Effect of prostaglandins on cell function and multiplication of parainfluenza 3 viruses in WISH cells.

Cytotoxic effect of prostaglandins E2 and F2alpha on cells grown in vitro and the influence of these compounds on multiplication of myxovirus parainfluenza 3 were investigated. The prostaglandins were added to culture medium (0-01-10 mug/ml) 24 hr before virus infection, or for 2 and 48 hr after inoculation with viruses. WISH cells and monkey kidney cell cultures were used. No direct cytotoxic effect of prostaglandins at concentrations 0-01-1 mug/ml was found (viability, supravital staining, phase-contrast system, Nitro-BT reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to partial injury of the cell population with symptoms of damage to mitochondria. Prostaglandins E2 and F2alpha inhibited multiplication of parainfluenza 3 virus at concentrations 0-1-10 mug/ml. The inhibitory effect was most pronounced if prostaglandins were added to medium for the whole period of virus multiplication i.e. for 48 hr but little or no effect was found if they were added prior to inoculation or for 2 hr after it. Inhibitory effect of prostaglandins on replication phase of viruses is suggested.


Histochemical localization of glucose-6-phosphatase and 5-nucleotidase in the digestive system of Ophiocephalus Channa punctatus.

The histochemical localization of G6Pase and 5-Nase in the digestive system of Ophiocephalus (Channa) punctatus was studied. The highest activities of these enzymes were found in the liver. Appreciable activity was also found in the anterior intestine (duodenum) and pyloric caeca. The activity faded toward the middle and posterior intestine and rectum. In the stomach the activity was moderate. The activity of 5-Nase was weaker than that of G6Pase. In the stomach the enzymes were localized in the mucosa and gastric glands. The absorptive columnar epithelial cells were the major sites of localization in the intestine. The goblet cells were negative. The G6Pase activity was associated with the cytoplasm, while the 5-Nase activity was found in the cell membranes and the nuclei.


Pancreatic inclusions and its relation to boric acid poisoning: review of the literature and report of a case.

It is known among forensic and pediatric pathologists that the demonstration of pancreatic inclusions is considered to have diagnostic significance in cases of boric acid poisoning. Fisher was the first to suggest a causal relationship between the inclusions and boric acid toxicity. Valdes-Dapena and Arey paid particular attention to the pancreatic inclusions in reviewing the literature of cases of boric acid poisoning. They found the inclusions in 6 of the 55 autopsies. This paper describes the finding of pancreatic inclusions in a fatal case of gastro-enteritis not associated with boric acid intoxication. The nature of the inclusion bodies was studied by light and electron microscopy. Ultrastructurally, the cytoplasmic inclusions represent a non-specific autophagic cell degenerative process. Toxicological examination of postmortem blood and viscera showed no evidence of boric acid toxicity. The desirability of toxicological analysis in cases of boric acid poisoning and the restricted value of pancreatic inclusions as a diagnostic tool are emphasised.


Interactions of 17beta-estradiol and L-norepinephrine on the rat uterus.

Norepinephrine increased the in vitro uptake of 3H-estradiol by the uterus of spayed rats. This effect was observed at 15 and 30 min but not at 90 min. Norepinephrine also increased the binding of 3H-estradiol by the nuclear (p less than 0.02) and the cytosol fractions (p less than 0.01) when incubated with uterine homogenates, suggesting that norepinephrine does not require the presence of the intact tissue to exert its effects. The in vivo uptake of 3H-estradiol and the determination of the number of binding sites were performed in the uterus of rats treated with estradiol and estradiol plus norepinephrine. Norepinephrine alone increased the uptake of 3H-estradiol and the number of binding sites. The highest increment in both parameters was observed in the uterus of rats treated with estradiol plus norepinephrine. The estradiol Ka of the rat uterus cytosol treated with estradiol alone or plus norepinephrine was higher than that observed in the group without estradiol, suggesting the presence of different proteins that bind estradiol. These results indicate that norepinephrine increases the entrance of estradiol into the rat uterus both in vitro and in vivo.


Interactions between the pituitary, thyroid and adrenal cortex during acute exposure to cold or to electric shocks in the rat.

In intact rats acclimated to 25 +/- 1 degrees C, acute exposure to cold resulted in simultaneous stimulation of TSH and ACTH secretion. The plasma TSH response to cold was identical at temperatures varying from +14 to -10 degrees C, whereas the adrenocortical response increased proportionally to the severity of cold. Acute stimulation of ACTH secretion by exposure to a stressful situation (electrical shocks) did not alter the TSH response to cold. Conversely, acute blockade of the pituitary-adrenocortical response by dexamethasone treatment did not enhance the TSH response to cold. Chronic stimulation of ACTH secretion resulting from adrenalectomy did not interfere with the TSH response during subsequent exposure to cold. However, a reduced adrenocortical response to cold was observed during chronic hypersecretion of TSH resulting from previous thyroidectomy. These findings do not support the hypothesis of an inverse relationship between TSH and ACTH secretions during acute cold exposure, but rather suggest that these secretions are independent.


Intramyofiber metastases in skeletal muscle.

Autopsy examination in a case of metastatic carcinoma revealed psoas muscle invasion with nests of tumor cells within individual myofibers. Such findings suggest that there is an intracellular route for the spread of malignant tumor in skeletal muscle.


Benign lymph node inclusions.

Whether obtained during a radical surgical procedure, or as the only tissue removed in various cancer staging procedures, or as incidental tissue, lymph nodes may yield pivotal histopathologic information relating to further disposition of the patient. Most important are those cases in which lymph node findings are relied upon to establish a diagnosis of malignant disease. The two cases presented here (in addition to the few reports in the literature) represent two heretofore unreported sites of occurrence of such benign inclusions. There is need for an awareness of the occurrence, benign nature, and potentially misleading associations with frequent sites of malignant change in which metastasis to regional lymph nodes is known to occur. Particularly with the growing use of mediastinoscopy the misinterpretation of such benign inclusions could prompt the insitution of incorrect and potentially hazardous therapy or a decision to withhold therapy.


Distribution of adenylate kinase and phosphoglucomutase isoenzymes in the population of the city of New York.

Blood samples from unrelated persons living in New York, N.Y., were examined for phosphoglucomutase (PGM) and adenylate kinase (AK) phenotypes, the sample consisting of 164 Caucasians, 133 Negroes, 129 persons of Spanish origin or descent, and 156 Chinese for PGM and 136 Caucasians, 134 Negroes, 136 persons of Spanish origin or descent, and 156 Chinese for AK. The PGM1 gene frequency was found to be 0.7774 for Caucasians, 0.8083 for Negroes, 0.7461 for Hispanic persons, and 0.7917 for Chinese. One Hispanic person had a very rare type, PGM 8-1-FAST. The AK1 gene frequency was found to be 0.9669 for Caucasians, 0.9813 for Negroes, 0.9779 for Hispanic persons, and 1.000 for Chinese.


Polymorphism of red cell phosphoglucomutase, adenylate kinase and adenosine deaminase in a Polish population.

Genetic variants of PGM1, AK and ADA were studied in a sample of unrelated individuals from the Polish population. The gene frequencies observed are: PGM1/1: 0.715, AK1: 0.962 AND ADA1: 0.940.


The production of IgE and IgGa antibodies in normal rats and rats infected with Nippostrongylus brasiliensis.

The time courses of production of IgE and IgGa homocytotropic antibodies were measured in Wistar rats during a primary and secondary response to egg albumin with pertussis or Freund's adjuvants. An anamnestic IgE antibody response occurred in animals previously sensitized to antigen with killed Bordetella pertussis as adjuvant. IgGa  antibodies were formed in the primary response with Freund's complete adjuvant only, but were found during the secondary response with all adjuvants used. The time courses of formation of IgE and IgGa antibodies were very different during the secondary response. The production of both classes of antibody to egg albumin was studed in Wistar and Hooded Lister rats infected with Nippostrongylus brasiliensis. IgGa antibody formation was not potentiated by the infection. However, increased levels of IgE antibody, formed during a secondary response to antigen in infected animals, were consistently higher in both strains than during a primary response.


Genetically determined resistance to infection by hepatotropic influenza A virus in mice: effect of immunosuppression.

Mice carrying the gene Mx were resistant to the lethal action of a hepatotropic line of avian influenza A virus. In resistant animals, foci of liver necrosis were self-limiting, and maximal virus titers reached were much below those in susceptible animals. Resistance could not be abrogated by immunosuppressive treatment with cyclophosphamide, methotrexate, or procarbazine, although such treatment prevented cellular infiltration at sites of virus replication and appeared to delay virus clearance. Silica and thorium dioxide, thought to inhibit macrophage function, likewise failed to abolish resistance. Regenerating liver tissue did not support more extensive virus replication than did intact adult liver.


Cross-reacting herpes simplex virus antigens in hamster and mouse cells transformed by ultraviolet light-inactivated herpes simplex virus type 2.

Murine and hamster cell lines, each transformed with a different strain of herpes simplex virus (HSV), were examined for cross-reacting antigens by in vitro and in vivo assays. A comparative study by the indirect immunofluorescence technique detected common cross-reacting viral antigens. Cytoplasmic fluorescence patterns were observed in the 333-8-9 hamster line, the H238 murine line, and the H238 clonal lines; these patterns were identical to the fluorescence pattern of HSV -2-infected controls when reacted with HSV antiserum. Tumor rejection studies in the BALB/c host indicated that each cell line provided immunity against a tumorigenic challenge of transformed mouse cells. The H238 clone EC1 3 provided a 53% immunity against itself at an inoculum of 10(6); the 333-8-9 line supported a 26% immunity. These data demonstrate a common HSV antigenicity between the murine and hamster transformed lines and further indicate that the HSV genome is involved in transformation.


Purification of hepatitis A antigen from feces and detection of antigen and antibody by immune adherence hemagglutination.

