 3 calves which had much lower IgM antibody titers than those attained in the primary response. Twenty-eight days after the second injection the titers of IgG were the same or only a 2-fold dilution less than their 7-day secondary titers, whereas IgM titers generally decreased considerably more than this. Guinea pig and rabbit sera were equally effective as C' sources in potentiating CRNAb, whereas bovine serum was a poor C' source.


Anti-viral activity against encephalomyocarditis virus and Semliki Forest virus and acute toxicity of poly I and poly C administered sequentially to mice. Mice are protected against lethal intraperitoneal and intravenous infection by encephalomyocarditis virus and Semliki Forest virus by sequential treatment with poly I followed by either polyC or poly5-hydroxyC without production of interferon when the treatments are 4 or more hours apart and by the intraperitoneal or intravenous routes. Maximum protection occurs around 4 hours before infection and is still significant 20 hours after infection. Treatments with combinations of other homoribopolynucleotides were not found to be anti-viral. Protection by sequential polyI, polyC treatment of mice is relatively short-lived and does not 'hypo-reactivate' the protective effect of polyI:C and shows approximately half the protective effect of polyI:C. The toxicity of sequential polyI, polyC treatment is lower than that of polyI:C particularly if poly5-hydroxyC is substituted for polyC. Silica treatment of mice indicates that stationary macrophages are required for protection by polyI followed by polyC but an effect on humoral or cell mediated immune responses does not appear to be involved. The effect appears to be a synergism between the protection conferred by polyI or polyC alone.


Potentiation of poly I:C induced viral resistance by polycationic modified polypeptides. The potentiation of polyI:C induced viral resistance by various side group modified polycationic polyglutamic acid derivatives was investigated. It was established that the efficacy of the various polycationic substances depends primarily on their macromolecular properties. Viscosity and the degree of cationic substitution have been found to be of paramount importance. The maximal potentiating efficacy, expressed as the minimum protective dose of polyI:C necessary for complete protection of cells in the presence of the polycations, was exerted by compounds of highest viscosity and degree of subsitution. The potentiating efficacy of the polycationic derivatives tested could only be observed in a relatively narrow range of concentration, depending on their viscosity and degree of substitution. In view of the extremely low minimal protective dose of polyI:C (10(-5) mug/ml) in the presence of our most effective compound it is assumed that the action of a few molecules of polyI:C may be sufficient to render a cell resistant against viral infection. Within the limits of the described experiments, the efficacy of polycationic derivatives did not seem to be influenced by the modification (e.g. quaternarization) of the cationic group.


Surface antigens on HeLa cells persistently infected with HVJ (Sendai virus). Surface antigens of HeLaHVJ cells, a cell line persistently infected with HVJ, were studied by fluorescent antibody staining. After absorption with concentrated HVJ virions and HeLa cells, anti-HeLaHVJ antiserum was able to demonstrate specific surface fluorescence on HeLaHVJ cells, while this serum no longer reacted with original HeLa cells nor with HVJ virions. During cytolytic infection of HeLa cells with HVJ, this specific surface antigen appeared at an early stage of infection prior to the appearance of newly synthesized HVJ viral antigens and moreover appeared in spite of the inhibition of viral protein synthesis. This antigen was detected neither on HeLa cells infected with other myxoviruses except HVJ nor on various other kinds of cells infected with HVJ. The specific surface antigen was still found on the HeLaHVJ cell surface after incubation at 38 degrees C for two days, while HVJ structural antigens on the cell surface no longer could be detected. Mild short-term treatment of HeLa cells with trypsin, neuraminidase from vibrio cholerae, phospholipase-C and hyaluronidase failed to expose specific antigen. The antigen was distinguishable from the Forssman and human blood type antigens. The mechanism of appearance of a new antigen on the surface of HeLaHVJ cells remains unclear.


Localisation on foot-and-mouth disease virus (FMDV) of an antigenic deficiency induced by passage in BHK cells. Passage of FMDV in BHK suspended cells was confirmed to induce an antigenic deficiency on the virion. By immunodiffusion experiments with complete virus, with trypsin-treated virus and with 12S virus fraction it was shown that the induced antigenic deficiency is located on the trypsin-removable part of the virion. These results were confirmed by absorption experiments.


Pinwheel inclusions in morphogenesis: a possible alternative to induction by viruses. Pinwheel inclusions (PWs) were found in cells of callus tissue derived from explants of secondary phloem parenchyma of carrot (Daucus carota) storage root and grown on a basal medium containing zeatin and indoleacetic acid or coconut milk, naphthalene acetic acid, or combinations of these. Preliminary attempts to demonstrate the presence of viruses in the callus tissue failed. The possibility that the tissles were infected by a low titer or unstable conventional virus or by a defective mutant has not been ruled out. However, two lines of evidence suggest that the PWs in these tissues may be a result of culture conditions and not of virus infection. First, no PWs or other cytoplasmic inclusions were found in cells of otherwise similar tissue cultured on basal medium alone, and multifibrillar bundles (MFBs) but not PWs were found when the tissues were cultured on a medium that stimulates differentiation and morphogenesis. Second, culture stimulated to differentiate and containing MFBs only were returned to the supplemented basal medium and subsequently found to contain both PWs and MFBs.


Bilateral sixth nerve palsy. Analysis of 125 cases. Bilateral abducens nerve pareses were nearly as common as unilateral cases in an inpatient setting (125:143). Cerebrospinal fluid abnormalities were more frequent among the bilateral cases, but generally the same causes produced unilateral and bilateral sixth nerve palsy. The relative ease of diagnosis was in contrast with the large number of undiagnosed or "vascular" cases in previous studies of outpatients. The degree of lateral rectus limitation proved to be of limited help in suggesting the cause or predicting recovery of oculomotor function. The etiology was of some prognostic value, with universal recovery of pressure palsies and rare improvement with tumor involvement. Myasthenia, orbital muscle entrapment, convergence spasm, divergence palsy, and pretectal pseudoconvergence entered into the differential diagnosis, but were only occasionally difficult to exclude.


Familial fingerprint body myopathy.

Familial fingerprint body myopathy. Muscle biopsy specimens from two half brothers with a congenital benign muscle disorder and from their mother, clinically unaffected, were studied by histochemistry and electron microscopy. In the children's biopsy specimens, the ultrastructural examination showed numerous fingerprint bodies located at the periphery of the muscle fibers. The histochemical pattern was different in these two specimens. In the mother's biopsy specimen, while fingerprint bodies were not found, the muscle fibers showed slight but definite changes. Even if the fingerprint bodies by themselves are not specific for a particular muscle disease, their occurrence in two half brothers is a valid argument in favor of the individalization of the fingerprint body myopathy, which has been previously described.


Herpes simplex virus in human cornea, retrocorneal fibrous membrane, and vitreous. A case of recurrent herpes simplex virus keratouveitis was studied using both electron microscopic and viral culture techniques. We describe a patient who had a particularly violent course, including three failed grafts, endophthalmitis, and wound leak. We report the identification of virus in a quiescent failed corneal graft and retrocorneal membrane; persistence of virus enabling its culture from the vitreous; and electron microscopic identification in the cornea removed at the third transplant. The identification of cases in which viral proliferation occurs is necessary for a greater understanding of the pathogenesis of herpetic corneal disease and for determining in which particular patient steroid therapy should be avoided.


Human conjunctivitis. II. Treatment.

Human conjunctivitis. II. Treatment. One hundred forty-three patients (207 eyes) with conjunctivitis or blepharoconjunctivitis were investigated to determine (1) the safety of topical corticosteroid therapy and the relative efficacy of formulations of increasing potency and (2) the effectiveness of a steroid-antibiotic preparation compared to each of its components alone and to a placebo. The corticosteroids were equally effective in suppressing conjunctival inflammation; all were more effective than the placebo. Active conjunctivitis was controlled more readily by those preparations containing a steroid, both alone and in combination. The corticosteroid alone (dexamethasone) was more effective in producing inactivation of conjunctivitis than the antibiotic alone ( a mixture of neomycin sulfate and polymyxin B sulfate). This observation remained unchanged when cases of Staphylococcus aureus conjunctivitis were analyzed separately. No serious complications resulted from any treatment regimen.


