Effect of 5-iodo-5'-amino-2',5'-dideoxyuridine on varicella-zoster virus in vitro.

The antiviral effect of the nucleoside analog 5-iodo-5'-amino-2',5'-dideoxyuridine (AIU) was tested with three isolates of varicella-zoster virus (VZV). AIU concentrations of 10 to 800 muM (3.5 to 288 mug/ml) reduced the number of plaques produced by VZV-infected cells and cell-free VZV from approximately 30 to 95%. Smaller plaque size was also observed in the presence of AIU. AIU was less effective than arabinofuranosylthymine in reducing VZV-induced plaques since as little as 5 mug of arabinofuranosylthymine per ml completely blocked plaque formation by cell-free VZV. Toxicity assays with human diploid embryo fibroblast cells were also carried out. Drug concentrations as high as 800 muM were not toxic to human diploid embryo fibroblast cells as determined by radiolabeling of cell deoxyribonucleic acid, ribonucleic acid, and protein.


Extragonadal germ-cell tumours.

The case histories of two patients with an extragonadal germ-cell tumour are discussed. One had a retroperitoneal embryonal carcinoma, and the other had a mediastinal choriocarcinoma-seminoma. The pathogenesis, symptomatology, diagnosis and treatment of these rare tumours are discussed in some detail.?


A year's experience of the rotavirus syndrome and its association with respiratory illness.

In a hospital study rotavirus was identified in 51% of 152 children with diarrhoea. These patients showed a clinical pattern that was distinct from patients in whom the diarrhoea was associated with bacteria, other viruses, or no pathogens. A respiratory illness was described in 66% of rotavirus patients and usually preceded the gastrointestinal symptoms. Vomiting lasted between one and 3 days and was curtailed by substituting the normal diet with clear fluids. Watery diarrhoes continued for 4 or 5 days, even when rehydration was by the intravenous rather than the oral route. Prolonged diarrhoea was rare. Most children infected with rotavirus were under 2 years of age, but dehydration was most severe in infants aged between 12 and 18 months. A clinician can thus recognise the rotavirus syndrome and expect spontaneous recovery if adequate rehydration is maintained for a critical few days.


Recurrent attack of rotavirus gastroenteritis after adenovirus-induced diarrhoea.

A 14-month-old baby boy suffered a second attack of rotavirus gastroenteritis within one month of the initial one. The second attack followed a diarrhoeal episode associated with adenovirus. Gastrointestinal symptoms in the second attack were more severe than those of the first. The adenovirus-associated enteritis was mild compared with the illness in both episodes of rotavirus infection.


[The leukocyte migration inhibition test (LMIT) in recurrent herpes simplex labialis. Comparison of the results of treatment with BCG and Levamisole (author's transl)].

Forty six patients suffering from recurrent herpes simplex labialis (HSLR) have been treated by either BCG or Levamisole. Therapy was equally successful in both groups: 10 patients remained free of symptoms during the observation period, 11 patients improved clearly and 2 patients did not respond to the therapy. Sensitization of lymphocytes toward herpes simplex virus antigen (HVS-AG) was determined in vitro by means of the leukocyte migration inhibition test (LMIT); test were performed before therapy and in certain intervals after the end of it. While lymphocytes of 10 control persons could be stimulated by HSV-AG, those of patients suffering from HSLR did not react to this antigen before onset of any therapy. Clinical success of therapy correlated well with the in vitro results of the LMIT: after therapy, the lymphocytes of symptom-free and improved patients also reacted in vitro to the antigen added.


Glucocorticoid effects in vitamin D intoxication.

Calcium balance studies and measurement of 25-hydroxyvitamin D3 (25[OH]D3) levels were performed on a vitamin D intoxicated, hypoparathyroid patient before, during, and after successful management of hypercalcemia with oral prednisone therapy. Prednisone effected a dramatic reduction in both mean serum calcium levels and mean 24-hour urinary calcium excretion within four days on two separate occasions. No changes were apparent in fecal calcium excretion. Calcium balance became less negative with prednisone treatment. Levels of 25(OH) D3 during the same period did not change. Decreased calcium mobilization from bone best accounted for the glucocorticoid-mediated amelioration of hypercalcemia.


Day treatment and psychotropic drugs in the aftercare of schizophrenic patients. A Veterans Administration cooperative study.

Schizophrenic patients referred for day treatment at the time of discharge from ten hospitals were randomly assigned to receive day treatment plus drugs or to receive drugs alone. They were tested before assignment and at 6, 12, 18, and 24 months on social functioning, symptoms, and attitudes. Community tenure and costs were also measured. The ten day centers were described on process variables every six months for the four years of the study. Some centers were found to be effective in treating chronic schizophrenic patients and others were not. All centers improved the patients' social functioning. Six of the centers were found to significantly delay relapse, reduce sumptoms, and change some attitudes. Costs for patients in these centers were not significantly different from the group receiving only drugs. More professional staff hours, group therapy, and a high patient turnover treatment philosophy were associated with poor-result centers. More occupational therapy and a sustained nonthreatening environment were more characteristic of successful outcome centers.


Substrate oxidation and energy production by Guerin epithelioma mitochondria.

The mitochondria from Guerin epithelioma and normal rat liver were isolated and their energy production abilities were compared. Both mitochondrial preparations were of the same degree of purity. The percentage of mitochondrial protein in tumour homogenates was three times lower than in rat liver. Guerin epithelioma mitochondria characterized a high respiratory control ratio with succinate and low with NAD dependent substrates in the presence of ADP. The ADP-ATP exchange rate was five times lower in tumour than in rat liver. Energy dependent potassium ions accumulation was about 45% of that found in rat liver mitochondria. The uptake of respiratory substrates was similar in tumour and liver mitochondria. These results indicate on the low efficiency of oxidative phosphorylation in the mitochondria of Guerin epithelioma.


Influence of ACTH on activity of some granulocytic proteases in patients with multiple sclerosis.

The effect of treatment with ACTH on activity of granulocytic acid and neutral proteases in patients suffering from multiple sclerosis was studied. Protease activity was distinctly higher in granulocytes from patients. ACTH normalized the activities investigated in patients with exacerbating course multiple sclerosis, but had no influence on hydrolases in the progressive form of the disease.


The influence of PGE1 and db-cAMP on permeability of lysosomal membranes of rat lymphocytes in the in vitro histamine test.

An attempt was made to develop as in vitro model of inflammation which would allow the study of the inflammatory process on the subcellular level and on the influence of PGE1 and db-cAMP on permeability of lysosomal membranes of liver lymphocytes. The results showed that the in vitro histamine test permits evaluation of early changes in the lysosomal membranes. Drugs such as acetylsalicylic acid, hydrocortisone, as well as PGE1 and db-cAMP exerted a stabilizing influence on the lysosomal membranes of rat lymphocytes.


Influence of insulin on pharmacokinetics of mannosulfan in rats.

The purpose of the present study was to investigate the possibilities of potentiation of the antitumor action of mannosulfan after its administration together with insulin. The pharmacokinetics of mannnosulfan were investigated after its administration separately and together with insulin. Concentrations of mannosulfan in the plasma were determined by gas chromatography. Insulin enhanced the rate of absorption of mannosulfan from the peritoneal cavity and prolonged its elimination from the body. It may be assumed that insulin enhances not only passage of mannosulfan from the peritoneal cavity to blood, but also from blood to tissues. Since increased antitumor effectiveness of mannosulfan was accompanied by its decreased toxicity, it may be concluded that insulin causes selective cummulation of the cytostatic only in some tissues, among others, in the tumor.


Electromyographic value of the tibialis posterior muscle.

Differentiation between peroneal palsy and L5 radiculopathy poses a diagnostic challenge. However, clinical weakness of the tibialis posterior muscle indicates that the involvement must lie outside the peroneal territory, potentially at a radicular level. Electromyographically this muscle should provide similar information but it has been relatively ignored, possibly for reasons of presumed inaccessibility. A retrospective study was undertaken to delineate the potential electrical discriminatory value of this muscle. Patients selected were those with clinical evidence of lower extremity neurogenic dysfunction in whom needle electrode examination had been performed on the tibialis posterior as well as other posterior and anterior compartment musculature. The parameters measured were the presence of abnormal spontaneous activity suggestive of denervation plus neurogenic motor unit alterations. Abnormalities were detected in the tibialis posterior which were reflected equally in the anterior but not posterior compartment muscles, indicating a shared L5 radicular innervation. Thus this muscle represents a useful source of electrical information assisting in the differentiation between peroneal and radicular dysfunction.


H-reflex latency in uremic neuropathy: correlation with NCV and clinical findings.

Sixty-two uremic patients on dialysis of varying durations were tested bilaterally for posterior tibial nerve H-reflex latency, at 3-month intervals. Bilateral nerve conduction velocities (NCVs) of the peroneal, tibial, and sural nerves were concomitantly determined in all subjects. Proprioception sense, vibration perception threshold at the great toes, and deep tendon reflexes at the knee and ankle were determined in all subjects on the day of electrodiagnostic testing. The sensitivity of the H-reflex latency in detection of the onset and severity of uremic neuropathy was assessed. H-reflex latency changes were compared to NCV and clinical test results. The following was found: (1) of the parameters studied, the H-reflex latency appeared to be the most sensitive indicator of early uremic polyneuropathies, (2) electrodiagnostic tests were more sensitive to the onset of neuropathies than the clinical testing parameters studied, and (3) the sural sensory nerve appeared to be involved earlier than peroneal and tibial motor nerves in neuropathies studied.


[Serologic study of 2 strains of foot-and-mouth virus, cultured in cell line IB-RS-2, clone 26-3].

The antigenic characteristics of the two FMDV plaque size variants ASQ-PG and ASQ-Pp were studied by serum neutralization kinetics. The results evidentiated that the antiserum ASQ-PG and ASQ-Pp neutralized both variants at the same extent. The plaque population of these variants after passage in laboratory animals was compared in IB-RS-2 cells.


[Effect of hormones on the susceptibility of the swine cell line IB-RS-2 to foot-and-mouth disease virus].

The actions of hydrocortisone and insulin on the multiplication of foot-and-mouth disease virus were studied. The data obtained showed that the infectivity and the synthesis of the virus nucleic acid as evaluated through the plaque assay method and the kinetics of Uridine-3H incorporation were increased or decreased by hydrocortisone (2 x 10(-6) M). The induction of both effects seems to be related to the carbohydrate metabolism: when the maintenance medium contained glucose or glucose plus calf serum there was an inhibition on virus multiplication and no effect was observed in the absence of both serum and glucose. This was found both when the hormone was added to the cell culture 24 hours before infection as well as when it was added immediately or thirty minutes after virus adsorption. The possibility that these effects could be related to the synthesis of viral RNA-polimerase is discussed. Insulin (50 ng/ml) did not induce any effect on foot-and-mouth disease virus multiplication. However this hormone antagonized the action of hydrocortisone on the infectivity and on the synthesis of foot-and-mouth disease virus nucleic acid.


