[Tumour antigens inducing immune reactions]. Antigens of viral tumours are the same for all the tumours due to the same virus. Antibodies in tumours bearing animals allow to detect antigens in nucleus, in cytoplasm and on the cell membrane which carries also embryonic antigens and the antigen responsible for tumour rejection by sensitized lymphcytes (TSTA or TATA). Is this antigen identical to the surface antigen shown by antibodies? Purification of membrane antigens will answer this important question. Chemically induced tumours bear tumour rejection antigens having an individual specificity, perhaps related to modified histocompatibility antigens, and embryonic antigens. Both give rise to antibodies and sensitized lymphocytes. Among human tumours, Burkitt lymphoma is strongly antigenic. Its viral origin is highly likely. Antibodies in sera of Burkitt patients react with antigens present in nucleus, cytoplasm and on the membrane of malignant or transformed cells. Sensitized lymphocytes in the peripheral blood recognize a membrane antigen probably different of that revealed by antibodies. Antibodies found in sera of patients with carcinoma react mainly with tissular antigens. In these cases, methods exploring delayed type reactivity, such as leukocyte migration inhibition and moreover skin testing with tumour extracts, gave some promising results.


[Superinduction of interferon and and a study of its messenger RNA]. Combined use of interferon inductor poly-IC and antibiotics (cycloheximide and actinomycin D) provided a significant increase (up to 1000 times) in interferon production by chick, mouse, monkey and human cells. Messenger RNA with matrix activity for interferon (mRNA-IF) was isolated from superinduced cells. On translation of mRNA-IF in homogenous and heterogenous cells the specificity of interferons produced was determined by the type of the cells from which mRNA-IF was isolated. Sedimentation analysis of various mRNA-IF revealed 2 peaks of activity: major (5--15S) and minor (25--30S).


Comparative study of the antiviral activity of acyl derivatives of 2,2'-anhydro-1-beta-D-arabinofuranosylcytosine and other nucleosides against encephalitis in mice. Anti-deoxyribonucleic acid virus activities of 3'-O-acyl derivatives of 2,2'-anhydro-1-beta-d-arabinofuranosyl cytosine (cyclo-C) and 1-beta-d-arabinofuranosylcytosine (Ara-C) were evaluated by using an in vivo test system in mice. Among the derivatives tested, 3'-O-decanoyl cyclo-C hydrochloride was the most effective against herpes simplex virus-induced encephalitis in mice when the drug was administered directly into infected brains of mice (target-organ treatment). A comparative study of the treatment of herpetic encephalitis in mice with 3'O-decanoyl cyclo-C and other nucleosides, including Ara-C, 9-beta-d-arabinofuranosyladenine (Ara-A), and 5-iodo-2'-deoxyrudine (IUdR), proved Ara-A to be more efficacious than the other nucleosides, followed by 3'-O-decanoyl cyclo-C, which was more active than Ara-C and IUdR. Administration of 3'-O-acyl cyclo-C's by intraperitoneal injection, however, failed to demonstrate activity against herpetic encephalitis in mice. The antivaccinial activity of 3'-O-decanoyl cyclo-C was also compared with that of other compounds against encephalitis and dermal tail lesions in mice caused by vaccinia virus infection. Interaperitoneally administered 3'-O-decanoyl cyclo-C and Ara-C also showed no significant activity against the diseases. Under these test conditions, N-methylisatin-beta-thiosemicarbazone (Marboran) was the most active compound, followed by Ara-A.


Experimental herpes simplex virus type 1 encephalitis: treatment with 5-trifluoromethyl-2'-deoxyuridine. 5-Trifluoromethyl-2'-deoxyuridine (F(3)dThd) was evaluated for its neurotoxicity and for its ability to increase the life span of mice injected intracerebrally with herpes simplex virus type 1 (HSV-1) and F(3)dThd simultaneously. F(3)dThd showed no neurotoxicity at the highest concentration tested (100 mg/kg). Mice injected intracerebrally with HSV-1 died within 5 days postinfection. However, all mice injected concurrently with HSV-1 and 100 mg of F(3)dThd per kg lived through the termination of the experiment (60 days). Protection of mice from HSV-1 encephalitis by F(3)dThd has been shown to be dose dependent, with 100, 75, 50, and 25 mg of F(3)dThd per kg yielding a survival rate of 100, 90, 50, and 10%, respectively. HSV-1 titers in mouse brains receiving HSV-1 and 100 mg of F(3)dThd per kg concurrently were 100- to 1,000-fold lower at 2 to 4 days postinfection than control mice receiving HSV-1 alone. F(3)dThd was shown not to stimulate interferon production. Encephalitis caused by a ribonucleic acid virus, encephalomyocarditis virus, was not modified by F(3)dThd treatment.


Amorphous ferrous sulfide as a reducing agent for culture of anaerobes. Amorphous ferrous sulfide, prepared by reacting ferrous ammonium sulfate and sodium sulfide, is an excellent reducing agent for the culture of anaerobes. It reduces resazurin and reacts much more rapidly with O2 than does either soluble sulfide (HS)- or cysteine. One of the end products of the oxidation of ferrous sulfide with O2 is red and serves as an indicator for the oxygen contamination of a culture medium. Amorphous ferrous sulfide served as a suitable reducing agent for the growth of species of Methanobacterium or Clostridium. Its use is recommended for enrichment or culture of anaerobes (e.g. autotrophs, fermentative organisms) from sediments and other habitats were organic reducing agents are undesirable and where soluble sulfide might be toxic.


Regeneration of pleated filters used to concentrate enteroviruses from large volumes of tap water. Pleated cartridge filters are capable of concentrating enteroviruses from large volumes (well over 2,000 liters) of tap water. These epoxy-fiberglass filters can be regenerated if they are treated with 0.1 N NaOH or autoclaved to inactivate any contaminating virus. The regenerated filters regained their ability to concentrate viruses from water at high flow rates.


Inactivation of polioviruses and coxsackieviruses in surface water. Inactivation rates of polioviruses 1 and 3 and coxsackieviruses A-13 and B-1 were determined in situ in the Rio Grande in southern New Mexico, using membrane dialysis chambers. Inactivation of the viruses was exponential, and the rates of inactivation were apparently affected principally by the water temperature. Stability of the viruses in river water differed, with poliovirus 1 and coxsackie B-1 being most stable. Typically 1-log reductions of infectivity at water temperatures ranging between 23 and 27 degrees C required 25 h for poliovirus 1, 19 h for poliovirus 3, and 7 h for coxsackie virus A-13. At water temperatures of 4 to 8 degrees C, the log reduction times for poliovirus 1 and coxsackievirus B-1 were 46 and 58 h, respectively. Results obtained with labeled poliovirus 1 and coxsackievirus B-1 and with infectious ribonucleic acid indicate that inactivation was due to damage to viral ribonucleic acid. Virus-inactivation rates were also affected by heat sterilization of river water, indicating the presence of a heat-labile or volatile inactivating factor. The inactivating factor in Rio Grande water was apparently present at a constant concentration over a 1-year period.


Degradation of malathion by salt-marsh microorganisms. Numerous bacteria from a salt-marsh environment are capable of degrading malathion, an organophosphate insecticide, when supplied with additional nutrients as energy and carbon sources. Seven isolates exhibited ability (48 to 90%) to degrade malathion as a sole carbon source. Gas and thin-layer chromatography and infrared spectroscopy confirmed malathion to be degraded via malathion-monocarboxylic acid to the dicarboxylic acid and then to various phosphothionates. These techniques also identified desmethyl-malathion, phosphorthionates, and four-carbon dicarboxylic acids as degradation products formed as a result of phosphatase activity.


Inactivation of clay-associated bacteriophage MS-2 by chlorine. The model system consisted of bacteriophage MS-2, bentonite clay, and hypochlorous acid (HOC1). Factors that influenced association of the bacterial virus with bentonite were the titer of unadsorbed viruses, clay concentration, cation concentration, temperature, stirring rate, and the presence of soluble organics. Variation of the kinetic adsorption rate constant with stirring speed indicates that phage attachment is a diffusion-limited process; the attachment reaction has an apparent activation energy of 1 kcal/mol. About 18% of clay-associated bacteriophages was recovered by mixing the suspension with an organic eluent. Inactivation data were obtained from batch reactors operated under those conditions in which loss of HOC1 was minimal during the reaction. Bacteriophages attached to clay were more resistant to HOC1 than were freely suspended phages; for equivalent HOC1 concentrations, clay-associated phages required about twice the time that freely suspended phages required for loss of 99% of the initial virus titer.


Autoimmune progesterone dermatitis.

Autoimmune progesterone dermatitis. Seven patients had autoimmune progesterone dermatitis. The morphological findings illustrate the polymorphous nature of the disease in which urticaria, erythema multiforme, and dyshidrosiform lesions were seen. Recurrence of the eruption five to ten days prior to the menses with spontaneous resolution following the menses was present in all cases. Intradermal skin testing to progesterone was done to confirm the diagnosis. Six of the seven patients has a history of use of artificial progestational hormones prior to the beginning of their eruption. It is postulated that the artificial progesterones may have been the trigger for the development of their autosensitivity. Treatment with conjugated estrogens resulted in remission of the disease in five of the seven cases reported.


The effect of methysergide and other receptor antagonists on fenfluramine-induced glucose uptake into the isolated rat hemidiaphragm. Fenfluramine in therapeutic concentrations causes a significant and dose related increase in glucose uptake into isolated rat and human skeletal muscle in the presence of insulin. Using fenfluramine, 100 ng/ml, on the rat hemidiaphragm preparation the effects of the following receptor blocking drugs in concentrations up to 250 ng/ml were investigated on this phenomenon: atropine, haloperidol, mepyramine, methysergide, propranolol and thymoxamine. Only methysergide, the 5-HT antagonist, reduced the action of fenfluramine in relatively low concentrations of the drug which were dose related (10 ng/ml causing approximately 40% inhibition). This suggests that the peripheral as well as the central actions of fenfluramine are mediated through 5-HT receptors.


The influence of adenylate cyclase inhibitors on the spontaneous activity of the cardiac pacemaker. The effects of adenylate cyclase inhibitors, haloperidol and chlorpromazine, on the spontaneous activity of the rabbit sinus node was studied by means of double sucrose gap and intracellular microelectrodes. Haloperidol and chlorpromazine arrested the spontaneous activity which could be restored with noradrenaline, in the presence of haloperidol, or with dibutyryl c AMP, in the presence of either inhibitor used. After arresting of the spontaneous activity, sinus node cells might utilize metabolic energy supplied from exogenous pyruvate. Pyruvate protected from depolarization or reestablished polarization in depolarized cells but was not able to restore the spontaneous activity. Discussion of these results suggests that c AMP may be directly involved in the spontaneous electrogenesis in the sinus node.