Hepatitis A antigen (HA Ag) was purified from feces collected during acute illness from patients with naturally occurring viral hepatitis, type A. Positive fecal specimens were identified by immune electron microscopy, but for detection of HA Agduring purification immune adherence hemagglutination  (IAHA) and microtiter solid-phase radioimmunoassay were used. Isopycnic banding in cesium chloride, rate-zonal separation in sucrose, and preparative zonal electrophoresis were used in various combinations for successive purification, and the purified antigen was successfully used in a test for antibody by IAHA. Seronconversions to HA Ag were demonstrated by IAHA in 20 instances of hepatitis A virus infection, but in none of six cases of type B hepatitis or three cases of post-transfusion hepatitis unrelated to heaptitis A or B viruses, nor in two individuals without hepatitis. In addition, the temporal pattern of antibody development during type A hepatitis was studied in serial sera from an experimentally infected chimpanzee. Antibody titers by IAHA correlated well with antibody ratings determined by immune electron microscopy.


Comparison of guinea pig cytomegalovirus and guinea pig herpes-like virus: growth characteristics and antigentic relationship.

The growth characteristics of guinea pig cytomegalovirus (GPCMV) and guinea pig herpes-like virus (GPHLV) in cell cultures were compared. Guinea pig fibroblast cells were highly susceptible to infection with both viruses, whereas guinea pig kidney cells were sensitive only to GPHLV. No cytopathic effect was observed in the latter cell system after infection with GPCMV,nor was there an increase in virus titer, although the cirus persisted in the kidney cells for 2 to 3 weeks postinfection. Electron microscope studies showed nonvirion tubular structures in GPCMV -infected fibroblast cells, but not in GPHLV- infected cells. Large packages of enveloped nuclear virus particles were commonly seen in GPHLV -infected cells, especially kidney epithelial cells, but none were found in the GPCMV -infected fibroblasts. Complete enveloped extracellular virus particles were present in both virus-cell systems. Both viruses showed narrow host spectra and replicated well only in guinea pig cells although GPHLV multiplied to some degree in rabbit cells. No antigenic relationship could be demonstrated between the two viruses using antisera specific for each virus that was produced in rabbits and guinea pigs. Rabbits produced high neutralizing antibody titers to GPHLV, whereas guinea pigs were the animals of choice for GPCMV antiserum production.


Comparison of guinea pig cytomegalovirus and guinea pig herpes-like virus: pathogenesis and persistence in experimentally infected animals.

The pathogenesis of guinea pig cytomegalovirus (GPCMV) and guinea pig herpes-like virus (GPHLV) in guinea pigs was compared. Animals were inoculated with the two viruses by different routes and sacrificed after varying periods of time. GPCMV was consistently isolated from salivary gland 2 weeks postinoculation and thereafter following intraperitoneal or subcutaneous incoulaton. Virus was less frequently found in other tissues including blood, spleen, and kidney. Intranuclear inclusions were seen in tissue sections of salivary gland after inoculation with GPCMV- infected tissue suspension, but were only rarely found after inoculation with tissue culture virus. In GPHLV-infected guinea pigs, consistent latent infection of leukocytes and other tissues was detected by cocultivation techniques. Intranuclear inclusions were not found in the spleen, salivary gland, or other infected tissues after GPHLV infection with either tissue culture virus or infected tissue suspension. Guinea pigs inoculated with GPCMV produced high titers of specific neutralizing antibody to the homologous virus; those inoculated with GPHLV developed long-term viremia accompanied by minimal neutralizing antibody levels to the virus.


Attachment of Mycoplasma pneumoniae to respiratory epithelium.

The attachment of radioisotope-labeled Mycoplasma pneumoniae to hamster tracheal rings in organ culture was examined by radioautography and liquid scintillation counting. Radioautographs of individual rings exposed for 8 h to (3H) thymidine-labeled virulent M. pneumoniae revealed a dense extracellular collection of emulsion grains along the luminal surface of epithelial cells. Similar exposure of rings to isotope-labeled avirulent M. pneumoniae resulted in no accumulation of emulsion grains. The numbers of attached virulent mycoplasmas, as measured by liquid scintillation counting of infected rings, were found to increase in a nearly linear fashion over an 8-h incubation period. Viability of the mycoplasmas and metabolic integrity of the tracheal rings were important for optimal attachment. Pretreatment of rings with neuraminidase or sodium periodate significantly impaired orgainism adherence. These data suggest a specificity of interation between virulent M. pneumoniae and tracheal epithelial cells that can be further examined through the use of isotopically labeled mycoplasmas.


Effect of ambient temperatures on multiplication of attenuated transmissible gastroenteritis virus in the bodies of newborn piglets.

Newborn piglets were found to be more resistant to infection with attenuated transmissible gastroenteritis virus when maintained at higher temperatures. This was attributed to a decreased rate of virus propagation and spreading in the bodies of the infected animals. The highest virus levels were detected in the tissues of piglets maintained at 8 to 12 C. In contrast, no virus was recovered from piglets maintained at 35 to 37.5 C. The virus was found only in the lymph nodes and respiratory organs in the piglets maintained at 20 to 23 C.


Modified hemolytic plaque technique for the detection of bluetongue virus antibody-forming cells.

A hemolytic plaque assay was developed for the detection of antibody-forming cells to bluetongue virus (BTV). Sheep erythrocytes (SRBC), onto which BTV had been absorbed, served as the indicator of lysis due to the presence of BTV antibody-forming cells. The ratio of BTV to SRBC was found to be critical for optimum hemolytic plaque formation. For routine use, 50 mul of 12% BTV SRBC, 0.1 ml of a spleen cell suspension, and 0.5 ml of 0.5% agarose in a balanced salt solution were mixed and plated on a microscope slide precoated with 0.1% aqueous agarose. Slides were incubated for 1 h at 37 C in a humidified incubator and subsequently flooded with 0.4 ml of a 1:15 dilution of complement. Incubation was continued for a further 2 h before the hemolytic plaques were scored. It was not possible to establish BTV serotype specificity by this technique.


Counteraction of poly(4-vinylpyridine-n-oxide) on the depression of viral interferon induction by coal dust.

The depressive activity of coal dust on interferon induction by influenza was markedly subverted when either coal dust or LLC-MK2 cell monolayers were pretreated with poly (4-vinylpyridine-N-oside). The polymer alone neither induced interferon synthesis, inhibited viral induction of interferon, influenced viral multiplication, nor affected cellular-induced resistance by interferon. Absorption of the polymer to coal dust not only occurred at a more rapid rate than to cell monolayers, but also less polymer was required to pretreat coal dust than cell monolayers to achieve comparable amelioration of interferon production. The polymer effectively negated the adverse activity of coal dust particles, irrespective of the latter's size (is less than 2.0 to 74.0 mum). Virus multiplication in the presence of coal dust=treated cell monolayers attained a level that was twofol higher than that noted with either polymer-pretreated coal dust or polymer-pretreated cell monolayers. Interferon production was almost completely inhibited in the presence of coal dust; pretreating coal dust or cells with the polymer abrogated this inhibitory activity of coal dust. It is tentatively suggested that coal dust particles per se directly interact with cell membranes to subvert interferon induction and that the formation of an absorbed polymerlayer on these complexes prevents their interaction.


Inhibition by zinc of hemolysis induced by bacterial and other cytolytic agents.

Zinc, cupric, and cadmium ions, in that order of effectiveness, inhibited lysis of washed, rabbit erythrocytes by the toxic bacterial product aerolysin. Hemolysis induced by a variety of other lytic agents was also inhibited by Zn2+ in approximately the same concentration as that, 0.33 mM, needed to inhibit aerolysin-induced hemolysis. Zinc ions did not inhibit osmotic lysis. Inhibition requires the continues presence of Zn2+ and apparently involves a readily reversible binding of Zn2+ to the cell surface, which, it is postulated is accompanied by a reversible alteration in the state of the lipid bilayer.


Antinuclear antibodies and elevated anti-Epstein-Barr virus titers in cancer patients.

One and one-half percent of human sera from patients seen at a clinic for treatment of cancer contained antibodies to the nuclei of chick kidney cells by indirect immunofluorescence tests. In the group of sera containing antinuclear antibodies, the geometric mean titer to Epstein-Barr virus (EBV) capsid antigen was significantly elevated. Sera obtained from normal adults or from patients with similar histological types of tumors that possessed no antinuclear antibodies contained lower levels of anti-EBV antibodies. The elevated titers to EBV were correlated with the presence or absence of antinuclear antibodies and not with a particular type or site of neoplastic disease.


Temperature-dependent inactivating factor of Pseudomonas aeruginosa exotoxin A.

The adenosine diphosphate ribosyl transferase activity of Pseudomonas aeruginosa exotoxin A(PA toxin) was found to be rapidly destroyed by heating at 45 to 60C but not by heating at 70 to 90C (for at least 30 min). This phenomenon has been previously described for other bacterial toxins (staphylococcal alpha-toxin and Vibrio parahaemolyticus hemolysin) and is termed an Arrhenius effect. In contrast, the Arrhenius effect was not seen when the PA toxin was heat-treated as above and tested for cell toxicity or mouse lethality. Although the PA toxin treated at 70C for 30 min retained a significant proportion (is greater than 70%) of its adenosine diphosphate ribosyl transferase activity, the cell toxicity and mouse lethality of the toxin were virtually abolished. A temperature-dependent inactivating factor that has proteolytic activity and is co-purified with the PA toxin was shown to be responsible for the Arrhenius effect. PA toxin separated from the factor by conventional disc gel electrophoresis or PA toxin preparations lacking the factor did not show the Arrhenius effect.


Pathogenesis of Sendai virus infection in the central nervous system of mice.