Malignant Glomus vagale: report of a case and review of the literature. Tumors of the chemoreceptor system are quite rare, and are usually considered to be benign, but locally invasive. We report a case of a glomus juxtavagale tumor with metastasis to cervical lymph nodes, lung, and bone. Review of the literature shows that 19% of similar cases reported also showed distant metastases, a rate significantly higher than for other chemodectomas. Clinically and histologically, benign and malignant tumors are identical, so only the demonstration of distant metastasis indicates malignant neoplasm. Primary treatment is surgical, but radiotherapy is useful for palliation.


Changes in plasma lipoproteins in subjects treated with the bile acid-sequestering resin polidexide (Secholex). The effects of a new anion-exchange resin, polidexide, 3-30 grams/day, on plasma lipids and lipoproteins have been studied in one normal subject and in three hyperlipidaemic patients. Plasma cholesterol concentrations were significantly reduced in all subjects over 80-120 days of therapy by amounts ranging from 14 to 20%. Plasma triglyceride concentrations increased in two patients with pre-existing hypertriglyceridaemia. Plasma apolipoprotein-B concentrations were reduced in all subjects. Cholesterol and apo-B concentrations in low density lipoproteins were reduced in all subjects, while triglyceride concentrations in very low density lipoproteins were increased in 3/4 subjects; Changes in high density lipoproteins were quite variable.


A mucosal disease virus infection of the pregnant ewe as a cause of a border disease-like condition. An experiment was designed to investigate whether a condition in Australian sheep with clinical and pathological similarites to Border Disease was caused by the infection of the pregnant ewe with a Mucosal Disease virus (MDV). Forty ewes, at 58 to 63 days after mating, were inoculated with material from lambs in which all, some or none of the tissues examined contained MDV. The clinical condition was observed only in lambs born to ewes inoculated with MDV-positive material and then only to ewes in the group which had serological evidence of MDV infection. It is concluded that the Border Disease-like condition in Australian sheep is caused by the infection of the pregnant ewe with a Mucosal Disease virus.


Space flight effects on the skeletal muscles of rats. Morphological and histochemical examinations of the skeletal muscles of rats flown for 22 d aboard the Cosmos-605 biosatellite have demonstrated atrophic and dystrophic developments in the soleus muscle accompanied by metabolic changes in the muscle tissue. The changes were reversible but did not disappear entirely 27 d postflight. Early atrophic developments were seen in the m. gastrocnemius, m. quadriceps, and m. ext. digitorum longus on the second postflight day. The comparative study of the muscles of flight and simulation rats has shown that pathology developed in the muscles as a result of diminished motor activity of animals being aggravated by weightlessness.


[Cystinosis (author's transl)].

[Cystinosis (author's transl)]. Cystinosis (syn. crystine storage disease) is inherited as an autosomal recessive trait. The severety of clinical symptoms may vary considerably. The most severe form of nephropathic cystinosis causes death of the affected patients at the age of 2 to 4 years (subacute course) or before puberty (primary chronical course). Three autopsy cases are demonstrated. Each of these children died from complications of chronic renal failure, caused by kidney contraction as a sequela of chronic interstitial nephritis. Cristalline cystine deposits were found in the renal interstium as well as in the RES cells of spleen and liver. Because of water solubility of L-cystine aqueous fixation and staining solutions must be avoided. Diagnostic doubly refractive brick- or needle-shaped cystine cristals can be demonstrated in frozen sections or tissue smears from spleen, liver, lymphnode and bone marrow.


Electrophoretic variability in island populations of Drosophila simulans and Drosophila immigrans. Genetic structure and variability were investigated in several Hawaiian populations of D. simulans and D. immigrans. Genetic variability is lower in Hawaiian populations of D. simulans than in Texas populations, and allelic differences exist as well. For D. immigrans, Hawaiian and Korean populations are similar in variability, allelic content, and gene frequencies. Several hypothesis are advanced to account for the patterns in gene variation observed between island and continental populations of these two colonizing species.


Substrate cycles in metabolic regulation and in heat generation. 1. The presence of substrate cycles in tissues has been demonstrated by direct isotope methods in recent years. This demonstration has provided the impetus for a reappraisal of the roles of substrate cycling in metabolic regulation and in heat production. These aspects of substrate cycling are discussed in this paper. The relationship between near-equilibrium reactions and substrate cycles is emphasized, since this provides a basis for the derivation of a function describing in precise quantitative terms the factors governing the amplification provided by substrate cycles in metabolic regulation. Some examples of the roles of substrate cycles in providing sensitivity in metabolic regulation are described. The importance of substrate cycling in heat generation in the flight muscle of the bumble-bee and in brown adipose tissue is discussed in detail. 2. We point out that the two possible roles of cycling, heat production and amplification, are intimately linked so that they must be discussed together. It is proposed that variable rates of substrate cycling may be possible so that, for short periods of time. sensitivity can be maximal without excessive heat generation. Variable rates over the long term may be involved in weight control, and the control of such variability in cycling rates may be impaired in obese subjects. Finally, the possibilities that substrate cycles provide explanations for the specific dynamic action of food and for alcoholic and accidental hypothermia are raised.


The affinity of low density lipoproteins for an arterial macromolecular complex. A study in ischemic heart disease and controls. Levels of serum cholesterol, triglycerides, lipoprotein pattern and the insolubilization of serum low density lipoproteins (LDL, betalipoprotein) by a factor present in arterial intima--media extracts was investigated in 55 patients with acute coronary heart disease and 50 healthy controls. In blood samples obtained 24 h after the episode, the serum betalipoproteins from male normotensive patients showed a high tendency to precipitate when incubated with the intima-media extracts, nearly twice the value measured in the control group. This affinity returned almost to control level after 21 days in the hospital. Hypertensive patients showed a serum cholesterol higher than the controls but almost no difference in LDL-arterial factor interaction. The composition of isolated LDL was studied in 7 patients and 8 controls and it was found that the total cholesterol/protein plus phospholipid ratio has a positive exponential correlation with the lipoprotein-arterial factor affinity. The results of LDL analyses suggest that a change in their composition, frequent in normotensive patients, can lead to an increased affinity of this lipoprotein for arterial intimamedia components. It is proposed that this process could be an additional risk factor contributing to the deposition of LDL in the arterial wall.


Serum lipid profiles in patients of myocardial infarction in the Chandigarh area (Northern India). To study the incidence of hyperlipoproteinaemia in patients with myocardial infarction (MI) in the Chandigarh area, estimations of various lipids have been carried out in 83 patients. A serial study has been carried out in 31 patients. Serum lipoproteins and uric acid were also estimated. Results show an incidence of only 18% hypercholesterolaemia in patients with MI and 15% in normal subjects. Age-wise distribution of hypercholesterolaemia was slightly higher in 41-60 years old patients when compared with other age groups. No other abnormality in lipid profile was observed. Hyperuricaemia was not observed. These results, therefore, differ markedly from those of similar studies published from the western world.


Comparative studies on fatty acid synthesis in atherosclerotic and hypoxic human aorta. The oxygen and glucose uptake, lactate formation, ATP/ADP and NADH/NAD ratios and incorporation of [14C]acetate and [14C]linolenic acid into lipids of early fatty streaks and more advanced complicated atherosclerotic lesions of human aorta were determined during aerobic and hypoxic incubation. Compared with grossly normal appearing sections of the aorta in intima and media preparations of early fatty streaks the oxygen uptake was increased while that in further developed atheroma was slightly diminished. Under aerobic incubation conditions the metabolic state of fatty streaks and atheroma was characterized by increased lactate formation, NADH/NAD ratio and incorporation of [14C]acetate and [14C]linolenic acid into the lipids, but by a lowered ATP/ADP ratio. More pronounced changes in these metabolic parameters were observed when the aortic tissue segments were incubated under hypoxic conditions. The analysis by argentation TLC of fatty acid methylesters derived from total lipids of aerobically incubated fatty streaks revealed an increased incorporation of [14C]acetate into the highly unsaturated long-chain fatty acids. In developed atherosclerotic lesions and in hypoxia the incorporation of radioacetate into the polyunsaturated fatty acids and the formation of 20:4 fatty acid from [14C]linolenic acid were, in contrast to the above finding, decreased while the synthesis of eicosatrienoic acid was increased. This finding suggests a block in the desaturation step of linoleic into 20:4 fatty acid in further developed atheroma and in hypoxia. In aerobically incubated atherosclerotic lesions and in hypoxia the palmitic acid was synthesized mainly by chain elongation while in grossly normal areas of the aorta at least part of this acid was synthesized de novo.