Pulmonary fibrosis from amorphous silica dust, a product of silica vapor. After prolonged occupation exposure to amorphous silica dust-a product of vaporized crystalline silica (quartz)-eleven out of forty workers showed reticular and/or nodular abnormalities in roentgenograms of the chest. From this group, we present three illustrative cases. These had widespread pulmonary disease with granulomatous nodules and fibrosis (documented histologically in two cases), but there was no demonstrable restrictive impairment of pulmonary function. We discuss the possible importance of this previously underestimated toxicity, especially in an industrial setting.


Insulin receptor status in disease states of man. When insulin or any peptide hormone binds to its receptor on the surface of a target cell, it initiates a series of biochemical steps that ultimately lead to the characteristic action of the hormone. The strength of the signal generated by the hormone depends equally on the hormone concentration, the receptor concentration, and the receptor affinity. Not only do hormone concentrations change rapidly and widely in vivo but so do receptor concentration and affinity. In hormone resistant states, any step in the biochemical pathway of hormone action at the target cell may be involved. Studies of insulin receptors in people indicate that the insulin receptor is altered in many common disorders such as obesity and diabetes, as well as in rare disorders such as extreme insulin resistance due to circulating antibodies directed at the insulin receptor itself. By responding to both intracellular and extracellular events, the insulin receptor is, therefore, a major site for the regulation of target cell responsiveness in vivo.


[Zollinger-Ellison syndrome and metastatic insulinoma; its response to streptozotocin]. A patient with pancreatic islet cell carcinoma metastatic to the liver developed Zollinger-Ellison syndrome. She presented hypoglycemic crisis afterwards, which were consequent to insulin excess. After streptozotocin treatment, she remained free of symptoms during the following 12 months.


[Post-partum myocardiopathy. Apropos of 25 cases]. The authors have studied 25 cases of PPC which were selected according to very strict criteria. These cases represent 1 case of PPC for every 2 687 confinements, and 37.8% of all cardiomyopathies in females of child-bearing age. The females with PPC belonged to the more deprived socio-economic population. Multiparity and twin pregnancies are predisposing factors. The clinical picture is usually one of biventricular of left heart failure. Four cases of embolism are included (16%, which is a high figure for Africa). The special investigations are important. Mechanographic studies show a very significant lengthening of the pre-ejection phase (especially QB1), and a reduced ejection phase; haemodynamic studies show in particular increased pressures on the right side in 4 cases out of 6. (3 of which had an arterio-capillary gradient). Angiocardiography, carried out in 4 cases, showed abnormalities especially of the left ventricle (especially dilation and hypokenesia). The outcome may be favourable, especially after 3 to 6 months have elapsed. However, complications may arise in the long-term (10, 14 and 37 months). There were two deaths (8%). Histologically, the lesions were mainly of a congestive type, with some areas of fibrosis, and rarely areas of inflammation (myocarditis). From the aetiological standpoint, it might be thought that during the posptpartum period, various abnormal conditions prevail or would be discovered (13 in our series); but it is also true that some cases of true PPC, with marked stasis and a tendency to thrombosis, as well as severe involvement of the myocardium (as witnessed especially by the mechanographic findings), to progress after 3 months either to resolution, death, or complications; the histological findings in the two fatal cases were of myocarditis.


Characterization of synchronized cultures of Bumilleriopsis filiformis. Changes in cytochrome-f photooxidation and fluorescence induction kinetics. Activity of the photosynthetic apparatus of synchronized cultures was studied with the xanthophycean alga Bumilleriopsis filiformis, following the kinetics of fluorescence induction and photooxidation of cytochrome f (= cytochrome c-553) of intact cells. During the beginning of the cell-division phase, minimum cellular photosynthetic activity is observed and a maximum after its completion, which is accompanied by corresponding changes in Hill reaction activity and re-reduction of cytochrome f by photosystem II light. At minimum activity, the level of steady state fluorescence was higher than at the maximum. This is due, at least in part, to the diminished electron flow between the two photosystems seemingly caused by decreased photosystem I activity. This explanation was suported by the kinetics of cytochrome-f photooxidation. Thus, electron transport activity of both photosystems appears to vary during the cell cycle.


Role of silicon in diatom metabolism. VIII. Cyclic AMP and cyclic GMP in synchronized cultures of Cylindrotheca fusiformis. Levels of the cyclic nucleotides, cAMP and cGMP, were determined in four species of pennate diatoms; changes in their levels and ratios were monitored in silicon-starved and light-dark synchronized cultures of Cylindrotheca fusiformis. Content of both cAMP and cGMP changed during the cell cycles: when silicate was added to starved cultures, cAMP, cGMP and DNA levels rose rapidly; cAMP and cGMP declined befor DNA synthesis was complete and continued to fall during the events leading to cell separation. In unstarved synchronies, net synthesis of DNA continued until cell separation; 1 h before cell separation cAMP levels fell while those of cGMP rose. The results support of the proposal that cAMP and cGMP may play a part in the process of cell division in the diatom, possible involving silicon.


Possible role of cyclic AMP in the synthesis of chlorophyll in Chlorella fusca. The intracellular concentration of cAMP in the green alga Chlorella fusca was in the range of 2.10(-9) to 10(-8) moles/g dry weight and was strongly dependent on the growth conditions. The cAMP level was high with high light intensity, low nitrate or glucose concentration. Intracellular cAMP increased only by factor of 2 when high amounts (up to 10(-3) M) of cAMP were added to the medium. Most of the given cAMP was converted to 5'-AMP. Addition of cAMP had little effect on the chlorophyll content of the cells, only at 10(-6) M some enhancement in photoautotrophic cultures was observed. On the other hand high amounts of cAMP in the medium increased the growth rate. DBcAMP* showed a positive effect on chlorophyll synthesis and growth rate at much lower concentrations compared to cAMP. Stimulation effects of exogenous cAMP on the synthesis of chlorophyll were also observed in mixotrophic cultures with a high glucose/nitrate ratio, conditions where chlorophyll synthesis is repressed. Similar to autotrophic conditions DBcAMP was more effective than cAMP. These data indicate that cAMP may act in a system controlling the chlorophyll content of the cells in response to nutrients or light.


Isolation and characterization of lipid globules from the zoospores of Blastocladiella emersonii. Lipid globules were isolated and characterized both chemically and morphologically. They were composed mainly of triglyceride and free sterol, which accounted for over 90% of the total globule content. Smaller amounts of diglyceride, carotenoid, free fatty acid, phospholipid and protein were found. No sterol esters or monoglycerides were detected. Morphologically, the isolated lipid globules resembled the lipid globules in situ. They were spherical, 0.4-1.5 mum in diameter and lacked a trilaminar membrane.


Alterations in motor activity, sleep, and biochemistry in a cycling manic-depressive patient. Biochemical and electrophysiological factors were studied longitudinally in a rapidly cycling manic-depressive patient. Slow changes in mood, motor activity, sleep, and urinary norepinephrine levels during the course of each depressed and manic episode are reported, as well as rapid alterations in many variables at the time of mood switch. Urinary concentrations of norepinephrine and its metabolite, 3-methoxy-4-hydroxyphenyl glycol (MHPG) were significantly lower in depression than in mania; norepinephrine but not MHPG excretion increased prior to the switch. We postulate that the slow behavioral and biological changes preceding switches in this patient are an important manifestation of the cyclic process in manic-depressive illness.


Lipoprotein lipase.

Lipoprotein lipase. The activity of lipoprotein lipase in the vascular bed represents the major pathway by which triglyceride fatty acid is cleared from the plasma and made available to the epripheral tissues. Recent studies on the properties of the enzyme, both solubilized and membrane-bound have provided new information on the regulation of its activity with the major triglyceride-rich lipoprotein substrates. A key role for this enzyme in the regulation of plasma triglyceride levels is indicated from studies of lipase levels in human adipose tissue and in blood plasma obtained after injection of heparin.


Vasoactive intestinal polypeptide and gastrin-producing islet cell carcinoma. A 61-year-old woman had watery diarrhea, hypochlorhydria, hypokalemia, and elevated serum gastrin levels. She had islet cell carcinoma of the body of the pancreas with multiple metastases to the liver. Radioimmunoassay and immunofluorescence demonstrated both vasoactive intestinal polypeptide (VIP) and gastrin in the surgically removed carcinoma and in a metastatic focus. Electron microscopical findings confirmed the presence of two cell types whose secretory granules had characteristics ascribed to these two hormones. Plasma prostaglandin E levels were also elevated above normal. Serum VIP levels became elevated to the Verner-Morrison range prior to her death of a bleeding duodenal ulcer two years after initial symptoms.


Nerve and muscle in steroid-induced weakness in the rabbit. The purpose of this study was (1) to produce a cortisone induced myopathy, (2) to find out whether or not there is an associated neuropathy, and (3) if a cortisone induced neuropathy is produced, to determine its temporal relation to the myopathy. Several corticosteroid preparations were administered to rabbits, in different dosages and for different periods of time in an attempt to produce maximal effects. Decadron and cortisone  acetate in doses of 0.8 mg/kg of body weight and 10 mg/kg of body weight respectively, proved most effective in producing a myopathy. Higher dosages of decadron were fatal, and hydrocortisome, 10 mg/kg of body weight, was not effective. Experimental animals receiving effective doses of decadron, cortisone acetate, and hydrocortisone lost weight, became less agile and, with continued administration of corticosteroids, became immobile. Hind limbs were affected earlier  than forelimbs. The muscle lesion consisted of Z-line irregularity and streaming, vacuolation, variation in fiber size, fragmentation and phagocytosis. Cytologic alterations consisted of Z-line streaming, mitochondrial and lipid aggregates and myofilamentous disarray and disorganization. Sciatic nerve histological and cytological findings were not different from controls. They consisted of minor degrees of thickening and tortuosity of myelin sheaths which occurred in both controls and experimental animals. The electron microscopic findings in the sciatic nerve following cortisone administration have not heretofore been reported in the literature. It is concluded that cortisone myopathy is due to a primary effect of cortisone on skeletal muscle and not secondary to a peripheral nerve lesion.


Sural nerve conduction: a standardized technique. Occasionally patients with symptoms and signs suggestive of mild peripheral neuropathy, after routine nerve conduction and electromyographic studies, are found to have values within normal limits. Several authors have suggested that sural nerve conduction studies might be more sensitive indicators of mild peripheral neuropathy. Normal values for sural nerve latencies, amplitudes and conduction velocities have been reported; however, the techniques used have not generally been rigidly standardized. This study describes a standardized and reproducible method of performing human sural nerve conduction studies and presents values obtained from systematically studying a series of 56 normal volunteers.


