[Necrotizing enteritis in suckling pigs (Clostridium perfringens type C enterotoxemia). II. Toxin formation, heat and drug resistance of Clostridium perfringens strains isolated from suckling pigs and broilers with necrotizing enteritis].

Nineteen Clostridium perfringens Type C strains and ten foreign control strains of subtypes C1, C3, and C4 were tested for their toxin formation and spore resistance to heat. The 19 Type C strains had been isolated from unweaned piglets in the context of necrotising enteritis outbreaks in the GDR. The Clostridium perfringens Type C strains formed beta-toxin, but they failed to form epsilon-toxin or gamma-toxin, alpha-toxin was successfully recorded from 15 of the 19 strains tested from unweaned piglets. The minor-lethal toxin fractions were also tested, with delta-toxin being recorded from all strains, non-alpha-delta-theta-toxin from six, theta-toxin from five, and K-toxin from one. Tests for delta-toxin, lambda-toxin, and mu-toxin were negative. The Clostridium perfringens Type C strains isolated in the GDR from unweaned piglets with necrotising enteritis were, basically, identical with those described in Denmark by von Hogh (1967) with regard to toxin formation. Clostridium perfringens strains cultured in broilers with necrotising enteritis were characterised by regular toxin production in the context of alpha, theta, delta as well as non-alpha-delta-theta. They failed to form beta, epsilon, gamma and lambda, while mu-toxin was formed by them quite irregularly. They, consequently, are Type A strains. Resistance to chloramphenicol and/or oxytetracycline was exhibited by 78.5 per cent of 237 tested Clostridium perfringens strains which had been isolated from unweaned piglets and broilers with necrotosing enteritis. Multiple resistance was recorded from 33.9 per cent. All strains were susceptible to penicillin, nitrofurantoin, and erythromycin.


[Electrocardiographic functional diagnosis of the vegitative nervous system in cattle. 2. Determination of the propranolol blockade effect on cardiac beta-receptors under application of maximum isoprenaline doses].

The blocking effects of 50 mg and 100 mg of propanolol, a beta-receptor blocker, upon the beta-adrenergic receptors of cattle heart were verified by means of 250 microgram of isoprenaline, a maximum dose. The 50-mg-propanolol dose inhibited between 77 and 80 per cent of the isoprenaline-caused QT reduction, whic action was considered as being sufficiently safe to rule out any beta-adrenergic cardiac effect. Blockade was further increased to the order of between 89 and 92 per cent by using 100 mg of propanolol, but certain side-effects occurred (expiratory dyspnoe, quinidinlike action in terms of PQ extension, and vagovasal collapse). Therefore, 50 mg of propanolol are recommended as the proper dosage with which to examine cardiac and extracardiac beta-adrenergic receptors in cattle. Application of propanolol to non-anaesthetised, nonatropinised cattle resulted in higher bloackade of isoprenaline-caused QT reduction, as compared to the parameters PQ, TP, TQ, time of cardiac action, PP, and momentary heart rate. This phenomenon is being analysed. With reference being made to the peculiarities of sympathico-parasympathetic interactions on the heart, QT time is considered as a more safe parameter by which to assess substrate competition between propanolol and isoprenaline at cardiac beta-receptors, while the ventriculodiastolic ECG recordings TQ, TP, and PQ as well as time of cardiac action, PP and momentary heart rate might come under a somewhat stronger influence of the parasympathetic system.


[Effects of the main derivatives of vitmain D in 3 siblings with "pseudo-deficiency" rickets].

Three siblings, respectively 20, 16 and 12 years old, presented with hypocalcemic vitamin D resistant rickets (Prader's type). Their clinical history included several periods of spontaneous cessation of therapy, with severe relapses. Since 1973, treatment was strictly observed, allowing to test the therapeutic effects of 25 OH D3, 1-25 (OH) 2 D3 and 1-alpha (OH) D3. The clinical effects are reported as well as biochemical data. Among them, an inactive form of hyperparathyroidism is emphasized, which may resemble some cases of pseudohypoparathyroidism. Simultaneous resistance to exogenous PTE was also demonstraded.


[Herpetic meningoencephalitis in infants. Study of 7 cases].

Seven children with herpes meningoencephalitis aged between 14 days and 13 months were studied. The herpes infection was confirmed by isolation of herpes virus from the CSF (one case), from the brain (one case), and in the 5 remaining cases by serology. The strains isolated were herpes type 2. The clinical features of herpes meningoencephalitis in the first year of life and the laboratory data are described.


[Characterization and solubilization of specific growth hormone binding sites in the rat liver].

The interaction of 125I-labeled human growth hormone (HGH) with microsomal membranes of female rat liver is a specific, saturable and reversible process; at saturation, about 0.9 picomoles of 125I-HGH are bound per mg of membrane proteins. Equilibrium is achieved after 90 minutes of incubation. The binding is dependent on membrane concentration and pH of the incubation medium. Membrane treatment by phospholipase A causes a 1.5 fold increase in binding capacity. 125I-HGH exposed to membranes or eluted from the membrane-receptor complex retains biological activity as tested by binding to liver membranes. The variation in binding with the age of the female rat is probably related to a change in the binding capacity and not in the binding affinity. Microsomal membranes can be solubilized by the use of Triton X-100; solubilized membranes retain their capacity to bind HGH and the properties of the interaction are similar to the ones of the particulate membranes. Lactogenic and somatogenic receptors are eluted from an acrylamide-agarose column in the same position; their apparent molecular weight is 350,000.


Estrogen therapy for severe persistent depressions in women.

Positive results are reported from a double-blind study of estrogen therapy administered to severely depressed, inpatient women who had failed to respond to various conventional treatments of depression. Large doses of oral conjugated estrogen were administered for a three-month period to 23 premenopausal and postmenopausal inpatient women. Placebos were administered for a comparable period to 17 similar patients. The posttreatment Hamilton ratings of depression were significantly reduced in the estrogen-treated group, but not in the placebo group. Possible physiological mechanisms are discussed. The risk-benefit ratio for estrogen therapy of depression in these patients was judged to be favorable. However, periodic endometrial biopsies are required to monitor the endometrial response of women receiving high doses of estrogens.


Cytochemical studies of ameloblasts and the surface layer of enamel of the rat incisor at the maturation stage.

In order to elucidate the cytochemical properties of the membranous structure between enamel and ameloblasts of the rat incisor at the maturation stage, chromic phosphotungstic acid (Cr-PTA) and periodic acid-silver methenamine (PA-silver) techniques for electron microscopy were employed in combination with a digestion test with hyaluronidase, neuraminidase, collagenase or trypsin. Also, acid phosphatase activity of ameloblasts at the maturation stage was examined with a modified GOMORI's metal salt method. An intensely Cr-PTA reactive band approximately 0.1 micron thick appeared along the surface layer of enamel at the transitional stage, and at the very beginning of the maturation stage another intensely Cr-PTA reactive band which was seen by uran-lead stain to be a delicate electron-dense membranous structure appeared as well between enamel and ameloblasts. A lot of cytoplasmic small vesicles or tubular structures, both intensely reactive to Cr-PTA, were observed near the apical membranes of the overlying ameloblasts indicating that those organelles must have been responsible for the secretion of the latter band. Acid phosphatase activity was clearly demonstrated at Cr-PTA reactive large vesicles in the cytoplasm of those cells. The PA-silver staining technique manifested a band heavily deposited with silver grains along the surface layer of enamel, i.e., where the former band existed, but showed no particular reaction at the latter, the band-like layer between enamel and ameloblasts. Hyaluronidase or neuraminidase treatment remarkably decreased the Cr-PTA reaction of the latter band. Trypsin or collagenase treatment, on the other hand, not only eliminated the Cr-PTA reaction but digested the band itself. These results suggest that the membranous structure between enamel and ameloblasts of a rat incisor is not so-called enamel cuticle but a basal lamina produced by overlying ameloblasts and that the basal lamina contains collagenous components even though it lies on enamel.


Anti-Gm and anti-Inv antibodies in children during the course of clinical infectious mononucleosis syndrome.

Forty-eight children with the symptoms of infectious mononucleosis were examined. In six of then anti-Gm(1) antibodies were discovered, in two anti-Gm (21), in one child anti-Inv (1).


Studies on cyclic AMP-cyclic GMP cooperation and on the influence of acetylcholine, adrenaline and thiols in lymphocyte reactivity.

In rat's lymph node lymphocytes, maximal stimulation of the response after suboptimal doses of PHA (1/2 of the optimal dose), measured by the incorporation of 3H-thymidine, after 10(-6) M acetylcholine and after 10 min interval between PHA and acetylcholine application was obtained. In these experiments inhibition of acetylcholine action by its muscarainic antagonists was not observed. It was shown that exogeneous cAMP as well as other factors, which increase cAMP level in lymphocytes had opposite effects to those, which could be caused by increasing of cGMP. Acetylcholine in optimal concentration (10(-6) M) as well as thiols, did not increase the level of endogeneous cAMP in rat lymphocytes.


Heterospecific activity of rat interferon.

In the experiments performed in vitro and in vivo it has been found that the rat and rat embryo fibroblasts cultured in vitro after the induction with virus produce interferon which displays the antiviral activity not only in the homologous cells but also in the heterologous ones. When analysed by chromatography on Sephadex G-100 it was shown that the rat serum contains two interferon populations differing in the molecular weight and both active in the homologous and heterologous cells. The interferon with the heterospecific activity has been used in the experimental therapy of mice infected with Encephalomyocarditis Virus (EMC), Vesicular Stomatitis Virus (VSV) and Influenza Virus, and was found to be effective.


Studies on the mode of action of IME--an inhibitor of interferon activity from mouse embryo tissues.

IME--inhibitor of interferon activity from mouse embryo tissues does not inactive interferon by direct contact; it does not inhibit the production of interferon and it does not affect the replication of challenge virus. IME shows no species-specific activity since it exhibits anti-interferon action not only in homologous interferon system but also in the heterologous rat, chick and human interferon systems. The interferon-induced antiviral state as well as the efficient cell's metabolism appear to be necessary for the expression of activity of IME.


Studies of RNA isolated from Piptoporus betulinus as interferon inducer.

Crude RNA isolated from Piptoporus betulinus and two fractions of it obtained after the chromatography contain single-stranded RNA. The crude RNA, but not its chromatographic fractions, are resistant to the pancreatic RNA-ase. The crude preparation of RNA from P. betulinus contains beside ribose, considerable amounts of glucose, galactose and mannose. The crude RNA from P. betulinus induces in the human fibroblasts interferon with specific activity of 400 units per mg of protein.


Application of antisera to interferon in studying oncogenic viruses.

Susceptibility of BALB/c mice to infection with Moloney sarcoma virus (MSV) and to Herpes simplex virus type 2 (HSV-2) was considerably increased by administration of sheep anti-mouse interferon (anti-IF) serum. The regression of the MSV-induced tumors was inhibited when the weanling (three-to-four-week-old) mice were injected with the anti-IF serum. Using the anti-IF serum it has been found that the antagonism between HSV-2 and Rauscher leukemia virus in the mouse is not mediated by interferon. It is suggested that interferon is an important factor controlling growth of virus and/or virus induced tumor cells in the mouse before it develops a strong immunological response.