Increased collagen synthesis and the kinetic characteristics of prolyl hydroxylase in tissues of rabbits with experimental arteriosclerosis. Increased aortic and liver prolyl hydroxylase activity has been suggested as an early biochemical indicator of the fibrotic changes which occur in rabbits with injury induced arteriosclerosis. Daily administration of epinephrine (0.025-0.050 mg/kg, i.v.) and thyroxine (0.050 mg/kg, i.p.) to rabbits for 3 weeks produced aortic fibrous plaques with a 4-fold increase in aortic prolyl hydroxylase and also a 5-fold increase in liver prolyl hydroxylase. Histopathologically, the livers of these rabbits show subcapsular areas of necrosis. When total prolyl hydroxylase related antigen was measured. the increase in liver prolyl hydroxylase activity accounted for only a small portion of the total prolyl hydroxylase antigen. However, in the aorta a majority of the increase in antigen is due to the increased amount of enzyme. DNA content per aorta was unchanged and RNA content increased in the aortic tissue of the arteriosclerotic rabbits. However DNA and RNA levels increased 60% in the livers of arteriosclerotic rabbits. In vitro incorporation of radioactively labeled proline into collagenase digestable protein was at least 2-fold greater in aorta and liver minces from arteriosclerotic rabbits. Michaelis--Menten kinetic parameters were obtained for the liver prolyl hydroxylase purified by affinity chromatography from arteriosclerotic rabbits. The Km for the enzyme from treated animals was not significantly different from control. However, the Vmax of the enzyme purified from diseased liver was 4-fold greater when compared to controls.


Stimulation of proliferation in stationary primary cultures of monkey aortic smooth muscle cells. Part 2. Effect of varying concentrations of hyperlipemic serum and low density lipoproteins of varying dietary fat origins. The outgrowth of medial explants of thoracic aorta from Rhesus monkeys has used to study the influence of hyperlipemic serum on cell proliferation. After 5-6 weeks of rapid growth in BME plus 10% normal serum, the cultures reach a stationary phase during which they show little mitotic activity. When it replaces 5% of the normal serum in the media, hyperlipemic serum induces another proliferative phase in the cultures, as measured by [3H1thymidine incorporation and increase in culture area. Low density lipoprotein (LDL) has the greatest stimulatory effect, while high density lipoprotein (HDL) has no effect. Hyperlipemic serum or its LDL still stimulates the cells even when diluted to achieve cholesterol levels comparable to the values with normal serum or LDL. Normal LDL has no effect, even when concentrated to increase its cholesterol level in the media. Thus it appears that hyperlipemic LDL has a stimulatory effect on arterial smooth muscle cells which does not depend on its higher lipid or cholesterol level.


Clearing factor lipase (lipoprotein lipase) activator. A method for the measurement of the net activating ability of human sera. The hydrolysis of an emulsified triglyceride substrate by clearing factor lipase (lipoprotein lipase) normally requires the presence of particular activating polypeptide species. These are present in serum, together with other inhibitory species, as part of the serum lipoproteins. The paper describes a method whereby the net activating ability of individual human sera may be measured routinely. In a normal population, this activating ability is shown to be correlated positively with the fasting serum triglyceride concentration. As the fasting triglyceride concentration increases, there is a rise in the proportion of the total activating ability that is associated with the very low density lipoproteins. A dietary fat load does not raise the total activating ability but does increase the proportion of the total that is associated with the serum lipoproteins of lowest density.


Mesoridazine and human sleep.

Mesoridazine and human sleep. 1 Mesoridazine, a phenothiazine of short half-life, and potentially useful as an hypnotic, has here been investigated using volunteers of late middle age. 2 The electrophsiological recording of all-night sleep was studied in seven subjects for a 7-week period during which ther received mesoridazine (10 mg nightly) for 3 weeks. The drug reduced the frequency of transitions into wakefulness and stage 1 (drowsiness) and reduced the time spent in stage 1; there was a withdrawal rebound. Mesoridazine increased REM sleep above baseline levels and a rebound fall below baseline occurred on withdrawal. The drug did not alter the amount of stage 3 + 4 slow wave sleep. 3 Subjective self-ratings were assessed in a 6-week study of sixteen subjects. Sleep quality improved on mesoridazine (10 mg nightly) but there was diminution of zest and freshness 20 min after rising. Daytime concentration and anxiety were rated as not affected either by administration or withdrawal.


Anxiety and sleep after fosazepam.

Anxiety and sleep after fosazepam. 1 Six volunteers of mean age 59 years received placebo for week, then fosazepam (60 mg) nightly for 3 weeks, then placebo for 3 weeks. Subjective ratings and all-night electrophysiological recordings were made. 2 Fosazepam administration improved subjective sleep quality nut impaired feelings of morning vitality. Its withdrawal was associated with anxiety, impaired concentration and continuing impairment of morning vitality. Measured sleep duration increased on fosazepam, sleep was less broken, slow wave sleep stages 3 and 4 diminished in duration and so did REM sleep. 3 Despite the short-life of fosazepam some drug effects persisted for several days after withdrawal, suggesting action of a long half-life metabolite.


Solid tumour models for the assessment of different treatment modalities: IV. the combined effects of radiation and 5-fluorouracil. Neither radiation alone (375 to 1500 rad) nor5-fluorouracil (FU) alone (50-250 mg/kg) is sufficient to prevent an increase in the volume of the solid tumour model hepatoma 3924A. However, as little as 750 rad with 100 mg/kg FU can reduce the tumour below the volume at the time of treatment for as long as 14 days. A series of combined FU and radiation doses given every 11 days should then result in successively smaller tumour volumes until the tumour is eradicated. Changes in tumour volume were analysed by two different methods: (1) tumours in each treatment mode were grouped together and the average response to treatment determined, and (2) tumour volume changes in individual tumours were analyzed utilizing the chi2 technique, which fits the logarithmic tumour volume change with time to polynomials. This two-directional method of analysis has the advantage of permitting both an overview of the main effects of treatment via the averages, and at the same time a detailed examination of the mechanism by which these effects occur through the analysis of individual response. The results suggest that, in addition to concentrating on the cellular response immediately after therapy, greater emphasis should be placed on the kinetic changes of the tumour 1-3 weeks after single or multiple modality therapy. These findings demonstrate how the sequencing of single and/or combined treatment modalities may be investigated in order to detemine how best to obtain maximum effects of treatment on different types of tumours following recovery of the host from the previous treatment series.


BCG treatment of malignant pleural effusions in the rat. Intrapleurally injected cells of an ascitic rat tumour produced intrapleural effusions and solid pleural deposits. BCG, or its methanol extraction residue (MER) injected into the pleural space, suppressed tumour development and prolonged survival. Treatment was effective if given a few days before or after tumour injection. In contrast, active specific immunotherapy by repeated s.c. injection of viable or radiation-attenuated tumour cells in admixture with BCG was unsuccessful, and  did not improve the response to intrapleural BCG treatment.