The Harlem regional stroke program: an overview. The Harlem regional stroke program has been a demonstration model designed to detect, treat, and follow up stroke and hypertension patients through collaboration with a municipal teaching hospital, community practitioners, local service organizations and a major medical school. Many aspects of the stroke program appear suitable for replication at the local or regional levels in varied settings. In particular, the program has demonstrated the need to link community outreach programs, stressing early detection and preventive-care education, with sustained treatment and follow-up programs. The stroke program has also suggested ways in which specialized programs can be incorporated into long-term comprehensive health planning and care facilities at local and regional levels.


Luteinizing hormone and testosterone during nocturnal sleep: relation to penile tumescent cycles. Variations of plasma LH and testosterone in relation to sleep-related penile tumescence were assessed. Plasma LH and testosterone were measured at 10-20 min intervals in five normal adult men during 2 nights of sleep. Blood samples were obtained by means of an indwelling venous catheter while sleep was monitored polygraphically and penile tumescence recorded with a penile mercury strain gauge. Tumescent episodes were recorded in all subjects during 9 of the 10 nights of the study, and the percentage of time during sleep, which was associated with tumescence, ranged from 13% to 34%. Simultaneous REM and tumescence comprised 57.7% of total tumescent time. Abrupt elevations of plasma LH and testosterone were observed during the night without a significant relationship to stages of sleep. The mean of all hormonal determinations for the five subjects did not show significant differences in plasma LH and testosterone between fully and partially tumescent episodes and nontumescent periods. Mean testosterone during REM sleep with tumescence, a state of autonomic activation involving sexual function, was consistently higher for the five subjects than during periods free from REM and tumescence.


Radical pancreatoduodenectomy for cancer of the papilla of Vater. Over a 22-year span, 87 patients with carcinoma of the papilla of Vater underwent radical pancreatoduodenectomy. No patient was lost to follow-up, and extended observation was possible in most cases: the definitive operation was at least five years earlier than this study in 87% and at least ten years earlier in 73%. Operative mortality was 11.5% among patients who had a single definitive operative procedure and 15.4% among those whose treatment involved reoperation after prior exploration elsewhere. Overall survival rates at two, five, and ten years were 56%, 34%, and 20% respectively. Factors associated with favorable survival were histologic differentiation (Broders grades 1 and 2), absence of nodal metastasis, and papillary histologic characteristics. Noteworthy is the fact that no patient having resection of an undifferentiated carcinoma (Broders grade 3 or 4) survived four years.


Expression of the human cytomegalovirus genome in mouse cells and in human-mouse heterokaryons. Mouse cells with an established human cytomegalovirus (HCMV) infection were fused with susceptible human embryonic fibroblast cells. CMV-specific early antigens could be demonstrated in the cytoplasm and cell-membrane of the heterokaryons. Treatment with 5-iodo-2'-deoxyuridine (IUdR) of the heterokaryons or of the latently infected mouse cells, prior to their fusion with human cells, could induce the appearance of immunoflourescent elements, characterised as late antigens, and of infectious virus. Our data show that the mouse cells, in the latent stage of infection, contain the whole virus genome and that the replication of the virus is controlled by a genetic mechanism of the host cells both in virus-harbouring mouse cells and in heterokaryons.


Altered sensitivity of Rous sarcoma virus transformed cells to inhibition of RNA synthesis by alpha-amanitin. Chick embryo cells transformed by Rous sarcoma virus (RSV) continue to synthesize 40--50 percent of control amounts of RNA following 12--24 hour exposure to 2 microng/ml of the toxin whereas normal chick embryo cells similarly treated synthesize less than 5 percent of control amounts of RNA. Analysis of cells treated with alpha-amanitin, or the resistant forms I and III polymerase, do not increase in infected cells over the levels found in uninfected control cells during the first 24 hours following infection indicating that increase in polymerase levels in infected cells does not account for the observed resistance. No significant difference was detected in the sensitivity to alpha-amanitin in the form II polymerase isolated from normal and transformed cells; The greater sensitivity of normal cells to alpha-amanitin can be reduced by growing the cells at low cell density but the resistance of RSV transformed cells is not significantly altered by changes in cell density. The results suggest that the resistance of RSV transformed cells may be related to altered control of density dependent contact-inhibition of the transformed cells.


The effect of arginine deprivation on DNA, thymidine kinase and RNA polymerase synthesis in simian virus 40-infected monkey kidney cells. In arginine-deprived cells infected with simian virus 40 (SV40), both viral DNA and viral structural proteins were synthesized but infectious virus was not produced. The syntheses of cellular DNA, thymidine kinase and DNA polymerase were induced in virus-infected cells deprived of arginine but the maximum rate of synthesis of these enzymes occurred much later than that in infected cells incubated in the presence of arginine.


Immunoprecipitation of herpes simplex virus type 1 antigens with different antisera and human cerebrospinal fluids. Rabbit convalescent and hyperimmune sera, human patient and blood donor sera, as well as cerebrospinal fluids of humans with herpes simplex virus encephalitis all recognize similar major antigenic components in herpes simplex virus infected rabbit or human cells as shown by electrophoretic analysis of immunoprecipitates. Besides the main glycoproteins with an apparent molecular weight of 100,000 (peak I) the antisera precipitate glycoproteins in a region of an apparent mol. wt. of 60,000--80,000 (peak II), which were resolved into distinct glycoprotein species only by antibody-containing cerebrospinal fluids. The peak II glycoproteins appear on the surface of the infected cell early, and absorb neutralizing antibodies, whereas the peak I glycoproteins are less accessible. Both antigens can be demonstrated in the cell as early as about 2 hours post infection. All major antigenic components studied were found to be glycosylated except one protein with an apparent mol. wt. of 110,000. The herpesvirus specificity of these antigens is demonstrated by a variety of control experiments. The antigens detected are virion components.


Activation of latent herpesvirus hominis in explants of rabbit trigeminal ganglia: the influence of immune serum. More than fifty albino rabbits were inoculated into the right scarified cornea with 10(7) PFU of the Kupka strain of human herpes virus type 1 (HHV-1). At intervals ranging from 4--280 days post infection (p.i.), both gasserian ganglia, both trigeminal nerve trunks and pieces from brain stem and from both corneas were explanted. Activation of the latent HHV-1 was found mainly in the homolateral ganglion tissue, but also in explants originating from the opposite ganglia. Within 24--72 hours, prior to the release of virus into the medium, one infectious unit of HHV was recovered from 10(4)--10(5) cells of the ganglion explant. In addition, a few neurons and satellite cells revealed the presence of virus-specific antigens when the explants were examined by immunofluorescence in serial sections. If the gangia were explanted in the presence of immune serum, the virus recovery rate was at least twice lower as compared to the virus activation in explants kept in the absence of immune serum.


In vitro and in vivo inhibition of virus multiplicaton by microwave hyperthemia. The effect of microwave hyperthermia (41 degrees and 43 degrees C) on virus multiplication have been explored in vitro (HSV-1 infected primary rabbit kidney cultures) and in vivo (mice infected with HSV-1 or vaccinia). In vitro the cells were inoculated with HSV-1 and heated to 41 degrees or 43 degrees C either before or after infection. Virus yields were significantly decreased when the cells were exposed to hyperthemia within the first few hours after infection, while hyperthemia was without effect when applied before infection or with several hours delay after infection. In mice inoculated intranasally with HSV-1, mortality due to herpes encephalitis was significantly reduced upon daily exposure to microwave hyperthermia from the day of infection onward. In mice inoculated intravenously with vaccinia, a significant decrease in the number of specific tail lesions was observed if the animals were exposed to microwave hyperthermia within the first three days after infection, while irradiation prior to infection or delayed until several days after infection did not exhibit an appreciable effect. Our data suggest that microwave hyperthermia interferes directly with the virus multiplication cycle both in vitro and in vivo.


Conjunctival impression cytology.

Conjunctival impression cytology. A new technique is presented using impression of the conjunctiva with a plastic device to study the conjunctival response in various conjunctival disease states. When used with a rapid acting stain the impression technique proved to be accurate, reproducible, and nondestructive when compared to the standard spatula scraping of the conjunctiva using Giemsa stain.


Drug interaction in the eye. Concurrent corticosteroid-antibiotic therapy for inflammatory keratitis. Concurrent instillation of individual preparations of a corticosteroid and an antibiotic resulted in significantly (P less than .05) lower peak corneal and aqueous humor steroid levels than those achieved by the steroid alone. Both the interval elapsing between instillation of the two drugs and the sequence in which they were administered influenced subsequent steroid bioavailability. Corticosteroid levels in the cornea after administration of a combination steroid-antibiotic preparation were not significantly different (P less than .05) from those detected after instillation of the same steroid alone, suggesting that, for the treatment of corneal disorders, use of a combination preparation may offer a method to circumvent certain drug interactions. The decrease in ocular steroid bioavailability could not be directly equated with differences in antinflammatory effectiveness, so that the therapeutic relevance of the demonstrated drug interaction is not known.


[Hormone therapy of the juvenile angiofibroma (author's transl)]. Four juvenile angiofibromas were examined with the electron microscope. Preceding the operation a hormonal therapy with stilbestrol disphosphate (Cytonal, Honvan) was carried out in the cases. The therapeutical effect after application of this drug is clearly correlated to especial changes in histology and cytology. The changes comprise reactions of the vascular component and of the stromal fibroblasts. Endothelial cells and pericytes show a markedly diminished cellular activity, a proliferation of vascular wall cells is not to be proved. The stromal fibroblasts may predominantly be classified as fibroblasts with histiocyte-like features, at which the organelle equipment speaks for a high activity of their metabolism. Furthermore an increased number of myofibroblasts and smooth muscle-like elements are encountered in the stroma. The striking diminution of lesion size and the reduction of disposition to bleeding from the beginning of the hormone therapy is suggested as a direct consequence of cellular contraction. Additionally alterations of synthesis of collagen fibers and ground substance by fibroblasts with histiocyte-like features contribute to a further gradual decrease of the growths.


Adjuvant systemic therapy in "early" breast cancer. "Early" breast cancer is a systemic disease in the majority of cases. Progress has been and is being made toward the determination of those women at risk of recurrence. Attractive as it seems, the value of systemic adjuvant therapy, given at the time of minimal tumour load i.e., after mastectomy, is not proven. Therefore, for the average case it is better to treat the cancer locally and to observe and leave further evaluation of systemic therapy to those centres which are able to conduct controlled, randomized trails.