The present study was aimed to clarify the pathogenesis of Sendai virus infection to the central nervous system (CNS) of mice. One-to 2-day-old suckling and 4-week-old mice were inoculated intracerebrally with the virus. The virus multiplied higher in suclings than in adults. Immunofluorescent studies in sucklings revealed that the viral antigens appeared initially in ependyma, choroid plexus epithelium, and meninges. Subsequently they spread to subependymal cells and finally were found in neurons of hippocampus for as long as 4 months postinfection. In adults, however, the viral antigens rapidly disappeared in the early stage. Most mice inoculated intracerebrally with Sendai virus appeared healthy, although hydropcephalus developed in a few mice. Virus-specific antibody and interferon production seemed to have no influence on the persistent infection of Sendai virus in the CNS of mice. One of the most significant findings may be that the viral antigens persist in the brain for as long as 4 months in a latent form. This may offer a useful model for the study of latent CNS infection of paramyxoviruses.


Quantitative cytophotometry evaluated as a method for analyses of herpes simplex viral deoxyribonucleic acid synthesis.

Feulgen deoxyribonucleic acid (F-DNA) microspectrophotometry was evaluated as a potential tool for quantification of herpes simplex virus type 1 (HSV-1) and 2(HSV-2) DNA synthesis in single cells. Since HSV DNA synthesis has been extensively studied using incorporation of radioactive precursors into viral and cellular DNA, microspectrophotometric measures were correlated with biochemical data obtained using tritium-labeled thymidine ([3H]TdR). It was established that: (i) viral-induced increaae in F-DNA can be cytophotometrically detected between 1 and 6 h postinjection (p.i.), which corresponds to the initial incorporation of [3H) TdR into viral DNA; (ii) peak F-DNA levels occurred 8 h p.i., which supported cytological observations of a more rapid development of an inclusion body in HSV-2-infected nuclei. Despite the fact that F-DNA cytophotometry is unable to distinguish between cell and viral DNA, the overall study supports the existence of a good correlation between data obtained using microspectrophotometric and conventional isotope methods. Furthermore, cytophotometry complements biochemical evaluations in that it permits analyses of DNA changes on a single-cell basis or the detection of infection in very small numbers of cells.


Efficacy of phosphonoacetic acid on herpes simplex virus infection of sensory ganglia.

Treatment of mice with phosphonoacetic acid markedly reduced the incidence of latent ganglionic infection with herpes simplex virus when administered within 24 h after viral inoculation, but had no effect on an already established ganglionic infection.


Tumour angiogenesis factor (TAF) in human and animal tumours.

Extracts were made from Walker 256 carcinoma, spontaneous rat mammary adenocarcinoma, Wilms' tumour, human neuroblastoma and human haemangioma. Chromatography of the extracts on Sephadex G-100 yielded four fractions, A, B, C and D. Injection of fractions B and C resulted in the growth of new capillaries in the subcutaneous fascia or rats. Controls, e.g. similar extracts of rat liver or human kidney, did not induce neovascularisation. The endothelium of newly-formed blood vessels contained many mitotic figures. A limitation of this method is that it is qualitative only. In order to develop a quantitative in vitro assay for a tumour angiogenesis factor (TAF), short-term primary cultures were initiated from adult rat brain white matter, as cells from such cultures were shown to be vascular in origin. Addition of fractions containing TAF (B and C) which were active in vivo failed to stimulate thymidine uptake by the cells. The possible reasons for this failure and the therapeutic potential of TAF in cancer control are discussed.


Establishment and characterization of a human histiocytic lymphoma cell line (U-937).

A human hematopoietic cell line (U-937) with exceptional characteristics was derived from a patient with generalized histiocytic lymphoma. The morphology of the cell line was identical to that of the tumor cells in the pleural effusion from which the line was derived. Since Epstein-Barr virus (EBV) carrying diploid lymphoblastoid cell lines unrelated to the tumor population often become established in vitro from non-Burkitt lymphoma explants, several parameters were studied to discriminate the U-937 from such lines: morphology in vitro, growth characteristics, cytochemistry, surface receptor pattern, Ig production, lysozyme production, beta2-microglobulin production, presence of EBV genome and karyotype. In all these respects U-937 differed from prototype lymphoblastoid cell lines. The histiocytic origin of the cell line was shown by its capacity for lysozyme production and the strong esterase activity (naphtol AS-D acetate esterase inhibited by NaF) of the cells. It is therefore concluded that the U-937 is a neoplastic, histiocytic cell line.


Activation in vitro by BUdR of a productive EB virus infection in the epithelial cells of nasopharyngeal carcinoma.

Material from a nasopharyngeal carcinoma (NCP) has been passaged in athymic (nude) mice to eliminate non-malignant infiltrating cells. The human origin and derivation from NPC malignant epithelial cells of the nude mouse tumours have been confirmed by chromosome examination, electron microscopy showing desmosomes and keratin fibrils, and postive EB virus nuclear antigen (EBNA) testing. Samples of the mouse-grown tumours were cultured and pure monolayers of epithelial cells were obtained which still expressed EBNA and contained desmosomes and keratin; these cultures grew well for about 3 weeks. Extensive electron microscope searches failed to reveal herpes virus particles. In contrast, cultures treated with BUdR showed typical immature and mature herpes virus particles in epithelial, keratin-containing cells, and immunofluorescence tests for virus capsid antigen with a battery of human sera identified this agent as EB virus. EB virus has thus, for the first time, been activated in NPC epithelial cells and shown to be capable of replication in a cell type other than a primate B-lymphocyte.


Localization of mouse satellite DNA on chromosomes of experimentally induced glioblastomas; non-centromeric lable in one glioblastoma producing C-type particles.

The chromosomal localization of satellite DNA in two tissue culture lines derived -rom malignant mouse CNS tumors was investigated by in situ hybridization of 3H single-stranded satellite DNA purified by isopynic centrifugation in alkaline CSC1. Both tumors were glioblastomas originally induced by a methylcholanthrene implantation into the cerebrum of C3H mice; both displayed aneuploid chromosomal constitutions. One of these glioblastomas (TC 541) revealed labelling only of centromeric portions of the chromosomes even in cells containing greater than 200 chromosomes and thus it had a pattern of satellite distribution comparable to that of normal cells. The other glioblastoma (TC 509), that produced C-type particles and had a decrease in satellite DNA, displayed interstitial and telomeric label in some chromosomes in addition to labelling of the centromeres. "Hoechst 33258" fluorescence showed some interstitial and telomeric bright bands as well as centromeric bright regions, though to be consistent with in situ studies. The localization of satellite DNA to the chromosome arms and its possible relation to C-type virus is discussed.


The effect of calcium chloride on the activity and inhibition of bacterial collagenase.

The enzymatic behavior and inhibition patterns of collagenase of Clostridium histolyticum in the presence of 0.5 M and 3.4 mM CaCl2 have been examined viscosimetrically. The more concentrated salt was found to enhance the rate of digestion of calfskin collagen when either measured viscosimetrically or colorimetrically by trinitrobenzenesulfonate. However, the rate of digestion of calfskin gelatin is unaffected by 0.5 M CaCl2 as determined colorimetrically. Calcium chloride also proved to have a marked effect on the inhibitory behavior of a series of imidazole compounds. Histidine (10mM) is about three-fold more effective as an inhibitor in 0.5 M CaCl2 than in 3.4 mM CaCl2, whereas a reverse effect is true for histamine, Imidazolylpropionate (10mM) was only weakly inhibitory (16%) in 0.5 M CaCl2 and not at all in 3.4 mM CaCl2. Inhibition by 10 mM imidazole was not detectable. These observations may be useful in the design of inhibitors for tissue collagenases which share a number of common characteristics with the bacterial enzyme.


Immunology of herpes simplex virus in fection.

The accompanying inflammation seen in ocular HSV infection-the result of interactions between viruses and the immune response-can be both beneficial and potentially harmful to the host. An understanding of the interaction of virus-immune cells is just evolving from current advances in basic research. Based on our studies on HSV keratitis [11], the control of ocular HSV infection appears to involve an early inflammatory phase with macrophage reactivity and elaboration of MIF. Transient virus-specific lymphocytes with effector reactivity, as well as neutrophils with chemotactic activity, occur during the stromal keratitis. Finally, antibody-dependent complement-mediated lysis provides another phase of restriction of infection. Thus, a rationale for effective management and therapy of occular HSV disease must be based on an understanding of (1) the immunological and immunopathological mechanisms of corneal inflammatory disease initiated by the virus; (2) the immunological mechanisms in recovery from the disease; and (3) the host's humoral and cellular immune status during virus persistence (latency) and during recurrent episodes of infection. We hope that new information obtained from assessment of roles of the humoral and cellular immune responses in recovery from disease and in recurrent disease will provide new approaches to the management of ocular HSV infections.


Localization of the major group-specific protein (p27) of avian tumor viruses by immunofluorescence in chicken cells and tissues.

An immunofluorescence technique was developed for the major group-specific (gs) p27 antigen of avian type C viruses. The localization of this antigen virus-infected in chick embryo fibroblasts was perinuclear, intracytoplasmic and at the cell surface in the majority of the cells, while it was at the cell surface only in some of the cells. No antigen was found in the nucleus. When chickens were experimentally infected with RAV-1 or a wild-strain avian leukosis virus (of subgroup A), the viral gs antigen was detected in lymphocytes of bursa of Fabricius and the spleen. Distinct specific staining was seen in the medulla of germinal centers in bursas of Fabricius and in the white pulp of the spleen where B lymphocytes are thought to be located. This is consistent with the possibility that B lymphocytes are the target cells in the infection of chickens with avian leukemia viruses.


Cytomegalovirus: nucleocapsid assembly and core structure.

New ultrastructual aspects of cytomegalovirus nucleocapsid assembly are illustrated in cells from a Kaposi sarcoma. The occurrence of viral deoxyribonucleoproteins in the form of strands and penetration of these strands into open capsids is illustrated. The geometrical arrangement of the core material of the nucleocapsid is aslo emphasized.


The induction of SV40-transformed chinese hamster and mouse kidney cells by mitomycin C.

