Tetramethyl-p-phenylenediamine oxidase reaction in Azotobacter vinelandii. It was possible to quantitate the tetramethyl-p-phenylenediamine (TMPD) oxidase reaction in Azotobacter vinelandii strain O using turbidimetrically standarized resting cell suspensions. The Q(O2) value obtained for whole cell oxidation of ascorbate-TMPD appeared to reflect the full measure of the high respiratory oxidative capability usually exhibited by this genera of organisms. The Q(O2) value for the TMPD oxidase reaction ranged from 1,700 to 2,000 and this value was equivalent to that obtained for the oxidation of the growth substrate, e.g., acetate. The kinetic analyses for TMPD oxidation by whole cells was similar to that obtained for the "particulate" A. vinelandii electron transport particle, that fraction which TMPD oxidase activity is exclusively associated with. Under the conditions used, there appeared to be no permeability problems; TMPD (reduced by ascorbate) readily penetrated the cell and oxidized at a rate comparable to that of the growth substrate. This, however, was not true for the oxidation of another electron donor, 2,6-dichloroindophenol, whose whole cell Q(O2) values, under comparable conditions, were twofold lower. The TMPD oxidase activity in A. vinelandii whole cells was found to be affected by the physiological growth conditions, and resting cells obtained from cells grown on sucrose, either under nitrogen-fixing conditions or on nitrate as the combined nitrogen source, exhibited low TMPD oxidase rates. Such low TMPD oxidase rates were also noted for chemically induced pleomorphic A. vinelandii cells, which suggests that modified growth conditions can (i) alter the nature of the intracellular terminal oxidase formed (or induced), or (ii) alter surface permeability, depending upon the growth conditions used. Preliminary studies on the quantitative TMPD oxidation reaction in mutant whole cells of both Azotobacter and a well-known Mucor bacilliformis strain AY1, deficient in cytochrome oxidase activity, showed this assay can be very useful for detecting respiratory deficiencies in the metabolism of whole cells.


Combinations of antimicrobial agents. I. The in vitro sensitivity of 100 strains of Pseudomonas aeruginosa to polymyxin B, colistin, carbenicillin, gentamicin and doxycycline and to various combinations of these antibiotics. The MICs for 100 strains of Pseudomonas aeruginosa were determined for polymyxin B, colistin, carbenicillin, gentamicin and doxycycline and various combinations of these antibiotics. This study was carried out by means of a tube dilution method. Additive actions were observed in the combinations of polymyxin B plus carbenicillin, polymyxin B plus gentamicin, polymyxin B plus doxycycline, carbenicillin plus doxycycline and gentamicin plus doxycycline. A synergistic action was observed for the combinations of colistin plus carbenicillin, colistin plus gentamicin, colistin plus doxycycline and carbenicillin plus gentamicin. In none of the combinations was any antagonistic action encountered. Numerous reasons for using combined antibiotic treatment are listed. The results were obtained exclusively in in vitro investigations.


Malignant hepatoma associated with acquired hepatic cutaneous porphyria. A 60-year-old woman with a three-year history of an acquired hepatic cutaneous porphyria was discovered to have a porphyrin-producing malignant hepatoma. An attempt was made to treat her with fluorouracil perfused through a hepatic artery catheter. It did not produce shrinkage of the tumor, but the patient remains alive and ambulant a year after the infusion, although her skin lesions (scarring, bulla formation, and hyperpigmentation) on the fingers, back of hands, and face persist and she continues to excrete abnormally high quantities of porphyrin in her feces.


[Electron microscopical investigation of rhinoscleroma (author's transl)]. A boy from Togo with a resistent Rhinoscleroma was observed. The specific granuloma was examinated by histological and electron microscopic methods. The plasma cells form fibrillary material or guttate Russel bodies in the rough endoplasmatic reticulum. The morphology of Mikulicz-cells and Klebsiella rhinoscleromatis is described. The cells are supposed to be (histiocytic) macrophages. The surface of the germs is coated with a fimbria-like radiate substance which in the vacuoles of the cells is crosslinked. This material could be the eliciting antigen for the formation of Russel bodies. These bodies then may represent immune substances bound within the cell.


Histophysiology of the vegetative peripheral nervous system of skin. Preterminal nerve fibers of the peripheral vegetative nervous system make inmediate contact (neuro-effector-areas) to interstitial cells (I.C.). This connection is characterized through a common glycocalyx with the nerve fiber. The I.C. are specific innervated cells and differ morphologically from Schwann-cells, fibrocytes, and histiocytes. The I.C. are able to come into morphologically different contacts with neighbouring cells by microvilli-like cell protrusions. These neighbouring cells then are able to contact other cells by themselves. The results are interpreted in the sense of electro-mechanical feed-back system of information processing in the vegetative periphery.


Estrogen receptor and natural course of breast cancer. The tumors from approximately 50% of patients with breast cancer contained estrogen receptor (ER). ER appeared more often and at higher levels in the tumors of postmenopausal women. Eleven out of 12 patients who had multiple ER assays from various metastatic sites showed no significant discrepancies in ER values. ER level appears to decrease as the duration of metastatic cancer increase. Patients with ER in the tumor more frequently have bone metastases than those without ER. Visceral metastases occurred more often with ER negative patients and appeared to have a more malignant course with significant shorter survival.


Some steric factors affecting smooth muscle relaxation by cAMP analogs. The inhibitory potency of equimolar concentrations of cyclic adenosine monophosphate (cAMP) and three of its butyrylated derivatives; N6, O2'-dibutyryl cAMP (DBcAMP), N6-monobutyryl cAMP (N6MBcAMP) and O2' monobutyryl cAMP (O2' MBcAMP) was determined in isolated guinea pig ileal and aortic smooth muscle. Substitution in the N6 position did not markedly alter the inhibitory action of cAMP in these smooth muscles. However, an increase in the effectiveness of low concentrations of epinephrine in aorta was noted in the presence of the N6 substituted derivatives (0.1 mM). Addition of a butyryl group to the O2' position greatly decreased the activity of cAMP in both intestinal and vascular smooth muscle. The character of smooth muscle relaxation shown by cAMP and its analogs is very similar to that shown by adenosine and its analogs. These substances all appear to be acting in the same manner to relax intestinal and aortic smooth muscle.


Acute idiopathic polyneuritis caused by cytomegalovirus. Cytomegalovirus was isolated from the PBBC from a patient with acute idiopathic polyneuritis. Viremia occurred during the early phase of the neurologic disease, preceding viruria, and was accompanied by specific CMV-IgM antibodies, which indicates a recently acquired infection. Complement fixing antibodies to CMV remained stable throughout the illness at a relatively low titer. Previously, CMV has been associated with acute idiopathic polyneuritis on the indirect evidence of rising or elevated titers of complement fixing antibodies or isolation of virus from urine or saliva or both. Our findings provide direct evidence for the causative role of CMV in acute idiopathic polyneuritis.


[Observations on tumor-associated fibrinolysis in human breast-cancer (author's transl)]. In 30 cases of breast-cancer, including 24 primary cases, the tumor-associted fibrinolysis was investigated using agar plate method. In 60% of the tissue sample fibrinolytic activity was found. No activity could be detected in the remaining 40%. There was evidence of a positive correlation between tumor-associated fibrinolysis and tumor size as well as the rate of axilla metastases. Among the fibrinolytic active cases there was a disproporionate number of adenocarcinoma, whereas in the inactive collective the solid and scirrhous carcinoma prevailed. There was no relation between the tumor-associated fibrinolysis and the percentage of tumor cells in the tissue. The firbinolytic inactive tumors showed a better histopathological adaptation to the surrounding tissue than the active ones. There was less small-cell infiltrate in the stroma of the fibrinolytic active tumors than in the inactive cases. No significant difference was found in the form of growth and the occurence of fibre structure. For clinical assessment a longer period of observation and a larger study are necessary.


Significance of tissue estrogen receptors in the strategy of breast cancer treatment. Using evidences of estrogen receptors in mammary tumor tissues it was tried 1. to find substances which inhibit the tumor growth by their binding to the estrogen receptors; 2. to predict responsiveness to endocrine therapy; and 3. to find a definition of an improved therapeutic strategy in breast cancer. There is a continuously increasing rate of receptor positive tumors, caused by an improved technique of determination and tissue handling. Estrogen receptors were detected in 73 per cent of all primary mammary tumors and 58 per cent of metastatic tumors respectively. As new therapeutic substances nafoxidine and Estracyt were tested in controlled clinical trials. To get exact data about hormonal sensitivity further quantitative investigations of receptor activities of mammary tumors and their therapeutical responsiveness have to be done. As a new step in the strategy of breast cancer therapy based on receptor determinations a combined therapy with hormones and cytotoxic chemotherapeutics has to be checked in clinical trials in uture.


Steroid hormone receptors in human breast cancer and clinical correlations. Human breast cancer was studied by in vitro techniques on tissue slices and extracts for a specific binding of estradiol and dihydrotestosterone. Estradiol receptors were found in 50 percent of patients in the premenopausal group and in 47 percent of patients in the postmenopausal group. The incidence of receptors in patients with metastatic disease was 32.5 percent. In 18 of 35 patients who were positive for receptor sites a remission was observed after a variety of endocrine treatment regimens. In contrast only 3 patients out of 43 with a negative binding pattern responded to treatment with nafoxidine for a very short time. The best correlation was found in the ovariectomized group. Between the responders and the nonresonders there is no difference in the quantitative receptor content. The same is valid in view of the duration of the remission.


Estimation of high affinity estradiol binding sites in the human breast cancer. The specific estrogen binding capacity of the cytosol has been investigated in 51 human breast cancers (49 women, 2 men). The estrogen binding sites of the cytosol has been analysed by Korenman's dextrancoated charcoal technique. Several methods have been used in order to distinguish the specific estrogen binding sites and the unspecific binding: temperature differentiation and saturation of the binding sites with nonlabelled estrogen or its antagonist Clomid. It has been concluded that the differentiation of the estradiol binding sites (specific or unspecific) with the elevated temperature effect or the additon of Clomid may be helpful only as a qualitative test. The elevated temperature of Clomid may influence the increase of the unspecific estradiol binding. The most suitable method to eliminate the unspecific binding of estradiol is the saturation of cytosol from control samples with non-radioactive estradiol-17-beta. The specific estrogen binding capacity of the cytosols of different tumours varied over a wide range (0--840 fM E2/mg protein). It has been found that among 51 persons there were 31 patients (61%) with measureable amounts of high affinity estradiol binding sites in cancerous tissues. In 20 cases (39%) the existence of estradiol specific binding sites could not be observed.