Fine structure of a virilizing adrenocortical adenoma. An adrenocortical adenoma associated with adrenogenital syndrome in a two-year-old boy was investigated light and electron microscopically. Urinary 17-ketosteroid excretion was considerably elevated and unresponsive to dexamethasone administration. The level returned to normal after surgical removal of the tumour. Adenomatous cells display striking cellular and nuclear pleomorphism. Megalocytes with huge nuclei and nucleoli frequently occur. Deep cytoplasmic indentations cause nuclear pseudoinclusions and bizarre shape of the nuclei. True nuclear inclusions are also seen, as well as nuclear fragmentation. Cytoplasmic organelles show striking morphological alterations. Mitochondria with lamellar and tubular cristae are transformed into round or ovoid organelles of vesicular type. Their internal compartment is reduced, matrix material increases relatively, and mitochondrial inclusion bodies develop. Mitochondrial inclusions are identified as corresponding to fuchsinophil (siderophil or argyrophil) granules seen in the light microscope. Their staining properties indicate their glycoprotein nature. Vesicular profiles of smooth endoplasmic reticulum predominate and stacks of rough endoplasmic reticulum are transformed into tubules and vesicles. In Golgi regions, only vesicular elements are enriched. Lipid droplets are scarce. It was not possible to demonstrate histochemically catalase activity in microbodies. Dense bodies only occur in small, undifferentiated tumour cells. Multivesicular bodies, autophagosomes and residual bodies are rare. Lipofuscin is absent. Tumour cells are thought to derive from a population of undifferentiated cells ("germinative tumour cells"). Their morphological features and organelle equipment during a hypothetical course of differentiation and following dedifferentiation is described and discussed with respect to exceeding androgen synthesis.


Pyrimidine biosynthesis in Serratia marcescens: a possible role for nonsequential enzyme interactions in mimicking coordinate gene expression. The coordinate expression of four sequential enzymes in the de novo pyrimidine pathway may result from the interaction of the various polypeptides of the pathway in Serratia marcescens rather than represent some unit of transcriptional regulation. These interactions were defined by examining the polypeptide association observed in extracts of parental and mutant strains in a series of pleiotropic pyrimidine auxotrophs. Extracts of pyrE auxotrophs [processing dihydroorotate (DHOase) activity but no orotidine-5'-monophosphate pyrophosphorylase (OMPppase) activity] stimulate OMPppase activity in extracts of pyrC auxotrophs (posessing reduced OMPppase activity but no DHOase activity). Separation by molecular weight on Sephadex G200 has suggested an aggregation between the final two enzymes, OMPppase and OMPdecarboxylase (OMPdecase), and the earlier enzyme, DHOase. The reduction of OMPppase activity in pyrC auxotrophs (encoding either a defective polypeptide or reduced levels) is explained by the lack of adequate levels of DHOase for aggregate formation. Such polypeptide interactions appear to mimic the coordinate formation of polypeptides which are controlled as a unit of regulation. The measurable levels of enzymatic activity vary in a quantitatively identical manner, but the variation does not result directly from the regulation of polypeptide formation.


The action of progesterone and diethylstilboestrol on the dehydrogenase and esterase activities of a purified aldehyde dehydrogenase from rabbit liver. A steroid-sensitive aldehyde dehydrogenase (EC 1.2.1.3) was purified from rabbit liver and is homogeneous by the criterion of electrophoresis in polyacrylamide gels with or without sodium dodecyl sulphate. The enzyme is tetrameric, of subunit mo.wt. 48 300, and contains no tightly bound zinc. The fluorescence of the protein is decreased in the presence of progesterone, which is inhibitory to the reactions catalysed by the enzyme. When NADH is bound to the enzyme, the fluorescence of the coenzyme is augmented to an extent independent of the presence of steroids or acetaldehyde. The purified enzyme catalyses the oxidation of acetaldehyde and glucuronolactone, and the hydrolysis of 4-nitrophenyl acetate. Each of these reactions is inhibited by progesterone in such a manner as to suggest the formation of a catalytically active enzyme-hormone complex. Diethylstilboestrol inhibits the hydrolysis of esters by this enzyme, but stimulates the oxidation of aldehydes, except at low aldehyde concentrations; the ligand is then inhibitory. NADH inhibits the hydrolysis of 4-nitrophenyl acetate by the enzyme in a partially competitive fashion.


A functioning complex between tryptic fragments of cytochrome c. A route to the production of semisynthetic analogues. We report the discovery of a functioning non-covalent complex between two peptides obtained from a limited tryptic digest of horse heart cytochrome c. We have used the phenomenon to produce three modified versions of the complex. We have replaced lysine-39 semisynthetically with ornithine in the first analogue, with p-fluorophenyl-alanine in the second, and removed it  entirely in the third.


Purification of glycolytic enzymes by using affinity-elution chromatography. 1. A systematic procedure for the purification of enzymes by affinity-elution chromatography is described. Enzymes are adsorbed on a cation-exchanger, and eluted with ligands specific for the enzyme concerned. 2. All of the glycolytic and some related enzymes present in rabbit muscle can be purified by the affinity-elution technique. The pH range for adsorption and elution of each enzyme was found, and the effects of minor variations of conditions are described. 3. A description of experimental conditions suitable for affinity elution of each enzyme is given, together with special features relevant to each individual enzyme. 4. Theoretical considerations of affinity elution chromatography are discussed, including its limitations, advantages and disadvantages compared with affinity-adsorption chromatography. Possible developments are suggested to cover enzymes which because of their adsorption characteristics are not at present amenable to affinity-elution procedures.


The disulphide-bonded nature of procollagen and the role of the extension peptides in the assembly of the molecule. 1. The molecular weights of chick tendon and cartilage procollagens, and their constituent polypeptides, were determined by gel filtration and gel electrophoresis. The values obtained are in good agreement and indicate that the mol.wts. of the secreted procollagens (types I and II) and their individual pro-alpha-chains are of the order of 405 000-445 000 and 137 000-145 000 respectively.2. Digestion of tendon procollagen with human rheumatoid synovial collagenase gave products consistent with the presence of large non-helical peptide extensions at both N-and C-termini. Electrophoretic analysis gave apparent mol.wts. of 17 500 and 36 000 for the respective N- and C-terminal extensions of pro-alpha1(I)-and pro-alpha2-chains, and inter-chain disulphide bonds were restricted to the C-terminal location. 3. During the biosynthesis of procollagen by tendon and cartilage cells a close correlation was observed between the extent of inter-chain disulphide bonding and the proportion of procollagen polypeptides having a triple-helical conformation. These processes appeared to commence in the rough endoplasmic reticulum and be completed in the smooth endoplasmic reticulum, but the rate at which they occur in cartilage cells is markedly slower than that found in tendon cells. 4. When the intracellular [14C]procollagen polypeptides present in the rough-endoplasmic-reticulum fractions of tendon and cartilage cells were analysed under non-reducing conditions on agarose/polyacrylamide composite gels, no significant pools of dimeric intermediates were detected. 5. In both cell types, inter-chain disulphide-bond formation occurred even when hydroxylation, and hence triple-helix formation, was inhibited. The presence of pro-alpha1- and pro-alpha2-components in a ratio of 2:1 in the disulphide-linked unhydroxylated procollagen isolated from tendon cells demonstrated that correct chain association occurs in the absence of hydroxylation. This observation is consistent with a model for the assembly of pro-gamma112-chains in which the recognition and selection of pro-alpha1-and pro-alpha2-chains in a 2:1 ratio are directed by the non-helical C-terminal extension peptides of tendon procollagen.


Preparation and properties of nylon-tube-supported nicotinamide--adenine dinucleotide kinase. Pigeon liver NAD kinase was covalently coupled to the inside surfaces of nylon tubes, both directly to the nylon and via polyamine specers Km values and the inactivation energy of the reaction changed upon immobilization, but the pH-dependence remained unaltered. The activities and stabilities of different preparations are compared.


Studies in vitro of the effects of adenosine 3':5'-cyclic monophosphate on the phosphorylation of nuclear proteins in isolated rat heart nuclei. Isolated rat heart nuclei were prepared by homogenization and sucrose-density-gradient centrifugation. The protein/DNA ratio of these nuclei was 3.1:1 (w/w), and the histones/non-histone proteins/DNA proportions were 1.4:1.6:1 (by wt.). Non-histone proteins were fractionated into six major groups by elution on a quaternized anion-exchanger (QAE-Sephadex A-50 column with increasing concentrations of NaCl in 5M-urea/0.01 M-Tris/HCl buffer (pH8.3). When isolated nuclei were incubated in a medium containing [gamma-32P]ATP, a differential distribution of 32P was observed in the six fractions of nonhistone proteins. The fractions eluted from the Sephadex column with 0.35M- and 0.6M-NaCl contained contained 80% of the total radioactivity incorporated into the non-histone proteins. This incorporation into the 0.35M- and 0.6M-NaCl fractions was increased by 66 and 112% respectively in the presence of cyclic AMP. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these two particular fractions showed a selective increase in labelling of five protein bands in the presence of cyclic AMP.


Human collagen 'fingerprints' produced by clostridopeptidase A digestion and high-pressure liquid chromatography. Samples (1-2mg) of purified human type I, II and III collagens and alpha1(I) and alpha2 chains were digested with clostridiopeptidase A and the released peptides analysed by ion-exchange high-pressure liquid chromatography. Specific 'fingerprints' were produced for each type of collagen. The reproducible nature of these 'fingerprints' and the reconstitution of the type I 'fingerprint' from the 'fingerprints' of the component alpha1(I) and alpha2 chains showed that the specificity of these 'fingerprints' was related to the primary structure of each type of collagen. In addition, some of the differences observed between the 'fingerprints' of the alpha1(I) and alpha2 chains of type I collagen were shown to be suitable for the quantitative analysis of these chains.


A latent form of collagenase in the involuting rat uterus and its activation by a serine proteinase. 1. The involuting rat uterus displays an extremely rapid breakdown of collagen. Collagenase activity can be assayed directly in the insoluble 6000g pellet of uterine homogenates. At 1 day post partum, about 85% of this collagenase activity is in a latent form. 2. This latent form can be activated by trypsin or by a serine proteinase present in the uterine pellets. 3. The activating enzyme of the tissue is inhibited by a wide spectrum of trypsin inhibitors, including Trasylol, soya-bean and lima-bean trypsin inhibitors, snail inhibitor and di-isopropyl phosphoro-fluoridate. Partial inhibition is produced by benzamidine, phenylmethanesulphonyl fluoride, epsilon-aminohexanoate, leupeptin, antipain and alpha1-antitrypsin. Ovomucoid, 7-amino-1-chloro-3-tosylamido-1-heptan-2-one and 1-chloro-4-phenyl-3-(N-benzyloxy-carbonyl)amino-L-butan-2-one are not inhibitory. 4. Extraction of uterine pellets with 0.1 M-CaCl2 at 60 degrees C releases both latent and active collagenase. Exclusion chromatography on Sephadex G-100 gives an apparent molecular weight of approx. 77000 for the latent form and 66000 for the active form. The latent form is suggested to be a zymogen of collagenase.