Immunological experimental arthritis in pigs. I. Propagation dynamics of the attenuated strain of Aujeszky's disease virus in the pig organism and the humoral immunity formation.

Studies on the propagation dynamics of Aujeszky's disease virus and the formation of general and local joint immunity were to provide information explaining the mechanism of experimental rheumatoid arthritis in animals. It was shown that: Aujeszky's disease virus administered intramuscularly caused viremy on 3-4th day after its application and disappeared from the blood circulation on 5-6th day, introduced into the pig ankle joint, the virus was eliminated from the articular fluid two hours after injection, but it appeared again 3-7 days later probably due to the propagation in the cells of the synovial membrane, and that first parenteral administration of the virus induced the formation of neutralizing antibodies in low titer (5-10 units); application of the virus for the second time brought about a considerable increase of antibodies titer within 40-80 units.


Immunological experimental arthritis in pigs. II. The level of complement in the pigs immunized with the Erysipelothrix rhusiopathiae and virus of Aujeszky's disease.

The level of complement was studied in 3 groups of pigs. One group was immunized with Erysipelothrix rhusiopathiae and two remaining groups with virus of Aujeszky's disease. The lowest titers, irrespective of the starting level were shown to exist in all the studied groups in the third week after the last dose of the virus.


Immunological experimental arthritis in pigs. III. Inflammatory reactions in the brain of pigs immunized with the attenuated virus of Aujeszky's disease.

Histopathological brains and spinal cords of pigs were examined at different times of immunization with the virus of Aujeszky's disease (AD). Temporary inflammatory reaction was found to have the features of non-suppurative disseminated inflammation in the second week after the second virus dose had been given. Perivascular and subependymal lymphocytic infiltrations were observed and the focal multiplication of microglia.


Immunological experimental arthritis in pigs. IV. Reaction of the synovial membrane of the non-immunized and immunized pigs after intraarticular administration of the virus of Aujeszky's disease.

7 days after intramuscular administration of the attenuated virus of Aujeszky's disease, strain TK 300 L, it was found: in non-immunized animals--immunological arthritis with fibrinoid necrosis and abundant infiltrations from lymphoid cells; in animals of low antibodies titer--only few infiltrations from lymphoid and plasmic cells round the sub-synovial vessels; in animals of high antibodies titer--vast areas of fibrinoid necrosis, abundant infiltrations from lymphoid cells and presence of gigantic cells.


Immunological experimental arthritis in pigs. V. Reaction of the synovial membrane in the pigs non-immunized and immunized with the virus of Aujeszky's disease after the intraarticular administration of the immunological complexes.

Immunized animals were given intraarticular complexes formed in vitro from the autologous serum and virus AD. 7 days after complex administration with excess of the virus, weak inflammatory reactions of the immunological type were noted. After neutral complex administration virulent immunological inflammation of the synovial membrane took place. The histological picture resembled rheumatoid arthritis in the man and the changes obtained after the virus administration to the joints with a high level of antibodies. Administration of complexes and homological serum alone to the joints of the nonimmunized animals caused the occurrence of superficial focuses of fibrinoid necrosis, but there was a lack of cellular reactions.


Studies on cell surface antigens of mouse leukemic and normal lymphocytes. II. Relationships between mammary leukemia, gross virus related and major histocompatibility antigens on L1210 leukemic lymphocytes.

The presence of ML antigen on leukemia L1210 cells was confirmed by different procedures. In addition, by applying appropriate absorptions and blocking techniques combined with ferritin labelled antibodies in electron microscopy (EM), the independent localization of ML antigen and murine leukemia virus Gross (MuLV-Gross) cellular antigen on L1210 cells was demonstrated. The ferritin labeled antigenic sites appeared to be free from budding viral particles. In further studies the 3M KCI extracts of L1210 leukemia cells purified by polyacrylamide gel electrophoresis (PAGE) and the antigenic material from leukemia L1210 cells purified by affinity chromatography were characterized. Their identity was verified by anti ML-sera. Using the modified lyso-strip method, the presence of ML antigen and H-2K and H-2D gene products on independent molecules on the surface of L1210 cells was shown. In addition, in preparative polyacrylamide gel electrophoresis (PAGE) of 3M KCL extracts of L1210 cells, ML and H-2d antigenic products were found in separate fractions.


Intestinal strangulation in germfree and monocontaminated dogs.

Earlier studies in the germfree rat demonstrated that the common intestinal bacteria vary greatly in their ability to cause death after intestinal strangulation. Some of these experiments were repeated in adult, germfree and monocontaminated beagles. Neither short closed-loop hemorrhagic nor long closed-loop ischemic strangulation killed the germfree dog. Either procedure rapidly killed the dog with a conventional bacterial flora or a dog monocontaminated with Clostridium perfringens. The dogs monocontaminated with Bacteroides fragilis died after several days, whereas Escherichia coli killed only one of three animals. These experiments demonstrate the usefulness of the germfree dog as a unique research subject, confirm our earlier studies on intestinal strangulation in germfree rats, and further emphasize the differing lethal potentials of the intestinal bacteria in intestinal strangulation.


Lewy bodies in the presence of Alzheimer's disease.

Large numbers of Lewy bodies in the substantia nigra were found in a case of Alzheimer's disease. Parkinsonian symptoms were not recognized. The patient appears to be an example of an association recognized by Woodard but not yet understood. The case is discussed in the context of reported relationships between Parkinson's disease or "Lewy body disease" and dementia, on the one hand, and reports linking various forms of psychoses with an unusually high incidence of Lewy bodies, on the other.


Facial nerve in recurrent benign pleomorphic adenoma.

Improvements in the treatment of benign and malignant tumors in the parotid gland have substantially reduced the incidence of recurrence. This has come about primarily be the abandonment of the enucleation techniques and the development of lateral lobectomy operation. The recurrence rate for benign mixed tumor in the parotid gland is variously reported in the ranges of 0.5% to 10%. Because the benign mixed tumor comprises approximately 65% of the tumors in this gland, this complication assumes an important and specific role. A review of this problem establishes the principles of management, extending from simple reexcision through total parotidectomy with preservation of the facial nerve, and radical parotidectomy with resection of the facial nerve and immediate nerve grafting.


[Morphology of a malignant parotideal lymphoma with paraproteinaceous cytoplasmic inclusions (author's transl)].

A female patient presented with a lump in the right parotedeal region. A needle aspiration biopsy was performed which revealed numerous monomorphous atypical lymphoid cells with PAS-negative cytoplasmic inclusions. This raised the suspicion of malignancy. Therefore, the tumour was excised. Histologically, a nodular lymphoma of low grade of malignancy was diagnosed. The tumour contained many epithelioid histiocytes, sometimes with PAS-positive material. The case was difficult to classify after the Kiel-nomenclature, whilst classification on the basis of Rappaport's system was much easier.


Idiopathic facial palsy. Viral, hereditary, or both?

Two cases of acute peripheral facial paralysis occurring in two siblings in a family of six members, who all developed influenza-like symptoms, are reported. Both patients with facial paralysis, as well as two other members of the family had elevated titers against herpes simplex virus, even though it was not possible to demonstrate any raise in titers between the acute and convalescent phase. The "epidemic" occurrence is from a statistical point of view more than just a coincidence, and it is concluded that these findings support the hypothesis that Bell's palsy may be caused by reactivation of herpes simplex virus, probably facilitated by some hereditary component. The authors suggest that a combination of infectious (most likely viral) and hereditary factors may be etiologically important for the occurrence of acute peripheral facial paralysis.


Infectious bursal disease virus infection attempts in turkeys and Coturnix quail.

Attempts failed to infect Coturnix quail with infectious bursal disease (IBD) virus by exposure at 7, 14, 21, and 31 days old. There were no clinical signs or gross and microscopic changes in the bursa of Fabricius, and serologic tests and virus isolation attempts from cloacal swabs were negative. Turkeys of two breeds exposed to IBD virus at 6 or 8 weeks old showed no clinical signs or lesions in the bursa of Fabricius, and the virus could not be isolated from cloacal swabs. They did respond serologically, however, by developing both precipitin and virus-neutralizing antibodies.


Virus-neutralization versus agar-gel precipitin tests for detecting serological response to infectious bursal disease virus.

The virus-neutralization (VN) test was found much more sensitive than the agar-gel precipitin (AGP) test for detecting prior exposure to infectious bursal disease (IBD). Many sera that were negative in the AGP test were found to have VN antibodies, and virtually all sera in a commercial flock were free of precipitin but had VN titers. VN titers varied widely on a flock basis, and revaccination of an 8-month-old flock through the drinking water did not alter the antibody titers. Inoculation of maternally immune chicks at 7 days of age with IBD virus resulted in a good serologic response by both the AGP and VN tests.


Preparation of inactivated oil-emulsion vaccines with avian viral or Mycoplasma antigens.

The influence of the composition of water-in-oil emulsions on their physical characteristics was determined by preparing experimental emulsions with various water-to-oil ratios and various emulsifiers. Emulsions containing Tween 80 in the aqueous phase and Arlacel A or Arlacel 80 in the oil phase were lower in viscosity than emulsions containing only an oil-phase emulsifier. Viscosity decreased as the concentration of oil increased. Oil-emulsion vaccines prepared with aqueous- and oil-phase emulsifiers had low viscosity, were stable for more than 12 weeks at 37 C, and induced a marked primary antibody response in chickens.


Experimental induction of hemorrhagic-aplastic anemia in chickens. I. Etiology.

Exposure to infectious bursal disease virus (IBDV) at 1 day old followed by inclusion body hepatitis virus (IBHV) inoculation at 36 days produced typical lesions of hemorrhagic-aplastic anemia syndrome (HAS). The lesions included severe anemia, widespread hemorrhages, and dermatitis. HAS could not be induced in the first 4 weeks of life in chickens inoculated at one day old with IBHV alone or in combination with IBDV. It was concluded that the immunosuppressive effects of IBDV failed to alter the pathogenicity of IBHV in chicks less than 4 weeks old. This resistance was considered to be age-related. Subcutaneous inoculation of day-old chicks with IBDV produced a more severe infection than did oral exposure. Serial passage of IBHV in day-old chicks had no significant effect on the viral pathogenicity.


Comparative pathogenicity of six avian adenovirus isolants in the liver.

The comparative pathogenicity of six avian adenovirus isolants for chickens was determined. The most virulent isolant was DPI-1, causing focal to massive hepatic necrosis with hemorrhage, while Tipton (Conn), Tipton (Purdue), GAL-1, and DPI-2 isolants were considered less pathogenic and produced only mild focal hepatic necrosis. The Indiana-C isolant was the least pathogenic and failed to produce any significant hepatic lesions. Intranuclear inclusions were observed in hepatocytes of all but the Indiana-C inoculated chickens on the third day postinoculation (PI), disappearing by the seventh day PI.