Estradiol receptor activity in tissues of human breast cancer. Using the method for receptor determination recommended by the EORTC breast, cancer group we found 33.8% of our tissue specimen experimentally estradiol sensitive. No relation could be seen between receptor binding ability of the tumor tissues and lymph node involvement, tumor histology, clinical stages of the disease and menopausal state of the patients, respectively. In most cases primary tumor tissues and tissues of invaded lymph nodes show a corresponding behaviour in relation to estradiol uptake. The differences observed in the effects of various antiestrogenic substances on estradiol binding ability can not be explained at present.


[Presence of RNA-and DNA-tumor viruses within a cell line from mouse spleen and thymus (author's transl)]. Cells of spleen and thymus from mice infected with Rauscher leukaemia virus were cultivated in vitro. A new cell type repeatedly grew out of this cell line. Electron-optically, RNA--viruses and DNA-viruses were found within the new lines, but there was no cell containing both viruses at the same time. According to experimental results in rats, the DNA virus is polyoma-like. The importance the presence of two different viruses within the same cell is discussed.


Influence of double infections on the induction of thymidine kinase by UV-irradiated herpes simplex virus types 1 and 2 and pseudorabies virus. Simultaneous infection of primary rabbit kidney cells with HSV type 1 TK+ and a TK- strain results in a mutual influence of both viruses on the induction of thymidine kinase (TK). TK+ virus has an enhancing and TK- virus a depressing effect on TK induction by a superinfecting TK+ virus. The enzyme induction depends on the ratio of multiplicities of both viruses. The mutual influence on TK induction depends further on the time of addition of the superinfecting virus: the effect of the second virus can still be observed when given 6 hours after primary infection. Identical phenomena can be observed using combinations with HSV type 2 or Pseudorabies viruses. The ability of HSV to induce TK is progressively inactivated with increasing the time of UV-irradiation. The depressing effect of a TK- strain and the stimulating effect of a TK+ strain on superinfecting TK+ strains is UV-sensitive: after 6 minutes of UV-irradiation neither inhibition nor stimulation of TK induction by a superinfecting TK+ strain can be observed. Infection by long-term (20 minutes) UV-irradiated TK+ strains results in a depression of TK induction by a superinfecting TK+ virus. Long-term irradiation of the TK- virus does not show this effect. Cytosine-arabinoside has no effect on the mutual influence of TK induction by TK+ and TK-strains; the phenomenon of mutual depression therefore has to be considered an early process.


Simian virus 40-chinese hamster kidney cell interaction. I. Relationship of chromosome changes to transformation. After approximately 20 in vitro passages, Chinese hamster kidney (CHK) cell cultures transformed upon exposure to different strains of SV 40 can show a diploid modal chromosome number of 22 with chromosome counts exclusively or essentially in the diploid range (20-25). In primary culture and at the 5th-7th subculture, tumors produced in nude mice by such cells can display a diploid modal value of 22 chromosomes. Numerical chromosome variations and karyotype abnormalities observed in CHK cells transformed upon infection with SV 40, are apparently indistinguishable from those that can be observed in uninfected CHK cells undergoing "spontaneous"-transformation. These  results indicate that polyploidization is not a necessary step either in the process of transformation or in that of tumorigenic conversion with SV 40.


Swinepox. Virus isolation, experimental infections and the differentiation from vaccinia virus infections. The isolation of swinepox virus in primary pig kidney cell cultures is reported. The differentiation from vaccinia virus was possible with challenge infections of convalescent pigs and the use of the agar gel diffusion precipitation (AGDP) test and immuno-electroosmophoresis (IEOP). Using both immune precipitation tests reactions of identity were obtained between the heterologous antigens of swinepox and vaccinia viruses. A total of 829 pig sera from the field were tested for precipitating antibodies with the IEOP. Antibodies were detected in 65 (=7.8 per cent) of these serum samples.


Ultrastructural aspects of experimental swinepox with special reference to inclusion bodies. A light and electron microscopic study was performed on pox-like epidermal lesions in an experimentally infected pig. Light microscopical investigation of semithin sections revealed the presence of nuclear vacuoles and of different types of cytoplasmic inclusions. In electron microscopical studies large numbers of both immature and mature virus particles and the cytological changes indicative of pox virus infection were observed. Various types of intra-cytoplasmic inclusions -- i.e. fibrillar inclusions, crystalloid-containing dense inclusions, complex membraneous inclusions and dense homogeneous inclusions -- were encountered in addition to viroplasms and nuclear vacuoles. Because of the presence of vacuoles in nuclei of stratum spinosum cells the diagnosis swine pox by swinepox virus was most probable. These nuclear vacuoles have not been described in swine pox caused by vaccinia virus, the only other known cause of pox in swine.


The polypeptide composition of avian infectious bronchitis virus. Avian infectious bronchitis virus grown in ovo was purified by differential centrifugation and isopycnic sedimentation in density gradients. The purified virus was analysed by SDS polyacrylamide gel electrophoresis and found to comprise up to sixteen polypeptides, four of which were glycopeptides. Bromelain treatment of the particles removed three polypeptides and two glycopeptides.


Purification of herpesvirus saimiri and properties of the viral DNA. Conditions for growth, concentration, and purification of Herpesvirus saimiri were determined. Optimal yields of infectious Herpesvirus saimiri (HVS) were obtained from infected owl monkey kidney (OMK) cells grown at 32.5 degrees C in medium containing 10 per cent fetal calf serum. Forth-five percent of the initial infectious HVS was recovered after an 18-fold concentration using 8 per cent polyethylene glycol 6000 in the presence of 0.5 M NaCl. Polyethylene glycol concentrated HVS was purified in an isopycnic-linear Renografin gradient (1.0-1.3 g/cm3. Ninety-six percent of the infectivity was recovered in a single 1.16 g/cm3 density region. DNA extracted from purified HVS was resolved into two distinct density classes by CsCl equilibrium centrifugation (1.727 and 1.709 g/cm3). DNase treated HVA virions yield four DNA species with densities of 1.727, 1.718, 1.712, and 1.706 g/cm3 in CsCl centrifugation.


Polykaryocytosis induced by vesicular stomatitis virus infection in BHK-21 cells. Cytopathological effects by vesicular stomatitis virus (VSV) infection were studied in several cell lines. Marked polykaryocyte formation was observed in monolayers of certain strains of BHK-21 cells infected with VSV. The BHK-21-KB cells were found to be the most susceptible to virus-induced cell fusion. This type of cell fusion was related to intracellular growth of the viruses, and strong cytolytic changes were found to occur following the development of large multinucleated giant cells. The cell-fusing activity was associated with the infectivity of VSV and was neutralized by anti-VSV immune serum. The viruses irradiated for 20 minutes or heated at 60 degrees C for 10 minutes lost completely both infectivity and cell-fusing activity. These experimental results indicate that virus replication was responsible for fusion of BHK cells.


The influence of substances changing the intracellular concentration of cyclic adenosine 3'5'-monophosphate on interferon synthesis in chick embryo cell culture. The influence of cyclic 3'5'-adenosine monophosphate (cAMP), adrenalin and theophylline on interferon synthesis induced by influenza B virus (Lee strain) in chick embryo cell cultures was studied. In 5-day-old cell culture, theophylline was shown to enhance the inhibiting effect of exogenous cAMP and adrenalin on interferon synthesis and in 1-day-old culture, on the contrary, to enhance interferon production whereas adrenaline under these conditions had no effect on interferon synthesis at all. In 5-day-old cultures the activity of adrenalin and theophylline was manifested when they were added to the maintenance medium not later than 4 hours postinfection, and was not associated with the influence on interferon inducer adsorption on to cells or on virus multiplication in sensitive systems. Treatment of cells with these substances had no effect on interferon release from the cells. In the concentration used, adrenalin and theophylline exerted no cytotoxic effect. Theophylline inhibited incorporation of 3H-uridine and 14C-leucine into the acid insoluble fraction of the infected cells in 1-day-old cultures, while in 5-day-old cultures this was observed only when adrenalin and theophylline were used together. It is suggested that endogenous cAMP is essential for control of interferon synthesis and that different cAMP levels in cells of different ages may be one of the causes of the varying potency for interferon synthesis in young and old cell cultures.


Immunoglobulins, antibodies and inhibitors of parainfluenza 3 virus in respiratory secretions of sheep. Virus neutralising and haemagglutination inhibiting (HI) activities were monitored in the serum, nasal secretions and tracheo-bronchial secretions of lambs infected with Parainfluenza virus type 3 (PI3). HI activity was found in the secretions of both control and infected lambs, whereas neutralising antibody was found only in nasal secretions from infected lambs. Fractionation revealed that most of the HI activity in the secretions was due to a large molecular weight protein which also inhibited the haemagglutination (HA) of Parainfluenza virus type 1 (PI1) and type 2 (PI2) and Newcastle disease virus (NDV). This inhibitory activity was partially sensitive to receptor destroying enzyme (RDE). IgA antibodies specific for PI3 were also found in the respiratory secretions. However no increase in IgA levels was detected in the nasal secretions of the infected lambs. It is suggested that in certain reports non-specific inhibitors present in the nasal secretions of calves, may have been confused with PI3 specific IgA antibody.


[Studies on the Relationship between immunity and the level of neutralizing antibodies in pigs vaccinated against foot-and-mouth disease (author's transl)]. The statistical relationship between the titers of neutralizing antibodies and the immunity of 706 pigs vaccinated against FMD was studied. This was done for each of four virus strains separately. Whereas no correlation between both test systems could be detected in case of the strains A5 Westerwald, C Detmold and O1Santander, a significant correlation was ascertained for the strain O1Kaufbeuren. Becuase of the different findings depending on the virus strain under study it was concluded that the antibody titer alone does not provide a useful measure for potency testing of FMD-vaccines for pigs.


Use of iodinated organic compounds for the density gradient centrifugation of viruses. Use of Urografin and Conray for the equilibrium centrifugation of viruses is described. These pharmaceuticals, which consist of iodinated arylic compounds, reach densities of 1.6 g/cm3 and have low intrinsic viscosities. Poliovirus, Newcastle disease virus, and lymphocytic choriomeningitis virus were centrifuged to equilibrium in gradients made of these substances. Viral infectivities were not measurably affected, which is especially noteworthy in the case of the very labile lymphocytic choriomeningitis virus. Buoyant densities were found to be significantly lower than densities obtained with gradients made of CsCl and sucrose.


Natural and experimental infections of Japanese tree sparrows with Japanese encephalitis virus. 20 to 37 per cent of wild Japanese tree sparrows had neutralizing antibodies to Japanese encephalitis virus (JEV). Sparrows free of antibodies were inoculated with 10,000 plaque-forming units of JEV. None of the birds became clinically ill. Virus could be detected in blood plasma during the first 3 days after inoculation but not thereafter. Neutralizing antibodies started to appear at 4 days after inoculation and the response was of variable duration, in some cases extending up to 200 days after inoculation.