Studies on the mechanism and kinetics of the 2-oxoglutarate dehydrogenase system from pig heart. 1. The kinetic properties of the 2-oxoglutarate dehydrogenase system were investigated. To this end, initial-velocity studies were carried out by the method of Fromm [(1967) Biochim. Biophys. Acta 139, 221-230]. Reciprocal plots of the results did not agree with those expected for the Hexa Uni Ping Pong mechanism previously proposed for the system. 2. The measured initial velocities were fitted to initial-rate equations corresponding to several possible mechanisms by using a computer optimization technique. Statistical analyses performed on the results of the optimization studies indicated that one mechanism was a significantly better fit to the experimental data than the other mechanisms tested. This mechanism is one in which there is a random order of binding of NAD+ and CoA and release of succinyl-CoA, although the binding of 2-oxoglutarate and release of CO2 is still given a Ping Pong mechanism, which precedes the binding of the other substrates. These conclusions were supported by NADH-inhibition studies. 3. The usefulness of the method of fitting initial-rate data to rate equations and the applicability of the proposed enzymic mechanism to the enzyme complex are discussed.


Adenosine diphosphate ribosylated histones. When rat liver nuclei were incubated with [adenine-3H]NAD, besides histone 1, histone 2A and especially histone 2B accepted 3H radioactivity. 3H radioactivity was also found on the non-histone proteins and on the small amounts of histones 1 and 3 released into the supernatant during incubation. [14C]Adenine uptake in vivo by liver and thymus nuclei showed radioactivity in histones 1 and 3. After digestion with Pronase and leucine aminopeptidase 14C- or 32P-labelled histone 3 released a serine phosphate-containing nucleotide, which on acid hydrolysis yielded ADP-ribose and serine phosphate. Serine phosphate was also found in the material from the nucleotide peaks from histones 2A and 2B. ADP-ribosylated histones 1 and 3 were more easily released from nuclei than their unmodified forms and showed higher [32P]Pi and [3H]lysine uptakes in vivo [Ord & Stocken (1975) FEBS Meet. Proc. 34, 113-125].


Cholera toxin requires oxidized nicotinamide-adenine dinucleotide to activate adenylate cyclase in purified rat liver plasma membranes. Activation of adenylate cyclase in isolated rat liver plasma membranes by cholera toxin was demonstrated. The activation requires the presence of NAD+ and ATP and is irreversible.


Factors affecting the binding of [3H]adenosine 3':5'-cyclic monophosphate to protein kinase from bovine adrenal cortex. Inorganic salts, several proteins and traces of protein precipitants were tested to find out by what mechanisms they modulate the binding of cyclic [3H]AMP to protein kinase (ATP-protein phosphotransferase; EC 2.7.1.37). The separation of free and bound cyclic AMP by (NH4)2SO4 precipitation was unaffected by the above agents and was more reliable than the Millipore filtration technique. Several binding sites for cyclic AMP were revealed in adrenal-cortex extract. When this extract was used as binding reagent in an assay for cyclic AMP, the standard curve was distorted in the presence of KCl because the salt affected the different binding sites to a varying extent. At high ionic strenth the protein kinase isoenzyme I dissociated and showed an extraordinarily high affinity for cyclic AMP. Trichloroacetate and perchlorate at very low concentrations were able to dissociate the protein kinase and modulate its binding characteristics as well. A progressive decrease in the cyclic AMP-binding capacity occurred on prolonged incubations. The binding protein was protected against inactivation by 2-mercaptoethanol, EDTA and several proteins. It was more resistant to denaturation when complexed to cyclic AMP. The enhancement of cyclic AMP binding by bovine serum albumin was investigated in some detail and appeared to be a pure stabilizing effect. It is proposed that the competitive-binding assays for cyclic AMP based on protein kinase be conducted at high ionic strength and in the presence of stabilizers (protein, EDTA, 2-mercaptoethanol). The interference from agents that may dissociate the protein kinase or influence its stability will thus be decreased.


Effects of prostaglandin on substrate uptake and cell division in human diploid fibroblasts. PG (prostaglandin) E1 inhibits the uptake of iridine, thymidine, 2-deoxy-D-glucose and L-isoleucine into human diploid WI38 fibroblasts. The inhibition occurs within seconds of the addition of the prostaglandin to the culture. PGE2, PGF1alpha and PGF2alpha behave similarly. Arachidonic acid and 8,11,14-eicosatrienoic acid also decrease uptake in the presence or absence of indomethacin. Other unsaturated fatty acids such as oleic acid, linoleic acid and linolenic acid are essentially inactive. Ricinoleic acid (the 9-hydroxyoleic acid), however, inhibits uptake to about the same degree, at concentrations similar to those of the prostaglandins. Results indicate that this rapid blockage by the prostaglandins and certain fatty acids is not cyclic AMP-mediated. For example, although PGF1alpha and PGF2alpha are much poorer stimulators of cyclic AMP formation than are PGE1 and PGE2, they are nevertheless effective inhibitors of substrate uptake. Adrenaline, a very effective stimulator of cyclic AMP formation in the cells, is not inhibitory. Also, the addition of 8-methylthioadenosine 3':5'-cyclic monophosphate (methylthio cyclic AMP) to the culture, methylthio cyclic AMP decreases the uptake of nucleotides into cultures undergoing active cell division, approximately to values found in quiescent cultures. PGE1 also has this effect on cells undergoing active growth. This gradual decrease is substrate uptake caused by PGE1 appears to be a separate event from its initial rapid inhibition of uptake.


Studies on sex-organ development. The hormonal regulation of steroidogenesis and adenosine 3':5'-cyclic monophosphate in embryonic-chick ovary. 1. We investigated the production of steroid hormones by the ovaries of the developing embryonic chick under conditions of organ culture. Radioimmunoassay techniques were used to measure the amount of steroid hormone released into the culture medium. Stimulation of the production of steroid hormones by choriogonadotropin from the urine of pregnant human was dose-dependent. Oestradio and testosterone production was optimal when 20 i.u. of gonadotropic hormone was present in the culture medium 2. During development, both left and right ovaries responded to gonadotropic hormone stimulation with a 2.5-3-fold increase in oestrogen production. However, the right ovary was twice as efficient in testosterone production as the left one. The presence of dibutyryl cyclic AMP in the culture medium of the embryonic ovaries mimicked the effect of the gonadotropic hormone. 3. The human choriogonadotropic hormone stimulated cyclic AMP production in the embryonic ovarian tissue. Thyrotropin, growth hormone and insulin had no stimulating effect. 3-Isobutyl-1-methylxanthine potentiated the gonadotropic hormone effect by increasing the concentration of cyclic AMP in the ovarian tissue. 4. The amount of cyclic AMP synthesized in the embryonic ovary was gradually increased (from 1.2 to 6.5 pmol/mg of tissue) when incubated with increasing doses of human choriogonadotropic hormone in vitro. The newly synthesized cyclic AMP reached the maximum concnentration after 30 min of incubation, then decreased at 2 h of incubation. A portion of the newly synthesized cyclic AMP was released into the culture medium. 5. At various developmental stages, both left and right embryonic-chick ovaries responded to stimulation by gonadotropic hormone with an increase in cyclic AMP production. The cyclic AMP concentration in the right ovary was 80% higher than that in the corresponding left ovary.


Hormonal and ionic control of the glycogenolytic cascade in rat liver. 1. A parallel dose-dependent activation of histone kinase, phosphorylase kinase and phosphorylase was observed in isolated hepatocytes incubated in the presence of glucagon; the effect of suboptimal concentrations of glucagon was antagonized by insulin. 2. An activation of phosphorylase which was not accompanied by a stable change in the activity of phosphorylase kinase was observed in hepatocytes incubated with phenylephrine, isoproterenol or vasopressin as well as on decapitation of unanesthetized animals. A dissociation of the two enzymic activities was also observed in hepatocytes incubated in the presence of a high concentration of glucose, in which phosphorylase was strongly inactivated with no change in the activity of phosphorylase kinase. 3. The activation of phosphorylase by phenylephrine in isolated hepatocytes was counteracted by insulin, greatly decreased by the absence of Ca2+ from the incubation medium, and completely suppressed by the replacement of Na+ by K+. 4. In a liver extract, phosphorylase kinase could also be activated by trypsin. Control, glucagon-activated or trypsin-activated phosphorylase kinase was inhibited by about 70% by EGTA and the activity was restored by the addition of Ca2+. 5. The mechanisms that control the activity of phosphorylase kinase and of phosphorylase are discussed.


The effect of insulin on the glycogenolytic cascade and on the activity of glycogen synthase in the liver of anaesthetized rabbits. 1. The administration of insulin to anaesthetized rabbits caused the inactivation of liver phosphorylase and phosphorylase kinase, but did not change either the hepatic concentration of cyclic AMP or the activity of cyclic AMP-dependent histone kinase. All measured parameters were increased by the subsequent administration of glucagon. 2. Activation of glycogen synthase by insulin was only observed when phosphorylase had been strongly inactivated.


Phosphorylation of rat thymus histones, its control and the effects thereon of gamma-irradiation. The phosphate content of rat thymus histones was determined. As expected for a replicating tissue, histones 1 and 2B were more phosphorylated and had higher 32P uptakes than did histones from resting liver nuclei; the other histones all showed 32P uptake, but the phosphate content and uptake of histone 2A was about half that for liver histone 2A. When thymus nuclei were incubated in a slightly hypo-osmotic medium, non-histone proteins and phosphorylated histones were released into solution; this was enhanced if ATP was present in the medium. [gamma-32P]ATP was incorporated into non-histone proteins, including protein P1, and into the ADP-ribosylated form of histone 1; negligible 32P was incprporated into the other, bound, histones. Histones 1 and 2B added to the incubation medium were extensively, and histones 2A and 4 slightly, phosphorylated. Histones released by increasing the ionic strength of the medium were phosphorylated. Added lysozyme and cytochrome c were neither bound nor phosphorylated, but added non-histone protein P1 was phosphorylated, causing other histones to be released from the nuclei, especially histones 2A and 3. The released histones were phosphorylated. gamma-Irradiation decreased 32P uptake into the non-ADP-ribosylated histones 1 and 4; phosphorylation of histone 1 in vitro was unaffected. The importance of non-histone proteins, ATP availability and nuclear protein kinases to the control of histone phosphorylation in vivo is discussed.