[Isolation and identification of the infectious bovine rhinotracheitis virus in a bovine fetus kidney (IBR/IPV)].

The authors describe the isolation and identification of a virus from bovine fetal kidney cells maintained in culture for 30 to 45 days. The virus was characterized as a herpesvirus of the infectious bovine rhinotracheitis in cell culture by the evidence of cytopathogenic effect in bovine kidney and testis cultures and serologically by a neutralization test using rabbit anti-infectious bovine rhinotracheitis sera.


[Isolation of foot-and mouth disease virus in swine with other diseases].

The authors took a survey of foot-and-mouth disease samples of myocardium and tonsil from swine which was died without clinicals signs of foot-and-mouth disease, with isolation of virus, type O, A and C. It was observed and accentuated relation between the incidence of hog cholera, pneumonia and atipic foot-and-mouth disease, especially from suckling pigs.


[Effects of polyionic compounds in the plaque formation of different strains of foot-and-mouth disease virus (FMDV) in the swine cell line IB-RS-2].

The effects of dithylaminoethyl (DEAE)-dextran (DD) and dextran-sulfate (DS) were studied in some strains of the foot-and-mouth disease virus (FMDV). The strains ASQ-PG, CR and O, large plaque variants in IB-RS-2 cells, when DD or DS were added to the agar overlay, the plaques were inhibited. However, the strains ASQ-Pp and CI, that are small plaque variants in IB-RS-2 cells, when to the agar overlay was added DD the plaques were greatly enhanced while when was added DS the plaques were inhibited.


A familial mitochondrial myopathy with central defect in neural transmission.

A family was found to exhibit progressive external ophthalmoplegia, congenital cataracts, variable somatic weakness, gonadal dysgenesis, and, in one member, an abnormal chromosomal pattern. Muscle biopsy specimens showed "ragged-red" fibers; electron microscopy showed widespread paracrystalline mitochondrial inclusions. Orbicularis oculi reflex testing in the propositus showed bilateral absence of the late response. The family was evaluated in relation to other familial mitochondrial myopathies; a central defect in brain stem neural transmission was suggested as a mechanism for the progressive external ophthalmoplegia.


[Swine cell sublines with different ploidies. III. Susceptibility to foot-and-mouth disease virus].

IB-RS-10-II subline with tetraploid level cells was more susceptible to the infection by the foot-and-mouth disease virus (FMDV) ASQ-PG strain than IB-RS-10-I subline with diploid level cells, when number and size of plaques and cytopathogenic effect of the virus were used as criteria. Besides, the virus yield in one-cycle of infection was almost the double in IB-RS-10-II than IB-RS-10-I cell subline and the near-tetraploid cells were more susceptible to be infected by the virus than the near-diploid cells. However, in relation to FMDV ASQ-Pp strain the cell subline with a diploid level was more susceptible than that with a tetraploid level. The effects of polyionic compounds under specific experimental conditions were peculiar to each cell subline. 1. Pretreatment of cell monolayers with DEAE-dextran increased the diploid level cells receptivity to the virus, whereas that of the tetraploid level cells was affected only slightly; pretreatment with dextran sulphate did not affect the receptivity of both cell sublines. 2. Pretreatment of the virus with the polycationic compound increased significantly its infectivity to the cells of both sublines; pretreatment of the virus with polyanionic compound decreased significantly its infectivity to the diploid level cells but did not affect that for the tetraploid level cells. 3. Treatment of the agar overlay with the polycationic compound induced increase on the plaque size formed in both cell sublines and treatment with the polyanionic compound inhibited plaque sizes in both cell sublines. A correlation was not found between the susceptibility of both cell sublines to the infection and the chromosomic alterations.


Primary aberrant oculomotor regeneration due to intracranial aneurysm.

Two elderly patients had unilateral ophthalmoparesis and retrobulbar pain. Both had subtle lid signs of aberrant regeneration of the third nerve without proceding acute oculomotor paralysis, and both were found to have intracranial aneurysms. Although primary aberrant oculomotor regeneration has been reported previously in patients with aneurysms and meningiomas, the diagnostic importance of this sign in the elderly has not been emphasized. The presence of lid elevation on downgaze associated with slowly progressive ophthalmoplegia in patients aged 65 or older suggests aneurysm in or near the cavernous sinus.


[Influence of a hypertonic medium on cell susceptibility to foot-and-mouth disease virus].

The influence of hypertonic medium on the relationship between two cell clones of IB-RS-2 swine line and the foot-and-mouth disease virus was studied. Although the number of infected cells was increased by the cell treatment with the hypertonic medium, before or during the time of virus adsorption and penetration onto the cells, showed by the plaque number, the viral replication was partially inhibited as showed by the plaque size as by the viral yield in one-cycle of infection. On the other hand, the cell treatment with that medium after virus adsorption and penetration time inhibited as the plaque number and size as the yield of the foot-and-mouth disease virus.


Topical antibiotic therapy of Pseudomonas aeruginosa keratitis.

The in vivo antibacterial effectiveness in the rabbit cornea of several commercially available ophthalmic antibiotic preparations was determined against a single strain of Pseudomonas aeruginosa isolated from a human corneal ulcer. Each antibiotic was instilled topically at hourly intervals, and the number of residual viable organisms in the cornea subsequently was ascertained. In vivo measurements correlated well with in vitro data and with generally held clinical impressions. Three antibiotics, gentamicin sulfate, polymyxin B sulfate, and colistin sulfate, suppressed corneal growth of P aeruginosa in commercially available concentrations. Gentamicin was slightly more effective than polymyxin B; both drugs were substantially more effective than colistin. Formulations of gentamicin and polymyxin B containing approximately four times the quantity of drug found in commercial preparations eliminated this P aeruginosa strain from the cornea much more rapidly than did the commercial preparations.


Vasopressin and isoproterenol activate adenylate cyclase in the guinea pig inner ear.

Cochleas from guinea pigs were perfused by isotonic buffer after punction of the carotid artery. The cochlea tissue was removed from the bony capsule and separated from the mediolus as band with a sharp needle under the microscope. Cell membranes were prepared subsequently from whole tissue. Purified membranes from the inner ear of guinea pigs contain adenylate cyclase which functionally is coupled with membrane receptors for vasopressin and beta-receptors for isoproterenol (epinephrine), respectively. Both hormones stimulate production of cyclic AMP at 37 degrees C. Furthermore, cyclase activity is increased by addition of Gpp (NH)p, a GTP analog. Possible relationships of these molecular events to cochlear events such as glycogenolysis, ionfluxes, transport and secretion mechanisms, and synaptic transmissions are discussed.


[Viruses, virus-like and virus-related structures in nasopharyngeal carcinoma (author's transl)].

Five cases of nasopharyngeal carcinoma with typically elevated EB-virus-antigen-titer were studied by light- and electron microscopic methods. Within the cytoplasm corona viruses in different forms, regular capsid-like particles with a diameter of 60 nm, tubuloreticular aggregates, and pathologic alterations of the membranes of the endoplasmatic reticulum could be found. The nuclei of the tumour cells often showed particles within the nucleoplasm with an average diameter of 100 nm containing a central core; the appearance of the particles is similar to that of herpes viruses as shown in the literature concerning EB-viruses. Atypic mitoses are common as well as free nucleoplasma-like condensates within the ground cytoplasm. Multiple doubling of the membranes of the endoplasmatic reticulum are characteristic for the tumor cell cytoplasm. Myelin figures are often situated within the enlarged tubules of the E. R., within mitochondria and within the nuclei. The results are discussed concerning the possible viral etiology of nasopharyngeal carcinoma. We do not believe that the particles found within the nucleoplasm have a relation to nuclear pores. The arguments are discussed.


Search for human tumour viruses by transfection: uptake of melanoma and Epstein-Barr virus DNA by human cells.

In a model system, consistent transfection of chick embryo fibroblasts (CEF) by DNA from the XC cell line occurred, with recovery of infectious Rous sarcoma virus. The techniques were then applied in attempts to recover possible human tumour viruses. Even with various modifications of the XC technique, DNA from three human malignant melanoma cell lines failed to infect adult or foetal human fibroblasts, although melanoma DNA was taken up into nuclei of target cells. XC DNA did not transfect human foetal fibroblasts and melanoma DNA was ineffective in CEF. DNA from the Raji (Epstein-Barr virus non-producer) and QIMR-WIL (producer) lymphoblastoid cell lines did not transfect human cord blood lymphocytes or amnion cells. These broadly applicable techniques therefore failed to recover EB virus, the putative melanoma retrovirus, or other potential tumour virus.


The prognosis of breast cancer based on histological assessment.

Histopathology data from 298 patients with breast cancer have been stored in a computer, and features influencing survival have been analysed by the life table method and presented in survival curves. Prognostic features assessed were the size, type, and grade of neoplasm, the amount of stromal elastosis and lymphocytic infiltrate, and the presence and number of axillary metastases. The most reliable indications of short survival are the presence of axillary metastases and a high tumour grade. These two features are correlated, as high-grade tumours are much more likely to have metastases at presentation. No statistical difference is found in the survival of patients with metastases in one or two axillary nodes compared with those with larger numbers involved. There are differences in survival in different types of carcinoma; the well-differentiated infiltrating duct carcinoma with tubular pattern has the best prognosis, followed by the medullary carcinoma. The size of the tumour has some prognostic significance, but this is outweighed by its grade.


Skin temperature changes in paradoxical sleep in man in the cold.

Mean skin temperature (Tsk) calculated from seven sites and rectal temperature (Tre) were recorded every minute for a total of 88 man-nights in eight young men sleeping at night in both cold (during the Artic winter) and neutral (laboratory) environments, and were related to the EEG stages of sleep, especially to paradoxical sleep (PS). In the neutral environment, Tre was always above 36 degrees C and Tsk increased during PS. In the cold conditions, during PS, Tsk increased when Tre was high, and decreased when Tre was below 36 degrees C. It was concluded that, although it is not known why a core temperature of about 36 degrees C is the critical point of change in the direction of Tsk variations during PS, the direction in which Tsk will vary during PS is dependent on the core temperature at the time.


Polymorphonuclear leukocyte-inhibitory factor of Bordetella pertussis. I. Extraction and partial purification of phagocytosis- and chemotaxis-inhibitory activities.