Inhibition of multiplication of parainfluenza 3 virus in prostaglandin-treated WISH cells. Influence of prostaglandins E2 (PGE2) and F2alpha (PGF 2alpha) on multiplication of myxovirus parainfluenza 3 was investigated. At concentrations of 0.01--1 mug/ml prostaglandins had no direct cytotoxic effects. PGE2 and PGF2alpha inhibited multiplication of parainfluenza 3 virus at concentrations 0.1--10 mug/ml. The inhibitory effect was most pronounced when the prostaglandins were added to medium for the whole period of virus multiplication (48 hours) while little or no effect was found when prostaglandins were added before virus inoculation or for 2 hours after infection. It is suggested that prostaglandins exert an inhibitory effect on the replication phase of the virus by influencing the growth of the WISH cells.


Triethyltin sulfate-induced neuropathy in rats. Electrophysiologic, morphologic, and biochemical studies. Adult rats given high orally administered doses of triethyltin (TET) sulfate lost weight, developed hind limb wasting, and became paraplegic or quadriplegic within three weeks of intoxication. A 33% reduction in the motor nerve conduction velocity (MNCV) of the sciatic nerve in the absence of significant demyelination was observed. There was observed, however, intramyelinic edema formation and an increased number of axonal neurofilaments and neurotubules; changes that paralleled the decrease in MNCV during the period of intoxication. Although the animals became asymptomatic and the MNCV normalized within two to three weeks of discontinuing the TET intoxication, the intramyelinic vacuoles and the increased numbers of neurofilaments and neurotubules persisted.


Experimental parainfluenza type 1 virus-induced encephalopathy in adult mice. Pathogenesis of chronic degenerative changes in the CNS. The pathogenicity of the 6/94 strain of parainfluenza type 1 virus, originally isolated from multiple sclerosis brain, was studied in adult mouse brains. Intracerebral inoculation of the virus caused mononuclear cell infiltration in the form of perivascular cuffing and a diffuse exudation into the parenchymal tissue, preferentially in cerebral white matter, that resulted in marked degeneration over a 90-day observation period. Immunofluorescent staining revealed viral antigen in the ependymal lining cells only during the first seven days after infection. No correlation was found between the severity of the brain lesions and the level of circulating antiviral antibody, preexisting or newly produced.


Lesions produced by human herpesviruses 1 and 2. Morphologic features in rabbit corneal epithelium. The morphologic features of corneal lesions produced in rabbits by human herpesviruses, types 1 and 2 (HH1 and HH2), were studied using light and electron microscopic techniques. Also, healing lesions produced by the HH1 virus and treated by idoxuridine were similarly investigated. Scanning electron micrographs showed that although the morphology of HH1 and HH2 lesions was similar in most respects. HH2 lesions typically were raised, whereas the edges of HH1 lesions were not elevated above the corneal surface. In both HH1 and HH2 lesions, infected epithelial cells first separated from neighboring cells, then became globular, and finally were removed, leaving a central excavation. During healing of HH1 lesions, normal epithelial cells invaded the central crater from all sides to cover damaged cells and reconstitute the epithelial surface.


Herpesvirus hominis encephalitis and retinitis. A previously healthy 44-year-old man died three weeks after the simultaneous onset of encephalitis and retinitis. Fundus changes were bilateral and included papillitis, rapidly progressive central retinal vein obstruction, and massive exudative retinal detachment. A 16-fold rise in herpesvirus hominis antibodies occured between the 10th and 20th days of illness. At autopsy, the brain showed changes characteristic of herpetic encephalitis, and cultures of the brain yielded Herpesvirus hominis type I. Intranuclear inclusion bodies typical of those produced by Herpesvirus were found by light microscopy in brain, optic nerves, retina, and choroid. Herpesvirus particles were found by electron microscopy in brain, optic nerve, and retina.


[Problems of the electric response audiometry (ERA) during the natural and artificial sleep (author's transl)]. The investigators carried out threshold determinations on 16 children and 6 adults in wakefulness, under general anaesthesia (we used chloral hydrate anaesthesia) and in sleep (stage II-III and stage REM). Falling asleep (stage I and initial stage of anaesthesia respectively) the latencies of the individual components of the acoustically evoked potentials are prolonged in mean of 30 msec. Simultaneously the amplitude of N1 significantly decreases and N2 becomes a prominent point (Fig. 1). The generation mechanisms of wave N2 are obviously different from those of wave N1. Its input-output curve takes a very steep course (Fig. 5) and the shortening of latencies increases with growing intensity of stimulus too (Fig. 4). Amplitude histogrammes demonstrated the dependency of the form of the acoustically evoked potential on the degree of synchronisation of EEG activity. While in the case of desynchronisation N1 appears more markedly, N2 does in the case of synchronisation. The mean deviation of the ERA threshold totals plus 3.8 +/- 6.9 dB (n = 41) under chloral hydrate anaesthesia, plus 4.9 +/- 6.7 dB (n = 37) in natural sleep in contrast to the wakefulness. With a 99% confidence there occur confidence intervals ranging from + 1 to + 7 dB and from +2 to +8 dB respectively. In identifying the threshold potentials error I (existing potential not recognized) occurred in 15-20%, error II (random wave seen as potential) in 20% of these studies. All these experiments showed significant lower variances for the latencies compared with variancies of amplitudes. The variance of amplitudes is smallest in children (Table 1) under general anaesthesia as well as in adults in wakefulness (Table 2). For the practical performance of ERA chloral hydrate is recommended for studies on children. A uniform EEG-state as well as a uniform depth of sleep are basic conditions for ERA during sleep, sedation or under anaesthesia. These conditions must constantly be controlled by EEG, EOG and EMG.


Galactose metabolism in relation to cataract formation in marsupials. Erythrocytic galactokinase and/or galactose-1-phosphate uridyl transferase activity were low in many species of marsupials. However, cataract formation was observed only in pouch-young members of these species when reared on cow's milk. The galactose tolerance of young kangaroos was found to be greatly impaired, but improved rapidly and markedly at the stage of which the definitive structure of the ruminant type of stomach as in adults is formed. The combination of high absorption of galactose and low levels of galactokinase and/or transferase thus appears to determine the predisposition of pouch-young marsupials to galactose cataractogenesis.


Prolonged control of hypoglycaemia by L-asparaginase in islet cell carcinoma producing insulin and gastrin. L-asparaginase (140,000 units) infused into the hepatic artery resulted in a remission from disabling hypoglycaemia for nine months in a man with islet cell carcinoma of the pancreas and hepatic metastases. The tumour produced insulin and gastrin with resulting hypoglycaemia and recurrent peptic ulceration which were unresponsive to other drugs. Following L-asparaginase there was a fall in both plasma and insulin and gastrin.


Insulinoma: aids to management.

Insulinoma: aids to management. Three cases of insulinoma seen in the past few months are reported in order to highlight recent advances in the management of this condition. These advances include serum insulin level determinations to correlate with blood sugar levels; selective arteriography as a means of localizing a tumour; and the monitoring of blood sugar levels during surgery.


A study of the interrelationship between the triacylglycerol and protein components of very-low-density lipoproteins using the perfused rat liver. High and low rates of very-low-density-lipoprotein triacylglycerol release from the perfused rat liver were achieved by using livers taken respectively from animals that had been given fructose for 48h or from animals that had been starved for 18h. 2. The higher rates of very-low-density-lipoprotein triacylglycerol release by the livers of the fructose-fed rats were associated with higher rates of very-low-density-lipoprotein protein release. 3. When the livers were perfused in the presence of [3H]leucine, radioactivity was incorporated into the very-low-density-lipoprotein apoproteins. The higher rates of very-low-density-lipoprotein triacylgycerol and protein release by the livers of fructose-fed rats were associated with a greater total incorporation of radioactivity into those apoproteins that entered the running gel during polyacrylamide-gel electrophoresis. However, the distribution of radioactivity among the various apoproteins was not significantly changed by the dietary treatments used.


A comparison of the regulation of pyruvate dehydrogenase in mitochondria from rat brain and liver. The total activity of pyruvate dehydrogenase in mitochondria isolated from rat brain and liver was 53.5 and 14.2nmol/min per mg of protein respectively. Pyruvate dehydrogenase in liver mitochondria incubated for 4 min at 37 degrees C with no additions was 30% in the active form and this activity increased with longer incubations until it was completely in the active form after 20 min. Brain mitochondrial pyruvate dehydrogenase activity was initially high and did not increase with addition of Mg2+ plus Ca2+ or partially purified pyruvate dehydrogenase phosphatase or with longer incubations. The proportion of pyruvate dehydrogenase in the active form in both brain and liver mitochondria changed inversely with changes in mitochondrial energy charge, whereas total pyruvate dehydrogenase did not change. The chelators citrate, isocitrate, EDTA, ethanedioxybis(ethylamine)tetra-acetic acid and Ruthenium Red each lowered pyruvate dehydrogenase activity in brain mitochondria, but only citrate and isocitrate did so in liver mitochondria. These chelators did not affect the energy charge of the mitochondria. Mg2+ plus Ca2+ reversed the pyruvate dehydrogenase inactivation in liver, but not brain, mitochondria. The regulation of the activation-inactivation of pyruvate dehydrogenase in mitochondria from rat brain and liver with respect to energy charge is similar and may be at least partially regulated by this parameter, and the effects of chelators differ in the two types of mitochondria.


Effect of protein-synthesis inhibitors on testosterone production in rat testis interstitial tissue and Leydig-cell preparations. Luteinizing-hormone-stimulated testosterone biosynthesis was inhibited by cycloheximide during incubation of rat testis intersitial tissue in vitro and also by puromycin and cycloheximide during incubation of Leydig-cell preparations, but not by chloramphenicol. These results suggest that a protein regualtor(s) formed by cytoplasmic protein synthesis is involved in steroidogenesis in the rat testis. The specific effect of cycloheximide and puromycin on protein synthesis rather than on other non-specific processes is suggested by the inhibition of protein synthesis and steroidogenesis with different doses of the inhibitors and the lack of effect of cycloheximide on luteinizing-hormone-induced adenosine 3':5'-cyclic monophosphate production. Stimulation of testosterone production by luteinizing hormone during superfusion of interstitial tissue was detectable within 10-20 min and reached a maximum of 120 min, and thereafter slowly decreased. Cycloheximide added at maximum steroid production caused a rapid decrease in testosterone synthesis which followed first-order kinetics (half-life 13 min), thus indicating that the protein regulator(s) has a short half-life. No effect of cycloheximide, puromycin or chloramphenicol on testosterone production in the absence of added luteinizing hormone was found, suggesting that the basal production of testosterone is independent of protein synthesis.