Collagenase is a component of the specific granules of human neutrophil leucocytes. Azurophil and specific granules were isolated from human polymorphonuclear neutrophil leucocytes. Collagenase was almost exclusively a component of the specific granules. This finding is in contrast with the distribution of other proteolytic enzymes, which are localized in the azurophil (or lysosomal) granules.


The effects of bathophenanthroline, bathophenanthrolinesulphonate and 2-thenoyltrifluoroacetone on mung-bean mitochondria and submitochondrial particles. The effects of bathophenanthroline, bathophenanthrolinesulphonate and 2-thenoyltrifluoroacetone on mung-bean mitochondria and submitochondrial particles were investigated. A variety of inhibitory effects on the oxidations of NADH, succinate and malate were observed. The results are discussed in relation to sites of inhibition and their relation to the effects on mammalian mitochondria.


Factors controlling the activities of phosphatidate phosphohydrolase and phosphatidate cytidylyltransferase. The effects of chlorpromazine, demethylimipramine, cinchocaine, norfenfluramine, mepyramine and magnesium ions. 1. Microsomal membranes from rat liver were incubated with ATP, CoA, Mg2+, [14C]palmitate, F- and sn-glycerol 3-phosphate in order to label them with [14C]phosphatidate. These membranes were isolated and used in a second incubation in which [3H]CTP was present, and the simultaneous synthesis of [14C]diacylglycerol and [3H]CDP-diacylglycerol was measured. 2. The addition of phosphatidate phosphohydrolase, which had been partially purified from the particle-free supernatant, supplemented the activity of the endogenous phosphohydrolase, but it did not alter the rate of CDP-diacylglycerol formation. 3. Adding EDTA inhibited phosphatidate cytidylyl-transferase activity and stimulated the activity of the phosphohydrolases by removing excess of Mg2+. 4. Increasing the concentration of Mg2+, norfenfluramine or chlorpromazine in the assay system stimulated cytidylyltransferase activity, but decreased the activities of both phosphohydrolases. 5. The mechanism for the stimulation of cytidylyl=transferase activity by the cationic drugs and Mg2+ was investigated with emulsions of phosphatidate and the microsomal fraction of rat liver. 6. There was a threshold concentration of about 5mM-MgCl2 below which no cytidylyltransferase activity was detected in the presence or absence of norfenfluramine. Just above this threshold concentration norfenfluramine stimulated cytidylyltransferase activity, but this stimulation disappeared as the Mg2+ concentration was raised to its optimum of 20mM. Norfenfluramine therefore partially replaced the bivalent-cation requirement. 7. At 30 mM-MgCl2 amphiphilic cationic drugs inhibited cytidylyltransferase activity at relatively high concentrations in a non-competitive manner with respect to phosphatidate. 8. The implications of these results are discussed with respect to the regulation of the synthesis of the acidic phospholipids compared with the synthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol.


Inhibition of lipogenesis by halothane in isolated rat liver cells. 1. Halothane at clinically effective concentrations [2.5 and 4% (v/v) of the gas phase of the incubation flask] was found to inhibit significantly lipogenesis from endogenous substrates, e.g., glycogen, or from added lactate plus pyruvate. This was accompanied by a decrease in the ratio of the free [NAD+]/[NADH] of the mitochondrion and the cytoplasm, as shown by the [3-hydroxybutyrate]/[acetoacetate] ratio and the [lactate]/[pyruvate] ratio. 2. Acetoacetate or pyruvate decreased the inhibitory effect of halothane and restored lipogenesis to control rates. They were reduced rapidly by 3-hydroxybutyrate dehydrogenase or lactate dehydrogenase respectively, with the concomitant oxidation of NADH and the generation of NAD+. 3. These results suggest that the mechanism by which halothane inhibits lipogenesis from glycogen or lactate is by inhibition of the oxidation of NADH; this results in inhibition of flux of carbon through pyruvate dehydrogenase and a shortage of acetyl-CoA for fatty acid synthesis. Thus when NADH acceptors are added in the presence of halothane, the concentration of mitochondrial NAD+ is raised so that the flux of carbon through pyruvate dehydrogenase increases and lipogenesis is restored.


Acetylcholine increases the breakdown of triphosphoinositide of rabbit iris muscle prelabelled with [32P] phosphate. 1. Paired iris smooth muscles from rabbits were incubated for 30 min at 37 degrees C in an iso-osmotic salt medium containg glucose, inositol, cytidine and [32P]phosphate. 2. One of the pair was then incubated at 37 degrees C for 10 min in unlabelled medium containing 10mM-2-deoxyglucose and the other was incubated in the presence of acetylcholine plus eserine (0.05mM each). 2-Deoxyglucose, which was included in the incubation medium to minimize the biosynthesis of triphosphoinositide from ATP and diphosphoinositide, decreased the amount of labelled ATP by 71% and inhibited further 32P incorporation from ATP into triphosphoinositide by almost 30%. 3. Acetylcholine (0.05mM) increased significantly the loss of 32P from triphosphoinositide (the 'triphosphoinositide effect') in 32P-labelled iris muscle. This effect was measured both chemically and radiochemically. It was also observed when 32Pi was replaced by myo-[3H]inositol in the incubation medium. 4. The triphosphoinositide effect was blocked by atropine but not by D-tubocurarine. Further, muscarinic but not nicotinic agonists were found to provoke this effect. 5. Acetylcholine decreased by 28% the 32P incorporation into triphosphoinositide, presumably by stimulating its breakdown. This decrement in triphosphoinositide was blocked by atropine, but not by D-tubocurarine. 6. The triphosphoinositide effect was accompanied by a significant increase in 32P labelling, but not tissue concentration, of phosphatidylinositol and phosphatidic acid. The possible relationship between the loss of 32P label from triphosphoinositide in response to acetylcholine and the concomitant increase in that of phosphatidylinositol and phosphatidic acid is discussed. 7. The presence of triphosphoinositide phosphomonoesterase, the enzyme that might be stimulated in the iris smooth muscle by the neurotransmitter, was demonstrated, and, under our methods of homogenization and assay, more than 80% of its activity was localized in the particulate fraction.


The oxidation-reduction potential of the reaction-centre chlorophyll (P700) in Photosystem I. Evidence for multiple components in electron-paramagnetic-resonance signal 1 at low temperature. The oxidation-reduction potential of the reaction-centre chlorophyll of Photosystem I (P700) in spinach chloroplasts was determined by using the ability of the reaction centre to photoreduce the bound ferredoxin and to photo-oxidize P700 on illumination at 20K as an indicator of the oxidation state of P700. This procedure shows that P700 is oxidized with Em (pH8.0)(mid-point redox potential at pH8.0)congruent to +375mV. Further oxidation of the chloroplast preparations by high concentrations of K3Fe(CN)6(10mM) in the presence of mediating dyes leads to the appearance of a large radical signal with an apparent Em congruent to +470mVA second, light-inducible, radical also appears over the same potential range. We propose that these signals are due to bulk chlorophyll oxidation and not, as was previously thought [Knaff & Malkin (1973) Arch. Biochem. Biophys. 159, 555-562], to reaction-centre oxidation. A number of optical techniques were used to determine Em of P700. Dual-wavelength spectroscopy (697-720nm) indicates Em congruent to +460-+480mV. The spectrum of the sample during the titration showed a large contribution to the signal by bulk chlorophyll oxidation, in agreement with the electron-paramagnetic-resonance results and those of Ke, Sugahara & Shaw [(1975) Biochim. Biophys. Acta 408, 12-25]. The light-induced absorbance change at 435 nm, usually attributed to P700, showed a potential dependence similar to that of bulk chlorophyll oxidation. Determination of Em of P700 on the basis of the appearance of the P700 signal in oxidized-versus-reduced difference spectra showed Em (pH8.0) congruent to +360mV. Measurements of the effect of potential on the irreversible photo-oxidation of P700 at 77K showed that P700 became oxidized in this potential range. We conclude that the reaction-centre chlorophyll of Photosystem I has Em (pH8.0) congruent to +375mV.


Alloxan cytotoxicity in vitro. Inhibition of rubidium ion pumping in pancreatic beta-cells. Exposing micro-dissected pancreatic islets of non-inbred ob/ob mice to 2-5 mM-alloxan for 10 min decreased the ability of the islets to accumulate Rb+. Rb+ accumulation in pieces of exocrine pancreas was unaffected by alloxan. When islets were treated with alloxan in the presence of 2-20 mM-D-glucose, the Rb+-accumulating ability was protected in a dose-dependent manner. The protective action of D-glucose was reproduced with 3-O-methyl-D-glucose but not with L-glucose or D-mannoheptulose; mannoheptulose prevented D-glucose from exerting its protective action. The inhibition of Rb+ accumulation was due to a decreased inward pumping, since alloxan did not affect Rb+ efflux from pre-loaded islets. The inhibitory effect of alloxan had a latency of about 1 min, as revealed by experiments with dispersed islet cells in suspension. Alloxan-treated islets showed only a marginal decrease in ATP and no change in glucose 6-phosphate concentration. Although alloxan slightly decreased the hydrolysis of ATP in a subcellular fraction enriched in plasma membranes, this effect could not be attributed to a ouabain-sensitive adenosine triphosphatase. The plasma membranes exhibited a K+-activated hydrolysis of p-nitrophenyl phosphate; this enzyme activity too was insensitive to alloxan. Glucose may protect the univalent-cation pump by preventing permeation of alloxan via a path coupled to the hexose-transport system. Inhibition of the pump may be fundamental to the induction of alloxan-diabetes.


A phosphodiesterase in rat kidney cortex that hydrolyses glycerylphosphorylinositol. 1. A phosphodiesterase, active at an alkaline pH, is present in the outer cortex of rat kidney and hydrolyses glycerylphosphorylinositol into glycerol and phosphorylinositol. Some inositol cyclic phosphate can also be formed indicating that the enzyme can act as a cyclizing phosphotransferase. 2. The enzyme is stimulated by Ca2+(2-3mM) whereas Mg2+ is inhibitory. 3. The activity is markedly stimulated by low concentrations of thiol reagents (1-2mM) such as cysteine or dithiothreitol. 4. The properties of the enzyme have been compared with glycerylphosphinicocholine diesterase (EC 3.1.4.2), which is also present in the isolated enzyme complex, and it is concluded that the enzymes have separate identities.