Growth of a transplantable lymphoma and its modification in mice infected with the inducing virus. The growth of a transplantable lymphoma was examined in normal mice and in mice previously infected with the lymphoma-inducing virus (ULV). Normal BALB/c mice respond to a footpad injection of X-irradiated lymphoma cells (ULMC) with popliteal lymph node (PLN) enlargement; mice previously infected with ULV do not. 106 viable ULMC injected into the footpads of ULV-infected mice grew progressively, and the animals died with disseminating malignant lymphoma. In contrast, this dose of cells injected into normal animals evoked strong host responses in the foot and draining lymph node, and no progressive growth of the lymphoma occurred. This increased susceptibility of the ULV-infected animals was also observed when ULMC were injected s.c. into the back or i.m. into the calf muscle, but not after s.c. injection of an unrelated 3-methylcholanthrene-induced sarcoma. Resistance to tumour growth after i.v. injection of ULMC is clearly ineffective, since 10 cells can grow and kill the animal, and in this case no increased susceptibility of ULV-infected animals was observed.


The induction of tumour cell adhesiveness and intercellular junctions by a glycoprotein of rat ascites hepatoma cell surface. Rat ascites hepatoma AH109A cells (present as a free form in vivo) can aggregate and then develop well-defined tripartite junctional complexes, including intermediate junctions, desmosomes and focal tight junctions, on incubation with a glycoprotein separated from rat ascites hepatoma AH136B cells (forming cell islnds in vivo). The development of binding structures was strongly inhibited by actinomycin D. AH109A cells or rat ascites hepatoma YS cells (present as a free form in vivo) previously treated with the glycoprotein for 24 h, when inoculated i.p., proliferated as free cells in the ascitic fluid, like the untreated cells. AH109A cells actively proliferating in the skin do not form any junctional complexes. The reason for the failure of island formation by AH109A cells or YS cells in vivo is discussed.


The effect of collagenase on surface markers of human peripheral lymphocytes. The effect of collagenase on the 'T' and 'B' markers of peripheral blood lymphocytes was studied in five healthy male blood donors. No significant difference between untreated and collagenase treated lymphocytes was found. Collagenase is recommended for use in liberating living cells from skin biopsy specimens.


The effect of labour on ACTH and cortisol levels in amniotic fluid and maternal blood. Levels of adrenocorticotrophic hormone (ACTH) and cortisol were measured in amniotic fluid during labour and in maternal blood during and after labour. There was a significant rise of maternal ACTH and cortisol levels during labour and a significant decrease after delivery in all 14 patients studied. There were no significant changes in amniotic fluid ACTH and cortisol levels during labour. The initial level of ACTH in amniotic fluid (162-7 pg/ml) was higher than that in maternal circulation (120-2 pg/ml). The correlation between maternal and amniotic fluid ACTH was not significant while the corresponding correlation for cortisol values was.


Purification and properties of guanosine 5', 3'-polyphosphate synthetase from Bacillus brevis. A ribosome-independent guanosine 5',3'-polyphosphate synthetase has been highly purified from Bacillus brevis (ATCC 8185). The enzyme has a molecular weight of 55,000, as measured by sucrose density gradient centrifugation. Like the ribosome-connected stringent factor of Escherichia coli, it catalyzes the synthesis of the guanosine 5', 3'-polyphosphates by a pyrophosphoryl transfer mechanism from adenosine triphosphate (ATP) to guanosine di- or triphosphates (GDP, GTP). It has an apparent Km of 0.14 mM for GDP and 0.77 mM for GTP, and is specific for the guanosine ribonucleotides as pyrophosphoryl acceptors. Several ATP analogues were tested for their ability to donate the pyrophosphoryl group. Mg2+ was required as a counter ion for the nucleotide substrate; however, an excess of Mg2+ was inhibitory. The property of the B. brevis enzyme is compared with the ribosome-linked enzyme of E. coli and an extracellular enzyme excreted by several types of Streptomyces reported upon recently.


Enzymatic synthesis of (15s)-[15-3h]prostaglandins and their use in the development of a simple and sensitive assay for 15-hydroxyprostaglandin dehydrogenase. The stereospecificity of swine renal NAD+-dependent 15-hydroxyprostaglandin dehydrogenase has been determined. It was found that the enzyme is a B-side specific dehydrogenase. (15S)-[15-3H]Prostaglandins were synthesized by stereospecific transfer of the tritium label of D-[1-3H]galactose to prostaglandins by coupling 15-hydroxyprostaglandin dehydrogenase with beta-D-galactose dehydrogenase, an enzyme of the same stereospecificity. A simple and sensitive assay for 15-hydroxyprostaglandin dehydrogenase was developed based on the stereospecific transfer of the tritium label of tritiated prostaglandins to glutamate by coupling 15-hydroxyprostaglandin dehydrogenase with glutamate dehydrogenase. The amount of prostaglandin oxidized is determined by the radioactivity of labeled glutamate present in the supernatant after charcoal precipitation of labeled prostaglandin. Concurrent assays with the present tritium release method and the thin-layer chromatography method indicated excellent correlation. The assay was employed to study some of the properties of swine renal 15-hydroxyprostaglandin dehydrogenase in crude extract and the distribution of enzyme activity in various tissues of rat. Enzyme activity was linear for the first 10 min studied and was nonlinear with increasing amounts of crude enzyme, indicating the possible presence of endogenous inhibitor(s). Apparent Km's for PGE2, PGF2alpha, and PGA2 were found to be 2.5, 12.5, and 3.9 muM, respectively. The distribution pattern indicated high levels of enzyme activity in gastrointestinal tract, lung, kidney, and spleen. The assay method may prove to be valuable for studying enzyme turnover and enzyme regulation by hormonal and pharmacological agents.


Guanosine tetraphyosphate and its analogues. Chemical synthesis of guanosine 3',5'-dipyrophosphate, deoxyguanosine 3',5'-dipyrophosphate, guanosine 2',5'-bis(methylenediphosphonate), and guanosine 3',5'-bis(methylenediphosphonate). A new procedure for the synthesis of the pyrophosphate bond has been employed in the preparation of nucleoside dipyrophosphates from nucleoside 3',5'-diphosphates. The method makes use of a powerful phosphorylating agent generated in a mixture of cyanoethyl phosphate, dicyclohexylcarbodiimide, and mesitylenesulfonyl chloride in order to avoid possible intramolecular reactions between the two phosphate groups on the sugar ring. That such reactions can readily occur was shown by the facile cyclization of deoxyguanosine 3',5'-diphosphate to P1,P2-deoxyguanosine 3',5'-cyclic pyrophosphate in the presence of dicyclohexylcarbodiimide alone. The phosphorylation reagent was initially tested in the conversion of deoxyguanosine 3',5'-diphosphate to the corresponding 3',5'-dipyrophosphate and was then used to phosphorylate 2'-O-(alpha-methoxyethyl)guanosine 3',5'-diphosphate, which had been prepared from 2'-O-(alpha-methoxyethyl)guanosine. In the latter case, the addition of the two beta phosphate groups was accomplished in 40% yield. Removal of the methoxyethyl group from the phosphorylated product gave guanosine 3',5'-dipyrophosphate, which was shown to be identical with guanosine tetraphosphate prepared enzymatically from a mixture of GDP and ATP. A modification of published procedures was also necessary to effect the synthesis of guanosine bis(methylenediphosphonate). Guanosine was treated with methylenediphosphonic acid and dicyclohexylcarbodiimide in the absence of added base. The product consisted of a mixture of guanosine 2',5' - and 3',5'-bis(methylenediphosphonate), which was resolved by anion-exchange chromatography. The 2',5' and 3',5' isomers are interconvertible at low pH, with the ultimate formation of an equilibrium mixture having a composition ratio of 2:3. The predominant constituent of this mixture has been unequivocally identified as the 3',5' isomer by synthesis from 2'-O-tetrahydropyranylguanosine.


Purification and characterization of the DNA untwisting enzyme from rat liver. The DNA untwisting enzyme has been purified approximately 300-fold from rat liver nuclei. The protein is greater than 90% pure as judged by sodium dodecyl sulfate-acrylamide gel electrophoresis. The native enzyme has a molecular weight between 64 000 and 68 000 and is composed of a single polypeptide chain. Evidence is presented that the protein can act catalytically. A trace amount of endonuclease activity associated with the most pure fraction could be a contaminant or it could be due to the action of the DNA untwisting enzyme itself.