The induction by mitomycin C (MC) of SV40 production in three lines of semi- or nonpermissive SV40-transformed cells is described. The production of virions by a line of semipermissive, virus-shedding Chinese hamster kidney cells is increased 1,000-fold by MC treatment; the proportion of infectious centers and V-antigen-containing cells is increased to a lesser degree, suggesting that both the burst per cell and the number of virus-producing cells are increased. MC treatment induces the production of small amounts of infectious DNA and virions in one line of nonpermissive mouse kidney cells, and infectious DNA apparently alone in another.


[Sturcture and morphogenesis of xanthomas in hyperlipoproteinemia type III. A morphologic, histochemical and electron microscopy study].

Histological, histochemical and electron-microscopical examinations have been performed on xanthoma of the skin and palmar creases in 5 patients with hyperlipoproteinemia type III, to follow the dynamics of xanthoma formation. The results were as follows: 1. There are no basic differences in histomorphology between xanthomas in type III-HLP and other types of HLP. Typical findings are: Rather dense distribution of relatively small foam cells within the dermis, only a few foam giant cells, great amounts of cholesterol intra- and extracellulary, and circumscribed necrobioses. 2. Xanthoma formation in type III-HLP is induced by pathological plasma lipoproteins. These seem to pass the endothelial barrier via pinocytosis or/and filtration. Between endothelial cells and their basal lamina plasma components sometimes could be observed. 3. The pathological lipoproteins induce a phagocytic reaction with foam cell formation in the dermal tissue. At first an augmentation of lymphocytoid or histiocytoid tissue macrophages could be detected in the dermis and especially in the perivascular region of venous blood vessels in the middermis. Perithelial cells as well as lymphocytoid and histiocytoid tissue macrophages engulve lipoproteins. Cytoplasmic enrichment of vacuoles with an electron opaque content (lipoproteins) are typical for type III-HLP; they lead to formation of typical foam cells. Within those cells lysosomal intracellular degradation of the engulved lipoproteins takes place; free cholesterol cristals, phospholipid-containing myelinic figures and residual bodies are endproducts of this process. They can lead to cellular necrosis with cellular remnants, lipoprotein vacuoles, free cholesterol and phospholipids lying free within the dermal tissue. 4. Factors are not well understood which control circumscribed xanthoma formation in type III-HLP. 5. These investigations show, that xanthomas are reactive new formations. The leading cellular substrate is the tissue macrophage, which is transformed into a foam cell by intake of lipoproteins. Foam cell formation, however, is not the result of a simple process of storage: after lipoprotein intake numerous, to a part lysosomal processes of chemical degradation and transformation, take place with an appearance of intracellular endproducts (free cholesterol, residual bodies, phospholipid-containing membranes). It is suggested that the dynamics of xanthoma formation might be importance in understanding of the process of atherosclerosis in those patients.


Comparative sensitivity of the BGM cell line for isolation of enteric viruses.

The BGM line of African green monkey kidney cells was less sensitive than primary rhesus monkey kidney cells or human fetal diploid kidney cells for isolation of certain echoviruses and adenoviruses from fecal specimens.


Fucolipid patterns of cell lines transformed by highly and weakly tumorigenic simian virus 40 and herpes simplex virus.

The fucolipids of simian virus 40 (SV40)-transformed Balb/c mouse and herpes simplex virus (HSV)-transformed hamster cells of varying degrees of tumorigenicity were examined. In the virus-transformed cell lines and in a spontaneously transformed cell line, as compared to non-transformed cell lines, there was a decrease in the incorporation of fucose radioactivity into fucolipid IV. Fucolipid IV is the least chromatographically mobile, presumably most complex fucolipid. Only in the highly transplantable SV40- and HSV-transformed cell lines was the decrease in incorporation of radioactivity in fucolipid IV as marked as that observed in oncornavirus-transformed cell lines. Similarly, only in the highly tumorigenic DNA-virus-transformed cell lines was there a significant increase in incorporation of radioactivity from radioisotopically-labeled fucose into fucolipid III as has been observed in oncornavirus-transformed cell lines.


Cross-protection between Tacaribe complex viruses. Presence of neutralizing antibodies against Junin virus (Argentine hemorrhagic fever) in guinea pigs infected with Tacaribe virus.

Cross-protection between Junin virus and five other Tacaribe complex viruses and the serological response of guinea pigs inoculated with Tacaribe virus are reported here. Previous infection with Tamiami or Pichinde viruses significantly delayed guinea pig deaths. A 58% survival rate was found among animals immunized with three doses of Amapari virus, while guinea pigs inoculated with one dose of Machupo or Tacaribe virus were fully protected against Junin virus. Neutralization tests performed in serum samples of guinea pigs immunized with five doses of Tacaribe virus showed that they developed monologous and heterologous neutralizing antibodies.


A major difference in the strategy of the calici- and picornaviruses and its significance in classification.

Pig kidney (1BRS-2) cells infected with vesicular exanthema virus (VEV), a calicivirus, did not contain any large precursor polypeptides similar to those found when they were infected with foot-and-mouth disease virus (FMDV). The largest induced protein found in the VEV-infected cells had a molecular weight identical with that of the virus structural polypeptide. This difference in strategy between VEV and FMDV, taken in conjunction with the morphological and structural differences described previously, provides strong evidence that the caliciviruses should not be included in the family Picornaviridae.


Soluble proteins and hydrolases during crown-gall induction in the tomato, Lycopersicon esculentum.

Soluble proteins isolated from tissues of the tomato Lycopersicon esculentum, after inoculation with Agrobacterium tumefaciens to induce tumours, have been examined by gel electrophoresis and cytochemically. Changes that occur include the suppression of host enzymes, the appearance of bacterial enzymes in the host tissues and the appearance of new enzyme bands in the affected cells. These  changes are detectable within 6 hr of infection and prior to evident morphological changes, and may be explained as derepression and repression of host genes, or the expression of released bacterial genes in the host cells.


Stabilization of configurational states and enzyme activities in subcellular fractions after fixation with extremely low concentrations of glutaraldehyde.

The activities of various enzymes in some subcellular organelle fractions were examined after fixation in glutaraldehyde of various concentrations. A high speed centrifuge was used to shorten the fixation time. At the lowest concentration (0.01%) glutaraldehyde stabilized instable configurational states of mitochondria as revealed by electron microscopy. In addition, at this concentration, at least 70% of the original monoamine oxidase, ATPase and cytochrome oxidase activities were preserved. The activity of acid phosphatase, on the other hand, was enhanced in a lysosomal fraction when fixed with the aldehyde at higher concentrations, e.g. 0.1% and 1.0%. It is possible that the aldehyde at higher concentrations has the same effects on the lysosomal membrane as freeze-thawing. Glucose-6-phosphatase activity was well-preserved in a microsomal fraction fixed with 0.01% glutaraldehyde but was decreased drastically when the concentration of the aldehyde was greater than 0.05%.


Collaborative study of an improved method for the enumeration and confirmation of Clostridium perfringens in foods.

A collaborative study was conducted in 10 laboratories to evaluate the performance of a new method for the enumeration of vegetative cells of Clostridium perfringens in foods. Results obtained by the new method were compared with results from the official first action method, 46.049-46.053. Per cent recoveries of 4 C. perfringens strains from inoculated roast beef samples were higher and more consistent in tryptose-sulfite-cycloserine (TSC) agar with or without added egg yolk than in sulfite-polymyxin-sulfadiazine (SPS) agar, specified in the official first action method. The confirmatory technique utilized in the new method was also found to be more reliable than the technique described in the official first action method. Based on the collaborative results, the new method with TSC agar for enumeration and a modified motility-nitrate medium together with a lactose-gelatin medium for confirmation of C. perfringens has been adopted as official first action to replace 46.049-46.053.


Studies on pig serum lipoproteins. I. Separation and properties of low-density lipoproteins.

The low-density lipoproteins in pig serum were separated into two subclasses (LDL1 and LDL2) by 2 to 7% pore size gradient gel electrophoresis. Preparative gel electrophoresis in 2 to 4% gradient gel made it possible to isolate these components as distinct entities. After delipidation by chromatography on Sepharose 4B in the presence of SDS, both apo-LDL1 and apo-LDL2 were found to have a molecular weight of 2.6X10(5). However, when these apoproteins were incubated in 10% sodium dodecyl sulfate, fragmentation occurred and the minimum fragment molecular weight was estimated to be 2.4X10(4). No essential difference was found in the amino acid compositions or fragmentation patterns of the apoproteins. However, the amounts of carbohydrates in the two apoproteins were different (7.09% in apo-LDL1 and 5.08% in apo-LDL2). The carbohydrate composition was 0.8% sialic acid, 2.38% N-acetyl-glucosamine, and 4.01% neutral sugars in apo-LDL1 and 0.5, 1.75, and 2.83% in apo-LDL2, respectively. In both apoproteins, mannose, galactose, and fucose were present in almost the same molar ratio of 4-5 : 2-3 : 1.


Glycolipids of the fish testis.

Glycolipids were purified from the total lipid extract of the testis or milt of a kind of puffer (Fugu rubripes rubripes) by adsorption column chromatography using silicic acid  and magnesium silicate and by preparative silica gel TLC. The glycolipids were identified as glucosylceramide (116 mug/g wet tissue) and galactosylceramide 26.7 mug/g). Seminolipid, a sulfagalactolipid specific to mammalian testis was not detected, but the presence of a small amount of sulfatide (15.2 mug/g) was demonstrated. The long-chain bases of both cerebrosides were mainly C18-sphingenine, but in sulfatide, C20-sphingenine was more abundant than C18-sphingenine. In both cerebrosides and sulfatide, the fatty acid compositions were similar, with nervonic acid as the predominant component. Two species of gangliosides were also obtained and were identified as N-acetylgalactosaminyl(1 leads to 4)[N-acetylneuraminyl(2 leads to 3)]galactosyl(1leads to 4)glucosylceramide (59.8 mug/g) and N-acetylneuraminyl(2 leads to 3)galactosyl(1 leads to 4)N-acetylglucosaminyl(1 leads to 3)galactosyl(1 leads to 4)glucosylceramide (45.0 mug/g). The long-chain bases of the two gangliosides consisted of C18-spingenine and C20-sphingenine, and the major fatty acids were palmitic and stearic acids.