Protein determinants of myelination in different regions of developing rat central nervous system. Measurements of several different protein determinants correlated with the time and rate of myelination in five areas of the central nervous system are presented. The deposition of protein in the subcellular fraction corresponding to the density of adult myelin, the appearance of basic protein characteristic myelin, the change in proportions of the individual myelin proteins, the appearance and distribution of the myelin marker 2':3'-cyclic nucleotide3'-phosphohydrolase, and the results of morphological studies of purified myelin are compared. According to these various criteria, and in agreement with the morphological observations of others, myelin appears earliest in the spinal cord, then in the brain stem, and latest in the cerebral hemispheres. Multilamellar myelin was observed in the rat brain stem and spinal cord as early as 5 days of age. The relative proportion of the individual myelin proteins changed with myelin maturation in all areas, with the larger basic protein decreasing reciprocally with increase of the smaller basic protein. The proportion of Wolfgram protein also decreased with maturation. Larger proportions of the enzyme 2':3'-cyclic nucleotide 3'-phosphohydrolase were located in the microsomal fraction at early ages. During development the enzyme activity gradually became associated more with a fraction of a density corresponding to adult myelin, suggesting the presence of precursor membrane fragments in microsomal fractions in the early stages of myelination before compact myelin formation. A significant proportion of the total nucleotide phosphohydrolase activity of the homogenate could not be recovered in subcellular fraction at early ages, but the recovers of the enzyme increased with maturation and the activity was found more in the myelin fraction.


Changes in enzymic activities of nucleoside diphosphate sugar interconversions during differentiation of cambium to xylem in sycamore and poplar. During the transition from primary wall formation to secondary thickening there is a marked shift in the synthesis of pectin, hemicellulose and cellulose. The activities of the enzymes [UDP-D-galactose 4-epimerase (EC 5.1.3.2)8 UDP-l-arabinose 4-epimerase (EC 5.1.3.5), UDP-D-glucose dehydrogenase (EC 1.1.1.22) and UDP-D--glucuronate decarboxylase (EC 4.1.1.35)] were measured in cambial cells, differentiating xylem cells and differentiated xylem cells isolated from sycamore and poplar trees, and phloem cells from poplar. At the final stage of the differentiation of cambium to xylem there was a decrease in activity of the enzymes directly involved in producing the soluble precursors of pectin (DUP-D-galactose 4-epimerase and UDP-L-arabinose 4-epimerase and an increase in those producing the precursors of hemicellulose (UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase). These results strongly suggest ahat the changes were correlated with the differences observed in the chemical composition of the wall during development. The changes found in the catalytic activity of the enzymes of nucleoside diphosphate sugar interconversion exert a coarse control over the synthesis of pectin and hemicelluloses. The tissues at all stages of development contained the necessary enzyme activities to produce all the precursors of pectin and hemicellulose, even at the final stage of differentiation when no pectin was formed.


Changes in enzymic activities of nucleoside diphosphate sugar interconversions during differentiation of cambium to xylem in pine and fir. A protein fraction [precipitate obtained between 40 and 65% (NH4)2SO4 satn.] prepared from cambial cells, differentiating xylem cells and differentiated xylem cells of pine and fir trees contained all the enzymes required for the nucleoside diphosphate sugar interconversions. By using UDP-D-[U-14C]glucose or UDP-D-[U-14C]galactose, UDP-D-[U-14C-]glucuronic acid and UDP-D-[U-14C]xylose as substrates, the activities of UDP-D-galactose 4-epimerase (DC 5.1.3.2), UDP-D-xylose 4-epimerase(EC 5.1.3.5), UDP-D-glucose dehydrogenase (EC 1.1.1.22) and UDP-D-glucuronate 4-epimerase (EC5.1.3.6), UDP-d-glucuronate decarboxylase (EC 4.1.1.35) were measured at different stages of cell-wall development. The specific activities and the activities per cell of these enzymes varied during differentiation of cambium to xylem according to the type polysaccharide synthesized. Variations were also found between the two species investigated. These data, compared with those obtained in out previous work on angiosperms [see the preceding paper, Dalessandro & Northcote (1977) Biochem. J. 162, 267-279], suggest that some control of polysaccharide synthesis operates at the level of the formation of the precursors of pectin and hemicellulose syntheses.


Effects of anti-microtubular agents and cycloheximide on the metabolism of chylomicron cholesteryl esters by hepatocyte suspensions. 1. Post-heparin plasma that promoted rapid hydrolysis of about 90% of the triacylglycerol markedly stimulated the uptake or binding of chylomicron cholesteryl ester by suspended hepatocytes. The net hydrolysis of chyle cholesteryl ester after the uptake by the cells was, however, slower than in vivo. 2. The cholesteryl ester uptake in the presence of post-heparin plasma was larger if the cells had been preincubated for 2h. It was inhibited by the presence of colchicine, vinblastine or cycloheximide during the preincubation, and by mild trypsin treatment of the preincubated cells. 3. The results suggested that the anti-microtubular agents, but not cycloheximide, also inhibited the hydrolysis of chyle cholesteryl ester after uptake or binding to the cells. 4. The uptake of isolated chylomicron remnant particles was more efficient than that of native chyle lipoproteins. It was, however, still stimulated by heparin alone and by post-heparin plasma. The heparin-stimulated uptake was markedly decreased if cycloheximide was present during the preincubation period.


Effect of thyroliberin on the concentration of adenosine 3':5'-phosphate and on the activity of adenosine 3':5'-phosphate-dependent protein kinase in prolactin-producing cells in culture. 1. The effects of thyroliberin were studied in cultured rat pituitary-tumour cells that synthesize and secrete prolactin (the GH4C1 cell strain). 2. Prolactin and cyclic AMP were measured by radioimmunological methods, and a cyclic AMP-dependent protein kinase was characterized by using histone as substrate. 3. Prolactin release was studied after 5-60min of treatment, and synthesis after 48h of treatment with thyroliberin. One-half maximum stimulation of release and synthesis were observed at 0.25 and at 4nM respectively. 4. Cyclic AMP was temporarily increased in cell suspensions after treatment with thyroliberin, and one-half maximum stimulation was observed at 25nM. 5. Dibutyryl cyclic AMP increased prolactin release and synthesis, one-half maximum effects being obtained at 20 micronM. 6. A cyclic AMP-dependent protein kinase, which was one-half maximally stimulated at 30 nM-cyclic AMP, was demonstrated. 7. An increase in the activity ratio (-cyclic AMP/+cyclic AMP) of the cyclic AMP-dependent protein kinase was observed after treatment with thyroliberin. Total protein kinase activity in the presence of cyclic AMP was unaltered. The time-course of enzyme activation was similar to that of cyclic AMP formation and corresponded to the time when prolactin release was first observed. 8. It is concluded that thyroliberin induces cyclic AMP formation, resulting in the activation of a cyclic AMP-dependent protein kinase.


Surface components involved in virally mediated membrane changes. 1. Virally mediated permeability changes are not brought about by neuraminidase or other enzyme. They are not mimicked by local anaesthetics such as benzyl alcohol or dibucaine. 2. Virus induces an increase in the exchange rate of membrane-bound Ca2+. The temperature-dependence, and sensitivity to concanavalin A, of the permeability change and of Ca2+ exchange are similar. 3. It is concluded that an increase in the exchangeability of membrane-bound Ca2+ constitutes an early and specific event after the attachment of Sendai virus to cultured cells.


The substrate specificity of adenosine 3':5'-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle. The known amino acid sequences at the two sites on phosphorylase kinase that are phosphorylated by cyclic AMP-dependent protein kinase were extended. The sequences of 42 amino acids around the phosphorylation site on the alpha-subunit and of 14 amino acids around the phosphorylation site on the beta-subunit were shown to be: alpha-subunit Phe-Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly-Gly-His-Ser-Leu-Gly-Ala-Asp-Leu-Met-Ser-Pro-Ser-Phe-Leu-Ser-Pro-Gly-Thr-Ser-Val-Phe(Ser,Pro,Gly)His-Thr-Ser-Lys; beta-subunit, Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-VALIle-Tyr-Glu-Pro-Leu-Lys. The sites on histone H2B which are phosphorylated by cyclic AMP-dependent protein kinase in vitro were identified as serine-36 and serine-32. The amino acid sequence in this region is: Lys-Lys-Arg-Lys-Arg-Ser32(P)-Arg-Lys-Glu-Ser36(P)-Tyr-Ser-Val-Tyr-Val- [Iwai, K., Ishikawa, K. & Hayashi, H. (1970) Nature (London) 226, 1056-1058]. Serine-36 was phosphorylated at 50% of the rate at which the beta-subunit of phosphorylase kinase was phosphorylated, and it was phosphorylated 6-7-fold more rapidly than was serine-32. The amino acid sequences when compared with those at the phosphorylation sites of other physiological substrates suggest that the presence of two adjacent basic amino acids on the N-terminal side of the susceptible serine residue may be critical for specific substrate recognition in vivo.


Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle. Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.


Specificity of a protein phosphatase inhibitor from rabbit skeletal muscle. A hear-stable protein, which is a specific inhibitor of protein phosphatase-III, was purified 700-fold from skeletal muscle by a procedure that involved heat-treatment at 95 degrees C, chromatography on DEAE-cellulose and gel filtration on Sephadex G-100. The final step completely resolved the protein phosphatase inhibitor from the protein inhibitor of cyclic AMP-dependent protein kinase. The phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities of protein phosphatase-III [Antoniw, J. F., Nimmo, H. G., Yeaman, S. J. & Cohen, P.(1977) Biochem.J. 162, 423-433] were inhibited in a very similar manner by the protein phosphatase inhibitor and at least 95% inhibition was observed at high concentrations of inhibitor. The two forms of protein phosphatase-III, termed IIIA and IIIB, were equally susceptible to the protein phosphatase inhibitor. The protein phosphatase inhibitor was at least 200 times less effective in inhibiting the activity of protein phosphatase-I and protein phosphatase-II. The high degree of specificity of the inhibitor for protein phosphatase-III was used to show that 90% of the phosphorylase phosphatase and glycogen synthase phosphatase activities measured in muscle extracts are catalysed by protein phosphatase-III. Protein phosphatase-III was tightly associated with the protein-glycogen complex that can be isolated from skeletal muscle, whereas the protein phosphatase inhibitor and protein phosphatase-II were not. The results provide further evidence that the enzyme that catalyses the dephosphorylation of the alpha-subunit of phosphorylase kinase (protein phosphatase-II) and the enzyme that catalyses the dephosphorylation of the beta-subunit of phosphorylase kinase (protein phosphatase-III) are distinct. The results suggest that the protein phosphatase inhibitor may be a useful probe for differentiating different classes of protein phosphatases in mammalian cells.