In vitro inactivation of cell-free Marek's disease herpesvirus by immune peripheral blood lymphocytes.

The effect of peripheral blood lymphocytes (PBL) on cell-free Marek's disease virus (MDV) was studied in vitro in PBL from intact or embryonal bursectomized (Bx) chicks infected with the apathogenic SB-1 isolate of MDV. The PBL were harvested purified and were mixed, 200 or 2,000 to 1, with cell-free GA-5 of MDV. Virus survival was assayed after incubation for 1 hour at 36 C by the standard assay for cell-free MDV. PBL from intact but not Bx chickens were able to inactivate virus. When the effective suspension of PBL was depleted of macrophages, inactivation no longer occurred, suggesting that it resulted from cooperation between macrophages and B-cells.


Experimental induction of hemorrhagic-aplastic anemia in chickens. II. Serum protein changes.

The serologic response of chickens to infectious bursal disease virus (IBDV) and inclusion body hepatitis virus (IBHV) was analyzed. Inoculation at one day old with either IBDV or IBHV significantly (P less than 0.05) reduced levels of serum gamma-globulins at 4 weeks postinoculation. This response was not elicited by inoculation of IBDV together with IBHV. Birds with experimentally induced or naturally occurring hemorrhagic anemia syndrome (HAS) had serum proteins quantitatively and qualitatively changed from those of controls. Serum protein profiles did not coincide, however, in experimentally infected and naturally infected chickens. Among naturally infected chickens, those that were IBHV-positive upon culture had significantly (P less than 0.05) lower hematocrit values.


Airsacculitis induced in broilers with a combination of Mycoplasma gallinarum and respiratory viruses.

Mycoplasma gallinarum was isolated from tracheas and air-sac lesions from broilers in flocks having higher than normal condemnations due to airsacculitis. A representative M. gallinarum isolant, given by aerosol or by air-sac inoculation, produced air-sac lesions in young chickens when given in combination with a vaccine combining Newcastle disease and infectious bronchitis or with a field strain of infectious bronchitis virus.


Immune response and pathogenicity of different strains of infectious bursal disease virus applied as vaccines.

Eight strains of infectious bursal disease virus (IBDV) were characterized by the criteria of immunity and pathogenicity engendered in young chickens. Some strains have been used commercially, and the others are potential candidates for vaccines. They were administered by drinking water, eyedrop, vent drop, and subcutaneous and aerosol routes. The viruses varied widely in pathogenicity in terms of bursal damage, morbidity, and mortality. Immunity induced with different routes of administration also differed markedly when measured by challenge, virus serum-neutralizing antibody titers, and the agargel precipitin test. Maternally derived immunity interfered with stimulation of active immunity by some of the less pathogenic strains. The more virulent strains, however, were able to provide protection under similar circumstances, although bursal damage was frequently severe. Two strains proved relatively virulent by the criteria employed. Characteristics desirable for IBDV strains to be given as vaccines under field conditions to young chickens are discussed briefly.


Studies on the polypeptides of poxvirus. I. Comparison of structural polypeptides in vaccina, cowpox and Shope fibroma viruses.

Radioactively labeled vaccinia, cowpox and Shope fibroma virions free from any detectable contamination with host cell protein, were dissociated into their constituent polypeptides, and these were then analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The profiles of constituent polypeptide bands of four strains of vaccinia virus (IHD-W, IHD-J, Lister and DIs) were almost the same, except that a polypeptide of about 41,000 daltons was not detectable in the autoradiogram of strain IHD-W which has no hemagglutinin. The profile of polypeptide bands of cowpox virions was also almost the same as that of vaccinia virions, except for several polypeptides of about 40,000 to 50,000 daltons, but the profile of Shope fibroma virions differed considerably from that of vaccinia or cowpox virions.


Status of endogenous avian RNA tumor virus in Marek's disease lymphoblastoid cell lines and susceptibility of those lines to exogenous RNA tumor viruses.

Three lymphoblastoid cell lines from Marek's disease (MD) tumors, two MD virus (MDV) producer lines (MSB-1 and HPRS-1), and a nonproducer line (RPL-1) were studied for the expression of avian leukosis sarcoma (ALS) viruses. The MSB-1 line was free of all known endogenous expressions, including replicating virus (RAV-O), group-specific (gs) antigens, and chick helper factor (chf). The RPL-1 and HPRS-1 were positive for gs antigens and chf. The RPL-1 and MSB-1 lines showed no evidence of an exogenous DNA provirus by nucleic acid hybridization (HPRS-1 line was not tested for that DNA). All three lymphoblastoid cell lines were susceptible to exogenous infection with both sarcoma and leukosis viruses of subgroup A but varied in susceptibility to subgroups B and C. All were resistant to subgroups D and E.


Avian adenoviruses--distribution of infection in commercial flocks of fowls in Argentina.

Group-specific avian adenovirus antibodies were surveyed in broiler, layer, and breeder chickens in three poultry-producing areas of Argentina: north, middle, and south. The method used was the agar double-immunodiffusion test. Of the flocks examined, 89% had antibodies to avian adenovirus, with the percentages higher in layers and breeders than in broiler chickens. A close relation was found between poorer hygienic condition and high adenovirus antibodies values.


Studies on the polypeptides of poxvirus. II. Comparison of virus-induced polypeptides in cells infected with vaccinia, cowpox and Shope fibroma viruses.

Virus-induced polypeptides in cells infected with vaccinia, cowpox and Shope fibroma viruses were examined by SDS-polyacrylamide gel electrophoresis followed by autoradiography. At least 42 vaccinia virus-induced polypeptides were identified among the polypeptides of cells pulse-labeled with [35S]-methionine and/or of fractionated cells labeled with [14C]-leucine for 24 hr. They consisted of 15 polypeptides (early polypeptides) which were synthesized even in the presence of cytosine-1-beta-D-arabinofuranosyl-HCl, and 27 polypeptides (late polypeptides) which were synthesized only in the absence of cytosine-1-beta-D-arabinofuranosyl-HCl. By the same procedure at least 40 cowpox virus-induced polypeptides (14 early polypeptides and 26 late polypeptides) and at least 31 Shope fibroma virus-induced polypeptides (13 early polypeptides and 18 late polypeptides) were identified. Comparative studies of virus-induced polypeptides on the basis of migration in SDS-polyacrylamide gel electrophoresis revealed that 11 polypeptides were early polypeptides common to both vaccinia and cowpox viruses; 21 were late polypeptides common to both vaccinia and cowpox viruses; 4 were early polypeptides common to both vaccinia and Shope fibroma viruses; 7 were late polypeptides common to both vaccinia and Shope fibroma viruses; 5 were early polypeptides common to both cowpox and Shope fibroma viruses; 9 were late polypeptides common to both cowpox and Shope fibroma viruses; 4 were early polypeptides common to all three viruses; and 7 were late polypeptides common to all three viruses.


Field experiments on live attenuated Japanese encephalitis virus vaccine for swine.

The efficacy of a live attenuated Japanese encephalitis virus (JEV) vaccine was examined in swine under conditions where natural infection could occur. The pigs immunized with the vaccine produced antibodies within one week after vaccination, and the antibody was retained until the end of the experiment, i.e. 36 days. However, the antibody titers in this group were lower than that in control group naturally infected with JEV. No virus was isolated from the five vaccinated pigs, but virus was isolated from all four untreated control pigs after natural infection, i.e., viremia was detected in all these animals. The duration of viremia in control pigs varied from one to four days. From these findings, it is concluded that immunization of swine with live attentuated JEV vaccine is useful in control of Japanese encephalitis (JE) in humans and some susceptible domestic animals.


Purification and characterization of subcomponent C1q of the first component of bovine complement.

Bovine C1q, a subcomponent of the first component of complement, was purified in high yield by a combination of euglobulin precipitation, and ion-exchange and molecularsieve chromatography on CM-cellulose and Ultrogel AcA 34. Approx. 12-16mg can be isolated from 1 litre of serum, representing a yield of 13-18%. The molecular weight of undissociated subcomponent C1q, as determined by equilibrium sedimentation, is 430000. On sodium dodecyl sulphate/polyacrylamide gels under non-reducing conditions, subcomponent C1q was shown to consist of two subunits of mol.wts. 69000 and 62000 in a molar ratio of 2:1. On reduction, the 69000-mol.wt. subunit gave chains of mol.wts. 30000 and 25000 in equimolar ratio, and the 62000-mol.wt. subunit decreased to 25000. The amino acid composition, with a high value for glycine, and the presence of hydroxyproline and hydroxylysine, suggests that there is a region of collagen-like sequence in the molecule. This is supported by the loss of haemolytic activity and the degradation of the polypeptide chains of subcomponent C1q when digested by collagenase. All of these molecular characteristics support the structure of six subunits, each containing three different polypeptide chains, with globular heads connected by collagen triple helices as proposed by Reid & Porter (1976) (Biochem. J.155, 19-23) for human subcomponent C1q. Subcomponent C1q contains approx. 9% carbohydrate; analysis of the degree of substitution of the hydroxylysine residues revealed that 91% are modified by the addition of the disaccharide unit Gal-Glc. Bovine subcomponent C1q generates full C1 haemolytic activity when assayed with human subcomponents C1r and C1s.


The assay of xylosyltransferase in cartilage extracts. A modified procedure for preparation of Smith-degraded proteoglycan.

A modification of the published method [Baker, Rodén & Stoolmiller (1972) J. Biol. Chem. 247, 3838--3847] for preparation of Smith-degraded proteoglycan is described. The new method is based on the finding that most of the chondroitin sulphate is cleaved from proteoglycan core protein by periodate oxidation. The borohydride reduction procedure was modified because the periodate-oxidized core protein is extensively degraded under the highly alkaline conditions previously used. The new method involves the separation of periodate-oxidized core protein from chondroitin sulphate by gel filtration on Sepharose 6B, and the reduction of the former in H3BO3/NaBH4 at pH 8.5 to produce the reduced species. Smith-degraded proteoglycan prepared by this method exhibited high acceptor activity for xylosyltransferase from embryonic-chick cartilage and had an apparent Km of 160 microgram/ml or 45 micrometer on a serine basis. In this assay system an apparent Km of 19 micrometer was obtained for UDP-xylose. The intermediate products periodate-oxidized core protein and reduced proteoglycen were inactive as xylosyltransferase acceptor substrates.


Low-temperature kinetics of the reactions of fully reduced membrane-bound cytochrome oxidase with oxygen in the Soret, alpha and near-infrared regions.

The kinetics of the reaction of fully reduced membrane-bound cytochrome oxidase with O2 obtained in the Soret, alpha and near-i.r. regions were analysed, and the contributions of the three intermediates of the reaction [Clore & Chance (1978) Biochem. J. 173, 799--810] to seven wavelength pairs (430--463, 444--463, 590--630, 608--630, 740--940, 790--940 and 830--940 nm) were determined. The nature of the intermediates is discussed on the basis of the data in the present paper together with data in the literature from optical wavelength scanning, e.p.r., i.r. and magnetic-susceptibility studies.