A new assay method for hydrogenase based on an enzymic electrode reaction. The enzymic electric cell method.

A new assay method for hydrogenase [EC 1.12.2.1] based on the enzymic electrode reaction of H2-H+ equilibrium has been established. The method is based on the experimental fact that the short-circuit current of the electric cell composed of an electrode with hydrogenase and methylviologen as the mediator of H2-H+ equilibrium and a saturated calomel electrode as the counter electrode, is practically proportional to the amount of hydrogenase in the cell. The new method is referred to as the "enzymic electric cell method." This technique has applications not only to routine activity assay but also to the direct determination of the time course of enzyme denaturation, which has not previously been possible.


Isolation and characterization of serologically active phosphatidylinositol oligomannosides of Mycobacterium tuberculosis.

Phospholipids extracted with hot methanol from Mycobacterium tuberculosis, strain H37Rv, were fractionated with solvent mixtures and by partition chromatography on silica-gel columns. Of 12 components detected on a thin-layer chromatogram of the total phospholipid fraction, 6 components were purified. They were all phosphatidylinositol oligomannosides: two hexamannosides (H-2 and H-3), one tetramannoside (H-4), one trimannoside (H-5), and two dimannosides (H-6 and H-7). The molar ratios of fatty acid to phosphorus were 3 for H-2, H-4, H-5, and H-6, and 4 for H-3 and H-7. In each lipid, more than 80% of the total fatty acids were palmitic and tuberculostearic acids. Each of the six lipids had serological activity to various degrees.


Mössbauer effect and electron paramagnetic resonance studies on yeast aconitase.

Mössbauer effect and electron paramagnetic resonance (EPR) were measured for yeast aconitase [EC 4.2.1.3] purified from the cells of Candida lipolytica (ATCC 200114). Mössbauer spectra suggested that yeast aconitase nostly contained two high-spin Fe(III) ions in an antiferromagnetically coupled binuclear complex that resembled oxidized 2 Fe ferredoxins, together with a small amount of high-spin Fe(II). EPR spectra recorded no signal at 77degreesK, but showed a slightly asymmetric signal centered at g=2.0 at 4.2degreesK, presumably due to the small amount of Fe(II) Fe(III) pairs.


Effects of ethyl p-chlorophenoxyisobutyrate on carbohydrate and fatty acid metabolism in rat liver.

1. Rats were fed on a diet containing ethyl p-chlorophenoxyisobutyrate (0.3%, w/w) for 14 days. 2. The alterations of contents of intermediates in the liver indicate that gluconeogenesis is inhibited at the reaction(s) between 3-phosphoglycerate and fructose 1,6-diphosphate. The [nad+]/([nadh] ratios in cytoplasm and mitochondria were increased about 3- and 4-fold, respectively. Marked increases in the contents of CoA and its thioesters were found. 3. Hepatic fatty acid synthesis increased about 3-fold. There was no evidence of inhibition of the acetyl-CoA carboxylase [EC 6.4.1.2] reaction by the drug.


Comparative specificity of geranylgeranyl pyrophosphate synthetase of Micrococcus lysodeikticus and pumpkin.

Comparative studies on the substrate specificity of geranylgeranyl pyrophosphate synthetase from Micrococcus lysodeikticus and from pumpkin seedlin revealed that geranyl pyrophosphate was the most active of the natural substrates for the pumpkin enzyme, whereas it was the least active for the bacterial enzyme. A marked difference was also observed between the enzymes from these two sources as regards the reactivity of 3-methyl-2-alkenyl pyrophosphates as a function of the size of the alkyl group.


Nucleoside diphosphate kinase and the flagellar movement of glycerinated spermatozoa.

Nucleoside diphosphate kinase [EC 2.7.4.6.] of sperm flagella and Tetrahymena cilia is detected mostly in the outer fiber fraction, suggesting an association of the enzyme with the outer fiber microtubules. The enzyme does not catalyze transphosphorylation between microtubule-bound GDP and exogenous ATP. Even when undulatory movement of glycerinated sperm is induced by MgATP, no phosphorylation is detected in the outer fiber fraction. These facts do not appear to support the hypothesis that the phosphorylation of microtubule-bound GDP is involved in the mechanism of flagellar movement.


Change in the ratio of cytochrome oxidase activity to nitrite reductase activity of Pseudomonas aeruginosa nitrite reductase with the kind of C-type cytochrome used as an electron donor.

The  ratio between the nitrite reductase and cytochrome oxidase activities of Pseudomonas aeruginosa nitrite reductase [EC 1.9.3.2.] varies with kind of C-type cytochrome used as the electron donor. Withe cytochrome c-548, 554 (Micrococcus sp.), the nitrite reductase activity is greater than the cytochrome oxidase activity, while the former is smaller than the latter with cytochrome c-554 (Navicula pelliculosa). The aerobic oxidation catalyzed by this enzyme of denitrifying bacterial ferrocytochrome c is greatly accelerated on addition of nitrite, while that of the algal ferrocytochrome c is not affected or is even depressed by the salt. An accelerative effect of nitrite is generally observed with many kinds of C-type cytochromes which react with the enzyme very or fairly rapidly. The difference in the ratio of the two activities of the enzyme seems to arise according to whether or not nitrite affects the interaction of C-type cytochrome with the enzyme.


Surface alterations in calf lymphocytes oxidized by sodium periodate.

In order to investigate alterations in surface structure in transformed lymphocytes, calf submandibular lymph node cell suspensions were oxidized with NaIO4. Oxidezed lymphocytes were morphologically transformed and had higher rates of DNA synthesis by 2 days after treatment. These results were prevented by reduction of the cell suspension with NaBH4, or by neuraminidase treatment of cells prior to oxidation. The amount of 125I-labeled Agaricus bisporus lectin bound to cells immediately after oxidation and the affinity constant for binding were increased over 2-fold, while cells immediately following oxidation and reduction showed decreased receptors with still higher affinity for the lectin compared to untreated cells. The amount of Phaseolus vulgaris lectin bound to oxidezed cells was also increased, but affinity was unchanged. Immediately following oxidation and reduction, these receptor sites were unchanged in number and affinity from untreated cells. In contrast, the number and affinity of receptors for concanavalin A were not changed immediately after oxidation or oxidation and reduction. In order to define the extent of compositional changes in surface glycoprotein receptors, plasma membranes were isolated from frozen calf submandibular lymph nodes. Compared to untreated plasma membranes, oxidezed membranes had similar contents of galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine, fucose, and amino acids. Sialic acid content of oxidized membranes was reduced when measured by thiobarbituric acid assay. Sialic acids of untreated plasma membranes co-chromatographed with N-glycolylneurominic acid and N-acetylneuraminic acid, while those of oxidized membranes co-chromatographed with N-glycolylneuraminic acid and 5-acetamido-3,5-dideoxy-L-arabino-7-aldehydo-2-heptulosonic acid. Therefore, specific surface conformational changes in certain classes of membrane glycoproteins are associated with mild Malapradian oxidation of membrane sialic acids. These temporally precede NaIO4-induced transformation of calf lymphocytes. This is consistent with an hypothesis of membrane-mediated stimulation of lymphocyte transformation.


Preparation and characterization of eukaryotic initiation factor EIF-3. Formation of binary (EIF-3-Met-tRNAf) and ternary (EIF-3-Met-tRNAf-GTP) complexes.

The 133,000 X g supernatant fraction prepared from ascites cells in 20 mM KCl (low CKl supernatant) contained the initiation factors EIF-1 and EIF-2 (and the elongation factore EF-1 and EF-2) but lacked EIF-3; thus, low KCl supernatant could be used to assay for EIF-3. EIF-3 was prepared from a crude initiation factor perparation (a 250 mM KCl extract of ascites cell ribosomes precipitated with 70% saturated ammonium sulfate) by chromatography on DEAE-Sephadex A-50 and hydroxylapatite. The EIF-O had no detectable EIF-1 and little or no EIF-2. Factor EIF-3 was required fro translation of encephalomyocarditis virus RNA. The molecular weight of EIF-3 was estimated by Sephadex G-200 filtration to be 139,000; the sedimentation coefficient was calculated to be about 5.8. EIF-3 formed a binary complex specifically with the initiator tRNA, Met-tRNAf, and if GTP was present the factor formed a ternary complex (EIF-3-Met-tRNAf-GTP). The EIF-3 preparation had no methionyl-tRNA synthetase activity to account for binding. Complex-formation was with eukaryotic Met-tRNAf and no other aminoacyl-tRNA. The binary and ternary complexes were retained quantitatively on Millipore filters (which was the most convenient assay), but they could also be demonstrated by filtration through Sephadex G-100 or by glycerol gradient centrifugation. GTP increased the rate, the amount, and the stability of complex formed; the ration of GTP to Met-tRNAf in the ternary complex appeared to be 1. The binary and the ternary complexes transferred Met-tRNAf to the 40 S ribosomal subunits, but not to 60 S subparticles. The factor-dependent binding of Met-tRNAf to the 40 S subunit did not require mRNA (or GTP). In the presence of 60 S subunits, the initiator tRNA bound to 40 S subunits was not transferred to 80 S ribosomes even if mRNA was added--that reaction may require another initiation factor. Treatment of EIF-3 with N-ethylmaleimide led to loss of its activity in complex formation and in support of the translation of encephalomyocarditis virus RNA. In addition to forming the binary and ternary complexes, and supporting the translation of encephalomyocarditis virus RNA, EIF-3 also increases the number of free ribosomal subunits by either preventing their association or causing dissociation of 80 S couples.


Complete amino acid analysis of proteins from a single hydrolysate.