Co-ordinate changes in enzymes of fatty acid synthesis, activation and esterification in rabbit mammary gland druing pregnancy and lactation. 1. The activities of fatty acid synthetase, acyl-CoA synthetase, glycerol phosphate acyltransferase and phosphatidate phosphatase were measured in the mammary glands of rabbits from day 16 of pregnancy to day 15 of post partum. 2. There were significant correlations between the increases in activities of these enzymes during this period. This was the case whether the activities were expressed per mg of homogenate protein, per g wet wt. of tissue or per total wet weight of the whole glands. The only exception was the lack of correlation between the activities of fatty acid synthetase and of phosphatidate phosphatase per g wet wt. of tissue. 3. These co-ordinate increases are discussed in relation to the changes which occur in fatty acid metabolism in the mammary gland during pregnancy and lactation.


Behavior of blood acetaldehyde in alcohol-treated rats following administration of thiurams. Single moderate doses of Na-dimethyldithiocarbamate and Na-diethyldithiocarbamate (thiocarb), respectively, did not influence the rate of elimination of ethanol i.p. in female rats, but there was a concurrent substantial increase in the blood acetaldehyde content. Tetramethylthiuram disulfide (thiram) produced a slight decrease in the rate of elimination of ethanol, while a comparable dose of tetramethylthiuram disulfide (disulfiram) failed to affect ethanol disappearance. At the same time, the blood acetaldehyde concentration showed a considerable increase which after thiram was much more significant than after disulfiram, Identical thiuram doses caused a significant reduction in the rate of elimination of acetaldehyde i.v., which suggests an inhibition of the aldehyde dehydrogenasel aldehyde oxidase and helps to explain the elevated blood acetaldehyde concentration which was observed in the presence of ethanol. It is concluded from these findings that thiuram activity decreases as the number of carbon atoms at the side-chains of the molecule is increasing.


[The protecting action of a combination of vasoactivating substances (CRP) on disturbances of the cerebral blood flow and metabolism and the cortical electrical activity (author's transl)]. The effect of anoxia and direct cortical stimulation was investigated in control, hypotensive and post reinfusional periods, before and after treatment with a combination of coumarin/rutin sulphate sodium salts/proxyphylline (CRP, Theokal). Theokal lowered the mean arterial pressure and a slight NADH oxidation appeared. Blood flow did not change after Theokal in spite of the 30 mm Hg blood pressure drop. This suggests an improved oxygen supply to the cortical cells. The increase in anoxic NADH reaction and the faster reoxidation kinetics of NADH following anoxia provided further evidence for the beneficial effect of Theokal on brain circulation and metabolism. It is to be emphasized that the described action of the drug can be observed only in hemorrhagic shock but not in normal animals.


[Human papilloma viruses (author's transl)]. Various types of human papillomas (warts) can be differentiated according to clinical parameters: Verrucae vulgares, plantares, planae and seborrheicae, in addition genital warts (Condylomata acuminata) and laryngeal papillomas. With the exception of Verrucae vulgares and plantares the epidemiology of these types of warts displays significantly different patterns. It was possible to demonstrate the heterogeneity of human wart viruses also by biochemical and serological means: we have identified 4 different types of human papilloma viruses so far, 3 of which are closely related with each other. Their potential role in the induction of malignant tumors in man is discussed.


Possible utilization of extracorporeal hemoperfusion in the treatment of coronary artery disease. A hypothesis and proposal for animal studies. It is proposed that in vivo immunoadsorption may be utilized in the treatment of coronary artery disease. The proposed method is based on binding of low density lipoproteins and cholesterol to antibodies directed against low density lipoproteins which are bound to a matrix in an extracorporeal hemoperfusion column. The various observations that suggest the hypothesis are presented. The feasibility of the proposed method and some of the problems that may be encountered in its utilization are discussed. The method should be evaluated in animal studies prior to consideration of clinical trials in patients.


Effect of essential oil of onion and garlic on experimental atherosclerosis in rabbits. The effects of the essential oils of onion (extracted from 2 g of raw onion per kg body weight) and garlic (extracted from 1 g of raw garlic per kg body weight) have been observed on experimental atherosclerosis produced by cholesterol feeding (0.5 g/kg) in rabbits. The rise in serum cholesterol and serum triglycerides was significantly reduced by both onion and garlic during the 4-month period of study. Cholesterol feeding significantly increased beta-(P less than 0.01) and pre-beta (P less than 0.001) lipoproteins while decreasing the alpha-fraction (P less than 0.001). Onion and garlic both prevented these changes. The beta/alpha ratio, which was initially 1.6 : 1, rose to 4.5 : 1 and 5.7 : 1 at the end of 2 months and 4 months of cholesterol feeding. However, this ratio did not increase significantly, both at the 2-month and 4-month period, when onion and garlic were added. Fibrinolytic activity significantly increased with onion (P less than 0.001) and garlic (P less than 0.001) while feeding only cholesterol actually decreased it (P less than 0.001). Onion and garlic reducec aortic atheroma by about half. It is suggested that the essential oils of onion and garlic protect against experimental atherosclerosis by preventing the fall in the alpha lipoprotein fraction and by enhancing fibrinolytic activity, as well as by lowering the serum cholesterol and triglyceride levels.


The intermediate coronary care unit. A stage in continued coronary care. The concept of continued and progressive coronary care rather than intermediate coronary care is proposed. At each clinical stage the patient may be at risk and his management needs to be planned appropriately--prevention of the development of coronary disease, prehospital care, acute coronary care, subacute coronary care, and late hospital stay. Meticulous continued care once the patient leaves the hospital and returns home may be needed for a long time. Although the benefit of an intermediate coronary care unit has not yet been proved, significant patient risk continues beyond 12 days of hospital admission. High risk patient subsets are emerging requiring careful continued monitoring and the ability to undertake emergency measures as needed, and this is particularly so in patients suffering large anterior infarction, in those with infarction associated with cardiac failure, when infarction is associated with fascicular block and other types of conduction disturbances, and in patients who continue with rhythm disturbances after their admission to the hospital. Electrocardiograph leads III and VI displayed simultaneously should be routinely monitored in patients with fascicular blocks and acute anterior infarction as a guide to instituting prophylactic transvenous pacemaking. The continuation of intensive patient care and monitoring beyond the usual 2 to 5 days in a coronary care unit allows early mobilisation of patients in safety, thus speeding their ultimate rehabilitation. There is, as yet, no satisfactory study documenting the need for intermediate coronary care units, but much presumptive evidence is available to indicate that this is so. A carefully controlled randomised study would be invaluable.


The actions of cyclic AMP, its butyryl derivatives and Na butyrate on the proliferation of malignant trophoblast cells in vitro. Cyclic AMP, and its derivatives N6-monobutyryl cyclic AMP and dibutyryl cyclic AMP, have been found to inhibit the proliferation of trophoblast cells of the BeWo cell line in vitro. Sodium butyrate (1 mM), a possible degradation product of the butyrate derivatives, also inhibited cell proliferation, giving similar growth rates to equimolar dibutyryl cyclic AMP. The inhibition by butyrate was however, not sufficient to account for the action of 1 mM N6-monobutyryl cycli AMP, which, like cyclic AMP, completely inhibited cell proliferation. The potency, specificity and toxicity of the substances were compared. The results suggest different modes of action for cyclic AMP and dibutyryl cyclic AMP.


Immunogenicity of tumour cells modified with various chemicals. Mouse tumour cells were treated with various chemical modifiers. The number of modifying groups per cell was determined with labelled reagents. The effects of the different modifying groups on the immunogenicity of the tumour cells was tested in syngeneic mice for tumour protection using a challenge dose of viable cells at 1000 or 10,000 time LD100. Best protection was obtained after immunization of animals with tumour cells modified with dimethylsulphate or acetic anhydride, or with glutardialdehyde-fixed cells treated with a carbodiimide and methylamine. Up to 40% of the animals remained tumour-free. The other animals exhibited a greatly increased mean survival time. The post-challenge sera showed no detectable amounts of antibodies against the tumour cells.


Cold lymphocytotoxic antibodies in nasopharyngeal carcinoma. Sera from patients with nasopharyngeal carcinoma (NPC), a disease associated with Epstein-Barr virus (EBV), were found to be cytotoxic at 15% degrees C in the presence of complement for a panel of human lymphocytes, with a higher frequency than those of matched controls. The cold lymphocytotoxic antibodies (LTA) responsible for this activity have the same properties as those described in sera from individuals with acute viral infections. The frequency and geometric mean titres (GMT) of LTA varied with the origin of the patient (Chinese larger than North African larger than Caucasian) and the stage of the disease (Stage IV larger than Stage I). A positive correlation between LTA and anti-EBV titres was found with regard to antibodies to the viral capsid antigen (VCA) and the EBV-specified nuclear antigen (EBNA). The absence of correlation between LTA and anti-early antigen (EA) titres probable reflects the complex relationships existing between viral infection and LTA production, but is compatible with the hypothesis that LTA acts as an immune regulatory mechanism in viral infections.


Effects of theophylline and dibutyryl cyclic AMP on proliferation and keratinization of human keratinocytes. Recent evidence suggested that cyclic adenosine 3',5'-monophosphate (cAMP) may be involved in the regulation of cell proliferation and differentiation. The effects of theophylline and dibutyryl cAMP on cell division and keratinization of human epidermal cultures were examined. Nine  to 12-day-old cultures were treated with these agents, separately and in combination, for various intervals. Both agents, either singly or in combination, depressed mitoses. The maximum mitotic inhibition was obtained in cultures treated with theophylline or theophylline plus dibutyryl cAMP. Tritiated thymidine studies showed that the test agents had no effect on labelling index (LI) at 4 h, but a 74% inhibition of LI was observed at 24 h. The maximum inhibition of LI (93%) occurred at 96 hours. In contrast to the control cultures, which rarely contain keratohyaline granules (KG), a marked increase in the production of these granules occurred in cultures treated with dibutyryl cAMP plus theophylline. The KG were present over the whole outgrowth. Theophylline alone also stimulated the production of KG, whereas dibutyryl cAMP had no effect. These data show that these agents inhibit cell division and this inhibition may be accompanied by an increased production of KG.


Hypertrichosis lanuginosa acquisita associated with pancreatic carcinoma. A patient with hypertrichosis lanuginosa acquisita associated with pancreatic islet cell carcinoma, probably D cell type, is presented. The hypertrichosis antedated other evidence of malignancy.