The maturation of the inner membrane of foetal rat liver mitochondria. A new method was devised for the isolation of foetal and neonatal rat lvier mitochondria, giving higher yields than conventional methods. 2. During development from the perinatal period to the mature adult, the ratio of cytochrome oxidase/succinate-cytochrome c reductase changes. 3. The inner mitochondrial membrane of foetal liver mitochondria possesses virtually no osmotic activity; the permeability to sucrose decreases with increasing developmental age. 4. Foetal rat liver mitochondria possess only marginal respiratory control and do not maintain Ca2+-induced respiration; they also swell in respiratory-control medium in the absence of substrate. ATP enhances respiratory control and prevents swelling, adenylyl imidodiphosphate, ATP+atractyloside enhance the R.C.I. (respiratory control index), Ca2+-induced respiratory control and prevent swelling, whereas GTP and low concentrations of ADP have none of these actions. It is concluded that the effect of ATP depends on steric interaction with the inner mitochondrial membrane. 5. When 1-day pre-partum foetuses are obtained by Caesarean section and maintained in a Humidicrib for 90 min, mitochondrial maturation is "triggered", so that their R.C.I. is enhanced and no ATP is required to support Ca2+-dependent respiratory control or to inhibit mitochondrial swelling. 6. It is concluded that foetal rat liver mitochondria in utero do not respire, although they are capable of oxidative phosphorylation in spite of their low R.C.I. The different environmental conditions which the neonatal rat encounters ex utero enable the hepatic mitochondria to produce ATP, which interacts with the inner mitochondrial membrane to enhance oxidative phosphorylation by an autocatalytic mechanism.


Conversion of thyroxine into tri-iodothyronine by rat liver homogenate. By using a highly specific radioimmunoassay the formation of tri-iodothyronine by the deiodination of thyroxine was studied in rat liver homogenate. Several observations suggest that the reaction observed is enzymic in nature. Pre-heating the homogenate for 30 min at 56 degrees C completely abolished conversion of thyroxine into tri-iodothyronine; the component of rat liver homogenate responsible could be saturated with substrate; iodotyrosines displayed competitive activity. Between 0 degrees and 37 degrees C, the tri-iodothyronine-production rate was positively correlated with incubation temperature. The addition of NAD+ enhanced conversion into tri-iodothyronine, which suggests that an oxidative mechanism is involved. 5-Propyl-2-thiouracil and 6-propyl-2-thiouracil, both known to prevent deiodination in vivo, greatly decreased the deiodiantion activity of rat liver homogenate.


Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system. Tthe properties of diphosphoinositide and triphosphoinositide phosphatases from rat kidney homogenate were studied in an assay system in which non-specific phosphatase activity was eliminated. The enzymes were not completely metal-ion dependent and were activated by Mg2+. The detergent sodium deoxycholate, Triton X-100 and Cutscum inhibited the reaction; cetyltrimethylammonium bromide only activated when added with the subtrates and in the presence Mg2+. Both enzymes had a pH optimum of 7.5. Ca2+ and Li+ both activated triphosphoinositide phosphatase, but Ca2+ inhibited and L+ had little effect on diphosphoinositide phosphatase. Cyclic AMP had no effect on either enzyme. The enzymes were three times more active in kidney cortex than in the medulla. On subcellular fractionation of kidney-cortex homogenates by differential and density-gradient centrifugation, the distribution of the enzymes resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), suggesting localization in the Golgi complex. However, the distribution differed from that of the liver Golgimarker galactosyltransferase. Activities of both diphosphoinositide and triphosphoinositide phosphatases and thiamin pyrophosphatase were low in purified brush-border fragments. Further experiments indicate that at least part of the phosphatase activity is soluble.


Poly(glucosylglycerol phosphate) teichoic acid in the walls of Bacillus stearothermophilus B65. 1. Walls of Bacillus stearothermophilus B65 contain a glycerol teichoic acid in which repeating structures consisting of 1-O-alpha-D-glucopyranosylglycerol phosphate are held together by phosphodiester linkage between the glycerol and glucose moieties of adjacent units. 2. The walls are not agglutinated on incubation with concanavalin A, nor does the isolated teichoic acid form a precipitate with this lectin. 3. No evidence was obtained of the presence of the glucosylated (1 leads to 2)-poly(glycerol phosphate) teichoic acid which has previously been reported to occur in walls of this bacterium.


A rapid NAD+-linked assay for microsomal uridine diphosphate glucuronyltransferase of rat liver and some observations on substrate specificity of the enzyme. 1. A new and rapid continuous assay of rat liver microsomal UDP-glucuronyltransferase (EC 2.4.1.17) has been developed. It is based on measurement of UDP production from UDP-glucuronate during the glucuronidation reaction; UDP production was continuously measured by coupling it to the conversion of NADH into NAD+ through pyruvate kinase and lactate dehydrogenase. This assay is independent of the acceptor substrate used; several findings confirm its applicability. 2. The glucuronidation rate of a series of phenol derivatives was determined with this assay, by using a Triton X-100-activated microsomal preparation as enzyme source. Conjugation of a series of nitrophenol derivatives was also investigated by the 'classical' assay (measurement of disappearance of the yellow colour of the nitrophenol during glucuronidation). The substrate with the highest conversion rate was 3-methyl-2-nitrophenol. 3. Both electron releasing and electron withdrawing ring substituents increased the glucuronidation rate of the phenol derivatives, as compared with phenol. 4. Lipid solubility seems important for determining the conversion rate: poorly lipid-soluble substrates were glucuronidated only at a low rate and high lipid solubility seems to be a prerequisite for high conversion rate. Glucuronidation of poorly lipid-soluble compounds may be limited by diffusion. 5. The consequences of these findings for the interpretation of studies on heterogeneity of the enzyme are discussed.


Proteoglycans of hyaline cartilage: Electron-microscopic studies on isolated molecules. Proteoglycan monomers from guinea-pig costal cartilage, bovine nasal and bovine tracheal cartilage were observed in the electron microscope after being spread in a monomolecular layer with cytochrome c. The proteoglycan molecule appeared as an extended central core filament to which side-chain filaments were attached at various intervals. The molecules from the three sources displayed great ultrastructural similarities. On average, the core filament was about 290 nm long, there were about 25 side-chain filaments per core filament, the side-chain filaments were about 45 nm long, and the distance between the attachment points of the side-chain filaments to the core filament was about 11 nm. With regard to the overall size of the molecules, no evidence of distinct subpopulations was obtained. Good correlation was found between ultrastructural data for the proteoglycan molecules and chemical data obtained by enzyme digestions and gel chromatography. Together these data strongly support the interpretation of the electron-microscopic pictures as indicating a central filament corresponding to the protein core and side-chain filaments corresponding to the chondroitin sulphate chain clusters of the proteoglycan monomers.


An improved method for purifying sialidase. An adsorbent specific for sialidase (EC 3.2.1.18) was prepared by coupling a glycoprotein containing glycosidically linked sialic acid to Sepharose. This adsorbent does not display the non-specific adsorption that occurs in previously reported methods.


A temperature-jump study of the reaction between azurin and cytochrome c oxidase from Pseudomonas aeruginosa. The electron-transfer reaction between azurin and the cytochrome oxidase from Pseudomonas aeruginosa was investigated by temperature-jump relaxation in the absence of O2 and in the presence of CO. The results show that: (i) reduced azurin exists in two forms in equilibrium, only one of which is capable of exchanging electrons with the Pseudomonas cytochrome oxidase, in agreement with M. T. Wilson, C. Greenwood, M. Brunori & E. Antonini (1975) (Biochem. J. 145, 449-457); (ii) the electron transfer between azurin and Pseudomonas cytochrome oxidase occurs within a molecular complex of the two proteins; this internal transfer becomes rate-limiting at high reagent concentrations.


Adenosine 3':5'-cyclic monophosphate-dependent and plasma-membrane-associated protein kinase(s) from bovine corpus luteum. Plasma-membrane fractions FI and FII isolated from bovine corpus luteum by discontinuous sucrose-density-gradient centrifugation, at sucrose-density interfaces of 1.14/1.16 and 1.16/1.18 respectively, contained membrane-associated protein kinases that phosphorylated both the structural proteins of membranes as well as exogenously added protein substrates. Both fractions were characterized with respect to endogenous and exogenous protein substrate specificity, pH-dependence, effect of bivalent metal ions and sensitivity toward cyclic nucleotides. These membrane-associated kinases showed an optimum pH of 6.0 and had an absolute requirement for bivalent metal ions such as Mg2+, Mn2+, or Co2+ that cannot be replaced by Ca2+. Both the activities were stimulated two- to four-fold by cyclic AMP in vitro with an apparent Km of 83 and 50 nM for fractions FI and FII respectively. Other cyclic 3':5'-nucleotides were effective only at higher concentrations, but even the most effective, cyclic IMP, showed a stimulation nearly an order of magnitude lower than that of cyclic AMP. In contrast, stimulation by cyclic dTMP and cyclic dAMP was very weak. Cyclic AMP showed no significant effect on the apparent Km value of both enzymes for histone and MgCl2 but it somewhat decreased the Km value for ATP. Nucleoside triphosphates like GTP, CTP and UTP inhibited the transfer of [32P]Pi from [gamma-32P]ATP into mixed histone catalysed by membrane-associated kinases either in the presence or in the absence of cyclic AMP. In addition to protein kinases, these membrane fractions also possessed cyclic AMP-binding activities. The apparent association constant (Kalpha) for cyclic AMP binding was 1.0 X 10(10) and 2.6 X 10(10) M for FI and FII membrane fractions respectively.


The investigation of substrate-induced changes in subunit interactions in glyceraldehyde 3-phosphate dehydrogenases by measurement of the kinetics and thermodynamics of subunit exchange. An investigation was made of changes in subunit interactions in glyceraldehyde 3-phosphate dehydrogenase on binding NAD+, NADH and other substrates by using the previously developed method of measurement of rates and extent of subunit exchange between the rabbit enzyme (R4), yeast enzyme (Y4) and rabbit-yeast hybrid (R2Y2) [Osborne & Hollaway (1974) Biochem. J. 143, 651-662]. The free energy of activation for the conversion of tetramer into dimer for the rabbit enzyme (R4 leads to 2R2) is increased by at least 12kJ/mol in the presence of NAD+. This increase is interpreted in terms of an NAD+-induced 'tightening' of the tetrameric structure probably involving increased interaction at the subunit interfaces across the QR plane of the molecule [see Buehner et al. (1974) J. Mol. Biol. 82, 563-585]. This tightening of the structure only occurs on binding the third NAD+ molecule to a given enzyme molecule. Conversely, binding of NADH causes a decrease in the free energy of activation for the R4 leads to 2R2 and Y4 leads to 2Y2 conversions by at least 10kJ/mol. This is interpreted as a NADH-induced 'loosening' of the structures arising from decreased interactions across the subunit interfaces involving the QR dissociation plane. In the presence of NADH the increase in the rate of subunit exchange is such that it is not possible to separate the hybrid from the other species if electrophoresis is carried out with NADH in the separation media. In the presence of a mixture of NADH and NAD+ the effect of NAD+ on subunit exchange is dominant. The results are discussed in terms of the known co-operativty between binding sites in glyceraldehyde 3-phosphate dehydrogenases.