A new factor that inhibited phagocytosis to opsonized targets and chemotaxis of PMN was extracted from B. pertussis cells, and named PMN-inhibitory factor (PIF). Cells in phase I produced 10 times more PIF than those in phase III, and like other phase I-associated components--the hemagglutinin, the histamine-sensitizing factor and agglutinogens--PIF showed degenerative, phenotypic variation during in vitro culture of phase I bacteria. PIF was partially purified by four steps, including adsorption chromatography on Dansyl-aminononamethylene Sepharose. The resulting fraction was heterogeneous but showed little histamine-sensitizing and cytotoxic activities and was free from LPS, the hemagglutinin and a leukocyte agglutinin. The inherent resistance of B. pertussis cells, in either phase I or III, as demonstrated also in the present study, and PIF-mediated defiance against immunological defense mechanism may constitute a complex host-parasite relation in experimental infections with B. pertussis.


The semisynthesis of fragments corresponding to residues 66-104 of horse heart cytochrome c.

We describe the N epsilon-acetimidylation of horse heart cytochrome c with retention of biological activity, the cleavage of the modified protein by CNBr, the separation of the fragments, and their further side-chain protection. We describe the manipulation of the amino acid sequences of the fragments by stepwise semisynthetic methods. We have prepared fragments corresponding to residues 66-78 and 66-79 of the protein, as well as the [Asp66] analogue of fragment 66-79. We have prepared the natural sequence and the [o-fluoro-Phe82] analogue of the fragment corresponding to residues 81-104 of the protein, and the [N epsilon-trifluoroacetyl-Lys79], the [N epsilon-dinitrophenyl-Lys79] and the [S-acetamidomethyl-Cys79] analogues of fragment 79-104, and the [N epsilon-Cbz-Lys81] analogue of fragment 80-104. We have coupled back the fragments of natural sequence to form a semisynthetic fragment corresponding to residues 66-104 of the protein. Modified fragments were also coupled to give analogues of the 66-104-residue sequence. In every case the homoserine residue representing methionine-80 was removed from the C-terminus of the 66-80-residue fragment and replaced by methionine on the N-terminus of the 81-104 residue fragment during the preparation of the fragments for coupling. The semisynthetic fragments are ready for specific deprotection and further coupling. We have coupled one such fragment to the (1-65)-peptide to produce semisynthetic [Hse65]cytochrome c. The product has satisfactory characteristics on chemical analysis, and on assay of its biological activity.


Differences in the degradation of native collagen by two microbial collagenases.

The early stages of degradation of native collagen by two bacterial collagenases were studied by electron microscopy and by automatic Edman degradation. The purified collagenase from Clostridium histolyticum was shown to cleave native collagen at several sites, but not progressively from the N-terminus, as had been previously suggested. The homogeneous collagenase from Achromobacter iophagus cleaves native collagen preferentially at two sites corresponding to the interbands 33-34 and 41-42. The latter lies within the region cleaved by the eukaryotic collagenases.


Calmodulin and myosin light-chain kinase of rabbit fast skeletal muscle.

1. It is confirmed that myosin light-chain kinase is a protein of mol.wt. about 80,000 that is inactive in the absence of calmodulin. 2. In the presence of 1 mol of calmodulin/mol of kinase 80-90% of the maximal activity is obtained. 3. Crude preparations of the whole light-chain fraction of rabbit fast-skeletal-muscle myosin contain enough calmodulin to activate the enzyme. A method for the preparation of calmodulin-free P light chain is described. 4. A procedure is described for the isolation of calmodulin from rabbit fast skeletal muscle. 5. Rabbit fast-skeletal-muscle calmodulin is indistinguishable from bovine brain calmodulin in its ability to activate myosin light-chain kinase. The other properties of these two proteins are also very similar. 6. Rabbit fast-skeletal-muscle troponin C was about 10% as effective as calmodulin as activator for myosin light-chain kinase. 7. By chromatography on a Sepharose-calmodulin affinity column evidence was obtained for the formation of a Ca2+-dependent complex between calmodulin and myosin light-chain kinase. 8. Troponin I from rabbit fast skeletal muscle and histone IIAS were phosphorylated by fully activated myosin light-chain kinase at about 1% of the rate of the P light chain.


Myocardial extraction of cyclic AMP during pacing-induced angina.

The application of cyclic AMP as a marker of myocardial ischemia in humans with coronary artery disease has been investigated during pacing-induced angina. Cyclic AMP was determined by a radioimmunoassay. In 15 patients myocardial lactate extraction at rest (20 +/- 12%) converted to production levels (-30 +/- 23%) during angina (p less than 0.0005). Insignificant changes occurred in coronary venous plasma cyclic-AMP levels. The mean myocardial cyclic-AMP extraction at rest (0.3 +/- 8.7%) converted to small release values (-6.0 +/-11%) during angina (= n.s.). No significant correlation was found between myocardial lactate and cyclic-AMP uptake or release. Therefore, cyclic AMP is an insensitive marker in the evaluation of myocardial ischemia.


Enzymes of myo-inositol and inositol lipid metabolism in rats with streptozotocin-induced diabetes.

Diabetes, with only mild ketosis, was induced in male rats by a single injection of streptozotocin. After 12 weeks the specific activities of enzymes concerned with the metabolism of inositol and of inositol lipids were measured in various tissues. Inositol 1-phosphate synthase (EC 5.5.1.4) was most active in testis and the activity was significantly less in diabetic rats than in controls on a similar diet. Inositol oxygenase (EC 1.13.99.1), which converts myo-inositol into glucuronic acid, was also less active in kidney from diabetic animals. CDP-diacylglycerol-inositol phosphatidyltransferase (EC 2.7.8.11) and phosphatidylinositol 4-phosphate kinase (EC 2.7.1.68) showed decreased specific activities in brain and sciatic nerve of diabetic rats. By contrast the diabetic state did not affect the specific activities of phosphatidylinositol kinase (EC 2.7.1.67) or phosphatidylinositol 4,5-bisphosphate phosphatase (EC 3.1.3.36) in these tissues. The results are discussed in relation to diabetic neuropathy.


Phosphorylation of threonine and serine residues of native and partially dephosphorylated caseins by a rat liver cyclic AMP-insensitive protein kinase.

The preferential phosphorylation of threonine residues of native casein fractions by a rat liver cyclic AMP-independent protein kinase (EC 2.7.1.37) is abolished by preliminary limited dephosphorylation of the substrates, which promotes a fall in the phosphothreonine/phosphoserine ratios from values higher than 1 to much less than 0.1. This finding and the identification of the threonine residues phosphorylated support the view that the liver protein kinase affects threonine residues only when suitable serine residues, which fulfil the structural requirements for attack by the enzyme but which are not yet phosphorylated, are not available.


The cell-wall phosphatase of cotton (Gossypium) is inhibited by kelthane.

Kelthane [4,4'-dichloro-alpha-(trichloromethyl)benzhydrol] was previously shown to decrease the limited tolerance of susceptible varieties of cotton (Gossypium) to Verticillium wilt. Kelthane was shown in the present study to inhibit the cell-wall p-nitrophenyl phosphatase of cotton. In view of information already establishing the cell wall as a primary site of action of Verticillium wilt, the data are interpreted as suggesting an as yet undefined interaction between Kelthane, cell-wall phosphatase and verticillium-resistance mechanisms of the cell wall.


Effect of fluocortin butylester and beclomethasone dipropionate on the adrenal gland in beagle dogs.

The effect of butyl-6 alpha-fluoro-11 beta-hydroxy-16 alpha-methyl-3,20-dioxo-1,4-pregnadien-21-oate (fluocortin butylester, Vaspit) administered intratracheally and of beclomethasone dipropionate given intratracheally or orally on the adrenal glands of beagle dogs was investigated. The adrenal function was evaluated using a standardized ACTH stimulation test including eosinophil counts 5 h after a single i.v. injection of 0.02mg (approximately 2 IU) ACTH/kg body weight were found as well as the histological and morphometrical examination of the adrenal cortex. The cortisol determination (with Clark's method) 1.5 h and eosinophil counts to be the optimum indices for the evaluation of adrenal function. Beclomethasone dipropionate given intratracheally at daily dose levels of 0.05; 0.1 and 0.5 mg/kg body weight lead to a dose dependent adrenal suppression on the basis of plasma cortisol concentration, eosinophil counts after ACTH stimulation and size of zona fasciculata and reticularis. A complete adrenal suppression was observed at the highest dose level of 0.5 mg/kg bw. Also the oral administration of 0.1 mg/kg bw./day of beclomethasone dipropionate had a definite adrenal suppressive effect comparable to that of 0.1 mg/kg bw. given intratracheally. However, intratracheal administration of fluocortin butylester even after a 320 times higher dose (2 X 8 mg/kg bw./day) had no suppressive effect on the adrenal gland of the beagle dog.


[Transplantable experimental tumor. Morphological and ultrastructural study (author's transl].

An experimental tumor which has been induced in newborn chicken, was transplanted into inbred BALB/c mice and then studied by the means of light and electron microscopy. Sendai virus fusion between MSV-H transformed rat cells and chickembryo cells provided the tumor. Histological pattern of this neoplasia seems to be superposable, both in chicken and in mouse, to that of anaplastic fibrosarcomas and shows some differences from either Rous sarcoma or BALB/c mouse H-6668 spontaneous rabdomyosarcoma patterns. tumor malignancy was evaluated onthe basis of rapid growth


Species differences in the activity of a serum triglyceride transferring factor.

Low density lipoproteins (LDL), endogenously labelled with 3H in the triglyceride moiety, were isolated from rabbit serum and subsequently incubated in vitro at 37 degrees C with unlabelled preparations of rabbit high density lipoproteins (HDL) or very low density lipoproteins (VLDL). In incubations performed in the presence of phosphate buffer, there was no significant transfer of [3H]triglyceride from LDL to either HDL or VLDL; but when rabbit lipoprotein-free serum (the dialysed 1.21 g/ml infranatant) was added, transfer was apparent to both HDL and VLDL. The triglyceride transferring activity of the lipoprotein-free serum was abolished by heating at 85 degrees C for 10 min; all the transferring activity was found in the fraction which precipitated with ammonium sulphate at a concentration of less than 50% saturation. In direct contrast to the rabbit studies, rat serum failed to show a comparable process of triglyceride transfer. In subsequent experiments, mixtures of labelled LDL and unlabelled VLDL isolated either from rabbits or from rats were incubated with lipoprotein-free rabbit, rat or human serum. The lipoprotein-free serum of both the rabbit and man was effective in promoting transfer of 30--50% of LDL [3H]triglyceride into VLDL, regardless of the species origin of the lipoproteins. By contrast the lipoprotein-free serum of rats was only slightly more effective than buffer alone in promoting such transfers. It has been concluded that rabbit and human serum contains a triglyceride transferring factor of far greater activity than that in rat serum.