Collagenase enzymes from Clostridium: characterization of individual enzymes. Four collagenases have been purified to apparent homogeneity from extracts of Clostridium histolyticum and partially characterized. The four purified enzymes are devoid of hydrolytic activity against casein and the synthetic substrate, benzolyarginine naphthylamide, but all retain activity against native collagen. The enzymes are initially spearated by isoelectric focusing where three of the enzymes show distinct isoelectric points: collagenase I = 5.50, collagenase II = 5.65, and collagenases IIIa and IIIb = 5.90-6.00. Collagenases IIIa and IIIb can be subsequently separated on diethylaminoethylcellulose. The four purified enzymes show single bands upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Calibration of the molecular weights on the basis of migration distance shows a marked dependence on gel porosity. At high acrylamide concentration, collagenases I, II, and IIIa appear to converge to a limiting molecular weight congruent to 81 000, while collagenase IIIb has a distinctly lower value congruent to 72 000. The similarity between these molecular weight values and those derived from the sedimentation and diffusion coefficients of the native enzyme indicates that each collagenase is a single polypeptide chain. All of the collagenases have comparable catalytic activities against a series of natural and synthetic substrates and are immunologically cross-reactive. Although all four enzymes are evident upon initial electrofocusing of the crude extract, it is possible that the multiplicity of forms is, at least in part, a consequence of lysis following initial secretion from the cell.


Hydrolysis of triphosphoinositides by a soluble fraction of Crithidia fasciculata. Homogenates of Crithidia fasciculata (a species of Trypanosomidae) were shown to contain a phosphatase (EC 3.1.3.36) and a phosphodiesterase (EC 3.1.4.11) which hydrolyse triphosphoinositides. Approximately 30% of the diesterase and most of the phosphatase are present in the soluble fraction. The triphosphoinositide phosphatase is specifically dependent upon Mg(2+) and is stable to storage with or without freezing. The triphosphoinositide phosphodiesterase requires Ca(2+) and is inactivated during storage. Both activities are maximal in the presence of cetyltrimethylammonium bromide and require protection or reactivation by GSH or dithiothreitol. Unlike similar mammalian enzymes the protozoal triphosphoinositide phosphatase does not hydrolyse diphosphoinositides. The two enzymes may be separated by (NH4)2SO4 fractionation and gel filtration on Sephadex G-200.


Localization of neutral glycosphingolipids in human plasma. 1. The localization of the neutral glycosphingolipids glucosylceramide, lactosylceramide, trihexosylceramide and globoside in human plasma was investigated. Glycosphingolipids were isolated and analysed by gas-liquid chromatography. 2. After Sephadex gel chromatography of human plasma, about 75% of the glycosphingolipids were found in the fraction containing most of the lipoproteins. 3. After fractionation of the lipoproteins by ionic precipitation, 15-25% of each glycosphingolipid was found in the very low-density lipoprotein + chylomicron fraction, 30-45% in the low density lipoprotein fraction and 40-50% in the high density lipoprotein fraction. 4. After fractionation of lipoproteins by density-gradient ultracentrifugation, 15% of each glycosphingolipid was found in the very low-density lipoprotein + chylomicron fraction and 85% in the low density and high density lipoprotein fractions. No glycosphingolipids could be detected in the ultracentrifugal residue which contains the bulk of the albumin.


Selective utilization of endogenous unsaturated phosphatidylcholines and diacylglycerols by cholinephosphotransferase of mouse lung microsomes. In the presence of CMP, cholinephosphotransferase of mouse lung microsomes catalyzes the conversion of endogenous phosphatidylcholines into 1,2-diacyl-sn-glycerols and CDPcholine. 2. In this conversion cholinephosphotransferase shows a distinct preference for those molecular species of phosphatidylcholine which contain an unsaturated fatty acid. The enzyme hardly utilizes endogenous depalmitoylglycerophosphocholine as a substrate. 3. Membrane-bound 1,2-diacyl-sn-glycerols were also prepared by treatment of mouse lung microsomes with a pure phospholipase C from Bacillus cereus. These 1,2-diacyl-sn-glycerols were subsequently utilized as substrate by cholinephosphotransferase in the formation of phosphatidylcholine. In the latter reaction, cholinephosphotransferase exhibited a pronounced preference for unsaturated 1,2-diacyl-sn-glycerols and hardly utilized the endogenous 1,2-depalmitoyl-sn-glycerol. 4. The low affinity of cholinephosphotransferase for either dipalmitoylglycerophosphocholine or 1,2-dip


Activity of cholinephosphotransferase, lysolecithin: lysolecithin acyltransferase and lysolecithin acyltransferase in the developing mouse lung. 1. The present study presents the activity profiles of cholinephosphotransferase, lysolecithin:lysolecithin acyltransferase and lysolecithin acyltransferase at different stages of development of the mouse lung. 2. The specific activity of cholinephosphotransferase, a key enzyme in the de novo synthesis of phosphatidylcholine, increases during the later stages of fetal development until it reaches a maximal value at a gestational age of 17 days, i.e. 2 days before term. Thereafter, the activity of the enzyme declines again until around term. 2. The specific activity of lysolecithin:lysolecithin acyltransferase which catalyzes the transesterification between two molecules of 1-acyl-sn-glycero-3-phosphocholine, appears to be much lower than that of cholinephosphotransferase at gestational ages below 18 days. However, around day 18, the specific activity of lysolecithin:lysolecithin acyltransferase increases dramatically until it almost equals the maximal activity of cholinephosphotransferase measured on day 17. 4. The specific activity of lysolecithin acyltransferase, which catalyzes the direct acylation of 1-acyl-sn-glycero-3-phosphocholine, does not change significantly during the prenatal development and is lower than that of either lysolecithin:lysolecithin acyltransferase or cholinephosphotransferase at all stages of development. 5. These results are discussed in view of the possible role of these enzymes in the biosynthesis of pulmonary 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.


High resolution preparative column chromatographic system for gangliosides using DEAE-Sephadex and a new porus silica, Iatrobeads. A new high-resolution preparative column chromatographic system was developed for efficient and rapid isolation of ganglioside molecular species. The system involved a combination of ion-exchange and adsorption chromatographies using DEAE-Sephadex A-25 and the newly developed, totally porous silica spheres, Iatrobeads. Using this system the brain gangliosides, GM1, GD1a, GD1b and GT1 were obtained in high purity and in milligram amounts, in a relatively short time, by simple procedures. The presence of a number of unidentified molecular species of gangliosides, which are present only in small amounts, was also demonstrated.


A rapid assay method of collagenase activity using 14C-labeled soluble collagen as substrate. A rapid assay method for vertebrate collagenase (EC 3.4.24.3) activity has been developed using 14C-labeled soluble collagen as substrate. The method is based on the incubation of collagen with enzyme in the presence of glucose to prevent collagen fibril formation followed by selective extraction of the enzyme digestion products into dioxane at a final concentration of 50%. The rate of reaction was about 10 times higher than that obtained by the conventional method using reconstituted collagen fibrils as substrate and the relationship between enzyme activity and concentration was linear over a wider range. When the method was applied to the assay of human granulocyte collagenase, the results showed good correlation with those obtained by the conventional gel method.


Particulate carbonic anhydrase in homogenates of human kidney. About 2% of human kidney carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been found in particulate fractions. Its distribution in the particulate fractions obtained by differential centrifugation suggests that it may be concentrated in the brush border. The particulate enzyme is like red cell carbonic anhydrace C in its susceptibility to inhibition by anions. Particulate carbonic anhydrase is firmly bound to the membrane and is not released by incubation at pH 10.6 and 37 degrees C or by addition of Triton X-100 or deoxycholate. In 10% Triton X-100 at pH 11.3 and 37 degrees C, the particulate enzyme is inactivated with a half time of about 20 min, and this is at least an order of magnitude slower than the inactivation of soluble enzymes in the presence or absence of membranes. The soluble enzymes are inactivated within a few minutes at 25 degrees C in 3-4% sodium dodecyl sulfate, but the particulate enzyme is relatively stable under those conditions, and its half-time of inactivation at 14 degrees C with a detergent-protein ratio of 25 was about 24 h. Gel filtration with Ultragel AcA-44 in sodium dodecyl sulfate indicates that the membrane carbonic anhydrase has a molecular weight of less than 66 000, so its stability is not due to association with large membrane fragments or vesicles. These results suggest that the membrane enzyme may be a different isozyme than the soluble carbonic anhydrases. Although present in relatively small amounts, its localization on the membrane could give it functional significance.