An analytical procedure which affords the precise amino acid composition of a protein or a peptide from a single hydrolysate is described. This method utilizes 4 N methanesulfonic acid containing 0.2% 3-(2-aminoethyl)indole, rather then 6N HCl as a catalyst for hydrolysis. The hydrolysis is carried out in vacuo (20 mu) at 115 degrees for 22 to 72 hours. Half-cystine is determined as S-sulfocysteine by treating the hydrolysate with dithiothreitol followed by an excess of tetrathionate. The values of all amino acids, including tryptophan and half-cystine, were close to the expected theoretical values for the proteins examined. The method has the advantage that the neutralized hydrolysate can be applied directly to an ion exchange column. Further, the method is capable of distinguishing between free sulfhydryl groups as S-carbosymethylcysteine and disulfides as S-sulfocysteine. A limitation of the procedure is that tryptophan remains sensitive to the presence of carbohydrate in the sample.


Investigations of equilibrium complexes of myoxin subfragment 1 with the manganous ion and adenosine diphosphate using magnetic resonance techniques.

Electron paramagnetic resonance spectroscopy and water proton relaxation rate (PRR) measurements were used to characterize a complex formed at the myosin subfragment 1 (S1) ATPase site with stoichiometric amounts of Mn(II) and ADP. In the absence of nucleotide, Mn(II) binding at the active site is very weak, although two other classes of sites for Mn(II) on subfragment 1 were identified which are not directly involved in the ATPase reaction. A high affinity Mn(II) site (termed L-site with KL = 3 muM) is associated with a region of the molecule which is susceptible to proteolysis (probably the LC2 light chain subunit) since its stoichiometry depends on the conditions employed for the preparation of subfragment 1 during the papain treatment of myosin. In addition there are a number of weak sites for Mn(II) (termed N-sites) probably associated with anionic groups on the surface of the molecule. In order to study the properties of Mn(II) and ADP bound at the active site by magnetic resonance techniques, subfragment 1 preparations virtually free of the L-site were used, since such an ancillary site competes for the available Mn(II). MnADP binds to subfragment 1 with an apparent dissociation constant, KT, of about 4 muM at 25 degrees. The resultant complex, S1-MnADP, has a low PRR enhancement factor (1.7 at 24.3 MHZ), and its frequency (magnetic field) dependence indicates that this is because there are no readily exchangeable water molecules within the first coordination sphere of Mn(II. Relaxation of the bulk solvent is mediated by protons bound transiently within the outer spheres (4 to 7 A) of the Mn(II). A nitroxide spin label attached to the reactive thiol group of subfragment 1 enhances the solvent PRR, and this property is sensitive to the binding of MgADP to the active site. However, no dipolar spin-spin interaction was detected between the nitroxide group and Mn(II) in the S1-MnADP complex, indicating that the metal ion and thiol group are well separated.


Cytochrome c1 of bakers' yeast. I. Isolation and properties.

Cytochrome c1 has been purified from mitochondria of the yeast Saccharomyces cerevisiae. The procedure involves solubilization withcholate, ammonium sulfate fractionation, disruption of the dytochrome b-c1 complex with mercaptoethanol and detergents, and chromatography on DEAE-cellulose. The final product is psectrally pure, contains up to 62 nmol of covalently bound heme per mg of protein and does not react with oxygen or carbon monoxide. Sodium dodecyl sulfate disaggregates the purified cytochrome into a single 31,000 dalton subunit carrying the covalently attached heme group. Many cytochrome c1 preparations contain in addition an 18,500 dalton polypeptide which is devoid of covalently bound heme. Since this polypeptide can be removed from the heme-carrying polypeptide by relatively mild procedures, it is probably not an essential subunit of cytochrome c1. Cytochrome c1 is extremely sensitive to proteolysis. If it si purified in the absence of protease inhibitors, a family of heme polypeptides with molecular weights of 29,000, 27,000, and 25,000 daltons is obtained. In the presence of the protease inhibitor phenylmethylsulfonylfluoride the purification yields predominantly a 31,000 dalton heme protein with only little contamination by a 29,000 dalton degradation product. In order to show that only the 31,000 dalton heme-polypeptide is the native species, yeast cells were labeled with the heme-precursor delta-amino[3H]levulinic acid, converted to protoplasts and directly lysed with dodecyl sulfate in the presence of protease inhibitors. Subsequent electrophoresis of the lysate in the presence of dodecyl sulfate reveals the covalently bound heme of cytochrome c1 as a single symmetrical peak at 31,000 daltons.


Cytochrome c1 of bakers' yeast. II. Synthesis on cytoplasmic robosomes and influence of oxygen and heme on accumulation of the apoprotein.

In the preceding paper (Ross, E., and Schatz, G. (1976) J. Biol. Chem. 251, 1991-1996) yeast cytochrome c1 was characterized as a 31,000 dalton polypeptide with a covalently bound heme group. In order to determine the site of translation of this heme-carrying polypeptide, yeast cells were labeled with [H]leu(be under the following conditions: (a) in the absence of inhibitors, (b) in the presence of acriflavin (an inhibitor of mitochondrial translation), or (c) in the presence of cycloheximide (an inhibitor of cytoplasmic translation). The incorporation of radioactivity into the hemeprotein was measured by immunoprecipitating it from mitochondrial extracts and analyzing it by dodecyl sulfate-polyacrylamide gel electrophoresis. Label was incorporated into the cytochrome c1 apoprotein only in the presence of acriflavin or in the absence of inhibitor, but not in the presence of cycloheximide. Cytochrome c1 is thus a cytoplasmic translation product. This conclusion was further supported by the demonstration that a cytolasmic petite mutant lacking mitochondrial protein synthesis still contained holocytochrome c1 that was indistinguishable from cytochrome c1 of wild type yeast with respect to molecular weight, absorption spectru, the presence of a covalently bound heme group, and antigenic properties. Cytochrome c1 in the mitochondria of the cytoplasmic petite mutant is firmly bound to the membrane, and its concentration approaches that typical of wild type mitochondria. However, its lability to proteolysis appeared to be increased. A mitochondrial translation product may thus be necessary for the correct conformation or orientation of cytochrome c1 in the mitochondrial inner membrane. Accumulation of cytochrome c1 protein in mitochondria is dependent on the abailability of heme. This was shown with a delta-aminolevulinic acid synthetase-deficient yeast mutant which lacks heme and any light-absorbing peaks attributable to cytochromes. Mitochondria from mutant cells grown without added delta-aminolevulinic acid contained at least 20 times less protein immunoprecipitable by cytochrome c1-antisera than mitochondria from cells grown in the presence of the heme precursor. Similarly, the respiration-deficient promitochondria of anaerobically grown wild type cells are almost completely devoid of material cross-reacting with cytochrome c1-antisera. A 105,000 X g supernatant of aerobically grown wild type cells contains a 29,000 dalton polypeptide that is precipitated by cytochrome c1-antiserum but not by nonimmune serum. This polypeptide is also present in high speed supernatants from the heme-deficient mutant or from anaerobically gorwn wild type cells. The possible identity of this polypeptide with soluble apocytochrome c1 is being investigated.


Regulation of glycogen metabolism in astrocytoma and neuroblastoma cells in culture.

The regulation of glycogen metabolism in C-6 astrocytoma and C-1300 neuroblastoma cells in culture has been investigated. Two modes of control of glycogen metabolism appear to be operative. The regulation of intracellular glycogen concentrations and the predominant forms of glycogen phosphorylase and glycogen synthase vary with (a) the available energy supply, and (b) altered intracellular concentration of cyclic adenosine 3':5'-monophosphate (cyclic AMP). Both cell lines respond to glucose in the medium; when glucose levels are high, glycogen is synthesized, glycogen phosphorylase a decreases, and glycogen synthase a increases. When glucose in the medium decreases to a critical level, the phosphorylase a increases and glycogen concentrations in the cells decrease in aprallel with the medium glucose. The critical glucose concentration is 2.5 mM for the astrocytoma cells and 4 mM for the neuroblastoma cells. Insulin promotes the conversion of phosphorylase to the b form and synthase to the a form in both cell lines. All of these changes occur without alteration in the intracellular cyclic AMP concentrations. When cyclic AMP concentrations are increased in either cell line, phosphorylase a is increased, synthase a is decreased, and glycogen concentrations decrease. Isobutyl methylxanthine is effective in promoting glycogenolysis in both cell lines. Norepinephrine is effective with the astrocytoma cells, and prostaglandin E1 is effective with the neuroblastoma cells.


Termination of procollagen chain synthesis by puromycin. Evidence that assembly and secretion require a COOH-terminal extension.

Embryonic chick fibroblasts were incubated with [14C]proline and puromycin in the low concentrations of 1 to 3 mug/ml. The molecular weight of the synthesized procollagen chains, as measured by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, was progressively reduced by increasing concentrations of puromycin in this range. For example, at 3 mug/ml the great majority of the [14C]proline was contained in procollagen chains having an average molecular weight of about 95,000 instead of the control value of 125,000. Associated with this decrease in molecular weight there was a marked decrease in the incorporation of cysteine although [14C]proline incorporation was relatively unaffedted. Disulfide bond formation was drastically inhibited as was triple helix formation as measured by resistance of the procollagen to pepsin digestion. Although the shortened procollagen chains were of normal hydroxyproline content, they nevertheless were secreted much more slowly than normal procollagen. Based upon these findings, we postulate that: (a) low concentrations of puromycin terminate procollagen chains before a COOH-terminal extension is completed, (b) these COOH-terminal extensions are required for normal assembly of the three individual procollagen chains and for triple helix formation, and (c) only assembled, triple helical procollagen molecules are selected for normal secretion.


Thermodynamic and EPR characteristics of two ferredoxin-type iron-sulfur centers in the succinate-ubiquinone reductase segment of the respiratory chain.