The reaction of Pseudomonas aeruginosa cytochrome c oxidase with carbon monoxide. The binding of CO to ascorbate-reduced Pseudomonas cytochrome oxidase was investigated by static-titration, stopped-flow and flash-photolytic techniques. Static-titration data indicated that the binding process was non-stoicheiometric, with a Hill number of 1.44. Stopped-flow kinetics obtained on the binding of CO to reduced Pseudomonas cytochrome oxidase were biphasic in form; the faster rate exhibited a linear dependence on CO concentration with a second-order rate constant of 2 X 10(4) M-1-s-1, whereas the slower reaction rapidly reached a pseudo-first-order rate limit at approx. 1s-1. The relative proportions of the two phases observed in stopped-flow experiments also showed a dependency on CO concentration, the slower phase increasing as the CO concentration decreased. The kinetics of CO recombination after flash-photolytic dissociation of the reduced Pseudomonas cytochrome oxidase-CO complex were also biphasic in character, both phases showing a linear pseudo-first-order rate dependence on CO concentration. The second-order rate constants were determined as 3.6 X 10(4)M-1-s-1 and 1.6 X 10(4)M-1-s-1 respectively. Again the relative proportions of the two phases varied with CO concentration, the slower phase predominating at low CO concentrations. CO dissociation from the enzyme-CO complex measured in the presence of O2 and NO indicated the presence of two rates, of the order of 0.03s-1 and 0.15s-1. When sodium dithionite was used as a reducing agent for the Pseudomonas cytochrome oxidase, the CO-combination kinetics observed by both stopped flow and flash photolysis were extremely complex and not able to be simply analysed.


The reversible delipidation of a solubilized sodium-plus-potassium ion-dependent adenosine triphosphatase from the salt gland of the spiny dogfish. A microsomal fraction rich in Na+, K+-ATPase (sodium-plus-potassium ion-dependent adenosine triphosphatase) and the corresponding K+-dependent p-nitrophenyl phosphatase from the rectal salt gland of the spiny dogfish was solubilized by treatment with deoxycholate at high ionic strength. On gel filtration through Sepharose 6B, the ATPase apoenzyme could be separated, in apparently soluble form, from the tissue-fraction phospholipids and was almost free of enzymic activity (2% of the p-nitrophenyl phosphatase activity and 0.2% of the ATPase activity being recovered). On mixing the apoenzyme with an activator consisting of cooked ox brain, a large proportion of the original enzymic activity was obtained. Specific activities of the re-activated enzyme were somewhat higher than in the material before gel filtration: values of 1300-1450 mumol and 250-290 mumol/h per mg of protein were obtained for the hydrolysis of ATP and of p-nitrophenyl phosphate respectively. The activity was inhibitible by ouabain.


Physicochemical characterization of the four-iron-four-sulphide ferredoxin from Bacillus stearothermophilus. 1. A stable ferredoxin was prepared from Bacillus stearothermophilus and purified by chromatography on DEAE-cellulose and by electrophoresis. 2. The minimum molecular weight determined from the amino acid composition was about 7900 and this was in reasonable agreement with a value of 8500 determined by polyacrylamide-gel electrophoresis. The ferredoxin contained four iron atoms and four labile sulphide groups per molecule. 3. The optical absorption, optical-rotatory-dispersion and circular-dichroism spectra are typical of ferredoxins containing 4Fe-4S clusters. 4. Oxidation-reduction titrations, combined with electron-paramagnetic-resonance (e.p.r.) spectroscopy, showed that the protein has a mid-point potential, at pH8, of -280 +/- 10mV, and that only one electron-accepting paramagnetic species is present. 5. The e.p.r. spectrum of the reduced ferredoxin is more readily saturated with microwave power at low temperatures than those of the eight-iron ferredoxins, indicating that there is another mechanism of electron-spin relaxation in the latter. 6. Mossbauer spectra of both redox states were observed over a range of temperatures and in magnetic fields. At high temperatures (77 degrees K and above) both redox states appear as quadrupole-split doublets; in the reduced state two resolved doublets are seen, suggesting appreciable localization of the additional reducing electron. 7. The average chemical shift indicates formal valences of two Fe3+ and two Fe2+ in the oxidized state and three Fe2+ and one Fe3+ in the reduced state. However, the spectra indicate that there are differing degrees of electron delocalization over the iron atoms. 8. At low temperatures (4.2 degrees K) the oxidized form shows no hyperfine magnetic interaction, even in an applied magnetic field, evidence that the oxidized ferredoxin is in a non-magnetic state as a result of antiferromagnetic coupling between the iron atoms. 9. At 4.2 degrees K the reduced form shows a broad asymmetric pattern resulting from magnetic hyperfine interaction. This contrasts with the reduced ferredoxin of Clostridium pasteurianum, which shows a doublet, suggesting that in the latter there may be interaction between the two 4Fe-4S centres. 10. In large applied magnetic fields, positive and negative hyperfine fields are seen in the Mossbauer spectra of the reduced ferredoxin, evidence for antiferromagnetic coupling between the iron atoms in the 4Fe-4S centre. The high-field spectra of the reduced ferredoxin of B. stearothermophilus are similar to those of the reduced ferredoxin of C. pasteurianum.


Initiation factors in protein synthesis by free and membrane-bound polyribosomes of liver and hepatoma. The activity of initiation factors obtained from free and membrane-bound polyribosomes of liver and of transplantable H5123 hepatoma of rats was investigated by using an assay of protein synthesis in vitro in which poly (U)-directed polyphenylalanine synthesis was measured. Initiation factors of membrane-bound polyribosomes prepared by using the anionic detergent deoxycholate exhibited less activity in incorporating [14C]phenylalanyltRNA into polypetides than did initiation factors of free polyribosomes. However, when membrane-bound polyribosomes were prepared after using the non-ionic detergent Triton X-100, no significant differences in activities in polyphenylalanine synthesis were observed between the initiation factors of free and membrane-bound polyribosomes. These results suggest that Triton X-100 is preferable to deoxycholate in the isolation of of initiation factors from polyribosomes. Initiation factors, prepared by using Triton X-100, of free polyribosomes of hepatoma exhibited greater activity in the stimulation of polyphenylalanine synthesis than did the initiation factors of free or membrane-bound polyribosomes of host livers or of membrane-bound polyribosomes of hepatomas.


Electron spin resonance study of changes during the development of a mouse myeloid leukaemia. II. The ascorbyl radical. The ascorbyl radical concentration has been observed, by means of electron spin resonance spectroscopy, in the blood, spleen and liver of RF/J female mice throughout the development of a myeloid leukaemia. Changes in the concentration of the radical were detectable from an early stage in the disease and did not appear to be directly due to the leukaemic cells but could possibly be due to a reaction against them. Changes in the concentration of the paramagnetic metal ions during the leukaemia have been reported previously and it was found that changes in some of these species correlated with changes in the ascorbyl radical concentration.


[Severe mixed immunodeficiency. Report of a case]. The case of a 3 month old child with severe combined sex linked immunodeficiency is presented. The diagnosis was well doccumented, during his life. The child presented as a case of mucocutaneous moniliasis resistant to treatment. There was a history of similar cases in the family; diagnosis was made at post-mortem in one cousin and death occurred at early age in all kins so affected. Blood marrow transplant was not feasible in our case because histocompatibility was lacking in the kins studied. Three units of transfer factor were given as well as hyperimmune plasma but the child died in respiratory failure. Autopsy demonstrated pulmonary infection by Pneumocystic carinii and generalized citomegalic inclussion virus infection; almost complete absence of immune tissue was also demonstrated.


Murine leukaemia virus expression in the AKR following thymectomy. Thymectomy effectively prevents the development of spontaneous lymphoma in the AKR but how this effect is achieved remains to be determined. One possible mechanism, namely suppression of genomic expression of the oncogenic murine leukaemia virus now seems unlikely since levels of the group specific MuLV antigen were in comparision with their sham operated controls unaltered in both neonatally and adult thymectomized AKR.


[Intraspinal tumors in children. Review of 24 cases]. The clinical cases of 24 patients with intraspinal neoplasms seen at the Neurology and Neurosurgery ward of the Hospital del Nino IMAN, in a period of four and a half years are reviewed. The majority of our cases were diagnosed as suffering from tumors of embryological origin like teratomas, dermoid cysts and neuroblastomas. The signs and symptoms were reviewed, noting the importance of weakness of the lower extremities, the alteration in reflexes and sensitivity as well as back pain. We emphasize the great help of the X ray examination  of the spinal colum, both plain and with contrast media. The treatment that our patients underwent was reviewed and the results were evaluated, pointing out the fact that life prognosis is related to malignancy of the lesions and the neurological sequelae are intimatly related with the early diagnosis and treatment of this type of lesions.


Serum immunoglobulin levels in primary liver cancer: relationship to underlying cirrhosis and hepatitis-B (surface) antigenaemia. Serum IgG, IgM and IgA levels were measured by the single radial diffusion method in 107 South African Negro patients with primary hepatocellular cancer (PHC) and 112 healthy Negro blood donors. The mean serum IgG ANd IgM concentrations were significantly higher (P less than 0-001) in the PHC patients. In those patients in whom PHC was associated with cirrhosis, the serum IgG level was greater (P less than 0-02) than in those without cirrhosis. However, the mean serum IgG concentration in the non-cirrhotic cancer patients was still significantly higher than the control value (P less than 0-001). Thus, while cirrhosis may contribute to the raised IgG levels in PHC, other factors must also be involved. There was no difference in the serum immunoglobulin concentrations in PHC patients with and without hepatitis-B antigenaemia.


The leucocyte adherence inhibtion test in cancer of the large bowel. A recently introduced assay of cell mediated immunity and humoral inhibitory factors has been evaluated in colorectal cancer patients. Using a perchloric acid extract of adenocarcinoma of the large bowel as antigen, 16/27 patients with colorectal cancer had significant cellular reactivity when their separated peripheral leucocytes were tested in homologous AB serum. In autologous serum only 7/27 had significant reactivity; 6/20 patients with a variety of other malignancies showed sensitization  to the colorectal antigen preparation. It is concluded that the leucocyte adherence inhibiton test may offer a simple method of assaying for serum blocking factors in sequential studies but will be of little value in the diagnosis of large bowel cancer.


The activities of some polycyclic hydrocarbons and their "K region" epoxides in an in vitro-in vivo carcinogenicity test system. Benz(a)anthracene, 7,12-dimethylbenz(a)anthracene, dibenz(a)anthracene and benzo(a)pyrene and their related "K region" epoxides were tested for carcinogenic activities using a system in which mouse lung tissue was incubated in the presence of the test compound for 30 min and then implanted into isologous mice. Only 7,12-dimethylbenz(a)anthracene showed any marked carcinogenic activity under the conditions used, but all the compounds tested produced extensive proliferative outgrowths in the implanted tissues that may represent specific responses to the carcinogens.