Prostacyclin-dependent differences in TXA2 formation by platelets from normal and atherosclerotic rabbits.

Intact platelets from atherosclerotic rabbits release more thromboxane B2 (TXB2) and 12-hydroxyeicosatetraenoic acid (HETE) than platelets from normal rabbits following incubation with [14C]arachidonic acid (AA). In contrast, no difference is found between homogenates of platelets from normal and atherosclerotic animals. PGI2 stimulates cAMP accumulation and inhibits TXB2 formation more potently in intact platelets from atherosclerotic than from normal rabbits, but has no effect on AA metabolism in homogenised platelets.


High density lipoprotein cholesterole levels in peripheral vascular disease and in women on oral contraception.

Male patients with peripheral vascular disease (PVD) had significantly lower high density lipoprotein cholesterol (HDL-Ch) levels than their corresponding controls, while no significant difference could be demonstrated for female patients with PVD. No significant difference of HDL-Ch was found between women who used oral contraception and those who did not.


[Avoidance learning in rats treated with estrogens].

Male and female rats were treated with estradiol benzoate in the 5 degrees day of life. The avoidance conditioning, tested after 65 days, was significatively impaired in males treated with estradiol, a significative retardation was observed in females treated, but no significant end-difference versus no-treated females. It is supposed that a hormone induced synaptic shaft/spines ratio in preoptic area may be involved in avoidance conditioning.


A light and electron microscope study on the origin of Foà-Kurloff cells.

Numerous Foà-Kurloff (FK) cells have been found in the circulation, spleen, bone marrow, thymus and placenta of guinea pigs under endogenous or exogenous oestrogenic stimulation. The origin of these cells is obscure. In the present experiment, the distribution of FK cells in organs other than those stated above was studied following gonadectomy and hexoestrol administration in guinea pigs of both sexes for either 1 or 10 weeks. More FK cells than those expected from the vascularity of the organ were found in the liver and lungs of male and female animals. The number of FK cells in these organs was larger after the long-term treatment with hexoestrol and it was accompanied by a significant decrease in the number of Kupffer cells of the liver. The latter observation is in contrast to previous reports of increased numbers of Kupffer cells in the liver of oestrogen-receiving mice, a species not producing FK cells. These observations suggest a relation between the two types of cells. No transformation of mature Kupffer cells into FK cells was seen with the electron microscope. However, other findings suggest that both Kupffer cells and FK cells may well be derived from a common precursor of MPS in the bone marrow.


The nature of the effector cells of cell-mediated immune responses to Sendai and Kunz virus infections in mice.

The development of virus-specific cell-mediated immune (CMI) responses to Sendai and Kunz virus infections of the respiratory tract of mice is described. In addition a heterotypic cell-mediated immune response develops during each of the virus infections. Cell depletion studies indicate that the effector cells of the virus-specific CMI responses are theta antigen bearing T lymphocytes. Similar studies of the heterotypic responses suggest that 2 cell types are involved, T lymphocytes and NK cells.


Proliferative epidermis of essential fatty acid deficient mice has reduced cyclic AMP content.

We have simultaneously measured epidermal DNA synthesis, epidermal thickness and cyclic nucleotide levels in essential fatty acid (EFA) deficient hairless mice skin. In vitro epidermal DNA synthesis and epidermal thickness were significantly increased in EFA deficient epidermis. Cyclic AMP levels were significantly reduced when expressed using three different data bases. Cyclic GMP levels were increased using two data bases but these increases were not statistically significant; Cyclic AMP/cyclic GMP ratios were significantly decreased in the EFA deficient skin. The results provide evidence that cyclic nucleotide levels are altered in an animal model of benign epidermal proliferation.


Modulation of transferrin receptors in bone marrow cells by changes in lipid fluidity.

The hypothesis that modulation of transferrin receptors during haematopoiesis is mediated by changes in the lipid fluidity of the cell membranes was tested in this study with bone marrow cells. Binding conditions were first established, under which internalization of fluorescently-labelled transferrin was reduced to a minimum during the binding assays. Cholesterol depletion, which was achieved by a mild physiological treatment, and which increased the membrane-lipid fluidity, resulted in a substantial increase in the average number of the available transferrin receptors per bone marrow cell. Cholesterol enrichment mediated a converse effect. These findings correlate well with the changes observed in the availability of transferrin receptors and membrane microviscosity during differentiation of erythroid cells. In line with the notion of passive modulation, it is proposed that the transferrin receptor is a cross-membrane protein with a substantial portion facing the cytoplasm.


Sex hormone binding globulin capacity and postmenopausal hormone replacement therapy.

The sex hormone binding globulin (SHBG) capacity was measured in 26 normal untreated postmenopausal women and 10 postmenopausal women taking different types of hormone replacement therapy. The patients on hormone replacement therapy had significantly higher levels of SHBG than postmenopausal women (p less than 0.001) and also significantly higher levels than 52 normal ovulating women studied previously (Pogmore and Jequier, 1979; p less than 0.02). This suggests that postmenopausal women on hormone therapy are being overtreated.


Tumours of the salivary glands: a histological and clinical review.

The aim of this paper is to try to identify points of correlation between the histological appearance and the clinical behaviour of minor salivary gland tumours. To this end 62 well-documented minor salivary gland tumours have been followed up for at least five years or until recurrence. The discussion is limited to the adenoidcystic carcinoma, the pleomorphic adenoma and the basal cell adenoma, since only these three tumours were represented in sufficient numbers to be analysed. For each tumour a number of histological pointers to prognosis are reviewed. In the case of the basal cell adenoma, a recently recognised tumour, the possibility of confusion with the adenoidcystic carcinoma is discussed.


Chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes: evidence that the two reactions occur at one active site.

The relationship between the sites for catalysis of two reactions by the bifunctional enzyme chorismate mutase--prephenate dehydrogenase has been investigated. The results are consistent with the occurrence of both reactions at one active site. Comparisons have been made between experimental data for the time course of the overall reaction and computer simulations, according to various models for the relationship between the mutase and dehydrogenase sites. A model based on a single active site is consistent with the time course data if a minor proportion of the chorismate that reacts can be converted through to (hydroxyphenyl)pyruvate without the intermediate release of prephenate. Consistent with this requirement, some channeling of radioactivity from chorismate to (hydroxyphenyl)pyruvate has been detected. A model based on two separate sites has also been considered; the simulations show that if this model applies there is no need to postulate any channeling of the intermediate, prephenate, between the sites and there must be marked inhibition of the dehydrogenase reaction by chorismate. Since channeling has been observed and chorismate increases the dehydrogenase rate under all conditions, the two-site model appears unlikely. Consistent with the one-site model are the observations that a variety of inactivating conditions cause parallel loss of mutase and dehydrogenase activity and that identical protection against inactivation of both mutase and dehydrogenase by iodoacetamide is afforded by prephenate.


Proton nuclear magnetic resonance study of the conformation and configuration of the cyclized pyridine nucleotide adducts.

We have closely examined by high-frequenty 1H nuclear magnetic resonance spectroscopy the structure of the adducts which form when various carbonyl compounds react with pyridine nucleotides at elevated pH. These studies show that the adducts of N-(2,6-dichlorobenzyl)nicotinamide-acetone, n-(2,6-dichlorobenzyl)nicotinamide-pyruvate, NMN-pyruvate, NAD-pyruvate, NAD-acetaldehyde, and NAD-oxaloacetate form with identical structural features as well as configuration. The following structural features are observed: (1) the adducts are pyridine N-4-substituted compounds; (2) a second six-membered ring forms by addition of the nicotinamide amido to the carbonyl group of the compound forming the addition complex; (3) cyclization occurs stereospecifically, indicating that the stereochemistry is predetermined by the initial attack at the N-4 position; (4) two diastereomeric forms are observed for each nucleotide adduct. Finally, the determination of configuration at all symmetric carbon atoms in these adducts will be discussed.


Cross-linking of iodine-125-labeled, calcium-dependent regulatory protein to the Ca2+-sensitive phosphodiesterase purified from bovine heart.

The calcium-dependent regulatory protein (CDR).Ca2+ sensitive cyclic nucleotide phosphodiesterase was purified to apparent homogeneity from bovine heart by using ammonium sulfate fractionation, DEAE-ceelulose chromatography, and CDR-Sepharose affinity chromatography. The enzyme was purifed 13 750-fold with a 10% yield and a specific activity of 275 mumol of cAMP min-1 mg-1. The purified enzyme ran as a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 57 000. Phosphodiesterase activity was stimulated 10-fold by Ca2+ and CDR with half-maximal activation occurring at 9 ng/assay. [125I]CDR was cross-linked to the purified phosphodiesterase by using dimethyl suberimidate Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cross-linked products revealed a number of discrete 125I-labeled bands. The molecular weights of the cross-linked products indicate that the stoichiometry of the phosphodiesterase complex is A2C2, where A is the phosphodiesterase catalytic subunit and C is the calcium-dependent regulatory protein.


Stereochemical course of the reaction catalyzed by 5'-nucleotide phosphodiesterase from snake venom.

The hydrolysis reaction of ATP alpha S by snake venom phosphodiesterase is highly specific for the B diastereomer and proceeds with 88% retention of configuration at phosphorus. Since this enzyme also catalyzes the hydrolysis of the S enantimoer of O-p-nitrophenyl phenylphosphonothioate, the absolute configuration at A alpha of ATP alpha S (B) is assigned as the R configuration provided the two substrates are processed identically. A mechanism for the hydrolysis reactions catalzyed by the venom phosphodiesterase involving at least a single covalent phosphoryl-enzyme intermediate is in accord with this result.


Magnesium ion dependent rabbit skeletal muscle myosin guanosine and thioguanosine triphosphatase mechanism and a novel guanosine diphosphatase reaction.