Two distinct ferredosin-type iron-sulfur centers (designated as Centers S-1 and S-2) are present in the soulble succinate dehydrogenase in approximately equivalent concentrations to that of bound flavin. Both Centers S-1 and S-2 exhibit electron paramagnetic resonance absorbance in the reduced state at the same magnetic field (gz = 2.03, gy = 1.93, and gx = 1.91) with similar line shape. Center S-2 is reducible only chemically with dithionite and remains oxidized under physiological conditions. Thus, its functional role is unknown; however, thermodynamic and EPR characterization of this iron-sulfur center has revealed important molecular events related to this dehydrogenase. The midpoint potentials of Centers S-1 and S-2 determined in the soluble succinate dehydrogenase preparations are -5 +/- 15 mV and -400 +/- 15 mV, respectively, while corresponding midpoint potentials determined in particulate preparations, such as succinate-cytochrome c reductase or succinate-ubiquinone reductase, are 0 +/- 15 mV and -260 +/- 15 mV. Reconstitution of soluble succinate dehydrogenase with the cytochrome b-c1 complex is accompanied by a reversion of the Center S-I midpoint from -400 +/- 15 mV to -250 +/- 15 mV with a concomitant restoration of antimycin A-sensitive succinate-cytochrome c reductase activity. There observations indicate that, during the reconstitution process, Center S-I is restored to its original molecular environment. In the reconstitutively active succinate dehydrogenase, the relaxation time of Center S-2 is much shorter than that of S-1, thus Center S-2 spectra are well discernible only below 20 K (at 1 milliwatt of power), while the resonance absorbance of Center S-1 is detectable at higher temperatures and readily saturates below 15 K. Over a wide temperature range the power saturation of Center S-1 resonance absorbance is relieved by Center S-2 in the paramagnetic state, and the Center S-2 central resonance absorbance is broadened by Center S-1 spins, due to a spin-spin interaction between these centers. These observations indicate an adjacent location of these centers in the enzyme molecule. In reconstitutively inactive enzymes, subtle modification of the enzyme structure appears to shift the temperature dependence of Center S-2 relaxation to the higher temperature. Thus the EPR signals of Center S-2 are also detectable at higher temperature. In this system a splitting of the central peak of the Center S-2 spectrum due to spin-spin interaction was observed at extremely low temperatures, while this was not observed in reconstitutively active enzymes or in paritculate preparations. This spin-spin interaction phenomena of inactive enzymes disappeared upon chemical reactivation with concomitant appearance of the reconstitutive activity. These observations provide a close correlation between the molecular integrity of the enzyme and its physiological function.


Thermodynamic and EPR characteristics of a HiPIP-type iron-sulfur center in the succinate dehydrogenase of the respiratory chain.

In addition to the two species of ferredoxin-type iron-sulfur centers (Centers S-1 and S-2), a third iron-sulfur center (Center S-3), which is paramagnetic in the oxidezed state analogous to the bacterial high potential iron-sulfur protein, has bwen detected in the reconstitutively active soluble succinate dehydrogenase preparation. Midpoint potential (at pH 7.4) of Center S-3 determined in a particulate succinate-cytochrome c reductase is +60 +/- 15 mV. In soluble form, Center S-3 becomes extremely labile towards oxygen or ferricyanide plus phenazine methosulfate similar to reconstitutive activity of the dehydrogenase. Thus, even freshly prepared reconstitutively active enzyme preparations show EPR spectra of Center S-3 which correspond approximately to 0.5 eq per flavin; in particulate preparations this component was found in a 1:1 ratio to flavin. All reconstitutively inactive dehydrogenase preparations that Center S-3 is an innate constituent of succinate dehydrogenase and plays an important role in mediating electrons from the flavoprotein subunit to most probably ubiquinone and then to the cytochrome chain.


Biochemical and ultrastructural hepatic changes during vitamin B12 deficiency in animals and man.

Vitamin B12 deficiency has been shown to result in an increase in content and activity of the hepatic cytosolic enzymes of fatty acid synthesis. The present study demonstrated that ATP citrate lyase, an enzyme whose activity has been positively correlated with rates of fatty acid biosynthesis, also increased in the livers of B12-deficient animals. Total and specific activity of hepatic citrate synthase, an enzyme whose activity is unaffected by a variety of dietary and hormonal changes, also was found to be increased in the B12-deprived state. By contrast, the activity of hepatic succinate-cytochrome c reductase, a portion of a multicomponent enzyme complex synthesized in part within the mitochondria, was unchanged in B12 deficiency. Vitamin B12 deprivation resulted in an increase in hepatic mitochondrial cristae membranes in both animals and man. Histochemical and chemical analysis demonstrated increased glycogen in the liver cells from B12-deficient animals and man. Thus, in the livers from vitamin B12-deficient animals there is an increased activity of the otherwise highly constant Krebs cycle enzyme citrate synthase, and in both animals and man there are increased mitochondrial cristae membranes.


Differences in properties of cytosol and membrane-derived protein kinases.

Adenosine 3':5'-monophosphate-dependent protein kinase present in membrane fractions of bovine brain, heart, liver, and muscle was solubilized with Triton X-100. Certain properties of the membrane-derived enzyme were compared with those of two adenosine 3':5'-monophosphate-dependent protein kinases present in the cytosol fractions from each of the same tissues. The properties studied included chromatographic behavior on DEAE-cellulose columns, specificity with respect to substrate proteins, and sensitivity to NaCl and Triton X-100. The membrane-derived enzyme from each tissue had properties similar to those of the membrane-derived enzyme from each of the other tissues. Moreover, the cytosol enzymes from each tissue had properties similar to those of the corresponding enzymes in the cytosol from each of the other tissues. However, for any given tissue, the properties of the membrane-derived enzyme differed from those of the cytosol enzymes, possibly reflecting different functional roles for the membrane-bound and cytosol enzymes.


6-phosphofructokinase (pyrophosphate). Properties of the enzyme from Entamoeba histolytica and its reaction mechanism.

The inorganic pyrophosphate-requiring 6-phosphofructokinase of Entamoeba histolytica has been further investigated. The molecular weight of the enzyme is approximately 83,000 and its isoelectric point occurs at pH 5.8 to 6.0. The divalent cation requirement for reaction was explored. In the direction of fructose 6-phosphate formation half-maximal rate required 500 muM magnesium ion; in the direction of fructose bisphosphate formation 8 muM magnesium ion sufficed. ATP, PPi, polyphosphate, acetyl phosphate, or carbamyl phosphate cannot replace PPi as phosphate donor for the conversion of fructose 6-phosphate to fructose bisphosphate. In the direction of fructose 6-phosphate formation arsenate can replace orthophosphate. Isotope exchange studies indicate that little or no exchange occurs between Pi and PPi or between fructose 6-phosphate and fructose bisphosphate in the absence of a third substrate. These findings appear to rule out phosphoenzyme formation and a ping-pong reaction mechanism. PPi, Pi, and fructose bisphosphate are competitive inhibitors of fructose bisphosphate, PPi, and fructose 6-phosphate, respectively. This argues against an ordered mechanism and suggests a random mechanism. Fructose 6-phosphate and Pi were noncompetitive with respect to each other indicating the formation of a dead end complex. These product inhibition relationships are in accord with a Random Bi Bi mechanism.


Studies on the role of cyclic guanosine 3':5'-monophosphate and extracellular Ca2+ in the regulation of glycogenolysis in rat liver cells.

Catecholamines increased guanosine 3':5'-monophosphate (cyclic GMP) accumulation by isolated rat liver cells. The increases in cyclic GMP due to 1.5 muM epinephrine, isoproterenol, or phenylephrine were blocked by phenoxybenzamine but not by propranolol. The possibility that cyclic GMP is involved in the glycogenolytic action of catecholamines seems unlikely since cyclic GMP accumulation is also elevated by carbachol, insulin, A23187, and to a lesser extent by glucagon. Furthermore, carbachol had little effect on glycogenolysis while insulin actually inhibited hepatic glycogenolysis. The rise in cyclic GMP due to carbachol was abolished by atropine and that due to all agents was markedly reduced by the omission of extracellular calcium. However, the glycogenolytic action of glucagon and catecholamines was only slightly inhibited by the omission of calcium. The only agent which was unable to stimulate glycogenolysis in calcium-free buffer was the divalent cation ionophore A23187. There was a drop in ATP content of liver cells during incubation in calcium-free buffer which was accompanied by an inhibition of glucagon-activated adenosine 3':5'-monophosphate (cyclic AMP) accumulation. The presence of calcium inhibited the rise in adenylate cyclase activity of lysed rat liver cells due to glucagon or isoproterenol but not that due to fluoride. These results suggest that the stimulation by catecholamines and glucagon of glycogenolysis is not mediated through cyclic GMP nor does it depend on the presence of extracellular calcium. Cyclic GMP accumulation was increased in liver cells by agents which either inhibit, have little affect, or accelerate glycogenolysis. The significance of elevations of cyclic GMP in rat liver cells remains to be established.


Studies on the poky mutant of eurospora crassa. Fingerprint analysis of mitochondrial ribosomal RNA.

Base sequence and methylation of mitochondrial ribosomal RNAs from wild type and poky strains of Neurospora crassa were compared to determine whether a mutational lesion exists in poky 19 S RNA. At the outset, new procedures were developed for the isolation of intact nucleic acids from Neurospora mitochondria based on the substitution of Ca2+ for Mg2+ in the isolation media to inhibit mitochondrial nuclease activity. Using these procedures, intact and highly purified 32P-labeled ribosomal RNAs were extracted from purified mitochondrial ribosomal subunits of wild type and poky and compared using three complementary fingerprinting systems: two-dimensional electrophoresis of T1 plus phosphatase digests and homochromatography of T1 and pancreatic RNase digests. In supplementary experiments, 32P-labeled wild type RNA was co-fingerprinted with 32P-labeled poky and ratios of 32P/33P radioactivity were determined in each fragment to detect possible differences in stoichiometry. In addition, levels and patterns of methylated nucleotides were compared using procedures based on in vivo labeling with [methyl-3H]methionine and [32P]orthophosphate. In all these experiments, no difference was detected between wild type and poky in base sequence or methylation of either 19 S or 25 S RNA. Levels of methylation of Neurospora mitochondrial ribosomal RNAs were extremely low (less than 0.1% of the nucleotides), and results based on fingerprint analysis and DEAE-cellulose chromatography of alkaline hydrolysates of the [3H]methyl-labeled RNA suggested that 25 S RNA contains two ribose methylations, while 19 S RNA contains no methylated nucleotides.