Purification and characterization of the ferredoxin component of 25-hydroxycholecalciferol 1 alpha-hydroxylase.

The chick kidney mitochondrial iron--sulphur protein (ferredoxin), a component of the NADPH--cytochrome P-450 reductase functional in the 1 alpha-hydroxylation of 25-hydroxycholecalciferol, was purified to homogeneity by chromatography on DEAE-cellulose, gel filtration on Sephadex G-100 and preparative electrophoresis on polyacrylamide gel. A novel NADPH--cytochrome c reductase assay utilizing crude renal NADPH--ferredoxin reductase was used for the detection of the ferredoxin. A mol. wt. of 53 000 was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by Sephadex G-100 gel filtration of the 125I-labelled ferredoxin. The ferredoxin has a sedimentation constant (S 20, w) of 2.66S, an A411/A280 of 0.4, and a molar absorptivity of 7300 cm-1 . M-1. The electron-paramagnetic-resonance spectrum after reduction with Methyl Viologen and dithionite was characteristic of ferredoxins with signals at g = 1.956 and 2.025. Two iron and two labile sulphur atoms per molecule of ferredoxin were released by acid. Ouchterlony immunodiffusion tests by using goat anti-(bovine adrenal ferredoxin) antiserum showed precipitin reactions with the bovine adrenal ferredoxin and the chick renal ferredoxin as antigens, suggesting that the renal ferredoxin shares antigenic determinants(s) with the natural adrenal antigen. Amino acid analysis showed that of the total number of residues per molecule of ferredoxin, glutamic acid and aspartic acid are the most abundant residues, comprising 17 and 15% respectively.


Prolyl 3-hydroxylase and 4-hydroxylase activities in certain rat and chick-embryo tissues and age-related changes in their activities in the rat.

Prolyl 3-hydroxylase activity, expressed per unit of extract protein, was much higher in rat kidney cortex than in the lung, liver or skin. A marked decrease in activity was found in the kidney cortex, liver and skin beyond 10 days of age. The ratio of prolyl 3-hydroxylase to 4-hydroxylase activity in the kidney cortex was 13--17 times that in the skin, that in the liver 6--8 times, and that in the lung about twice the value for the skin, there being no changes in this ratio with age. In 16-day chick embryos the highest ratios of prolyl 3-hydroxylase to 4-hydroxylase activity were found in the liver, heart, lens, aorta and kidney, and the lowest ratios in tendon, cartilage, cartilaginous and membranous bone and skin. The results suggest that the differences in the extent of prolyl 3-hydroxylation between various collagens can in part be explained by differences in the amount of prolyl e-hydroxylase activity among different cells.


Effects of food deprivation on ketonaemia, ketogenesis and hepatic intermediary metabolism in the non-lactating dairy cow.

1. The aim of this work was to investigate why non-lactating dairy cows are less susceptible to the development of ketonaemia during food deprivation than are dairy cows in early lactation. 2. The first experiment (Expt. A) consisted of determining the effect of 6 days of food deprivation on the concentrations of ketone bodies, and of metabolites related to the regulation of ketogenesis, in jugular blood and liver of non-lactating cows. 3. During the food deprivation, blood ketone-body concentrations rose significantly, but to a value that was only 16% of that achieved in lactating cows deprived of food for 6 days [Baird, Heitzman & Hibbitt (1972) Biochem. J. 128, 1311--1318]. 4. In the liver, food deprivation caused: a rise in ketone-body concentrations; a fall in the concentration of glycogen and of various intermediates of the Embden-Meyerhof pathway and the tricarboxylic acid cycle; an increase in cytoplasmic reduction; a decrease in the [total NAD+]/[total NADH] ratio; a decrease in energy charge. These changes were all qualitatively similar to those previously observed in the livers of the food-deprived lactating cows. 5. There appeared therefore to be a discrepancy in the food-deprived non-lactating cows between the absence of marked ketonaemia and the occurrence of metabolic changes within the liver suggesting increased hepatic ketogenesis. This discrepancy was partially resolved in Expt. B by the observation in two catheterized non-lactating cows that, although there was a 2-fold increase in hepatic ketogenesis during 6 days of food deprivation, ketogenesis from the splanchnic bed as a whole (i.e. gut and liver combined) declined slightly owing to cessation of gut ketogenesis.


Relationship between hormonal activation of phosphatidylinositol hydrolysis, fluid secretion and calcium flux in the blowfly salivary gland.

The addition of 5-hydroxytryptamine to the isolated blowfly salivary gland stimulates fluid secretion, transepithelial calcium transport and the breakdown of 32P- or 3H-labelled phosphatidylinositol The breakdown of [32P]phosphatidylcholine and [32P]-phosphatidylethanolamine was not stimulated by 5-hydroxytryptamine. In salivary glands incubated with myo-[2-3H]inositol for 1--3 h, more than 95% of the label retained by the tissue was in the form of phosphatidylinositol. The addition of 5-hydroxytryptamine resulted in an increase in the accumulation of label in intracellular inositol 1:2-cyclic phosphate, inositol 1-phosphate and free inositol along with an increase in the release of [3H]inositol to the medium and saliva. The release of [3H]inositol to the medium served as a sensitive indicator of phosphatidylinositol breakdown. The release of [3H]inositol was not increased by cyclic AMP or the bivalent-cation ionophore A23187 under conditions in which salivary secretion was accelerated. The stimulation of fluid secretion by low concentrations of 5-hydroxytryptamine was potentiated by 3-isobutyl-1-methylxanthine, which had no effect on inositol release. The stimulation of fluid secretion by 5-hydroxytryptamine was greatly reduced in calcium-free buffer, but the breakdown of phosphatidylinositol continued at the same rate in the absence of calcium. These results support the hypothesis that breakdown of phosphatidylinositol by 5-hydroxytryptamine is involved in the gating of calcium.


How does insulin stimulate glycogen synthesis?

One of the important effects of insulin on intracellular metabolism is its ability to stimulate the synthesis of glycogen in muscle and liver. It does this by promoting a net decrease in the extent of phosphorylation of glycogen synthase, the rate-limiting enzyme in the pathway of glycogen synthesis, which increases its activity. Several years ago glycogen synthase was shown to be phosphorylated and inactivated by cyclic AMP-dependent protein kinase in vitro, suggesting that the effect of insulin on glycogen synthesis, and perhaps other intracellular processes, might be explainable in terms of the ability of the hormone to decrease the concentration of tissue cyclic AMP. However, the subsequent failure to detect a decrease in cyclic AMP concentration in muscle under conditions where glycogen synthase activity was stimulated by insulin, coupled with the discovery of a second glycogen synthase kinase whose activity is unaffected by cyclic nucleotides, now suggests the possibility that insulin may regulate the activity of a different class of protein kinase, through its own "second messenger". The identification and characterization of glycogen synthase kinase-2 and recent information about the regulation of glycogen synthase by phosphorylation-dephosphorylation in vitro and in vivo are presented.


Hormonal control of adipose-tissue lipolysis.

Adipose-tissue triacylglycerol is the major energy store in man. The physiological importance and biochemical mechanism of the hormonal control of lipolysis in white adipose tissue is reviewed. Rates of lipolysis and fatty acid release observed when adipose tissue is incubated in vitro are compared with rates of triacylglycerol turnover in man. It appears that enhanced rates of lipolysis in vivo, for example during fasting and exercise, may be a substantial fraction of the maximum obtainable by hormone stimulation in vitro. There is considerable species variation in the hormonal sensitivity of adipose tissue. Some hormones that stimulate lipolysis in vitro may not be significant lipolytic agents at physiological concentrations in vivo. In man and rat, the most important acutely acting lipolytic and anti-lipolytic hormones are catecholamines and insulin respectively. The sympathetic nervous system may play a role at least as important as circulating catecholamines in the mobilization of stored triacylglycerol. The effects of acute lipolytic hormones are modulated in the long term by corticosteroids and thyroid hormone. Stimulation of lipolysis is believed to be mediated by the increased intracellular cyclic AMP concentration that occurs after interaction of hormones with specific receptors in the plasma membrane. The properties of membrane receptors, adenylate cyclase, cyclic AMP phosphodiesterase, cyclic AMP-dependent protein kinase and triacylglycerol lipase, as studied in rat and human adipose tissue, are discussed. Several features of the action of lipolytic hormones in vitro are difficult to account for by the hypothesis that cyclic AMP is the only "second messenger" regulating lipase activity. These include anomalous effects of hormones at high concentrations and the possible existence of feedback inhibition limiting the accumulation of cyclic AMP and the stimulation of lipolysis. The mechanism of the anti-lipolytic action of insulin is at present unknown.


Effect of proxyphylline and benzopyrones on the cerebrocortical NAD/NADH redox state and reflectance in haemorrhagic shock.

The effect of 7-(2-hydroxypropyl)theophylline (proxyphylline) and coumarin (Theokal) on cerebrocortical NAD/NADH redox state and vascular volume was investigated in cats in haemorrhagic shock. The influence of these drugs on cortical NADH and UV reflectance responses induced by nitrogen breathing and electrical stimulation of the cortex was also studied. The results are summarized as follows: 1. Proxyphylline administration does not change the cortical vascular volume but shifts the NAD/NADH redox state towards oxidation. This occurs in spite of the significant arterial blood pressure drop. 2. Coumarin dilates the cerebrocortical vessels and increases the cerebral blood flow. Since NAD reduction was obtained though CBF increased, it is presumed that coumarin increased the rate of glycolysis and the lactate/pyruvate ratio. 3. Proxyphylline increased, coumarin decreased the amplitude of the anoxic NAD reduction. The stimulation induced NAD and reflectance reactions were not effected by coumarin. 4. Finally it is concluded that the beneficial effect of Theokal on brain circulation and metabolism in haemorrhagic shock is related with different mechanisms to both substances, proxyphylline and 5,6-benzo-alpha-pyrone.


[Biochemical and clinical studies on the influence of 2-mercaptopropionylglycine on liver cell function (author's transl)].

2-Mercaptopropionylglycine was used for the treatment of chronic hepatitis. The drugs showed very good clinical results. In biochemical studies it was found that there was no effect on phosphate transport. Instead, there was an increase of reactive SH-groups sensitive to oligomycin in the membrane of rat liver mitochondria. The ATPase activity in mitochondria was diminished, while ATP-contents of the mitochondrial suspensions were increased by the drug. In the presence of ATP-MG++ there was an increase of oligomycin-sensitive contraction of the mitochondria. In aged mitochondria, the P/O ratios were improved. Reversal of electron transport with formation of NADH by succinate in the presence of rotenone and ATP was, concentration dependently, improved by MPG. Thus, MGP obviously acts at some specific sulfhydryl groups of the mitochondrion. It is concluded that the action of the drug in chronic hepatitis may be interpreted via an improvement of mitochondrial function.