The mechanism of the Mg2+-dependent myosin subfragment 1 catalyzed hydrolysis of GTP and 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-triphosphate (thioGTP) has been investigated by rapid-reaction techniques. The myosin was isolated from rabbit skeletal muscle. The steady-state intermediate of these reactions consists pre-dominantly of a protein-substrate complex unlike the myosin subfragment 1 ATPase reaction which has a protein-products complex as the principal steady-state component. The mechanism of GTP hydrolysis catalyzed by subfragment 1 has other marked differences from the ATPase mechanism. The second-order rate constant of binding of GTP to subfragment 1 is tenfold greater than that for GDP binding. The dissociation rate constant of GDP from subfragment 1 is 0.06 s-1 compared with the subfragment 1 catalytic center activity for GTP hydrolysis of 0.5 s-1 at pH 8.0 and 20 degrees C. This shows that GDP bound to subfragment 1 forms a complex which is not kinetically competent to be an intermediate of the GTPase mechanism. GDP is hydrolyzed in the presence of subfragment 1 to GMP and Pi. The subfragment 1 GTPase mechanism has a nuber if features in common with that of the elongation factor Tu GTPase of the protein biosynthetic system of Escherichia coli.


Allosteric cooperative interactions among redox sites of Pseudomonas cytochrome oxidase.

Anaerobic reductive spectrophotometric titrations of Pseudomonas aeruginosa cytochrome oxidase were performed. Both types of hemes (C and D) of the dimeric enzyme were monitored. The reduction process was found to involve cooperative allosteric and spectroscopic interactions between the two subunits. The model fitting the data best involves the following features. (1) The redox potential of heme C is about 60 mV higher than that of heme D. (2) In the electron uptake, a positive cooperativity of about 30 mV exists between the two D-type hemes residing in the two subunits. (3) A negative cooperativity of the same magnitude (30 mV) is found between the two C-type hemes bound to two subunits. (4) No interaction was found between heme C and D in the same subunit or in the different subunits. (5) It is suggested that the reduction of the heme, of each kind, has about twice the spectral change compared to that observed upon reduction of the second one. The possible significance of this model for the mechanism of action of the enzyme is discussed


Influence of temperature and cholesterol on the rotational diffusion of band 3 in the human erythrocyte membrane.

Band 3 rotation in the human erythrocyte membrane is measured by observing flash-induced dichroism of eosin probes. The decay of the absorption anisotropy is found to be strongly dependent on temperature. The results are analyzed on the assumption that rotation of band 3 only occurs about the membrane normal. It is deduced that both fast and slowly rotating forms of band 3 coexist in the membrane. The equilibrium between these forms is temperature dependent, the slowly rotating species becoming increasingly dominant as the temperature is reduced. Plots of the fractional distribution of the different species against temperature show a marked change of slope at around 37--40 degrees C. The effects are essentially reversible over the range 1--45 degrees C and independent of the presence of the spectrin--actin network. The results could be due to temperature-dependent protein--protein associations mediated either by a protein conformational change or by lipid phase segregation. In further experiments, the cholesterol content of the erythrocyte membrane is varied by incubation with lipid vesicles. No significant changes in the rotational diffusion of band 3 are observed following variation of membrane cholesterol/phospholipid mole ratios over the range 0.34--1.66. This is a surprising result in view of the well-known effects of cholesterol on lipid fluidity.


Microsecond luminescence emission from copper cytochrome c.

The luminescence of Cu-substituted cytochrome c exhibits temperature-dependent decay modes and spectral shifts at temperatures below 80 K. By comparison with the data from metal-substituted porphyrins, we determined intramolecular rate parameters. The observed tripdoublet decay time is 13 +/- 1 mus at 77 K and the quartet decay time is 12 +/- 5 mus below 30 K. We also investigated the luminescence emission from Cu cytochrome c in the presence of cytochrome c depleted mitochondria and in the presence of soluble cytochrome c oxidase; upon binding, the intensity and decay time of the emission are altered. The results indicate an interaction between cytochrome c and its mitochondrial binding site which depends on the electronic state of cytochrome c.


Binding of reduced and oxidized nicotinamide adenine dinucleotide to pig heart supernatant malate dehydrogenase.

The association reactions of NADH and NAD+ with dimeric pig heart supernatant malate dehydrogenase (s-MDH) have been measured at pH 6 and 8 by calorimetric and fluorescence methods, and the thermodynamic parameters describing these reactions have been evaluated. Coenzyme binding is associated with the uptake of 0.55 mol of H+/mol of NADH at pH 8 and 0.19 mol of H+ at pH 6. No significant effect of NAD+ binding on proton binding was observed. Increase in ionic strength strongly affects the free energies of binding of NAD+ and NADH. No cooperativity was observed in the enthalpy or free energy changes for binding of NAD+ or NADH. The differences in free energy of binding of NAD+ and NADH and the effect of pH on binding of NADH are entropy based. These effects are interpreted as reflecting a small number of interactions within the active site that are predominantly ionic.


Purification and subunit structure of nicotinamide adenine dinucleotide specific isocitrate dehydrogenase from Neurospora crassa.

Neurospora crassa nicotinamide adenine dinucleotide specific isocitrate dehydrogenase (EC 1.1.1.41) has been purified to homogeneity by the criteria of disc gel electrophoresis and sedimentation equilibrium. Purification of the enzyme is facilitated by the presence of phenylmethanesulfonyl fluoride and by the use of a ribose-linked adenosine 5'-monophosphate affinity column. The enzyme appears to be composed of nonidentical subunits of molecular weights 42 800 and 38 300 as estimated by polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate. From the intensity of each band and the native molecular weight, it is concluded that the enzyme is composed of either six or eight subunits, three or four of each type, respectively. The availability of pure enzyme will allow clarification of the structure of the enzyme by ligand binding studies.


Mechanism of phenylalanyl-tRNA synthetase of Escherichia coli K. 10. Modulation of catalytic properties by magnesium.

The association of phenylalanylptRNA and Mg2+ follows a biphasic concentration dependence as indicated by the active site directed fluorescent indicator 2-p-toluidinyl-naphthalene-6-sulfonate. The macroscopic dissociation constants are 0.16 +/- 0.03 and 4.1 +/- mM. The effect of Mg2+ on the association of enzyme and MgATP, on the synergistic binding of MgATP and L-phenylalaninol, and on the pre-steady-state synthesis and pyrophosphorolysis of the enzyme-phenylalanyladenylate complex in the absence and the presence of tRNA Phe has been measured by established equilibrium and stopped-flow techniques using 2-p-toluidinylnaphthalene-6-sulfonate. At 10 mM Mg2+, the association of enzyme and MgATP is biphasic with dissociation constants of 0.25 +/- 0.03 and 9.1 +/- 1.7 mM. At 2 mM Mg2+, a single dissociation constant of 5.0 +/- 0.5 mM is indicated. The coupling constant of the synergistic reaction is 15 at 1 mM Mg2+ and 290 at 10 mM Mg2+. The Hill constant of the sigmoidal dependence is 3.6. The strengthening of the synergism is believed to reflect a Mg2+-dependent coupling of the synergistic reactions at the two active sites of the enzyme, the coupling being negligible at 1 mM and maximal at 10 mM Mg2+. The pre-steady-state rate of adenylate synthesis is accelerated by the presence of Mg2+. The effect is to decrease the value of the Michaelis-Menten constant of MgATP. Another effect is to increase the rate constant when tRNA Phe is present. At subsaturating [MgATP], the [Mg2+] dependence of the observed rate constant is hyperbolical in the absence and sigmoidal (Hill constant, 3.5) in the presence of tRNA Phe. The rate of the pyrophosphorolysis is enhanced by a decrease of the Michaelis-Menten constant of MgPPi. The effects on the thermodynamics and kinetics parallel the occupancy of the low-affinity Mg2+-binding sites of the enzyme.


Characterization of a novel collagen chain in human placenta and its relation to AB collagen.

A novel collagen chain, termed alpha C, has been isolated from human placenta by limited pepsin digestion. The collagen containing the alpha C chain copurifies with placental AB collagen during selective salt precipitation but is virtually absent from fetal birth membranes, which contain relatively larger amounts of AB. Both native AB and alpha C-containing collagens are resistant to human skin collagenase under conditions that support cleavage of type I by greater than 90%. The alpha C chain was separated from alpha B by phosphocellulose chromatography and subsequently from alpha P by chromatography on CM-cellulose. Its amino acid composition is distinct from alpha A and alha B although all three chains posses compositional features in common; the carbohydrate content of the alpha C chain was intermediate between those of alpha A and alpha B. Analysis by NaDodSO4-polyacrylamide gel electrophoresis of peptides produced by CNBr cleavage and by limited digestion with the enzyme mast cell protease indicated different and unique products for the alpha A, alpha B, and alpha C chains. The data support the existence of another collagen chain which is related to the alpha A and alpha B chains but which is structurally unique. The proteins containing these chains may in turn comprise a subfamily of collagen isotypes which represents a divergence from and/or specialization of the type IV basement membrane collagens.


Interaction of a paramagnetic analogue of oxaloacetate with citrate synthase.

Electron paramagnetic resonance studies have indicated that nitrosodisulfonate binds to pig heart citrate synthase. Titration of the enzyme with nitrosodisulfonate revealed several binding sites for the probe per subunit with one site (KD approximately 0.1 mM) having a greater affinity than the others. The substrate, oxaloacetate, competed very effectively for one of the nitrosodisulfonate binding sites (KD less than 10(-2) mM) at the same time eliminating the weaker probe binding sites. Citrate and (R)- and (S)-malates also displaced the probe. Failure to resolve low- and high-field shoulder in the high gain--high modulation electron paramagnetic resonance spectra of the enzyme--nitrosodisulfonate system indicated that the bound probe was "weakly immobilized". However, the electron paramagnetic resonance spectrum of the bound probe changed to one typical of a "strongly immobilized" nitroxide upon the addition of a saturating concentration of the substrate acetyl coenzyme A (acetyl-CoA) to the enzyme--nitrosodisulfonate system, indicating the formation of a ternary acetyl-CoA-enzyme-probe complex. Titration of the acetyl-CoA saturated enzyme with the probe indicated one binding site per subunit (KD = 0.37 mM). Thus, nitrosodisulfonate may be considered as a paramagnetic analogue of oxaloacetate in its interaction with citrate synthase. These results are compared with our previous studies with this enzyme, employing a spin-labeled acyl coenzyme A (acyl-CoA) derivative [Weidman, S. W., Drysdale, G. R., & Mildvan, A. S. (1973) Biochemistry 12, 1874--1883].


Isolation and characterization of cardiac sarcolemma.