The role of microsomal hydroxylases in the modification of chloroform hepatotoxicity in rats. Male rats have a greater microsomal amidopyrine N-demethylase activity per unit weight of liver, a shorter hexobarbitone sleeping time and are more susceptible to the hepatotoxic effect of chloroform than female rats. Phenobarbitone sodium, phenylbutazone and DDT induce microsomal amidopyrine N-demethylase in the liver of the male rat and reduce hexobarbitone sleeping time. Chlorpromazine is less effective in this respect. Phenobarbitone, phenylbutazone and, to a lesser extent, chlorpromazine potentiate the hepatotoxic effect of chloroform in male rats. SKF 525A, sodium diethyl-dithiocarbamate and carbon disulphide increase hexabarbitone sleeping time in male rats and protect to varying degrees against the hepatotoxic effect of chloroform.


The surface properties of the lung in rats with alveolar lipo-proteinosis. The physical behaviour of the intact lungs and of lung extracts from rats affected by alveolar lipo-proteinsosis as a consequence of silica inhalation, was studied by means of pressure volume relations and surface tension area loops. Air inflation of diseased lungs occurred at a lower pressure and collapse was less on deflation than in control specimens, although there appeared to be little change in elastic forces. When saline was used dusted rat lungs showed at higher lung volumes a peculiar hysteresis effect which is attributable to consolidation of alveoli by the lipid material. Extracts from affected lungs showed differences from controls in respect of maximum and minimum surface tensions and stability index but all the values fell within the accepted limits of normal. However, with extracts from pathological lungs the area of the hysteresis loop increased, the shape of the surface tension area curve was abnormal and the percentage compression required to reduce the surface tension to 120 muN/cm fell. Extracts from diseased lungs depressed the maximum surface tension of normal lung extracts and increased the hysteresis area but had little effect on the minimum tension, the stability index or the % compression to achieve 120 muN/cm. The response was thus mainly that of an extract from a dusted rat. The surface activity of phospholipids may be affected by neutral lipid, cholesterol and the products of cell breakdown, all of which occur in the alveolar material. The occurrence within the same lung of compunds which reduce surface tension and others which modify the same lung of compounds which reduce surface tension and others which modify this property suggests that their relative concentrations may determine the overall effectiveness of the lung lining.


The significance of plasma lipoproteins on erythrocyte aggregation and sedimentation. Increased erythrocyte aggregation can be induced by high concentrations of human lipoproteins. The dependence of aggregate formation on lipoprotein concentration was recorded by determination of erythrocyte sedimentation rate (ESR), by electrical measurement of the erythrocyte aggregation index (EAI) and by scanning electron microscopy. The lipoprotein concentrations necessary to induce a significantly increased ESR in an otherwise normal human plasma are much too high to be encountered in physiologic or even severe pathologic states. Therefore hyperlipoproteinaemia by itself cannot explain a raised ESR. In cases where the ESR is raised due to the presence of increased amounts of other erythrocyte aggregating plasma proteins (agglomerins), hyperlipoproteinaemia can contribute to a limited extent to the increase in ESR. The possible pathophysiological significance of the demonstrated erythrocyte aggregating capacity of human lipoproteins in a microvascular environment is noted.


A comparison of membrane glycoconjugates from mouse cells transformed by murine and primate RNA sarcoma viruses. Kirsten murine sarcoma virus (Ki-MSV) transformed Balb/eT3 mouse cells (K-Balb) were found to have altered membrane glycoconjugates compared to normal Balb/3T3 cells. There were reduced amounts of mono- and disialogangliosides, GM1 and GD1a, and activity of the specific galactosyltransferase required for synthesis of these gangliosides was reduced to between 0 and 18.5% of normal in the several K-Balb clones examined. When fucose-labeled glycopeptides derived from the surfaces of Balb/3T3 and K-Balb cells were compared by gel filtration chromatography, the glycopeptides from the transformed cells were enriched in earlier eluting components. These differences were also observed when the glycopeptides were derived from the entire cell and were diminished when the surface or cellular glycopeptides from Balb/3T3 and K-Balb were digested with neuraminidase prior to chromatographic analysis. Changes in these membrane sialoglycolipids and sialoglycopeptides were not influenced by Rauscher leukemia virus infection. In marked contrast, these changes in membrane glycoconjugates were not observed in Wooley monkey sarcoma virus (WSV) transformed Balb/3T3 cells (W-Balb). Although W-Balb cells like K-Balb were transformed by tissue culture criteria, their ganglioside composition, galactosyltransferase activity, and glycopeptide patterns were similar to normal Balb/3T3. These findings have potential implications concerning the role of these complex carbohydrates in the phenotypic alterations of transformed cells.


2' Derivatives of guanosine and inosine cyclic 3',5'-phosphates. Synthesis, enzymic activity, and the effect of 8-substituents. A series of representative derivatives of guanosine cyclic 3',5'-phosphate (cGMP) and inosine cyclic 3',5'-phosphate (cIMP) which contained modifications in either the 2' position or the 8 and 2' positions were synthesized. Three types of derivatives were investigated: (1) derivatives in which the 2' position has been altered to produce a 2'-deoxynucleoside cyclic 3',5'-phosphate or a 9-beta-D-arabinofuranosylpurine cyclic 3',5'-phosphate; (2) 2'-omicron-acyl derivatives; and (3) doubly modified derivatives containing a 2' modification [as in (1) and (2)] and an 8-substitution. 2'-Deoxyinosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosylhypoxanthine cyclic 3',5'-phosphate were obtained by HNO2 deamination of 2'-deoxyadenosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosyladenine cyclic 3',5'-phosphate (ara-cAMP), respectively. Treatment of 8-bromo-2'-omicron-(p-toluenesulfonyl) adenosine cyclic 3',5'-phosphate with NaSH yielded the intermediate 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoadenine cyclic 3',5-phosphate, which was converted directly to 2'-deoxyadenosine cyclic 3',5'-phosphate (dcAMP) by treatment with Raney nickel. 8-Bromo-2'-omicron-(p-toluenesulfonyl) guanosine cyclic 3',5'-phosphate was converted to 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate, and the latter was desulfurized with Raney nickel to give 2-deoxyguanosine cyclic 3',5'-phosphate. Ara-cAMP, 9-beta-D-arabinofuranosylguanine cyclic 3',5'-phosphate, and 9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate have been previously reported (Mian et al. (1974), J. Med. Chem. 17, 259). 8-Bromo-2'-omicron-acetylinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]-2'-omicron-acetylinosine cyclic 3',5'-phosphate were produced by acylation of 8-bromoinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]inosine cyclic 3',5'-phosphate, respectively; while 8-bromo-2'-omicron-butyrylguanosine cyclic 3',5'-phosphate was synthesized by bromination of 2'-omicron-butyrylguanosine cyclic 3',5'-phosphate.


Magnetic resonance studies of the proximity and spatial arrangement of propionyl coenzyme A and pyruvate on a biotin-metalloenzyme, transcarboxylase. A spin-labeled ester of CoA, R-CoA (3-carboxy-2,2,5,5-tetramethyl-1-pyrolidinyl-1-oxy CoA thioester), has been shown by competition studies using electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) to bind specifically to the propionyl-CoA binding sites of transcarboxylase. Titrations indicate 0.7 +/- 0.2 binding site for R-CoA per enzyme-bound biotin with a dissociation constant of 0.33 +/- 0.12 mM. Propionyl-CoA binds to this site with a 1.3-fold lower disonable agreement with kinetically determined inhibitor constants of CoA and propionyl-CoA and propionyl-CoA (D. B. Northrop (1969), J. Biol. Chem. 244, 5808). The bit of this spin-label on 1/T1 of water protons. The formation of a ternary transcarboxylase-R-CoA-pyruvate complex is suggested by the failure of pyruvate to displace R-CoA from the tight site and is established by the paramagnetic effects of enzyme-bound R-CoA on the relaxation rates of the protons and 13C atoms of enzyme-bound pyruvate. From the paramagnetic effects of R-CoA on the relaxation rates of the methyl protons of pyruvate at 40.5 and 100 MHz, and on the 13C-enriched carbonyl and carboxyl carbon atoms of pyruvate at 25.1 MHz, a correlation time of 7 nsec and distances from the bound nitroxide radical to the methyl protons, the carbonyl, and carboxyl carbon atoms of bound pyruvate of 7.9 +/- 0.7, 10.3 +/- 0.8, and 12.1 +/- 0.9 A, respectively, are calculated. These distances establish the close proximity of the CoA ester and keto acid sites on transcarboxylase. Together with the previously determined distances from the enzyme-bound (Co(II) to the methyl protons and 2 carbon atoms of bound pyruvate and to 12 protons and 3 phosphorus atoms of bound propionyl-CoA, the present distances are used to derive a composite model of the bound substrates in the overall transcarboxylation reaction. In this model the distance from the methyl carbon of pyruvate and the methylene carbon of propionyl-CoA, between which the carboxyl transfer takes place is only approximately 7 A. Depending on the detailed mechanism of the carboxyl transfer, the distance through which the carboxybiotin must migrate is therefore between 0 and 7 A. Hence the major role of the 14-A arm of carboxybiotin is not to permit a large carboxyl migration but, rather to permit carboxybiotin to traverse the gap which occurs at the interface of three subunits and to insinuate itself between the CoA and keto acid sites.


5'-Terminal and internal methylated nucleotide sequences in HeLa cell mRNA. The 5'-terminal oligonucleotides m7G(5')ppp(5')NmpNp and m7G(5')ppp(5')NmpNmpNp were isolated by DEAE-cellulose column chromatography after enzymatic digestion of 32P- or methyl-3H-labeled poly(A)" HeLa cell mRNA. The recovery of such oligonucleotides indicated that a high percentage of mRNA has blocked termini. The dimethylated nucleoside, N6, O2'-dimethyladenosine (m6Am), as well as the four common 2'-O-methylribonucleosides (Gm, Am, Um, Cm) were present in the second position linked through the triphosphate bridge to 7-methylguanosine (m7G) whereas little m6Am was in the third position. The only internal methylated nucleoside, N6-methyladenosine (m6A), was found exclusively as m6ApC and Apm6ApC after digestion with RNase A, T1, and alkaline phosphatase. Digestion with RNase A and alkaline phat pyrimidines are present in much smaller amounts or absent from this position. These results imply a considerable sequence specificity since there are thousands of different mRNA species in HeLa cells. Our studies are consistent with the following model of HeLa cell mRNA in which Nm may be m6Am, Gm, Cm, Um, or Am and one or more m6A residues are present at an unspecified internal location: m7G(5')ppp(5')Nm-(Nm)---(G or A)-m6A-C---(A)100-200A.