Cyclic AMP-stimulated phosphorylation of bovine tracheal smooth muscle contractile and non-contractile proteins. 1. Various proteins isolated from bovine tracheal smooth muscle were examined as phosphate acceptor substrates for a cyclic AMP-dependent protein kinase isolated from the same tissue. A fraction prepared in a manner similar to that of skeletal muscle troponin was the best substrate of the presumptive contractile proteins isolate. Actomyosin and tropomyosin were relatively poor substrates. 2. An assay was developed for the rapid detection in a large number of samples of the muscle specific substrate for the protein kinase on which we reported previously. 3. Using this assay, the muscle specific substrate found in bovine tracheal smooth muscle was partially purified resulting in a preparation which when resolved by polyacrylamide gel electrophoresis showed a single peak of 32P incorporated, and which could be further characterized. 4. Our findings suggest that the substrate contains a protein subunit of molecular weight 19 000, which can be phosphorylated at serine and threonine residues, in the presence of cyclic AMP and protein kinase. The phosphate is in a covalent ester linkage with these residues. 5. A phosphoprotein phosphatase was isolated from the bovine tracheal smooth muscle. 6. Bovine tracheal smooth muscle contains cyclic AMP dependent protein kinase and phosphoprotein phospahatase activity as well as the muscle specific substrate, suggesting that these elements may be part of a mechanism which regulates smooth muscle tone.


Binding of nitroxide stearate spin labels to bovine serum albumin. 1. 12-Nitroxide stearate binds to bovine serum albumin at about four independent and equivalent binding sites with an association constant of about 10(6) M-1. The binding at these high affinity binding sites is significantly reduced by addition of unlabeled stearate. These data suggest that nitroxide stearates probe the high affinity binding sites for long-chain fatty acids. 2. Qualitative analyses of the ESR spectra of 5-, 12- and 16-nitroxide stearate bound to bovine serum albumin and measurements of the interaction of these compounds so bound with ferricyanide ion provide a rough description of the binding site as follows: the polar headgroup of the spin-labeled fatty acid is rigidly fixed, but fairly accessible to paramagnetic ions. The middle part of the hydrocarbon chain of bound stearate spin label also is rigidly fixed but differs in being shielded from the solvent, presumably by a hydrophobic cleft. The methyl terminus shows greater motion, appearing to move within a narrow cone, and also appears to be somewhat accessible to paramagnetic ions.


Effects of guanidine hydrochloride on human plasma high density lipoproteins. Denaturation of human plasma high density lipoproteins during ultracentrifugation in guanidine-HCl is characterized by: dissociation of apoA-I, in the range of 2-3 M guanidine-HCl, and dissociation of apoA-I and apoA-II in 5-6 M guanidine-HCl. Denaturation of high density lipoprotein species, during a sequence of timed exposure to guanidine-HCl followed first by removal of the denaturant by dialysis and then by ultracentrifugation, is characterized by:dissociation of lipid-poor apoA-I, which follows a time course similar to denaturation-related changes in reported spectroscopic parameters; and apparent formation of lipoprotein aggregation products depleted in apoA-I and relatively enriched in apoA-II. These studies indicate differential properties of the major apoproteins in stabilizing high density lipoprotein structure and characterize a mode of lipoprotein transformation and degradation which apparently results from apoprotein dissociation coupled with aggregation of denatured lipoprote species.


Haem accessibility in cytochrome P-450 from rabbit liver. A proton magnetic relaxation study by stereochemical probes. Cytochrome P-450 was solubilized from phenobarbital induced rabbit liver and purified by affinity chromatography. The longitudinal proton magnetic relaxation rates of this ferric, low-spin sample (as confirmed by ESR) in 20% glycerol aqueous solution are very large compared with low-spin methaemoglobin and myoglobin derivatives. Similarly high rates were measured in a deuterated solution using the aliphatic protons of glycerol as stereochemical markers, which strongly suggests that the haem iron in cytochrome P-450 is much more accessible to the solvent than in harmoglobin or myoglobin. Type I substate (Spasman) produced small but significant increases in NMR rates both in the H2O and in the 2H2O solution, while binding of aniline (Type II substrate) doubled the rates.


Radioimmunoassay of apolipoprotein A-I of rat serum. A double antibody radioimmunoassay technique was developed for quantification of apolipoprotein A-I, the major apoprotein of rat high density lipoprotein. Apo A-I was labeled with 125I by the chloramine-T method. 125I-labeled apo A-I had the same electrophoretic mobility as unlabeled apo A-I and more than 80% of the 125I was precipitated by rabbit anti apo A-I antibodies. The assay is sensitive at the level of 0.5-5 ng, and has intraassay and interassay coefficients of variation of 4.5 and 6.5% respectively. The specificity of the assay was established by competitive displacement of 125I-labeled apo A-I from its antibody by apo A-I and lipoproteins containing apo A-I, but not by rat albumin and other apoproteins. Immunoreactivity of high density lipoprotein and serum was only about 35% of that of their delipidated forms when Veronal buffer was used as a diluent. Inclusion of 5 mM sodium decyl sulfate in the incubation mixture brought out reactivity equivalent to that found after delipidation. Completeness of the reaction was verified by comparison with the amount of apo A-I in chromatographic fractions of the total apoprotein of high density lipoprotein. Content (weight %, mean values +/- S.D.) of immunoassayable apo A-I was: 62.3 +/- 5.9 in high density lipoprotein; 1.7 +/- 0.3 in low density lipoprotein; 0.09 +/- 0.03 in very low density lipoprotein and 25.0 +/- 5.0 in lymp chylomicrons. Concentration in whole serum was 51.4 +/- 8.9 mg/dl and 33.6 +/- 4.1 mg/dl for female and male rats, respectively (p less than 0.002), equivalent to the sex difference in concentration of high density lipoprotein. 95% of the apo A-I in serum was in high density lipoprotein, 5% in proteins of d greater than 1.21 g/ml and less than 1% in lipoproteins of d less than 1.063 g/ml.


Repair replication in cultured normal and transformed human fibroblasts. Repair replication in response to ultraviolet irradiation has been studied in normal human diploid fibroblast cultures, W138, and an SV40 transformant, VA13. Quantitative comparisons have been made using the combined isotopic and density labeling method for assaying repair replication. We find no significant difference in the amount of repair replication performed its dose response, or the time course between growing and confluent W138 cells, early passage and senescent cells, or normal W138 cells and the transformed VA13 cells. When [3H]dThd was employed as the isotopic label in the presence of a 30-200 fold excess of unlabelled BrdUrd, apparent differences in repair replication were seen between W138 cells shortly after subcultivation and cells which had been allowed to reach confluence. These differences were the same over a wide dose range and regardless of the passage number of the cells, but could be influenced by using different serum lots. The differences were not seen, however, when [3H]BrdUrd provided the isotopic label; thus they reflect either impurities in the [3H]dThd or a slight discrimination by some cellular process.


Caffeine, cyclic AMP and postreplication repair of mammalian cell DNA. The methylxanthines, caffeine and theophylline, inhibit postreplication repair of DNA in mammalian cells. Because they also inhibit cyclic AMP phosphodiesterase, it was thought that there might be some connection between concentrations of cyclic AMP and postreplication repair. We tested this possibility by performing DNA sedimentation experiments with a cyclic AMP-resistant mouse lymphoma cell mutant and its wild-type counterpart. The results show that there is no connection between cellular cyclic AMP concentrations and the rate of postreplication repair. Therefore, it is more likely that caffeine and theophylline inhibit postreplication repair by some other means, such as by binding to DNA.