A procedure was developed for the isolation of cardiac sarcolemmal vesicles. These vesicles are enriched about ten-fold (with respect to the tissue homogenate) in K+-stimulated p-nitrophenylphosphatase, (Na+ + K+)-ATPase, 5'-nucleotidase activities and sialic acid content, all of which are believed to be components of the sarcolemma. The sarcolemma of tissue culture cardiac cells were radioiodinated and the distribution of this radioiodine paralleled the distribution of the other membrane markers above. There was very little contamination of the sarcolemmal fraction by sarcoplasmic reticulum (as judged by Ca2+-ATPase and glucose-6-phosphatase activities) or inner mitochondrial membranes (as judged by succinate dehydrogenase activity). There may, however, be some contamination by outer mitochondrial membranes (as judged by monoamine oxidase and rotenone-insensitive NADH cytochrome c reductase activities) which have rarely been monitored in cardiac sarcolemmal preparations. The purity of this preparation is good when compared with other cardiac sarcolemmal preparations. This preparation should be very useful in studying the roles of the cardiac sarcolemma (e.g. in excitation contraction coupling and Ca2+ binding).


Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12.

Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43 degrees C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20 degrees C, but in a liquid crystalline state when cells were grown at 37 and 43 degrees C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.


Synaptic membrane proteins as substrates for cyclic AMP-stimulated protein phosphorylation in various regions of rat brain.

Synaptosomal plasma membranes from mammalian brain contain protein kinase activity which phosphorylates endogenous membrane proteins and is stimulated by cyclic AMP. Using polyacrylamide gel electrophoresis it was shown that at least ten proteins in the synaptosomal plasma membrane fraction could be phosphorylated by endogenous cyclic AMP-stimulated protein kinase activity. The number of proteins whose phosphorylation was stimulated by cyclic AMP was strongly influenced by the pH and Mg2+ concentration used in the phosphorylation reaction. A complex pattern of cyclic AMP-stimulated protein phosphorylation was obtained only with synaptosomal plasma membranes and a crude microsomal fraction. Mitochondrial and myelin fractions exhibited no cyclic AMP-stimulated protein kinase activity. Investigation of the distribution of substrates for cyclic AMP-stimulated phosphorylation among various brain regions failed to reveal any regional differences.


Direct photoaffinity labeling of the primary region of the ouabain binding site of (Na+ + K+)-ATPase with [3H]ouabain, [3H]digitoxin and [3H]digitoxigenin.

The tritiated cardiotonic steroids, ouabain, digitoxin, and digitoxigenin are shown to photolabel the large polypeptide but not the glycoprotein or proteolipid component of the (Na+ + K+)-ATPase when they are bound to the inhibitory site and exposed to light of 220 or 254 nm. The extent of photolabeling is low, less than 1%, and is limited by photocross-linking of the enzyme. The mechanism of photoincorporation does not appear to be either photolysis of the lactone ring in ouabain or photolysis of tryptophan or tyrosine residues in the polypeptide.


The interaction of 3,3',4',5-tetrachlorosalicylanilide with phosphatidylcholine bilayers.

1. The interaction of the germicide 3,3',4',5-tetrachlorosalicylanilide (T4CS) with vesicles and dispersions of egg phosphatidylcholine has been studied by gel permeation chromatography, electron microscopy, electron spin resonance spin labelling and ion permeability measurements. 2. Incorporation of T4CS into vesicles of egg phosphatidylcholine gives rise to a large increase in the permeability rate of the paramagnetic cation N,N-dimethyl-N-(1'-oxyl-2',2',6',6'-tetramethyl-4'-piperidyl)-2-hydroxyethylammonium chloride through the lipid bilayer but has no significant effect on the vesicle sizes as measured by gel permeation chromatography or electron microscopy. 3. ESR studies using a spin-labelled fatty acid have demonstrated the presence of two different environments for the spin label when T4CS is incorporated into phosphatidylcholine bilayers. These two environments are identified as (a) highly ordered areas of the bilayer, rich in T4CS and (b) areas with very similar ordering to that in pure egg phosphatidylcholine. 4. The effectiveness of very low concentrations of the germicide in increasing vesicle permeability is explained in terms of its clustering to give rigid patches, rich in T4CS, rather than being evenly distributed throughout the bilayer. It is proposed that the increased ion permeability arises from leakage at the interfaces between the rigid and flexible regions of the lipid bilayer. 5. Comparisons between the effective levels of T4CS in phosphatidylcholine vesicles and its minimum inhibitory concentration with a Gram-positive bacterium confirm the validity of phospholipid vesicles as a model for studies of germicidal activity.


Smooth muscle cell sarcolemma. Purification and properties of plasma membranes from the rat uterus.

A membrane fraction with sarcolemmal properties was purified from the smooth muscle layers (myometrium) of rat uterus by successive differential and equilibrium centrifugation in sucrose. The putative sarcolemmal fraction was identified by iodination with [125I]iodosulfanilic acid, had an equilibrium density of 1.15, and was enriched in enzyme activities usually associated with the plasma membrane including 5'-nucleotidase (EC 3.1.3.5) and (Na+ + K+) ATPase (EC 3.6.1.3). These membranes were free of mitochondrial or nuclear membrane contamination, suggesting the relative enrichment of sarcolemmal membranes in the fraction. Proteins of the membranes were heterogeneous with respect to molecular weight, but only a few were labelled when intact muscle was radioiodinated. Uniform resistance of sarcolemmal proteins to trypsin digestion and salt extraction suggested many are tightly bound or intrinsic membrane proteins and was a further indication of the homogeneity of membranes in this fraction.


Isolation of neuronal plasma membranes from the crayfish Procamburus clarkii, with an aqueous two phase polymer system followed by sucrose density gradient centrifugation.

An aqueous two phase polymer system (Dextran-polyethyleneglycol system was developed for isolation of plasma membrane fraction from nerves of the crayfish, Procamburus clarkii. The polymer system effectively reduced both mitochondrial and endoplasmic reticulum marker enzyme activity from a crude membrane fraction. The similar enrichment of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) was shown by the polymer system as well as by the sucrose density gradient centrifugation. The purified plasma membrane fraction (PM) was obtained using the polymer system followed by sucrose density gradient centrifugation. The PM fraction had a high specific activity of (Na + K+)-ATPase of up to 17 times that in the homogenate, with smaller contamination by mitochondria and endoplasmic reticulum enzyme activities than any other membrane fraction. Electron micrographs of the PM fraction also supported the above evidences. The protein recovered from the PM fraction amounted to 1.1% of the total protein in the homogenate. The specific activity of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) in the membrane fractions was less increased than that of (Na+ + K+)-ATPase. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis suggested that polypeptide chains of estimated molecular weight 115,000 and 31,000 were enriched in the plasma membranes of the crayfish nerves.


Purification and partial characterization of aldehyde dehydrogenase from human erythrocytes.

Human erythrocyte aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) was purified to apparent homogeneity. The native enzyme has a molecular weight of about 210,000 as determined by gel filtration, and SDS-polyacrylamide gel electrophoresis of this enzyme yields a single protein and with a molecular weight of 51,500, suggesting that the native enzyme may be a tetramer. The enzyme has a relatively low Km for NAD (15 microM) and a high sensitivity to disulfiram. Disulfiram inhibits the enzyme activity rapidly and this inhibition is apparently of a non-competitive nature. In kinetic characteristic and sensitivity to disulfiram, erythrocyte aldehyde dehydrogenase closely resembles the cytosolic aldehyde dehydrogenase found in the liver of various species of mammalians.


Subunit interactions in rabbit-muscle glyceraldehyde-phosphate dehydrogenase, as measured by NAD+ and NADH binding.

1. The binding parameters for NADH and NAD+ to rabbit-muscle glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) have been measured by quenching of the flourescence of the protein and the NADH. 2. The fact that the degree of protein fluorescence quenching by bound NAD+ or NADH, excited at 285 nm and measured at 340 nm ('blue' tryptophans), is not linearly related to the saturation functions of these nucleotides, leads to a slight overestimation of the interaction energy and an underestimation of the concentration of sites, if linearity is assumed. 3. This is also the case for NADH, but not for NAD+, when the protein fluorescence is excited at 305 nm and measured at 390 nm ('red' tryptophans). 4. The binding of NAD+ can be described by a model in which the binding of NAD+, via negative interactions within the dimer, induces weaker binding sites, with the result that the microscopic dissociation constant is 0.08 microM at low saturation and 0.18 microM for the holoenzyme. 5. The binding of NADH can be described on the basis of the same model, the dissociation constant at low saturation being 0.5 microM and of the holoenzyme 1.0 microM. 6. The fluorescence of bound NADH is not sensitive to the conformational changes that cause the decrease in affinity of bound NAD+ or NADH. 7. The binding of NAD+ to the 3-phosphoglyceroyl enzyme can be described by a dissociation constant that is at least two orders of magnitude greater than the dissociation constants of the unacylated enzyme. The affinity of NAD+ to this form of the enzyme is in agreement with the Ki calculated from product inhibition by NAD+ of the reductive dephosphorylation of 1,3-diphosphoglycerate.


Determination of binding parameters of cyclic AMP and its analogs to cyclic AMP-dependent protein kinase by the fluorescent probe method.

The method for determination of dissociation constants for cyclic AMP and its analogs bound to cyclic AMP-dependent protein kinase from pig brain is described. The technique for measuring the binding parameters of the ligands is based on the changes in the fluorescent spectrum of etheno cyclic AMP once it is bound to protein kinase. The dissociation constants for a number of nonfluorescent cyclic AMP analogs were determined in the competitive displacement of etheno cyclic AMP by these analogs. The number of cyclic AMP-binding sites in the pig brain protein kinase was found to be 2.2; no cooperativity was observed upon binding. The holoenzyme complex (Mr = 180,000) of the protein kinase under study was established to have the stoichiometry of R2C2 type under native conditions.


The role of ferrichrome reductase in iron metabolism of Ustilago sphaerogena.

Ferrichrome, the ferric ionophore for Ustilago sphaerogena, can serve as a source of iron for the enzyme ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) in this organism, but only after enzymatic removal of the iron from its carrier. U. sphaerogena contains a specific ferrichrome reductase (NADH:ferrichrome oxidoreductase) which catalyzes cellular dissociation of the complex by reduction of the metal to the ferrous state. A spectrophotometric assay was developed based on trapping of the ferrous ion produced by ferrozine. There is an apparent inhibition by oxygen which is thought to be due to re-oxidation of the metal under the assay conditions. The close structural analogue, ferrichrome A, is not a substrate, nor is the ester type siderochrome ferric hexahydro-N,N',N"-triacetylfusarinine C. Aluminum desferriferrichrome is inhibitory. The importance of this enzyme for the metabolism of iron in this organism is discussed.


Improved in vitro light activation and assay systems for two spinach chloroplast enzymes.

An improved system for the in vitro light activation of the chloroplast enzymes fructose-1,6-bisphosphatase and NADP-dependent malate dehydrogenase is described. Through the presence of the monothiol beta-mercaptoethanol in the reaction mixtures the activated forms of the enzymes can be stabilized and their activity determined spectrophotometrically.