Nucleotide clusters in deoxyribonucleic acids. Comparison of the sequences of the large pyrimidine oligonucleotides of bacteriophages S13 and phiX174 deoxyribonucleic acids. The large pyrimidine oligonucleotides from the DNAs of the two related bacteriophages phiX174 and S13 have been sequenced. The largest pyrimidine oligonucleotide present is unique to S13 DNA and is the undecanucleotide C5T6, sequence C-T-T-C-C-T-C-T-T-C-T. Considerable sequence homology has been found between the pyrimidine oligonucleotides of the two phage DNAs. Out of 14 oligonucleotide sequences from S13 DNA (120 bases) at least ten are identical with sequences of oligonucleotides from phiX174 DNA (92 bases) and two are closely related (17 bases), the only difference being a single thymine to cytosine transition in each sequence (a total of 107 identical bases). The pyrimidine oligonucleotides of each phage DNA show extensive internal sequence homology among each other with up to eight bases identical in sequence in pairs of different oligonucleotides. Another interesting observation is the occurrence of symmetrical sequences (true palindromes) which read the same forwards as backwards. The longest symmetrical sequence is the nonanucleotide C4T5 sequence, C-T-C-T-T-T-C-T-C, present in both S13 and phiX174 DNAs. The extensive sequence homology observed between the pyrimidine oligonucleotides of S13 and phiX174 supports the close relationship of the two phages and provides further evidence that they were derived from recent common ancestors.


Synchronous digestion of SV40 DNA by exonuclease III. We have established an optimal condition for the synchronous digestion of SV40 DNA with Escherichia coli exonuclease III. Electron microscopy and polyacrylamide gel electrophoresis were used to obtain accurate measurements on the lengths of DNA before and after exonuclease III digestion. Based on this finding, a new method for determining the sequence of long duplex DNA can be realized. It involves (a) the synchronous digestion of the DNA from the 3' ends with exonuclease III, followed by (b) repair synthesis with labeled nucleotides and DNA polymerase, and (c) sequence analysis of the repaired DNA.


Conformational equilibria accompanying the electron transfer between cytochrome c (P551) and azurin from Pseudomonas aeruginosa. The redox reaction between cytochrome c (Cyt c) (P-551) and the blue copper protein azurin, both from Pseudomonas aeruginosa, was studied using the temperature-jump technique. Two relaxation times were observed in a mechanism assumed to involve three equilibria. The fast relaxation time (0.4 less than tau less than 8 ms) was ascribed to the electron exchange step. The slow relaxation time (tau congruent to 37 ms) was assigned to a conformational equilibrium of the reduced azurin that was coupled through the electron exchange step to a faster conformational equilibrium of the oxidized Cyt c (P551). But because the Cyt c (P551) isomerization, being very rapid, was uncoupled from the two slower equilibria, and was assumed to involve no spectral change, the amplitude of its relaxation time (tau congruent to 0.1 ms) would be zero. At 25 degrees C and pH 7.0 the rate constants for the oxidation and reduction of Cyt c (P551) by azurin were 6.1 X 10(6) and 7.8 X 10(6) M-1 s-1, respectively; for the formation and disappearance of the reactive conformational isomer of azurin they were 12 and 17 s-1, respectively. The rates for the Cyt c (P551) isomerization could only be estimated at approximately 10(4) s-1. The thermodynamic parameters of each reaction step were evaluated from the amplitudes of the relaxations and from Eyring plots of the rate constants. Measurements of the overall equilibrium constant showed it to be temperature independent (5-35 degrees C), i.e. deltaHtot = 0. This zero enthalpy change was found to be compatible with the enthalpies calculated for the individual steps. In the electron exchange equilibrium, the values of the activation enthalpies were two to three times higher than the values published for various low molecular weight reagents in their electron exchange with copper proteins, yet the rate of exchange between Cyt c (P551) and azurin was some hundreds of times faster. This was explained in terms of the measured positive or zero entropies of activation that could result from a high level of specificity between the proteins particularly in areas of complementary charges. The mechanism of electron transfer was considered as essentially an outer sphere reaction, of which the rate could be approximated by the Marcus theory.


Cleavage of Type II and III collagens with mammalian collagenase: site of cleavage and primary structure at the NH2-terminal portion of the smaller fragment released from both collagens. Collagenase cleavage of human Type II and III collagens has been studied using a highly purified preparation of rabbit tumor collagenase. Progress of the reactions in solution was followed by viscometry and the results indicated that under the conditions employed Type III collagen molecules were cleaved at approximately five times the rate of Type II molecules. Cleavage products of the reactions were isolated in denatured form by agarose molecular sieve chromatography. The molecular weights and amino acid compositions of the products demonstrated that Type II and III molecules had been cleaved at the characteristic three-quarter, one-quarter locus, giving rise to a large fragment derived from the NH2-terminal portion of the molecule and a smaller fragment representing the COOH-terminal region. The amino acid sequence at the NH2-terminal portion of the smaller fragment derived from Type II collagen was determined to be Ile-Ala-Gly-Gln-Arg, and the corresponding region from Type III collagen was found to have the sequence Leu-Ala Gly-Leu-Arg. These sequences for alpha1(II) and alpha1(III) chains adjacent to the site of collagenase cleavage along with previous data for alpha1(I) and alpha2 chains indicate that the minimum specific sequence required for collagenase cleavage is Gly-Ile-Ala or Gly-Leu-Ala. Inspection of the available sequence data for collagen alpha chains indicates that the latter sequences are found in at least three additional locations at which collagenase cleavage does not occur. Each of the sequences which are apparently not substrates for collagenase, however, are followed by a Gly-X-Hyp sequence. We suggest, then, that a minimum of five residues in collagen alpha chains COOH-terminal to the cleavage site comprise the substrate recognition site.


Translation of collagen messenger RNA in a cell-free system derived from wheat germ. A cell-free system for synthesizing protein from wheat germ was used to translate the messenger RNA extracted from 16-day embryonic chick calvaria. A part of the product had properties similar to collagenous peptides and served as a substrate for prolyl hydroxylase, an enzyme specific for collagen. The level of potassium was critical for the synthesis of high molecular weight products with properties similar to pro-alpha-chains. The potassium concentration for optimal protein synthesis, as judged by maximum incorporation of [3H]proline into acid precipitable material, was considerably lower than the concentration required for the synthesis of high molecular weight collagenous peptides.


Subunit dissociation of mitochondrial malate dehydrogenase. Fluorescence polarization studies of porcine mitochondrial malate dehydrogenase labeled with fluorescein isothiocyanate or fluorescamine indicated a concentration-dependent dissociation of the dimeric molecule with a KD OF 2 X 10(7) N at pH 8.0. These results were confirmed by the concentration dependence of the stability of the enzyme at elevated temperatures and the creation of hybrid molecules with fluorescein and Rhodamine B labeled subunits, in which energy transfer was observed. The binding of NADH resulted in a small shift of the subunit dissociation curve toward monomer, demonstrating that monomer has twice the affinity for reduced coenzyme. NAD+ binding prevented dissociation of the dimer, even at concentrations below 10(-8) N. These results indicate that binding of reduced or oxidized coenzymes results in different conformation changes, which are transferred to the subunit interface.


Estrogen receptors in the rat uterus. Retention of hormone-receptor complexes. The retention pattern and biochemical characteristics of estrogen receptors in the nuclei of uterine cells were studied as a function of time after the in vivo injection of estradiol (E2) to immature female rats. One hour after the injection of 0.1 mug of tritiated E2, approximately 0.20 pmol per uterus of receptor bound hormone is retained in uterine nuclei. This dose of E2 produces a maximal uterotrophic response. Six hours after E2 administration, uterine nuclei retain 0.04-0.08 pmol of hormone per uterus. Hormone receptor complexes extracted from uterine nuclei 1, 3, and 6 h after in vivo injection of hormone have similar structural and binding characteristics. Receptors extracted at all three times sediment at 5S in high salt gradients and have a dissociation binding constant of approximately 3 nM for E2. The wash-out curves of receptors as a function of salt concentration are identical for uterine nuclei from animals treated for 1 or 6 h with estradiol, suggesting that the nature of the nuclear binding of receptors is not altered during this time interval. Experiments utilizing the injection of unlabeled estradiol, followed by an in vitro exchange procedure with tritiated estradiol, indicated that the total nuclear estrogen receptor sites, i.e., filled and vacant, decreased similarly.


Chemical modification of the tryptophan residue in adrenocorticotropin. The single tryptophan residue in the pituitary hormone adrenocorticotropin was modified selectively by reaction with a variety of substituted o-nitrophenylsulfenyl chlorides. In addition to quantitative modification of the tryptophan residue, the reaction invariably resulted in partial oxidation of the methionine residue to the sulfoxide. The methionine sulfoxide derivative could be separated from the desired product by partition chromatography on Sephadex G-50 in the solvent system 1-butanol-pyridine-0.1% acetic acid (5:3:11). Thus, the 2,4-dinitrophenylsulfenyl, 2-nitro-4-carboxyphenylsulfenyl, and 2-nitro-4-carbamidophenylsulfenyl derivatives of adrenocorticotropin were prepared and characterized. Modifications in the isolation of adrenocorticotropin from ovine pituitaries are also described. The melanocyte stimulating activities of the native hormone and the analogues are discussed.


Preparation of radioiodinated simian virus 40 DNA for use in DNA - DNA reassociation kinetics experiments. A method is described for the preparation of 125I-labelled SV40 DNA. Using this method, SV40 DNA can be routinely labelled to 15 - 10(6) dpm per mug; much higher specific activities are easily obtained by minor modifications of the method. Once incorporated, the radioactive label dissociates from DNA exceedingly slowly at 4 degrees C or at 68 degrees C. Iodinated SV40 DNA is shown to be useful in the quantitation of viral nucleic acid sequences in SV40-transformed 3T3 cells by DNA - DNA reassociation kinetics.


Partial purification and properties of a DNA-binding protein from nuclei of cells infected with polyoma virus. A DNA-binding protein has been purified from nuclei of 3T3 cells infected with polyoma virus. The assay used to detect this activity measures the amount of double-stranded DNA retained on a nitrocellulose membrane filter in the presence of binding protein. The interaction between DNA and protein is salt dependent and occurs optimally at 0.8 M NaCl. The isolated protein can bind to both circular and linear duplex DNA. Incubation of the binding protein with PM2 or polyoma DNA results in the formation of a fast sedimenting DNA structure in neutral sucrose gradients. The isolated binding protein is also capable of producing a considerable stimulation of both Escherichia coli (Pol I) and T4 DNA polymerase activities when either single-stranded or intact, native T7 DNA is used as the template. The binding protein itself is free of detectable DNA polymerase or nuclease activity.