Induction of alpha type DNA polymerases in human cytomegalovirus-infected WI-38 cells. Productive infection of WI-38 cells with human cytomegalovirus (HCMV) induced the increase in the activity of DNA polymerases as well as the synthesis of viral and cellular DNA. Sedimentation analyses in sucrose gradients of high ionic strength showed that the HCMV infection caused marked increase in the activity of alpha-type polymerases (resolved into alpha1, 8 S, and alpha 2, 6 S, in the present experiments), while the infection little affected the level of beta-type polymerase (about 3.5 S) activity in both the nuclei and cytoplasm. Such increase in alpha-type polymerases was also observed when DNA synthesis in WI-38 cells was enhanced by SV40 infection or by an increased concentration of serum in medium. Phosphonacetate, which selectively blocked the synthesis of HCMV DNA, did not significantly affect the HCMV-mediated induction of DNA polymerases. However, phosphonoacetate added in the reaction mixture for DNA polymerase assay inhibited the activity of the HCMV-induced polyperase alpha, but not of the polymerases alpha2 and beta. These results support the idea that alpha-type polymerases are involved in the replicative synthesis of cellular and viral DNA.


The study of rous sarcoma virus-transformed baby hamster kidney cells using fluorescent probes. The fluorescent probes pyrene, pyrene butyric acid and N-phenyl 1-naphthylamine have been used to investigate the changes that accompany  in vitro transformation of a baby hamster kidney cell line using Rous sarcoma virus. The fluorescent probes which reside in the membrane were used to compare the changes in microviscosity and polarity of the membranes of normal cells with two transformed cell lines. The spectrofluorimetric data indicate that following transformation the probe N-phenyl 1-naphthylamine resides in a more polar environment. However, using the probe pyrene, the yield of excimer indicates decreased mobility of this probe in the membrane of transformed cells. The data also indicate differences between the two transformed cell lines. Laser photolysis was used to study the lifetime of the pyrene probes and the quenching of the pyrene fluorescence in the membrane by several different quenching molecules. The data indicate differences between the three cell lines and suggest that transformation decreases movement within the membrane.


Preparation and properties of vesicles of a purified myelin hydrophobic protein and phospholipid. A spin label study. Lipophilin, a hydrophobic protein purified from the proteolipid of normal hupid and protein in 2-chloro-ethanol followed by dialysis against buffer. This method resulted in homogeneous incorporation of the protein into lipid vesicles as judged by sedimentation on a sucrose gradient and freeze fracture electreter and the freeze fracture faces contained intramembrane particles. The effect of lipophilin on the organization of the lipid was studied by use of spin label probes. Two distinct components were present in the spectrum of fatty acid spin labels in the lipid-protein vesicles. One was immobilized presumably due to the presence of boundary lipid around the protein and the second component waicles and probably due to a lamellar phase but with a slightly greater order parameter. Lipophilin was found to increase the order parameter linearly with increasing concentration of protein incorporated into the vesicles. However, the phase transition temperature as measured from the 2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPO) solubility parameter was unchanged.


Hormonal control of uterine contraction. Characterization od cyclic AMP-dependent membrane properties in the myometrium. A mitochondria-free membrane fraction prepared from rat myometrium accumulated 45Ca2+ in the presence of oxalic acid and ATP. The rate of transport of Ca2+ into the membranous vesicles was increased by greater than 50% in the presence of 3',5'-cyclic AMP, but not by 2',3'-cyclic AMP or 5'AMP. Membrane ATPase activity was stimulated by Mg2+; slight additional stimulation was obtained in the presence of Na+ and K+ but not in the presence of Ca+2. Despite the cyclic AMP sensitivity of membrane ATPase activity, the absence of any effect of inhibitors of Ca2+-transport suggest it has little to do with Ca2+ accumulation by the membranes. Cyclic AMP-induced increase in Ca2+-transport and membrane ATPase activity was duplicated in vivo by incubating uteri in 10(-4)M isoproterenol prior to membrane isolation. Isoproterenol has been previously shown to increase myometrial cyclic AMP levels, and changes in Ca2+-transport by cell membranes in relation to intracellular cyclic AMP levels may be the mechanism through which hormones modulate uterine contractility.


Oxidation and reduction of membrane-bound cytochrome c in Hemophilus parainfluenzae. Reaction with oxygen, hydrogen peroxide and nitrate. Cytochromes of the a-, b-, c- and d-type become reduced when intact cells of Hemophilus parainfluenzae have become anaerobic following respiration with substrates such as formate or succinate, as shown previously (J. Biol. Chem. (1970) 254, 5096-5100). In the presence of formate after depletion of O2, there is an unusual two-step time course of reduction of the membrane-bound cytochrome c. The proportion of the cytochrome c which is reduced during the second stage is oxidizable by either nitrate or H2O2 and is reduced again when the nitrate or H2O2 have been depleted. We conclude that the observed two-stage reduction of cytochrome c results from the presence of an oxidant, probably H2O2, produced by reaction of formate dehydrogenase with O2. This was shown by the effects of cyanide, catalase and O2. In addition, no evidence for the production of the oxidant is seen when succinate is the substrate oxidized. Although measurements of absorption spectra indicated only one species of cytochrome c, kinetic evidence is presented for some separation of the cytochrome c into more than one electron transport pathway.


Electron paramagnetic resonance studies of cytochrome P-450 and adrenal ferredoxin in single whole rat adrenal glands. Effect of corticotropin. Low and high spin ferric cytochrome P-450 and reduced adrenal ferredoxin (adrenodoxin) have been directly studied by EPR techniques in whole rat adrenal glands. The spectra obtained correspond closely to those obtained from sub-cellular fractions except in the case of low spin ferric cytochrome P-450, where there are differences in the shape of the g = 2.41 line. The relative magnitudes of these peaks in anaerobic and aerobic rapidly frozen adrenals from control and corticotropin stimulated hypophysectomised rats were used to investigate the control and rate limiting steps in adrenal steroid biosynthesis via cytochrome P-450. All adrenals showed a close to maximal level of reduced adrenodoxin and aerobic and anaerobic glands from control rats and aerobic glands from corticotropin stimulated rats showed similar quantities of low spin ferric cytochrome P-450. On anaerobiosis the quantity of low spin ferric cytochrome in adrenals from corticotropin stimulated rats dropped to 30--40% of the aerobic level. Treatment of the rats with cycloheximide prior to administration of corticotropin prevented these changes. Approximately 0.4% of the total cytochrome P-450 was high spin ferric in control adrenals and in aerobic stimulated adrenals this rose to approximately to 0.6%. These results demonstrate that association of substrate with cytochrome P-450 is the rate limiting step in adrenal steroidogenesis via cytochrome P-450. It is suggested on the basis of these and mitochondrial optical and EPR experiments that the limiting step being observed is cholesterol binding to cholesterol side chain cleavage cytochrome P-450, and that the rate of this association is stimulated by corticotropin.


[Hydrocarbon metabolism in a marine bacterium]. The marine bacterium L.16.1 (Alcaligenes sp.) grows preferentially on alkanes (C10 to C18) with a very high growth yield (98 per cent); optimal growth depends strictly on the presence of a well-defined NaCl concentration (100 mM). Our strain is constitutive for the enzymatic systems responsible for the oxidation of alkanes to fatty acids, i.e. NADH-dependent hydroxylase, alcohol and aldehyde dehydrogenases, the latter of which located at the cytoplasmic membrane level. The aerobic oxidation of primary alcohols by particulate extracts prepared in the presence of 400 mM NaCl is NAD+-dependent (Km = 0.082 mM, Vmax = 238 with decanol). With extracts prepared in the absence of NaCl, Vmax undergoes a very strong decrease. On the contrary , the NAD+ (P)+-dependent oxidation of aldehydes is carried out anaerobically by the same extracts irrespective of the presence or the absence of added Na+ in the solutions used for the preparation of these extracts. A possible explanation for our results could be that Na+ acts on the enzymatic systems for which the maintenance of the membrane integrity is essential. This interpretation is consistent with the slowing down of the growth speed accompanying the decrease of NaCl concentration in the growth medium. With regard to alcohol and aldehyde-dehydrogenases, it is noteworthy that these enzymes behave like similar enzymatic activities induced by alkanes in other microorganisms.