Characterization of undecaprenol kinase from Lactobacillus plantarum.

A membrane-bound undecaprenol kinase from Lactobacillus has been identified by observing the ATP-dependent phosphorylation of [14C]undercaprenol. The product of this reaction was shown to be [14C]undecaprenyl monophosphate by comparison of its chromatographic mobilities with authentic undecaprenyl monophosphate. It was shown that 32P from [gamma-32P]ATP was incorporated into undecaprenyl monophosphate. The kinase was partially solubilized by a variety of methods utilizing Triton X-100. Both the membrane-associated and solubilized enzymes required Mg2+, Triton X-100 and dimethylsulfoxide for activity. The enzyme preferentially phosphorylated the C34, C50 AND C 55 polyprenols. Geranylgeraniol (C20) and dolichol (C100), however, were utilized only 6% and 13% as well as undecaprenol, respectively. Despite the 8-fold difference in apparent V values, the apparent Km values for dolichol and undecaprenol were both 14 microM. The apparent Km for the nucleotide cosubstrate, ATP, was 2 mM. No other nucleoside triphosphate could substitute for ATP.


Purification and characterization of a 15-ketoprostaglandin delta 13-reductase from bovine lung.

15-Ketoprostaglandin delta 13-reductase from bovine lung has been purified using affinity chromatography to apparent homogeneity, as judged from polyacrylamide gel electrophoresis with and without sodium dodecyl sulphate. Valine was identified as tne N-terminal aumino acid, and the isoelectric point was estimated at pH 7.8. Molecular weights of 56,000 and 39,500 were found by the use of gel filtration and SDS-polyacrylamide gel electrophoresis, respectively. The enzyme was found to be specific for the 15-keto group, thus 15-ketoprostaglandin E4 (apparent Km = microM) is a substrate, in contrast to prostaglandin E1. The enzyme was active with both NADH (apparent Km = 88--94 microM) and NADH (apparent Km = 5--9 microM) as coenzyme, but the V max with NADH was more than twice that obtained with NADPH. The enzyme did not catalyze the reversed reaction: 13,14-dihydro-15-keto-prostaglandin E1 to 15-ketoprostaglandin E1. The turnover number of the enzyme was determined to be either 60 or 42 min-1. The low value of the turnover number is compensated by a high concentration (96.4 mU/g tissue) of the enzyme in lung tissue, resulting in a high metabolic capacity. Thus, 15-ketoprostaglandin delta 13-reductase together with 15-hydroxyprostaglandin dehydrogenase ensures an irreversible catabolism of prostaglandins.


Regulation of lipogenesis in adipocytes. Independent effects of thyroid hormones, cyclic AMP and insulin on the uptake of deoxy-D-glucose.

Thyroidectomy is known to enhance fat cell phosphodiesterase activity; as a result, the response to lipolytic hormones is markedly reduced. Thyroidectomy also stimulates overall lipogenesis and the uptake of glucose: the present experiments investigated whether there was a correlation between cyclic AMP and glucose uptake. The parameter measured was the transport and phosphorylation (uptake) of deoxy-D-glucose in the presence of two modifiers of the cyclic AMP pool: phosphodiesterase inhibitors and the analogue, dibutyryl cyclic AMP. The inhibition by methylxanthines and dibutyryl cyclic AMP of deoxy-D-glucose uptake observed, was the same in fat cells from normal and thyroidectomized rats: the latter nonetheless still maintained their enhanced glucose uptake. It was therefore concluded that thyroid hormones and cyclic AMP control this step by different, separate pathways. Insulin, well known for its lipogenic effect, enhanced deoxy-D-glucose uptake in fat cells from both normal and thyroidectomized rats to the same extent (about 40%). An additive effect of thyroidectomy and insulin on glucose uptake was thus demonstrated. These results imply that glucose uptake in the adipocyte is controlled by at least three factors: thyroid hormones, cyclic AMP and insulin, each of which can act independently. Maximum glucose uptake is achieved in the presence of a combination of low concentrations of cyclic AMP, of insulin, and in the absence of thyroid hormones.


Differential effects of GTP on the coupling of beta-adrenergic receptors to adenylate cyclase from frog and turkey erythrocytes. Application of new methods for the analysis of receptor-effector coupling.

A detailed comparison of the interaction of beta-adrenergic receptors with adenylate cyclase stimulation and modification of this interaction by guanine nucleotides has been made in two model systems, the frog and turkey erythrocyte. Objective analysis of the data was facilitated by the development of new graphical methods which involve the use of logit-logit transformations of percent receptor occupancy versus percent enzyme stimulation plots (coupling curves). Receptor-cyclase coupling in turkey erythrocyte membranes demonstrates a proportional relationship between receptor occupancy and adenylate cyclase activation and is unaffected by exogenous guanine nucleotides. By comparison, the proportional relationship of receptor occupancy and adenylate cyclase activation observed in frog erythrocyte membranes in the absence of guanine nucleotides is modified by the addition of exogenous guanine nucleotides such that a greater fractional enzyme stimulation is elicited by low receptor occupancy. Methodological criteria crucial for valid comparison of receptor occupancy and adenylate cyclase activity are delineated. In addition, the possible molecular mechanisms of receptor-cyclase coupling which might give rise to the coupling curves observed are discussed.


Responses of nuclear glucose-6-phosphatase to diabetes and to hydrocortisone administered to normal and diabetic rats differ from those of the microsomal enzyme.

The effect of alloxan-diabetes, and of pharmacological doses of hydrocortisone administered to normal and diabetic rats, on carbamyl phosphate:glucose phosphotransferase and D-glucose-6-phosphate phosphohydrolase (EC 3.1.3.9) activities of isolated hepatic nuclei and microsomes were studied by assay at pH 7 in the absence and presence of deoxycholate. Hormonally related alterations both in activity levels and in the activation by the detergent (i.e. latency) of activities of the two cellular structural elements differed significantly. Most strikingly, (a) a 3--4-fold increase in the levels of activities of nuclei was seen in response either to diabetes or to hydrocortisone administered to normal rats whether or not detergent was added to preparations prior to assay; (b) the normally low degree of stimulation by detergent of activities of nuclei was unaltered in diabetes, and (c) administration of the glucocorticoid to diabetic rats decreased activity levels and increased their activation by detergent. Directly contrasting responses were noted with isolated microsomal preparations. Fundamental differences in the enzymes in these two organelle preparations are thus demonstrated. It appears that both synthetic and hydrolytic activities of this enzyme of nuclei may be manifest in the presence of requisite substrates, and that activities of this organelle may become increasingly prominent under certain hormonally perturbed conditions.


Role of cyclic AMP and related enzymes in rat lung growth and development.

Cyclic AMP levels in rat lungs showed phasic elevations which peaked during fetal, neonatal and late postnatal periods of development. Lung phospholipids showed major alterations in their levels during fetal and early neonatal life. Alterations in glycogen levels were accompanied by parallel changes in phosphorylase a/total phosphorylase activity which may be related to changes in cyclic AMP during development. Cyclic AMP levels were dependent on the relative activities of adenylate cyclase and cyclic AMP phosphodiesterase which also changed with age. Activation of adenylate cyclase by norepinephrine and NaF, and of cyclic AMP phosphodiesterase by calcium, was maximum neonatally and declined variably thereafter. These data suggest a relationship between cyclic AMP, glycogen and phospholipids during rat lung development.


Cell density-dependent increase in prolyl hydroxylase activity in cultured L-929 cells requires de novo protein synthesis.

Prolyl hydroxylase activity in cultured L-929 cells was found to increase when cells grew from log phase to stationary phase and when cells were harvested at the mid-log phase and replated at higher cell densities. Cycloheximide and actinomycin D inhibited the cell density-dependent increase in prolyl hydroxylase activity indicating that the increase in prolyl hydroxylase activity required de novo synthesis of protein and RNA. Prolyl hydroxylase was purified from cultured L-929 cells and antibodies against the protein were raised in rabbits. The antibodies were used to demonstrate that L-929 cells contained two forms of prolyl hydroxylase: an enzymatically active, tetrameric form consisting of two alpha and two beta polypeptide chains and an enzymatically inactive form containing immunologically cross-reacting protein. The polypeptide chains alpha, beta and cross-reacting protein were obtained by immunoadsorption. Peptide map analysis indicated that cross-reacting protein was similar if not identical to beta in primary structure, and alpha was different from both beta and cross-reacting protein. The results suggested that the prolyl hydroxylase levels in cells or tissues may be regulated by new protein and/or RNA synthesis.


Inhibition of the degradation of receptor-bound human choriogonadotropin by leupeptin.

The ability of leupeptin to block the degradation of receptor-bound human choriogonadotropin has been studied. It was found that this compound inhibited hormone degradation and intracellular cathepsin B activity in a parallel fasion, without affecting hormone-stimulated steroidogenesis.


[ESR of adsorbed chlorophyll a].

Oxygen and water vapour effect on the dark ESR signal of chlorophyll a adsorbed on silica in vacuum has been studied. It has been determined that when O2 and H2O are absent chlorophyll a adsorption is accompanied by an increase of the paramagnetic states portion (PSP) of the pigment. The PSP is increasing when oxygen is admitted. Water vapour does not increase the PSP of the adsorbed chlorophyll a. It has been supposed that the ESR signal of chlorophyll a in solution belongs to the chlorophyll-oxygen complex and that any strengthening of the pi-pi-interactions results in intensification of the ESR signal. It has been concluded that the ESR signal of adsorbed chlorophyll a belongs to the charge transfer complex of the molecular oxygen and the chlorophyll aggregate with strong pi-pi-interactions.


[Effect of inhibitors of free radical processes on the "dark" paramagnetic centers of the melanoprotein granules of the pigment epithelium of the eye].

Effect of three inhibitors of free radical processes (IFRP) differing in antiradical activity (2-tret. butyl-6-methyl-3-oxipyridin, 2-ethyl-6-methyl-3-oxipyridin and 3-oxipyridin) on paramagnetic centres of melanoprotein granules of pigment epithelium was studied by ESR method at room temperature. It was shown that IFRP decreased the dark signal and that the reaction of paramagnetic particles dissociation proceeded according to the equation of the first order reaction. The rate constants of these reactions were calculated. It was found that the relative amount of reacting and non-reacting paramagnetic particles with the inhibitor depended on the nature and concentration of IFRP.


Postnatal changes in carbamylphosphate synthetase-I and ornithine transcarbamylase activities after normal birth, premature delivery and prolonged gestation in rat liver: effects of actinomycin D and glucose.