Early androgen action in kidney of normal and androgen-insensitive (tfm/y) mice. Changes in RNA polymerase and chromatin template activities. Intranuclear accumulation of testosterone was compared with early changes in transcriptional events in kidneys from normal female and androgen-insensitive (tfm/y) mice. Following a subcutaneous injection of [3H] testosterone, total nuclear uptake of the steroid was maximal at 30 min and declined to about 40% of the peak value by 4 h after hormone administration. After a single subcutaneous dose of testosterone, RNA polymerase activity assayed in intact nuclei in the presence of Mg2+ and alpha-amanitin (nucleolar RNA polymerase I), as well as the enzyme activity sensitive to low concentration of the toxin (nucleoplasmic RNA polymerase II), increased within 15 min and attained peak values at 2 and 1 h, respectively. The activity of both polymerases declined almost to the control level by 4 h and then increased again with a second peak at 20 and 12 h for RNA polymerase I and II, respectively. Similarly, the template capacity of mouse kidney chromatin, as measured with mammalian RNA polymerase II, increased by 15 min, reached a peak at 1 h and returned to control level by 4 h following hormone treatment. A second dose of testosterone given at the nadir (4 h) was not capable of stimulating renal chromatin template activity significantly as compared to the effect observed after the initial hormone treatment. Contrary to the testosterone-stimulated changes in transcriptional events observed in normal female mice, androgens elicited no response in androgen-insensitive tfm/y mice, animals lacking cytosol androgen receptors. These results strongly support the contention that hormone-specific receptors are obligatory to steroid-mediated modifications in gene transcription.


Interactions of proteins and cholesterol with lipids in bilayer membranes. Mixtures of lipids and protein, the ATPase from rabbit sarcoplasmic reticulum, were studied by freeze-fracture electron microscopy and by measurement of the amount of fluid lipid with the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl (TEM-PO). In dimyristoyl phosphatidylcholine vesicles the protein molecules were randomly distributed above the transition temperature, Tt, of the lipid and aggregated below Tt. For mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine the existence of fluid and solid domains were shown in the temperature interval predicted from earlier TEMPO measurements. When protein was incorporated into this lipid mixture, freeze-fracture particles were randomly distributed in fluid lipids, or aggregated when only solid  lipids were present. In mixtures of dimyristoyl phosphatidylcholine with cholesterol the protein was distributed randomly above the transition temperature of the phosphatidylcholine. Below that transition temperature the protein was excluded from a banded phase of solid lipid in the case of 10 mol% cholesterol. In mixtures containing 20 mol% cholesterol, protein molecules formed linear arrays, 50-200 nm in length, around smooth patches of lipid. Phase diagrams for lipid/cholesterol and lipid/protein systems are proposed which account for many of the available data. A model for increasing solidification of lipid around protein molecules or cholesterol above the transition temperature of the lipid is discussed.


Phosphorus nuclear magnetic resonance of Acholeplasma laidlawii cell membranes and derived liposomes. 1. The 129 MHz 31P-NMR spectrum of Acholeplasma laidlawii membranes is very similar to the spectrum of the derived liposomes and is a typical "solid state" spectrum in which the major contribution to the linewidth is made by the chemical shift anisotropy. From the value of the chemical shift anisotropy an order parameter of 0.15 is estimated for the lipid phosphates in both membranes. 2. The 31P-NMR spectrum of the A. laidlawii membrane is insensitive to pronase digestion of 4-60% of the membrane proteins and subsequent cytochrome C binding. These results indicate that either no strong lipid polar headgroup-protein interactions occur in the membrane or that the lipid-protein "complexes" in the membrane have a fast rotation (Tc shorter than 10(-6)S) along an axis perpendicular to the plane of the membrane. 3. Phospholipase A2 degrades all the phosphatidylglycerol in the membrane. The resulting membrane contains a phosphoglycolipid as the sole phosphorus-containing compound. The 31P-NMR spectrum of these membranes is identical to the spectrum of the native membranes suggesting a similar motion for the phosphate groups in both lipids. 4. Ca2+ binding to liposomes prepared from either the total polar lipids or the total phosphorus-containing lipids isolated from the A. laidlawii membrane does not affect the 21P-NMR spectrum. 5. The 31P-NMR spectrum of the membranes and derived liposomes, however, is sensitive to lipid phase transitions. When the membrane lipids are in the gel state a broadening of the 31P resonance occurs demonstrating that the polar head group motion in a biological membrane is more restricted below the lipid-phase transition temperature.


Comparative study of the milk fat globule membrane and the mouse mammary tumour virus prepared from the milk of an infected strain of Swiss albino mice. Milk fat globule membranes and mammary tumour virus particles (d=1.17 g/cm3) have been obtained from the milk of a Swiss albino mice strain. Comparative biochemistry shows that these two structures differ significantly in the phospholipid, polypeptide and glycopolypeptide patterns and enzymatic activities. However, the lipid profile and the morphology of both structures suggest a filiation with the plasma membrane. Density fractions obtained from the crude virus preparation have been thoroughly investigated. The results suggest that most of these fractions represent degraded virus and/or atypical virus assembly.


Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell. Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various lectins is Ricinus communis greater than wheat germ greater than or equal to concanavalin A greater than or equal to soybean greater than Limulus polyphemus. No agglutination was noted for Ulex europaeus. Using 125I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites for concanavalin A and soybean lectins. Sodium deoxycholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity columns. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.


Correlation between protein kinase-mediated stimulation of calcium transport by cardiac sarcoplasmic reticulum and phosphorylation of a 22000 dalton protein. Increases in protein kinase-catalyzed phosphorylation of a 22000 dalton protein correlated closely with increases in phosphate-facilitated calcium transport measured concurrently in canine cardiac sarcoplasmic reticulum under similar conditions in the presence of varying concentrations of bovine cardiac protein kinase. A correlation coefficient of 0.93 and a P value of less than 0.001 were obtained. Protein kinase-catalyzed phosphorylation of the 22000 dalton microsomal protein may mediate the abbreviation of systole seen in the mammalian heart in response to inotropic agents like catecholamines.


Steroid-protein interactions. XXXIV. Chemical modification of alpha1-acid glycoprotein for characterization of the progesterone binding site. The nature of the steroid binding site in alpha1-acid glycoprotein (orosomucoid) was investigated by chemical modification of individual amino acids and subsequent examination of the binding affinity for progesterone. Equilibrium dialyses were performed under conditions that excluded contact with human skin. Reaction of the lysyl residues with trinitrobenzenesulfonic acid or arylisocyanates resulted in a reduction of active sites. In an alternate approach, one lysyl residue of alpha1-acid glycoprotein was protected from modification by trinitrobenzenesulfonic acid when progesterone was present to form the complex with alpha1-acid glycoprotein. We conclude that a lysyl residue is located in the binding site. Reaction of tetranitromethane with the tyrosine groups in alpha1-acid glycoprotein also reduced the number of active binding sites for progesterone. Again, a partial protection of this modification was seen in the presence of progesterone and other delta4-3-ketosteroids. The progesterone binding activity observed in the tyrosine-modified alpha1-acid glycoprotein by equilibrium dialysis and by fluorescence quenching titration can be interpreted best by the presence of one tyrosyl residue in the binding site, and involvement of a second tyrosine nearby. Modification of tryptophan in alpha1-acid glycoprotein by mild acid hydrolysis, N-bromosuccinimide, hydroxynitrobenzylbromide, and formic acid resulted in a decreased steroid binding; the formylation reaction was fully reversible. The approximate distance between progesterone and the tryptophan involved in the binding was calculated to be between 9.1 A and 14.1 A. When alpah1-acid glycoprotein was cleaved by the cyanogen bromide procedure according to Ikenaka et al. (1972, Biochemistry 11, 3817-3829), both the amino and the carboxyl fragment had a weak progesterone binding affinity which could be measured in 4 M NaCl. This result thus failed to specify the location of the steroid binding site in alpha1-acid glycoprotein. However, the closeness of tryptophan, lysine and tyrosine in the primary and presumably the tertiary structure of alpha1-acid glycoprotein is in agreement with the properties of the binding site suggested by our studies.


A putative glucocorticoid receptor and a transcortin-like macromolecule in pituitary cytosol. This study describes the binding of [3H]corticosterone and [3H] dexamethasone to soluble macromolecules in the pituitaries of adrenalectomized rats perfused at sacrifice to remove blood contamination. (1) Unlabeled dexamethasone competes only slightly for [3H] corticosterone binding when the steroids are added to cytosol as well as when the steroids are present from the time of disruption of the tissue. In the latter case, corticosterone is as good a competitor as dexamethasone for [3H]dexamethasone binding. (2) Gel permeation chrmatography with BioRad A-5M reveals the presence of a [3H] corticosterone-macromolecular complex with an elution volume comparable to transcortin as well as a very large hormone-binding complex eluting in the void volume of the column...


Hepatic accumulation of vitamin D3 and 25-hydroxyvitamin D3. Concomitant intravenous administration of 25-hydroxycholecalciferol and [3H] vitamin D3 to vitamin D-depleted rats did not affect the conversion of [3H] vitamin D3 to 25-OH-[3H] vitamin D3 as indicated by a serum 25-OH-[3H] vitamin D3 to content at 3 and 24 h identical to those observed in animals receiving [3H] vitamin D3 alone. Similarly, pre-dosing with 25-OH vitamin D3 24 h earlier did not affect the conversion. Co-administration to vitamin D depleted rats of vitamin D2 or D3, at 200-fold higher doses than a control group receiving tracer [3H] vitamin D3 alone, resulted in serum 25-OH vitamin D levels that were 15-20 fold higher than the control, indicating a similar metabolic fate for synthetic and natural vitamin D in rats and the ability of increased substrate to overwhelm hepatic constraints on 25-OH vitamin D production. Following intravenous administration of 25-OH-[3H] vitamin D3 to vitamin D depleted rats, hepatic 3H content decreased in parallel with serum radioactivity. Hepatic accumulation of intravenously administered vitamin D3 ([14C] vitamin D3) alone or with 25-OH-[3H] vitamin D3, by vitamin D-depleted rats revealed a marked preference for vitamin D3; the hepatic accumulation of [14C] vitamin D3 increased to 35% of the dose by 45 min, at which time 25-OH-[3H] vitamin D3 hepatic content was 7-fold less, and decreasing. Chromatography of extracts of hepatic subcellular fractions revealed more [14C] vitamin D3 than 25-OH-[3H] vitamin D3 in the microsomes, the reported site of calciferol 25-hydroxylase. Circulating 25-OH vitamin D, therefore, has comparatively minimal potential for hepatic accumulation. Product inhibition of the calciferol 25-hydroxylase must, therefore, result from recently synthesized hepatic 25-OH vitamin D, and is not affected by exogenous 25-OH vitamin D3.