In vivo study of free and esterified cholesterol turnover in various tissues of the rat. Rats were infused for 3.5 to 10 hrs with either red cells or plasma previously labelled in vivo by [3H]-cholesterol. Cholesterol specific radioactivities were measured in plasma, HDL, LDL and VLDL, and various tissues. Red cell infusions led to a higher labelling of free than of esterified cholesterol in the plasma of infused rats. The opposite situation was observed following plasma infusion. Comparison of free and esterified cholesterol specific radioactivities in each tissue showed that esterified cholesterol was transferred from plasma to all the tissues, except the adrenals. Study of the ratios of cholesterol specific radioactivities from one experimental group to the other in each tissue, made it possible to demonstrate clearly the occurence of hydrolysis within all the studied tissues except 5 of them where its existence remains uncertain (lung, heart, kidney, tendon, muscle) and of esterification in 3 tissues (adrenal, liver lung). In addition, ratios of cholesterol radioactivities (free/ester) were found to be identical in plasma and in 4 tissues, where neither hydrolysis nor esterification were detected (heart, muscle, kidney, tendon). This finding is an argument in favor of a simultaneous transport of free and esterified cholesterol from plasma into these 4 tissues and suggests that the entire lipoprotein particles can penetrate these tissues, with no specificity of one special class. In adrenal, unlike all other tissues: 1) the turnover of esterified cholesterol was achieved mostly by hydrolysis and esterification in situ; 2) a preferential lipoprotein class (LDL) was responsible for the transport of free cholesterol from the plasma.


[Kinetic properties and heat resistance of the peritoneal leukocyte myeloperoxidase of white mice]. The dependence of the myeloperoxidase-catalyzed reaction in the concentration of the enzyme and that of both substrates (hydrogenperoxide and o-dianizidine) was investigated. The substrates--velocity curves appeared non-hyperbolic, an inhibitory effect was registered for both substrates. Hill coefficient with a varying value was employed to characterize the deviation of the curves from Michaelis--Menten hyperbolas apparently caused by the interaction of non-identical subunits composing the enzyme molecule. The effect of the preincubation temperature on the enzyme activity and the kinetics of thermoinactivation of the enzyme are analysed, the purified enzyme preparation being more thermostable than the crude one.


[Participation of 5'-deoxyadenosyl-B12 in regulating methylation of tRNA]. The effects of different forms of cobalamines on the activities of tRNA-methylases of Zajdela ascite hepatoma were studied. Of six cobamides studied 5'-deoxyadenosyl-B12 and factor B containing as a ligand HSO3 in the concentrations of 2.4-10(-5) and 4.8-10(-5) M inhibited the tRNA-methylase activity by  21% and 15% correspondingly. The inhibitory effect of 5'-deoxyadenosyl-B12 is probably dependent on the adenosyl part of the molecule. 5'deoxyadenosyl-B12 exerted a selective effect of Zajdela ascite hepatoma tRNA-methylases, inhibiting largely the activity of 5-methyl cytosine methylase during the methylation of the E. coli K12W6 tRNA and yeast tRNA1 Val.


[Hysteresis, alternative stationary states and auto-oscillations in an open futile cycle one of the reactions of which is substrate inhibited]. In connection with the problem of regulation of futile (energy-dissipating) cycles in cell metabolism, a kinetic model has been investigated of an open cycle S1 (see article) S2, in which one of the enzymes (E-) is inhibited by the excess of its substrate S2. The quasi-stationary net velocity of the utilization of substrate S1 in the cycle as a function of its concentration is shown to be of a hysteretic character. Owing to this the alternative stationary states and self-oscillations may occur in the cycle. Under certain conditions the transition from one alternative state to another may reverse the direction of the net flux of conversion from S1 to S2 or vice versa. The self-oscillations are associated with a periodical change in the net flux direction. It is suggested the participation of glycogen (starch) in the self-oscillatory mechanism of the futile cycle formed by the phosphofructokinase and fructose bisphosphatase reactions may give rise to oscillations with the period of 10(3)-10(4) min, which may serve as the basis for the cell clock.


[Cytochrome oxidase of rat liver nuclear membranes and its interaction with mitochondrial cytochrome oxidase]. The percent of mitochondrial protein contamination in nuclei decreased 10-fold (from 18 to 1.8%) under purification of protein-labelled mitochondria before their introduction into nuclei-free homogenate, cytochromoxidase activity being unchanged. Thus, cytochromoxidase activity of nuclei does not correlate with the amount of nuclei-adsorbed mitochondrial protein, which demonstrates the presence of nuclear cytochromoxidase independent on mitochondrial protein. Radioactivity of protein-labelled mitochondria is proportially distributed between globuline, deoxyribonucleoprotein, acid and residual nuclear proteins, as it is shown under fractionation of nuclei isolated from protein-labeled mitochondria containing homogenate. The comparison of mitochondrial protein contamination of nuclear membranes and their possible contamination with cytochromoxidase and suecinate-cytochrome-c-reducatase activities revealed that cytochromoxidase activity of nuclear membranes is twice higher and succinate-cytochrome-c-reductase activity is considerably lower than it can be referred to mitochondrial protein contamination. The ratio of cytochrome-c-oxidase and succinate-cytochrome-c-reductase activities in isolated nuclear membranes is 4-7 times as high as that in mitochondrial membranes under the same isolation procedure. The data obtained make possible to consider the cytochromoxidase activity of nuclear membranes to be really nuclear enzyme, and not a contominant of nucleipreparation with mitochondrial membranes.


[Concentration of components of the adenylate system in heavy and light fractions of the sarcoplasmic reticulum of skeletal muscles and sensitivity of these fractions to the effects of imidazole-containing compounds and caffeine]. Fragments of sarcoplasmic reticulum from rabbit sceletal muscles sedimented within the range from 2000 g to 8000 g (heavy fraction) and 8000 g to 40000 g (light fraction) and washed with 0.6 M KCl, were practically free of adenylatecyclase activity. Phosphodiesterase cAMP was not found in the light fraction, while its activity in the heavy fraction was 500 pmol of cAMP/min per mg of protein. Both fractions contain bound cAMP (1-2 pmol/mg of protein) and specific sites of cAMP binding, the binding constant being approximately 10(6)M-1. The number of binding sites is 60 pmol/mg of protein for the heavy and 30 pmol/mg of protein for the light fractions. The level of phosphodiesterase activity in the heavy fraction correlates with its sensitivity to imidazole, anserine and caffeine. Imidazole and anserine increase in 1.5-1.8 times the value of Ca2+/ATP in the heavy fraction and produce no effect on Ca2+ transport by the light fraction. Caffeine decreases almost twice the Ca2+/ATP value in the heavy fraction and has practically no effect on Ca2+ absorption by enzymes of the light reticulum fraction. Imidazole and anserine activate membrane-bound phosphodiesterase, while caffeine inhibits it. It is suggested that structural rearrangements of membrane-bound phosphodiesterase under the effect of caffeine, imidazole and anserine are responsible for changes in the efficiency of Ca2+ transport by fragments of the heavy reticulum fractions.


[Extramitochondrial energy dependent synthesis of inorganic pyrophosphate in yeasts]. In yeast Saccharomyces N.C.Y.C. 644SU3 the simplest energy donor, inorganic pyrophosphate, can be formed not only in the processes coupled with the respiratory chain, but also under conditions when mitochondrial synthesis of the compound is inhibited (glucose repression, anaerobiosis). Stimulation of inorganic pyrophosphate formed after exogeneous addition of glycolytic system components and inhibition studies suggest that under those conditions the non-mitochondrial energy-dependent formation of inorganic pyrophosphate is due to glycolytic phosphorilation.