[Effect of metoclopramide on growth hormone (GH) release induced with ACTH].

We examined 10 volunteers subjects, sound and exempt from endocrine disease. The rapid injection of 0,25 mg of ACTH (Synacten) was followed by a distinct increase of plasma Growth Hormone (GH) within 30 or 60 min in 9 of 10 normal volunteers. The test were then repeated in the 12 "responders" after previous subministration of Metoclopramide and the GH response was totally abolished.


[Effect of TRH on growth hormone (GH) release induced with ACTH].

We examined 14 volunteers subjects sound and exempt from en docrine disease. The rapid injection of 0,25 mg of ACTH (Synacten) was followed by a distinct increase of plasma Hormone (GH) within 30 or 60 min in 12 of 14 normal volunteers. The test were the 12 "responders after previous subministration of 200 g of TRH and the GH response was totally abolished.


Further studies on antitumour responses induced by short-term pretreatment with syngeneic tumour cells.

The ability of s.c. injected tumour cells to specifically inhibit the growth of similar cells injected i.v. 2 days later has been confirmed. The capacity of tumour cells to elicit this effect varies form tumour to tumour. Furthermore, it is more readily achieved with cultured than with freshly excised tumour cells. The superior effect elicited by cultured tumour cells was not overcome by treating them with trypsin or pronase. The protection achieved was impaired in T-cell-depleted mice and mice which had been irradiated (400 rad) prior to pretreatment. In contrast, it was not affected by administration of silica, sodium aurothiomolate or cortisone acetate. The results imply that T-cell-dependent responses are involved in the protection conferred by pre-injecting tumour cells shortly before i.v. challenge.


Ablation of murine jejunal crypts by alkylating agents.

The gut microcolony assay has been used to measure damage to intestinal crypts by single and split doses of 3 alkylating agents: mechlorethamine hydrochloride (HN2), bis-chloroethyl-nitrosourea (BCNU) and isopropyl methane sulphonate (IMS). The single-dose survival curves for whole crypts were distinguished by extrapolation numbers (3.0, 176 and 1.5 respectively) that were lower than most previously published values for assay by irradiation. Significant sparing of crypts occurred when doses of HN2 or BCNU, but not IMS, were given in 2 equal fractions separated by more than 2 h. Deduced D0 values for those cells from which crypts regenerate were 1.9 mg/kg HN2, 19 mg/kg BCNU and 487 mg/kg IMS.


Synthesis of herpes simplex virus DNA in isolated chromatin.

Herpes simplex virus DNA synthesis was studied in isolated chromatin (HSV chromatin) of African green monkey kidney (RC-37) cells after HSV type 1 infection. After optimizing the in vitro system, HSV chromatin was shown to synthesize both viral and cellular DNA at ratios identical with those seen in vivo. After 30 min of DNA synthesis in vitro, the DNA products were identical in size to the prelabeled parental DNA. More than 60% of the newly synthesized single-stranded DNA fragments sedimented with a sedimentation constant of greater than 10 S. HSV DNA polymerase was found to be responsible for the synthesis of 80% of all in vitro made viral and most likely also cellular DNA sequences.


Photoreductive titration of the resonance Raman spectra of cytochrome oxidase in whole mitochondria.

A photoreductive titration of the resonance Raman (RR) spectra of cytochrome c oxidase in whole mitochondria was recorded by exploiting the preferential enhancement of the Raman signals of reduced cytochrome oxidase excited at 441.6 nm. When the sample was cooled to about--10 degrees C, it was possible to slow down the photoreductive effect of the laser and to record RR spectra at various states of reduction. Compared to the earliest recorded scan (most oxidized), the dithionite-reduced sample shows the appearance of new bands at 216, 363, 560, and 1665 cm-1. At intermediate stages of photoreduction, the 216- and 560-cm-1 bands appear before the 363- and 1665-cm-1 bands; photoreduction induces full intensity in the former bands, whereas the latter bands are photoreduced to 50% of the dithionite-reduced intensity. The relative intensities of a doublet at 1609--1623 cm-1 are affected by reduction: the band at 1609 cm-1 is weaker in the earlier scans; in later scans this band has grown to equal intensity with the 1623-cm-1 band. We conclude that this reductive titration of the RR spectrum of cytochrome c oxidase reflects three states in its reduction. The behavior of the doublet at 1609--1623 cm-1 suggests that the two hemes are nonequivalent but interacting. The band at 216 cm-1 may be indicative of an iron-copper interaction that is affected by the presence of external ligands.


Quenching of intrinsic tryptophan fluorescence in membranes of rat pituitary cells.

The GH4C1 strain of hormone-producing rat pituitary cells has specific receptors for the tripeptide thyrotropin-releasing hormone (TRH). Membranes prepared from GH4C1 cells show intrinsic tryptophan fluorescence which was quenched by low concentrations (10--100 nM) of TRH and Ntau-methyl TRH but not by biologically inactive analogs of TRH. Membranes from GH4C1 cells were subjected to thermal denaturation. A conformational transition was noted above 40 degrees C and an irreversible denaturation was observed at 52 degrees C. TRH-induced quenching of intrinsic fluorescence was lost completely in membranes previously incubated for 10 min at 30 degrees C while loss of [3H]-TRH binding was only about 20% at this temperature. Collisional quenching by iodide revealed that about 38% of the tryptophanyl residues in GH4C1 membranes were exposed to solvent. Quenching by TRH occurred with a shift in wavelength maximum from 336 to 342 nm suggesting that few of the tryptophanyl residues quenched by the tripeptide are totally exposed. Membranes prepared from cells preincubated with 20 nM TRH for 48 h, in which TRH receptors were decreased to 30% of control values, showed no quenching of tryptophan fluorescence in response to freshly added TRH. We conclude that the TRH-receptor interaction in GH4C1 cells is associated with a change in membrane conformation that can be measured by differential spectrofluorometry of intrinsic tryptophan fluorescence.


Exchangeability and rate of flip-flop of phosphatidylcholine in large unilamellar vesicles, cholate dialysis vesicles, and cytochrome oxidase vesicles.

Three model membrane systems have been characterized in terms of their interaction with phospholipid exchange proteins. Large unilamellar vesicles of phosphatidylcholine prepared by ether vaporization are shown to be homogeneous by gel filtration. Phospholipid exchange proteins from three sources are capable of catalyzing the rapid exchange of approximately half of the phospholipid from these vesicles. The remaining pool of radioactive phospholipid is virtually nonexchangeable (t1/2 of several days). Small unilamellar vesicles of phosphatidylcholine prepared by cholate dialysis also exhibit two pools of phospholipid (65% rapidly exchangable, 35% very slowly exchangeable) when incubated with beef liver phospholipid exchange protein. Cytochrome oxidase vesicles prepared both by a cholate dialysis method and by a direct incorporation method have been fractionated on a Ficoll discontinuous gradient, and tested for interaction with beef heart exchange protein. Two pools of phospholipid are once again observed (70% rapidly exchangable, 30% nonexchangeable), even for vesicles which have incorporated the transmembranous enzyme at a phospholipid to protein weight ratio of 2. The size of the rapidly exchangeable pool of phosphatidylcholine for each of the vesicle systems is consistent with the calculated fraction of phospholipid in the outer monolayer. The extremely slow rate of exchange of the second pool of the second pool of phospholipid reflects the virtual nonexistence of phospholipid flip-flop in any of these model membranes.


Divalent cation binding to phospholipid veiscles. Dependence on temperature and lipid fluidity.

Mn2+ binding to vesicles prepared from several different species of anionic phospholipids was determined as a function of temperature by electron paramagnetic resonance (EPR). The Mn2+ affinities of phosphatidylserine, cardiolipin and egg yolk phosphatidylglycerol all increased monitonically with temperature. Vesicles prepared from hydrogenated and natural (bovine) phosphatidylserine were monitored with respect to hydrocarbon chain fluidity as well as Mn2+ binding. Contrary to expectations based on surface potential considerations, the affinity of phosphatidylserine for divalent cations was apparently not lowered in going from the gel state to the lipid crystalline state of the bilayer. The results are instead consistent with an enhancement in cation affinity with increased lipid fluidity. Dipalmitoyl phosphatidylglycerol vesicle fluidity and Mn2+ binding were also studied with EPR. A large reduction in the measured Mn2+ affinity accompained melting of the phospholipid, but observed hysteresis in the temperature dependence of the binding render uncertain any simple explanation based on changes in surface potential. Supplementary light scattering data indicated that vesicle aggregation was involved in the hysteresis phenomena.


Analysis of Dictyostelium discoideum plasma membrane fluidity by electron spin resonance.

Dictyostelium discoideum grown axenically in media containing polyunsaturated fatty acids exhibited normal growth rates but impaired differentiation (Weeks, G. (1976) Biochim. Biophys. Acta 450, 21--32). Since cell-cell contact is vital for differentiation but unnecessary for growth we have examined the isolated plasma membranes of these cells. The lipids of the plasma membranes of cells grown in the presence of polyunsaturated fatty acids contain considerable quantities of these acids, but the total phospholipid and sterol contents of the plasma membrane are close to normal. Electron spin resonance studies using 5-doxyl-stearic acid as the spin probe reveal two things. Firstly, there are no detectable characteristic transition temperatures in the plasma membranes of D. discoideum. Secondly, the plasma membranes of cell grown in the presence of polyunsaturated fatty acids have essentially the same fluidity as that of the control cells. The possible significance of this result to impaired cell-cell interaction is discussed.


Disappearance of calcium-induced phase separation in phosphatidylserine-phosphatidylcholine membranes caused by protonation and by electric current.

Disappearance of Ca2+-induced phase separation in phosphatidylserine-phosphatidylcholine membrane has been studied under several conditions by monitoring electron spin resonance spectrum of spin-labeled phosphatidylcholine. The membranes were prepared in Millipore filters. Electron micrographs of the pre parations showed formation of multilayered structures lined on the pore surface. The phase separation was disappeared when the membrane was soaked in non-buffered salt solution (100 ml KCl, pH 5.5). It was markedly contrasting that when the bathing salt solution was buffered no disappearance was observed. Disappearance of the phase separation was also observed when the Ca2+-treated membrane was transferred to acidic salt solutions (less than or equal to pH 2.5) or to low ionic strength media (less than or equal to mM) buffered at pH 5.5, and then to the buffered salt solution (100 mM KCl, pH 5.5). These are due to replacement of Ca2+ by proton, proton-induced separation, followed by disappearance of the phase separation in the buffered salt solution. Biological significance of the competition between Ca2+ and proton for the phase separation or domain formation in the membranes was emphasized.


The purification of nuclease-free T4-RNA ligase.