The development of two urea cycle enzymes, carbamylphosphate synthetase-I and ornithine transcarbamylase was examined in neonatal rat liver. Normal birth on day 21.5 caused a marked increase of both activities as early as 4 h of extrauterine life. That increase occurred later in newborns delivered by cesarian section on day 21.5, but they exhibited a greater hepatic urea level. Premature delivery on day 20.5 caused a marked increase of both activities and hepatic urea level while prolonged gestation abolished these increases. The postnatal increase in both activities and in hepatic urea level was therefore dependent on a factor associated with birth. The in vivo administration of actinomycin D (2 microgram) at birth did not abolish the postnatal increase of both activities. Moreover, the administration of glucose (25 mg every 2 h) to newborns delivered by cesarian section on day 21.5 abolished the postnatal increase of ornithine transcarbamylase activity and hepatic urea level 23 h later. It seems that the transient hypoglycemia and appearance of gluconeogenesis at birth were the physiological mechanisms involved in the postnatal induction of ornithine transcarbamylase activity.


[Formation of nitrosyl complexes of nonheme iron (2.03 complexes) in animal tissues in vivo].

Formation or dinitrozyl non-haem iron complexes (2.03 complexes) in white rats' tissues in vivo has been studied. The formation of these complexes has been examined in the liver kidneys and small intestines when injecting p/o NaNO2 to the rats. Most of the complexes have been found in the liver. They have not been detected in the heart, spleen, muscle, cerebrum and marrow in vivo. 3-4-fold content of 2.03 complexes has been determined while introducing iron and NaNO2 in running water. This effect has not been found when introducing iron and NaNO2 simultaneously. The mechanism of 2.03 complex formation in the animal tissues in vivo has been proposed. According to this mechanism these complexes are formed in the tissues when Fe-NO from nitrozyl non-haem from complexes originating in blood passes into SH-group of protein in tissues. The formation of 2.03 complexes in vivo has been found in blood uniform elements.


[Free radical centers in the chromatophores and preparations of Rhodospirillum rubrum reaction centers].

The light-induced free radical ESR signals in chromatophores and reaction center preparation of R. rubrum were studied. It has been shown that the properties of the signals in both systems were strictly identical. At the microwave powers higher than 10 mW the ESR signal at g approximatley 2.00 is saturated and its asymmetry arises. Changes of the ESR signal shape may be explained by the superposition of P870+ signal with that from the ubisemiquinone. Addition of ferricyanide to chromatophores induces in the dark a new ESR signal characterized by a linewidth of 4.5 Oe which presumably arises from the antenna bacteriochlorophyll aggregates, because this signal is absent in the reaction center preparations.


Perinatal development of glucoeneogenic enzymes in rabbit liver.

Activities of the 4 hepatic gluconeogenic enzymes: glucose-6-phosphatase, fructose-1,6-diphosphatase, pyruvate carboxylase, particulate and cytosolic phosphoenolpyruvate carboxykinase (PEPCK) have been measured in fetal rabbits (22, 25, 28, 30 and 31 days of gestation) and in fasted or suckling newborns (1 and 2 days after birth). Between days 25 and 31 of gestation, fructose 1,6-diphosphatase and particulate PEPCK activities represent 50% of adult (pregnant female) activities, while pyruvate carboxylase is present at adult values during the same period. Glucose-6-phosphatase is low and cytosolic PEPCK absent in fetal liver until 30 days of gestation and increase significantly during the day preceding birth. Al the enzymes show a further increase after birth independently of the nutritional status of the animals (starved or suckling).


The effect of fluphenazine upon social and vocational functioning in remitted schizophrenics.

Thirty-six remitted schizophrenics who participated in an outpatient study of fluphenazine decanoate or oral fluphenazine vs placebo given for a year were examined for the effect of drug treatment upon social and vocational functioning. Only the period prior to any clinical relapse was evaluated. We found no difference between those on drug or placebo, and conclude that antipsychotic drugs, at least in the context of an aftercare clinic offering a rich spectrum of nonpharmacological services, and when combined with antiparkinson medication, do not interfere with social and vocational functioning.


Antibacterial effect of some combinations of pesticides of pure cultures of microorganisms.

The investigations conducted showed that the antibacterial effect of dinitro-o-cresol, cuprozane, and trichlorfon is due to their chemical structure and to the species of microorganisms on which they act. Dinitro-o-cresol and Cuprozane possess selective toxicity with respect to B. anthracis and Rh. gracilis and cuprozane with respect to Ch. perfringens. The method of evaluating the effect of combinations of chemical compounds consisting of two or more components which have been developed made it possible to detect the antibacterial effect of mixtures consisting of the indicated pesticides. It was established that the combinations studied possess synergic and additive effects, which also depend on the chemical structure of the pesticides and the species of microorganisms.


In vitro transforming activity of Epstein-Barr virus (EBV). II. Differences between M81 and B95-8 EBV strains.

Lymphocytes from 38 human cord blood and 9 adult circulating blood were aliquoted and infected in parallel either with the B95-8 EBV strain (produced by cotton-top marmoset lymphocytes transformed by EBV originating from an infectious mononucleosis line) or with the M81 EB virus (produced by callithrix jacchus marmoset lymphocytes transformed by EBV originating from a nasopharyngeal carcinoma (NPC) derived line). Significant differences were observed in the lymphoblastoid lines obtained and involved cell morphology, cell growth and synthesis of viral antigen. Cord blood lymphocytes infected with M81 virus resulted in lymphoblastoid lines where EA and VCA synthesis and production of virions took place, whereas this was not observed in B95-8 induced lines.


Chemotaxis of human leucocytes: II. Effects of lectins, colchicine, cytochalasin B, cyclic nucleotides and immuno-stimulatory products.

Drugs or products acting as chemo-modulators were assayed for their effect on the chemotactic activity of human normal neutrophils by a method of direct microscopic observation. Lectins (PHA-P and Con A) have an inhibitory effect at the level of 10 micrograms/ml. Colchicine and Cytochalasin B have also an inhibitory effect at the level of 0.25.10(-6)M/ml and 0.20 micrograms/ml. For the cyclic nucleotides: dibutyryl cAMP and products such as norepinephrine, aminophylline that raise the cellular level of cAMP have an inhibitory effect, and whereas cGMP and carbamylcholine have a stimulatory effect Imidazole and levamisole, have some enhancement of chemotaxis but it is not significant. Lectins by binding to cell surface receptors which assume the recognition of a chemotactic gradient in the environment, colchicine and cytochalasin B by interfering with the cytoskeletal elements (microtubules and microfilaments) cyclic nucleotides involved in the metabolic activity during the cell motility. The data show that the integrity of one of these three sequences is necessary for the normal activity of chemotasxis. Animal and human models (i.e. mutant mouse bg/bg and Chediak-Higashi syndrome) give some clue of their interrelation.


Virus induced diabetes and the immune system. II -- Evidence for an immune pathogenesis of the acute phase of diabetes.

The infection of normal DBA2 and BALB/C nu/ + mice with 2 X 10(2,6) TCID50 of the M variant of the EMC virus induced pathological mean glucose values of 426 +/- 99 mg/100 ml in DBA2 mice and 292 +/- 130 mg/100 ml in BALB/C nu/ + mice on day five following infection. As diabetic animals died afterwards, mean glucose values decreased in the surviving animals on day seven and fourteen. The infected immunodeficient BALB/C nu/nu mice with thymus aplasia did not show abnormal mean glucose values or higher standard deviation of the means (114 +/- 37 mg/100 ml) when compared to uninfected controls (117 +/- 23 mg/100 ml). This demonstrates that a complete thymus-dependent immune system seems to be necessary for the development of the acute stage of virus-induced diabetes in the mouse.


Hepatitis A infection and primary hepatocellular carcinoma.

The prevalence of antibody to the hepatitis A antigen (anti-HAV) has been studied in Senegal, among patients suffering from primary hepatocellular carcinoma (PHC) and among healthy blood donors. Anti-HAV was found in 62.5% of 64 cases of PHC as compared to 64% of 50 blood donors. Anti-HAV was as prevalent among PHC patients who evidenced chronic hepatitis B infection as among those without markers of chronic hepatitis B infection. These data suggest that hepatitis A infection is not associated with PHC in Senegal.


Abnormal red cell membrane phosphatase activity in an unidentified congenital hemolytic anemia.

The erythrocyte membrane contains a neutral phosphatase, which was studied with p-nitrophenyl-phosphate as the substrate. The enzyme was investigated in four members of the same family, suffering from a congenital spherocytic hemolytic anemia. Although the condition was transmitted as a dominant trait, it was not identified to the common hereditary spherocytosis. The phosphatase, instead of having a Michaelis-Menten kinetics, displayed a dramatically biphasic kinetics: substrate excess generated partial inhibition of the enzyme. We consider that such an abnormality, which was never encountered before, is a distinctive feature of a given type of congenital hemolytic anemia.


Electron spin resonance (esr) investigations on blood of patients with leukemia.

ESR investigations of lyophilized blood of patients with acute lymphatic leukemia exhibit an increase in spin concentration and an additional peak not present in control samples. This peak disappears almost completely concomittantly with a reduction in spin concentration after treating the patients with prednisolone, vincristine, and doxorubicin or after addition of 5mM of CuCl2 to blood samples. The results show that the ESR spectra of lyophilized blood samples can be utilized to follow the effect of therapy. Moreover, they suggest that the leukemic blood possesses a high concentration of antioxidants.


Lithium side effects in a routine lithium clinic.

Approximately two thirds of a group of bipolar patients attending the routine lithium clinic suffered sid effects. These patients tended to have a lower plasma and erythrocyte lithium concentration and a lower erythrocyte/plasma lithium ratio than those who had no side effects. Explanations of this unexpected finding are discussed.


The antidromic compound action potential of the hypothalamo-neurohypophysial tract, a tool for assessing posterior pituitary activity in vivo.

A sensitive technique was developed for measuring continuously the summated neural activity of the rat hypothalamo-neurohypophysial tract. Large compound potentials of 4-9 mV peak amplitude were recorded from the fibre tract in response to electrical stimulation of the neurohypophysis. It was estimated that all fibres in the tract were electrically activated and contributed to the compound potential. It was shown that a decrease of peak amplitude could be used as a measure of changes in endogenous neural activity. The decrease in peak amplitude during an increase of endogenous neural activity was due to fatigue and temporal spread of the compound potential, and also to collision of antidromic and orthodromic action potentials. Decrease of the compound potentials was related to the intensity of vaginal distension, haemorrhage and plasma hyperosmolality, and it correlated with neurohypophysial hormone release. This method may usefully complement hormone assays and single cell analysis.