RNA ligase has been highly purified in good yields from bacteriophage T4-infected Escherichia coli by a rapid and reproducible procedure. The enzyme is free of phosphomonoesterase and ribonuclease activities and is therefore suitable for the synthesis of oligoribonucleotides and for the labeling of the 3'-terminus of RNA. Greater than 90% of the protein in the enzyme preparation migrates as a single band on gradient polyacrylamide gels containing sodium dodecyl sulfate during electrophoresis. For use as a DNA synthesis reagent the enzyme may be reliably freed of deoxyribonuclease activity by an additional chromatographic procedure using a commercially avialable resin.


Dimethyl-10,12-benz(a)acridine; evidence for differential effects on the synthesis of RNA of mammalian or avian fibroblasts and some RNA viruses.

We have studied the differential effect of dimethyl-10,12-benz(a)acridine (DBMAcr) on the synthesis of RNA of chicken or mouse fibroblasts in culture and that of some RNA-containing viruses such as Rous sarcoma virus and Mengovirus. DMBAcr at low concentrations blocks the cell multiplication of both normal and Rous sarcoma virus-transformed chicken fibroblasts in culture; it affects transformed cells more than normal ones. The cell growth inhibiting effect of DMBAcr is reversible after short periods of incubation. DMBAcr depresses the synthesis of cellular DNA and RNA in parallel. Concurrently the synthesis of protein proceedes at a relatively high rate in DMBAcr-treated cultures. Its inhibitory effect on cellular RNA synthesis is mostly due to a block in the formation of 28 S and 18 S ribosomal RNA species; in contrast, the synthesis of 45 S ribosomal RNA precursor is proceeding at almost control rate. Also, the synthesis of heterogeneous nuclear RNA is not blocked by DMBAcr. The production of Rous sarcoma virus in transformed fibroblasts is not affected by DMBAcr. Since this is correlated with persisting high rates of protein and heterogenous nuclear RNA synthesis, the effects of DMBAcr suggest that the synthesis of Rous sarcoma virus-RNA shares the specificity of messenger and heterogeneous nuclear RNA. DMBAcr inhibits the synthesis of viral RNA of Mengovirus under conditions where the synthesis of total cellular RNA is not appreciably depressed, suggesting its differential effect on the DNA-directed and the RNA-directed RNA synthesis.


The absence of a role for the carbohydrate moiety in the binding of apolipoprotein B to the low density lipoprotein receptor.

The binding of low density lipoprotein (LDL) to fibroblasts occurs through apolipoprotein B, a glycoprotein. The role of the carbohydrate in binding was assessed in two ways: (1) LDL, freed of sialic acid and most of the glucosamine and hexoses by digestion with a mixture of glycosidases, bound to fibroblasts as does native LDL. (2) The glycopeptides liberated from apoprotein B by trypsin and pronase failed to inhibit LDL binding to fibroblasts. Apparently the carbohydrate moiety of LDL does not interact with the plasma membrane receptor.


Adenosine 3',5'-monophosphate-dependent protein kinase(s) in diploid and SV40 transformed human fibroblasts.

Cyclic AMP-dependent protein kinases (EC 2.7.1.37; ATP:protein phosphotransferase) in the human diploid fibroblast WI-38 and an SV40-transformant WI-38-VA13-2RA (VA13) have been compared on the basis of their concentrations in cells, isoenzyme composition and susceptibility to hormonal activation. In high population density cultures, total soluble cyclic AMP-dependent kinase activities measured with histone were essentially the same in WI-38 and VA13. Two soluble protein kinase forms separated by chromatography on DEAE-cellulose were present in both cell lines. The concentration of cyclic AMP required for half-maximal activation of both enzyme forms was 10-30 nM. Overall kinase stimulation was greater for the Peak I enzymes. Kinase activation induced in the presence of 0.5 M KCl was more rapid and complete for the Peak I enzymes. Under conditions which elevated the concentration of cyclic AMP in WI-38 and VA13 cells the activities of the soluble histone kinases were increased. Incubation of the cells with either of 5.7 micronM prostaglandin E1 or 1 micronM isopropylnorepinephrine induced complete activation of the cyclic AMP-dependent histone kinases within 5 min and maintained the effect for 20 min. When intracellular cyclic AMP levels were raised by prostaglandin E1, activation of glycogen phosphorylase (assayed-AMP) suggested that this enzyme cascade involving cyclic AMP-dependent protein kinase(s) was intact and responsive in both cell lines.


Alpha- and beta-adrenergic receptors of the rat salivary gland. Elevation after chemical sympathectomy.

Binding of [3H]dihydroergokryptine and [3H]dihydroalprenolol to membrane preparations from rat submaxillary gland was measured to characterize the alpha- and beta-adrenergic receptors, respectively. Kinetic analysis of the data revealed a high affinity binding site for each radioligand. Inhibition of binding at each site was stereospecific for the active isomer of the catecholamine used. The greater ability of a beta1 than beta2 specific beta-adrenergic antagonist to displace [3H]dihydroalprenolol binding indicated that this binding site was of the beta1 type. Chemical sympathectomy with reserpine or 6-hydroxydopamine resulted in a significant increase in both [3H]dihydroalprenolol and [3H]dihydroergokryptine binding in the rat submaxillary gland. 3scatchard analysis of the data indicated that these increases in binding were due to a change in total number of binding sites for [3H]dihydroergokryptine and [3H]dihydroalprenolol with little change in apparent affinities. This suggests that changes in alpha- and beta-adrenergic receptor density may be important in the development of supersensitivity in salivary glands after reserpine and 6-hydroxydopamine treatment.


Bone matrix studies. Influences of parathyroid extract, calcitonin, and cholecalciferol and of rickets and its treatment.

Bones from young rats were incubated with radioactive glucosamine and proline. The concentrations and specific activities of matrix glycosaminoglycan fractions, prepared by a cetylpyridinium chloride method, and the specific activity of insoluble collagen hydroxyproline were determined. Acute parathyroid extract treatment increased labelling of hyaluronic acid and a glycopeptide fraction. These effects were partially blocked by calcitonin treatment which had no effect by itself. Parathyroid extract inhibited collagen synthesis and this effect was not blocked by calcitonin. Effects of these two hormones on labelling of chondroitin sulfate fractions were more variable. Vitamin D-3 caused an increase in labelling of all matrix fractions measured in bone from thyroparathyroidectomized rats, but its stimulating effect upon collagen synthesis was blocked by parathyroid extract. Bones from rats made rachitic on a phosphorus and vitamin D-deficient diet were incubated in vitro with radioactive glucosamine and proline. Over a three-week period rachitic bone exhibited a progressive fall in concentration and labelling of a glycopeptide-hyaluronic acid fraction, while pair-fed animals supplemented either with phosphorus alone or with phosphorus and vitamin D-3 not only remineralized their bones, but the bones showed a pronounced increment in concentration and labelling of this fraction. Both treatment regimens also enhanced chondroitin sulfate and collagen labelling.


[Spike transmission in statistical neuronal ensembles. Induced epileptic focus in a model of hippocampal field CA3].

In a spatially heterogeneous model the transition to supercritical phase was investigated as to the parameter characterizing the activation level of the pyramidal cells related to one another assuming the nonuniformity radius to be R0 = const. This is a transition from spontaneous activity to epileptoid bursts. Before the onset of epileptoid bursts the region of stochastic nonequilibrium of solutions is developed likely to produce pathologic dynamic patterns. With further increase of the activation parameter the system comes to epileptoid state. This synchronized firing of pyramidal cells is accompanied with phases of inhibition. A decrease in the nonuniformity radius leads to the formation of an epileptic focus. It is a dissipative structure. The evolution of it is not further followed, since the transport equations do not include its dynamics.


[Molar extinction of pigment in the reaction center of photosystem II chloroplasts].

The absolute spin number in the Photosystem I chloroplast fragments from Vicia faba plant photooxidized in the presence of silicomolybdate has been carefully measured by means of camparison of ESR signals of P680-centers and of nitroxyl free radical aqueous solution. The data obtained together with simultaneous measurement of light-induced bleaching centered at 680 nm have been used to determine the molar extinction of reduced P680-center epsilon680 = 0.66 . 10(5) M-1 cm-1.


[Study of the transmembrane potential of mitochondria using cyanine dyes].

Changes in fluorescent intensity of cyanine dyes dis-C3-(5) and dis-C2-(5) were measured in suspension of rat liver mitochondria upon the development of the energized state by adding succinate or ATP. It has been shown that these dyes possess a rotenone-like effect but they do not damage the inner mitochondrial membrane. The data on ATP synthesis and Ca2+ transport correspond to the basic aspects of the chemiosmotic conception.


Amino acid sequence of rat thymus histone H2B and identification of the in vitro phosphorylation sites.

The amino acid sequence of rat thymus histone obtained in highly purified form by preparative electrophoresis, was determined. This sequence is identical to the sequence of calf thymus histone H2B. The in vitro phosphorylation of the rat histone with a cyclic AMP-dependent protein kinase isolated from rat pancreas led to the identification of four sites of phosphorylation: two major ones, at serine residues 32 and 36, and two minor ones, specific of the rat protein kinase, at serine residues 87 and 91.


Studies of the plasma membrane during maturation of the mammalian erythrocyte.

Plasma membranes were obtained from a homogeneous population of rabbit red blood cells at different maturation periods. Minor modifications in membrane proteins and membrane phospholipids and significant decreases in membrane glycoproteins and total lipids were observed with the age of the cell. The ouabain inhibited (Na+K+)-ATPase and the adenylate cyclase decreased with maturation but acetylcholinesterase and 5'-nucleotidase remained almost unchanged. The apparent activation energy of the ATPase increased with maturation. The results indicate that structural and functional modifications of the plasma membrane occur concomitantly with the ageing processes of the red cell.


[Cardiotropic effects of luliberin. Effects of luliberin on the activities of phosphorylase A and ornithine decarboxylase and concentration of 3', 5'-AMP].

The level of luliberin, the luteinizing hormone-releasing hormone (LH-RH), and the possibility of the hormonal control of metabolic and biosynthetic processes in the heart were studied. It was shown that the rat heart contains a factor, which is immunochemically and chromatographically related to LH-RH (19--46 picograms per organ). Intraperitoneal injection of LH-RH increases the activities of phosphorylase A and ornithine decarboxylase and the concentration of 3',5'-AMP and potentiates the stimulating effect of adrenaline on ornithine decarboxylase.


[Effect of linoleic acid and products of its free radical oxidation on succinate dehydrogenase].

Effects of linoleic acid and its oxidation products (with 75--80% content of hydroperoxide) on isolated succinate dehydrogenase were studied by means of spectrophotometry and ESR-spectroscopy. It was shown that unlike the unoxidized acid, low concentrations of its oxidation products increased catalytic activity, while its high concentrations of its oxidation products increased catalytic activity, while its high concentrations brought about a decrease of SH-group content and the amplitude of ESR signal of reduced nonheme iron in the enzyme.


[Conformational isomerization of lactate dehydrogenase complexes formed by pyruvate and coenzyme analogs].

The kinetics of LDH-catalyzed reduction of pyruvate involving APADH were studied. It was shown that under conditions of a single turnover reaction the first order rate constant is equal to 37+/-4 sec-1. The reaction rate (vo) did not change when a deutero-coenzyme was used. The relationship between vo and pyruvate concentration is hyperbolic. It is concluded that isomerization of the ternary LDH-APADH-pyruvate complex limits the reaction rate. The spectral properties and the kinetics of formation and dissociation of abortive LDH complexes with pyruvate and NAD analogs (APAD and PAAD) were studied. The participation of the carboxamide group of NAD in conformational isomerization of the LDH-NADH-pyruvate and LDH-NAD-pyruvate complexes was studied.


[Peculiarities of phosphorylation of skeletal muscle troponin under some forms muscle pathologies].

Phosphorylation of rat and rabbit troponin from normal skeletal muscles and from skeletal muscles of animals under avitaminosis, denervation and hypokinesia was studied. Phosphorylation was carried out by cAMP-dependent protein kinase with [gamma-33P] as substrate. The incorporation of labelled phosphorus into troponin T of the damaged muscles was decreased as compared to normal. After preliminary dephosphorylation of troponin by alkaline phosphatase immobilized on Sepharose 4B, the ability of damaged muscle troponin for subsequent phosphorylation was also decreased as compared to the control. It may be thus assumed that there exist conformational changes of troponin under muscular system pathologies.


[Interrelationship between metabolic and genetic regulation of alkaline phosphatase and poly- and pyrophosphatases].

The effects of orthophosphate and mutations in the regulatory genes of alkaline phosphatase on the activities of pyrophosphatase and polyphosphatase of E. coli were studied. It was shown that orthophosphate represses the synthesis of alkaline phosphatase as well as that of polyphosphatase without having any effect on pyrophosphatase. The genes phoR and phoS are involved in the formation of a repressory complex both for alkaline phosphatase and polyphosphatase. The gene phoT is probably involved in a partial repression of pyrophosphatase synthesis.


[Mechanism of catalysis by o-diphenol oxidase].

The reaction of terminal oxidation of the substrate (catechol) by molecular oxygen catalyzed by o-diphenoloxidase (o-diphenol: oxygen oxydoreductase; EC 1.10.3.1) is found to occur via a free radical mechanism. The copper of the active center changes its valency during the reaction. The spectra of substrate radicals and of the Cu2+ ions were registered by means of a high sensitivity ESR-spectrometer and their concentrations were determined.


Deacylation-reacylation of phospholipids by rat embryos and their associated placentas. I. Placental acyltransferase.

Homogenates and subcellular fractions of rat chorioallantoic placentas obtained on the 14th day of gestation were assayed for acyl-CoA: lysophospholipid acyltransferase activities using (14C)-linoleic acid, ATP, and CoA, or (14C)-oleoyl-CoA as acyl donor and 1-acylglycerophosphorylcholine or 1-acylglycerophosphorylethanolamine as acyl acceptor. We found that: (1) the Lands' deacylation-reacylation cycle is present in the rat placenta, (2) the enzymes were concentrated in the microsomal fraction, and (3) the optimal conditions for in vitro assay of those enzyme activities were similar to those reported for adult tissues. Comparison of the acyl composition of phospholipids in maternal blood with those in placental homogenates revealed similarities which are consistent with a role for this cycle in vivo in placental uptake of phospholipids. That this cycle may function also in placental transformation of phospholipids in vivo, is suggested by differences between the acyl composition of phospholipids in placental homogenates and microsomal fractions.


Large-scale isolation of equine liver alcohol dehydrogenase on a blue agarose gel.

Equine liver alcohol dehydrogenase (EC 1.1.1.1) has been purified by a new scheme using a blue agarose gel (Blue Sepharose) as an affinity sorbent. Starting amounts of 0.6 to 10 kg liver have been processed to enzyme possessing 1.5 U/mg average specific activity, in about three to four days. Some parameters concernining adsorption of enzyme to the blue gel as well as recovery therefrom have been explored.


Preparation and properties of an immobilized derivative of penicillinase.

Penicillinase (beta-lactamase I, EC 3.5.2.6) secreted by Bacillus cereus, strain 569/H, was covalently attached to aminoethyl cellulose via glutaraldehyde. The immobilized derivative shows increased thermostability and decreased susceptibility to conformational changes induced by certain substrates of penicillinase. The decline in the rate of hydrolysis of such substrates was consequently suppressed by immobilization. A marked increase in Km was observed with all substrates except for the unsubstituted 6-aminopenicillanic acid. The altered properties of the new derivative are attributed to the constraint imposed by immobilization on the conformational flexibility of the enzyme molecule. Thus, apart from obvious technological interest, immobilized penicillinase provides a useful model for the study of the role of flexibility in the function of an enzyme.


[Changes in the proliferative response of human lymphocytes in vitro on exposure to subcellular components of Bordetella pertussis].

Different components of B. pertussis were found to have a similar inhibitory effect on thymidine-3H incorporation caused by phytohemagglutinin (PHA) in the culture of lymphocytes taken from donors immunized with tetanus toxoid. However, the same fractions of B. pertussis produced differential effects on the reaction of lymphocyte proliferation in response to tetanus toxoid: murein-"ontaining membranes enhanced thymidine-3H incorporation, RNA-containing fractions reduced it and water-soluble components of disintegrated B. pertussis produced no effect on lymphocyte proliferation.


Glucocorticoid effects on peripheral blood lymphocytes in cows infected with bovine leukemia virus.

We examined the effects of glucocorticoids on peripheral blood lymphocytes (PBL) in lymphoproliferative conditions associated with bovine leukemia virus (BLV): persistent lymphocytosis (PL) and lymphosarcoma cell leukemia (BLSL). The effects of hydrocortisone 21-sodium succinate (HSS) on spontaneous incorporation (SI) and mitogen-stimulated incorporation of radiolabeled-thymidine and the effects of intramuscular administration of prednisolone acetate were studied. An expanded population of B lymphocytes in cows with PL was remarkable sensitive to glucocorticoids in vitro and in vivo. SI was markedly inhibited by concentrations of HSS as low as 10(-7) M. These results correlated well with in vivo observations, where an 80%-90% decrease in PBL occurred during the course of glucocorticoid administration. The decrease in total lymphocytes was accounted for almost entirely by a decrease in the expanded B lymphocyte population. Steroid-sensitive lymphocytes together with steroid-resistant cells were observed in cows with BLSL. The reduction in the steroid-sensitive lymphocytes was associated with rapid disease progression in cows with lymphosarcoma. Steroid-sensitive lymphocyte populations in cows with BLSL may include the same reactive B-cell population found in cows with PL. Glucocorticoids may prove to be a useful tool for study of the immune response to the oncogenic virus and lymphoma in BLV-infected cattle.


Influence of malathion (O,O'-dimethyl dithiophosphate of diethyl mercaptosuccinate) on body enzymes in dermal subacute toxicity studies in Bubalus bubalis species.

The effect of daily dermal spray of malathion for four weeks in recommended (0.5 and 1.0 per cent) and higher (5.0 per cent) concentration on various enzymes in Bubalus bubalis species were studied. The higher concentration of 5.0 per cent showed lethal effect after 2 to 3 exposures. The cholinesterase activity in both RBC (RChE) and plasma (PChE) were inhibited with all the concentrations. There was also significant (P less than 0.05) elevation in the activities of serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase with 1.0 and 5.0 per cent spray and enzyme activities remained altered even during post-medication. The extent of various biochemical changes were dose and time dependent.


Effects of concanavalin A on 5-hydroxytryptamine uptake by rabbit blood platelets and on their ultrastructure.

1. Effects of concanavalin A (Con A) and other lectins on 5-hydroxytryptamine (5-HT) uptake by rabbit blood platelets and on their ultrastructure were studied. 2. Uptake of [3H]-5-HT by platelets was decreased by application of Con A, E-PHA (lectin from Phaseolus vulgaris) and lentil-PHA (lectin from Lens culinaris), but not by wheat germ agglutinin (WGA). Con A induced specific changes in the ultrastructure of platelets, causing (i) a change in external appearance from a discoid to an irregularly spherical shape, (ii) re-arrangement of the canalicular system and formation of a concentric structure. These effects of Con A on platelets were antagonized by pretreatment with alpha-methyl-D-mannoside (alpha-MM), a specific inhibitor of Con A binding to glycoprotein. 3. The inhibition of 5-HT uptake by Con A was antagonized by colchicine, vinblastine and sodium nitroprusside (SNP), but not by cytochalasin B. 4. Theophylline, papaverine and dibutyryl cyclic adenosine 3',5'-monophosphate (db cyclic AMP) antagonized the effect of Con A on 5-HT uptake, but dibutyryl cyclic guanosine 3',5'-monophosphate had no effect. Theophylline and db cyclic AMP did not influence the effect of Con A on the ultrastructure of platelets. 5. It is suggested that binding of Con A to specific receptor glycoproteins can inhibit the 5-HT uptake system of platelets. Microtubules, contractile protein and the membrane adenylate cyclase system of platelets may also be regulatory factors in this mechanism.


The characteristics of [3H]-clonidine binding to an alpha-adrenoceptor in membranes from guinea-pig kidney.

1. [3H]-clonidine binds to membranes prepared from guinea-pig kidney. 2. At 25 degrees C the binding is rapid and saturable. 3. Scatchard analysis of the binding data showed that the Kd for [3H]-clonidine binding in kidney membranes is 8.54 nM and the density of binding sites 12.5 pmol/g wet wt. tissue. 4. Hill plots of the binding data showed that there were no cooperative site interactions associated with binding. 5. [3H]-clonidine binding could be displaced by drugs, the most potent being drugs with a high affinity for the alpha-adrenoceptor. The neuroleptic drugs (+)-butaclamol, cis-clopenthixol and cis-flupenthixol at high concentration also displaced [3H]-clonidine binding. 6. Drugs acting as agonists or antagonists of beta-adrenoceptors, histamine receptors, acetylcholine receptors as well as prostaglandins E1, E2, F1alpha and F2alpha, angiotensin II, arginine vasopressin, naloxone, nalorphine and pargyline had little effect on binding. 7. It is likely that the binding site labelled by [3H]-clonidine in guinea-pig kidney membranes is an alpha-adrenoceptor similar in some pharmacological aspects to an alpha2-adrenoceptor.


The effects of opioid drugs and of lithium on steroidogenesis in rat adrenal cell suspensions.

1. The effects of opioid drugs and of Na+ replacement on steroidogenesis in rat adrenal cell suspensions were investigated. 2. In medium containing normal Na+ (156 mM), opioid antagonists but not opioid agonists reduced the steroidogenic response to adrenocorticotrophic hormone1-24 (ACTH1-24) but not to dibutyryl adenosine 3',5' cyclic monophosphate (db cyclic AMP). 3. Replacement of 50% Na+ in the medium by choline had no effect on steroidogenesis, but further reductions in Na+ content reduced the steroidogenic activity of both ACTH1-24 and db cyclic AMP. 4. In 50% Na+ medium both opioid agonists and antagonists inhibited ACTH1-24 induced steroidogenesis. 5. Addition of therapeutic concentrations of lithium to otherwise normal medium inhibited the steroidogenic response to ACTH1--24 but not to db cyclic AMP. 6. The selective inhibition of ACTH1--24-induced steroidogenesis by opioid drugs suggests some similarity between the opioid and ACTH receptors. 7. The relevance of the potent inhibitory effect of lithium to its therapeutic actions is discussed.


Drug-induced peripheral neuropathies.

Review of the various drugs in current clinical use showed that over 50 of them may cause a purely sensory or mixed sensorimotor neuropathy. These include antimicrobials, such as isoniazid, ethambutol, ethionamide, nitrofurantoin, and metronidazole; antineoplastic agents, particularly vinca alkaloids; cardiovascular drugs, such as perhexiline and hydrallazine; hypnotics and psychotropics, notable methaqualone; antirheumatics, such as gold, indomethacin, and chloroquine; anticonvulsants, particularly phenytoin; and other drugs, including disulfiram, calcium carbimide, and dapsone. Patients receiving drug treatment who complain of paraesthesie, pain, muscle cramps, or other abnormal sensations and those without symptoms who are receiving drugs that are known or suspected to be neurotoxic should undergo neurological examination and studies of motor and sensory nerve conduction. This will allow the incidence of drug-induced peripheral neuropathy to be determined more precisely.


Immunity of children to diphtheria, tetanus, and poliomyelitis.

A survey of titres of diphtheria and tetanus antitoxins and of antibodies to polioviruses in the sera of 291 schoolchildren aged 15, 11, and 7 years showed that high immunisation rates can evoke protective concentrations of tetanus antitoxin in 98% of children and protective levels of the antibodies to diphtheria and all three types of poliomyelitis in 85% of children. Reinforcing immunisation at school entry appeared to be necessary to maintain adequate titres of diphtheria antitoxin in children up to 15 years of age, not essential to maintain adequate titres of tetanus antitoxin, and to have little effect on the titres of antibodies to poliomyelitis.


High-density lipoprotein in chronic renal failure and after renal transplantation.

Serum high-density-lipoprotein (HDL) cholesterol and apoprotein (apo) A concentrations were significantly reduced at the time of renal transplantation in 26 patients with chronic renal failure. In a prospective evaluation the behaviour of HDL concentrations after grafting was found to depend on renal function. Graft function was sustained in 19 patients, in whom HDL-cholesterol and apo A concentrations increased to lie within normal limits by six months after the operation. Successful transplantation also restored to normal the lipid and protein content of HDL as expressed by the ratio of HDL cholesterol to apo A. When transplant function was not sustained (seven patients), however, no changes in HDL were observed up to the time of the graft loss. Such changes in HDL with successful kidney grafting augur well in a population with many risk factors for coronary heart disease.


Relation between plasma hormone profiles, symptoms, and response to oestrogen treatment in women approaching the menopause.

Out of a consecutive series of 300 patients seen at a menopause clinic, 82 complained of symptoms generally associated with the climacteric, although they were still menstruating. Vasomotor disturbances were absent in 42 of these patients (group 1) and present in 40 (group 2). Headaches, insomnia, and dyspareunia were the most common complaints among the women with vasomotor symptoms, whereas loss of libido and depression predominated in those without. Conjugated equine oestrogens (Premarin) 1.25 mg daily given for three weeks out of four relieved nearly all symptoms in group 2, but in group 1 the response was disappointing. The mean plasma oestradiol concentration in women with vasomotor symptoms was significantly lower than that observed during days 1-10 of the menstrual cycle, but plasma testosterone values were not significantly different from those observed in younger women. Plasma follicle-stimulating hormone (FSH) and luteinising hormone  (LH) concentrations were similar to those seen after the menopause. Concentrations of these hormones in the women without vasomotor symptoms were similar to those in the younger, regularly menstruating women. After six months of oestrogen treatment patients in group 2 had a 2.1-fold increase in mean plasma oestradiol concentration, and plasma FSH and LH concentrations were reduced to 39% and 66% of their pretreatment values respectively; in group 1, however, no such pronounced changes occurred. High concentrations of FSH were present in patients with oestrogen-responsive symptoms, 15 U/1 being the diagnostic cut-off point. This measurement in the presence of characteristic symptoms therefore constitutes the best method of selecting patients for oestrogen-replacement therapy.


Neutral and acid sphingomyelinases: somatotopographical distribution in human brain and distribution in rat organs. A possible relationship with the dopamine system.

Acid and neutral sphingomyelinase activities have been measured in 22 regions of human brain, and in several rat organs. In general, acid sphingomyelinase activity was similar in most brain regions examined. By contrast neutral sphingomyelinase activity decreased 30-fold between the globus pallidus and white matter. In grey matter structures activity decreased in the order globus pallidus greater than substantia nigra greater than or equal to putamen greater than head of caudate greater than thalamus greater than cortical structures. Under the conditions of assay and in the presence of several possible donors or acceptors, there was no evidence of transfer of phosphoryl-choline to other lipid acceptors. Acid sphingomyelinase was ubiquitously distributed in all rat tissues examined, highest in liver and lowest in adipose tissue. Neutral sphingomyelinase activity was highest in brain; activity from 25 to 10% of that in brain was observed in testis, adrenal gland and aorta. Activity in the other organs examined was less than 10% of that in brain. We suggest that the neutral enzyme serves a special function in brain, perhaps related to the dopaminergic systems.


ACTH-like neurotropic peptides: possible regulators of rat brain cyclic AMP.

The influence of behaviorally active, N-terminal fragments of ACTH on the accumulation of cAMP in rat brain investigated in broken cell preparations of subcortical tissue, in slices of neostriatum and in vivo. ACTH1--24 has a biphasic effect on the activity of adenylate cyclase in broken cell preparations of rat brain subcortical tissue: concentrations below 25 micrometer stimulated, whereas concentrations of 0.1 mM and higher inhibited adenylate cyclase activity. The magnitude of the stimulation was dependent on the concentrations of ATP and Mg2+ in the incubation medium. Structure activity studies revealed that at a concentration of 10(-4) M ACTH1--16-NH2 and ACTH4--7 also inhibited the activity of adenylate cyclase, whereas ACTH11--24, ACTH1--10, ACTH4--10, [D-Phe7]ACTH1--10 and [D-Phe7]ACTH4--10 were inactive in this respect. Addition of 0.8 mM EGTA but not of 0.25 mM Ca2+ prevented the inhibition by 10(-4) M ACTH1--24. GMP-N-P (10(-5) M), naltrexone (10(-3) M) and ergometrine (10(-3) M) did not influence the inhibitory effect. ACTH1--24 enhanced the accumulation of cAMP in slices from rat brain neostriatum in a dose-dependent manner. This effect was already maximal 7.5 min after the addition of the peptide and was potentiated by isobutylmethylxanthine, a potent inhibitor or phosphodiesterase. Intraventricular injection of 1 microgram ACTH1--16-NH2 in rats significantly elevated (+ 27%) the concentration of cAMP in the septal region 60 min after the injection of the peptide. The results are discussed in terms of a possible involvement of cAMP as a second messenger in the central nervous system for N-terminal fragments of ACTH.


Mineral substance of bone tissue and of experimental cutaneous calcinosis in rats: chemical analysis and ESR study.

The evolution of the mineral constituents of subcutaneous calcinosis induced in rats by topical calciphylaxis was studied by the method of quantitative chemical analysis, and after treatment with excited gases by electron spin resonance (ESR) analysis. Chemical data show that the genesis of the subcutaneous calcinosis does not significantly alter the concentration of Ca, P, F, CO3, Mg, and Fe in the mineral phase of the femoral bone of calciphylactic rats. In the calcinosis an important increase of the fluoride concentration is noticed in function of the time after challenging. There is also a high concentration of Mg2+ ions in the early stages of the experimental calcification. Iron injected for the challenging is continuously present in the calciphylactic tissue after this treatment. This suggests that subcutaneous calcinosis might be a means of fixing certain heavy metal ions. After treatment with excited gases, the proportions of the trapped CO33- and O3- radicals are of the same order of magnitude in calciphylactic tissue after 12 days and observations in bone mineral. These suggest that after 12 days the mineral of the calciphylactic tissue has a crystalline state close to that of bone.


Acetylcholinesterase: characterization of native and proteolytically derived forms and identification of structural protein components.

The assymmetric 18S and 14S forms of acetylcholinesterase (EC 3.1.1.7) from Electrophorus electricus purified by affinity chromatography on N-methylacridinium Sepharose 2B were subjected to trypsin or collagenase proteolysis and changes in the enzyme composition and structure were monitored by sucrose gradient sedimentation, gel chromatography, and sodium dodecyl sulphate - polyacrylamide gel electrophoresis. A distinction between autolytic and tryptic degradation products is described and the generation of two new forms of acetylcholinesterase from the 18S and 14S enzyme by collagenase proteolysis is reported. The species derived from the 18S form of acetylcholinesterase has a sedimentation coefficient of 21.1S and a Stokes radius of 12.9 nm while the 14S form gives rise to a 17.3S species with a Stokes radius of 11.1 nm. The proteolytically sensitive component ('tail') of the asymmetric forms of acetylcholinesterase is identified with a subunit of 45 000 daltons on sodium dodecyl sulphate - polyacrylamide electrophoresis gels.


Protein kinase activity and ribosome phosphorylation in ethionine-treated rats.

The regulation of protein synthesis at the level of the ribosome was investigated using the model system of ethionine-induced inhibition of protein synthesis. The phosphorylation of ribosomal protein S6 was examined in vivo during ethionine intoxication and during the adenine-induced reversal of ethionine intoxication. The extent of phosphorylation of S6 correlated well with protein synthetic activity observed after ethionine, and ethionine followed by adenine treatments. No clear correlation was observed in the ethionine system between cyclic adenosine 3':5'-monophosphate concentration or the activity of ribosomal protein kinase and the phosphorylation of ribosomal protein S6. A role for a cyclic adenosine 3':5'-monophosphate-dependent ribosomal phosphoprotein phosphatase is postulated.


Thermally induced changes in reconstituted and membranous cytochrome c oxidase.

The Arrhenius plots of electron transport activity in cytochrome c oxidase reconstituted with well-defined phospholipids have been shown to display a change in slope at 20--25 degrees C regardless of the chemical nature of the incorporated lipid. In native membranous cytochrome c oxidase, the discontinuity in Arrhenius activity plot occurred at 16--18 degrees C. These temperature breaks were found to correlate with changes in spin-label mobilities but not with the bulk lipid transition observed by differential scanning calorimetry. Temperature-dependent reciprocal equilibrium between the immobilized and fluid pools is demonstrated. It is suggested that the changes in kinetic and spin-label spectral characteristics in cytochrome c oxidase membranes are related very likely to a lipid-protein interaction prompted by a thermally induced change in the physical state of the lipids that does not involve a gel to liquid crystalline transition.