Acetate kinase from Veillonella alcalescens. Regulation of enzyme activity by succinate and substrates.

Acetate kinase of Veillonella alcalescens has been shown to be highly regulated enzyme exhibiting two levels of control: the requirement for succinate as a heterotropic allosteric effector, and cooperative binding at the substrate level. Succinate addition was necessary for enzymatic activity in both the direction of acyl phosphate synthesis and that of ATP synthesis. Control at the substrate level was apparent in the cooperative binding (Hill coefficients of 2) of acetyl phosphate, ATP, and ADP. Typical Michaelis kinetic data were observed for succinate (Ka = 20 mM for acetyl phosphate synthesis, 0.4 mM for ATP synthesis), acetate, and propionate. The primary effect of succinate was to increase the apparent Vmax of the enzymatic reaction for the variable substrates, ATP, ADP, and acetyl phosphate. The results are interpreted as evidence that, as a heterotropic effector of the acetate kinase reaction, succinate may regulate levels of propionyl-CoA (produced from propionyl phosphate by action of phosphotransacetylase), a compound required for the conversion of succinate to propionate. Acetase kinase has been shown to be a probable dimeric protein composed of two subunits of molecular weight 44,000 each.


Characterization of a virion protein kinase as a virus-specified enzyme.

Antibodies which completely inhibited the enzymatic activity of the protein kinase associated with virions of frog virus were obtained by immunization of rabbits with the purified enzyme. This inhibition provided a specific probe for the frog virus protein kinase, since this antiserum had no inhibitory effect on a variety of other protein kinases, including the activity of uninfected cells, or the protein kinase associated with vesicular stomatitis virus or vaccinia virus cultivated in the same cell line as frog virus. The frog virus protein kinase was characterized as a virus-specified protein on the basis of the following observations: (a) the virion protein kinase was antigenically distinct from essentially all of the protein kinase expressed in uninfected cells; (b) following infection by frog virus more than a 15-fold increase was detected in the specific activity of intracellular protein kinase and most of this activity was antigenically related to the virion enzyme; (c) when frog virus was grown in cells derived from widely different species, the antigenic and biochemical specificities of the virion protein kinase remained identical; and (d) screening of cells infected with different temperature-sensitive mutants of frog virus indicated that certain viral mutants failed to synthesize this protein kinase when cultivated at the nonpermissive temperature.


Skeletal alterations following irradiation for Wilms' tumor: with particular reference to scoliosis and kyphosis.

The roentgenographic changes in the axial skeleton after irradiation for Wilms' tumor were studied in eighty-one patients. In addition to the initial alterations found in the individual vertebrae within the field of irradiation, spinal deformity subsequently developed in fifty-nine patients (pure scoliosis in thirty-eight, kyphoscoliosis in nineteen, and pure kyphosis in two). Unitl the adolescent growth spurt these deformities tended to remain slight, but some progression did occur. In seven patients the scoliosis became severe enough to require spine fusion. A Milwaukee brace used in three patients failed to correct the curve. The trapezoidal shape of the vertebrae and scarring of the soft tissues within the concavity made correction difficult. Recognizable roentgenographic alterations failed to develop in twenty-two patients who, in general, were older and had received less irradiation. There appeared to be a correlation between the amount of irradiation and the severity of the spinal deformity (p is less than 0.05) and between the age of irradiation and the amount of deformity (p is less than 0.02).


Skeletal immaturity in Perthes' disease.

Skeletal age was estimated by examination of radiographs of the carpus in 182 children suffering from Perthes' disease after the reliability of the Greulich and Pyle Atlas had been checked for a control group of British children. A striking tendency to delayed skeletal maturation was shown in the children with Perthes' disease. This trait was also found in ninety-three unaffected siblings of the patients. The velocity of skeletal ageing as the disease progressed was estimated. In some patients the carpal skeleton failed to mature at all for periods of up to three years and the term "skeletal standstill" is applied to this phenomenon. The significance of these findings is discussed and it is suggested that the maturation defect may have aetiological significance.


Developmental changes in mitochondria during the transition into lactation in the mouse mammary gland. II. Membrane marker enzymes and membrane ultrastructure.

The activity of cytochrome oxidase (an inner mitochondrial membrane marker) in mouse mammary gland homogenates was found to increase five- to sixfold from late pregnancy to day 8 of lactation, while that of monoamine oxidase (an outer membrane marker) increased only about 25%. The specific activity of cytochrome oxidase in the isolated mitochondria decreased slightly over the same period while the specific activity of monoamine oxidase decreased fivefold. This reflects the fact that both cytochrome oxidase and mitochondrial protein are increasing at a much greater rate than is monoamine oxidase activity. Mixing experiments preclude the possibility that the release or removal of an inhibitor or stimulator produces the changes in enzymatic activity. The cytochrome oxidase to monoamine oxidase ratio was followed throughout the pregnancy-lactation cycle in total mammary homogenates, isolated mammary parenchymal cells, and isolated mammary mitochondria. In each preparation the pattern was the same with little change in the ratio until late pregnancy; and then a three- to fourfold increase occurred and the values reached a maximum by day 8 of lactation. These experiments were interpreted as demonstrating that the observed enzymatic changes are reflective of alterations in the mitochondria of the mammary parenchymal cell population. Electron micrographs of mid-pregnant and mid-lactating mammary parenchymal cells in situ were prepared, and distinct changes in the mitochondrial morphology noted. The most significant and obvious change is the large increase in the number of inner membrane cristae and an increase in matrix density in the lactating gland cell. Therefore, both enzymatic and morphological studies support the concept of an expansion of the mitochondrial inner membrane during presecretory differentiation in the mouse mammary parenchymal cell.


Hepatic organelle interaction. IV. Mechanism of succinate enhancement of formaldehyde accumulation from endoplasmic reticulum N-dealkylations.

Further evidence for organelle interaction during drug metabolism by the liver is presented. The apparent stimulation by succinate of formaldehyde accumulation in the medium, which was reported to occur with liver slices and homogenates as well as with mitochondria plus microsomes, has been shown to be the result of succinate inhibition of mitochondrial aldehyde dehydrogenase. The mechanism of succinate inhibition is shown to be by reverse electron transport, and an increase in the NADH to NAD+ ratio in the mitochondria; the aldehyde dehydrogenase requires the oxidized form of the pyridine nucleotide as its cofactor. Studies on in vitro N-demethylation by liver microsomes and endoplasmic reticulum segments which cosediment with the mitochondria indicate that formaldehyde produced by the mixed function oxidase is handled differently from formaldehyde added to the medium. The latter is mainly retained in the medium containing 5 mM semicarbazide, while the generated formaldehyde is more than 50% consumed by the mitochondria. Electron microscopy has indicated that the microsomes and the endoplasmic reticulum fragments have a tendency to align themselves close to the mitochondria when present in the same medium. Consequently, it is possible that formaldehyde released to the medium adjacent to the mitochondria, as by N-demethylation, would be exposed to semicarbazide for shorter periods than that added directly to the medium. In agreement with this suggestion, complexing of formaldehyde with semicarbazide was observed spectroscopically not to be an extremely rapid reaction even at 37 degrees C. This is believed to be the reason for the greater extent of consumption of formaldehyde generated by the endoplasmic reticulum.


The Klippel-Trenaunay syndrome. A case report and review.

The Klippel-Trenaunay syndrome presents with a vascular nevus extending over an entire extremity, ipsilateral varicose veins dating from infancy and hypertrophy of all the tissues of the affected limb. This paper illustrates the problems that can be encountered in the management of this disorder. Congenital angiodysplasias should be suspected in all young individuals presenting with unilateral varicose veins. We recommend that modern methods of investigation be used in these cases to precisely define the anatomic pathology. In this manner, the appropriate treatment may be tailored to the patient's specific disorder.


Effects of nuclease and protease digestion on the ultrastructure of Paramecium basal bodies.

The action of deoxyribonuclease, ribonuclease, perchloric acid, and pronase on the fine structure of basal bodies of sectioned Paramecium was observed as part of a more extensive autoradiographic electron microscope analysis directed toward the problem of basal body DNA. DNase was found to have no detectable effect on basal body fine structure. Pronase first solubilized the linkers and C tubules of the triplets, then attacked the protein portion of the axosome, a localized portion of the ciliary axoneme adjacent to the distal end of the basal body, the rim fiber, and newly described lumen spiral complex. Prolonged pronase treatment disrupted the remaining microtubular elements, basal body plates, and cartwheel. RNase removed material from the axosome and the lumen complex, a conspicuous structure occupying the central portion of the basal body and consisting of a twisted or looped 90-A diam fiber or, more probably, pair of fibers, in association with large, dense granules. The apparent removal of both RNA and protein from this basal body structure by either of the two corresponding enzymes suggests an unusual organization of the two components. Observations from this and other laboratories suggest that the basal body RNA is single stranded. Its function is unknown but alternatives are discussed.


Purine and phosphoribosylpyrophosphate synthesis in differentiating murine virus-induced erythroleukemic cells in vitro.

Phosphoribosylpyrophosphate synthetase activity was determined in Friend virus-inducted erythroleukemic cells in culture, stimulated to differentiate in the presence of dimethylsulfoxide. The activity of phosphoribosylpyrophosphate synthetase did not decrease in cells which had acquired the specialized function of hemoglobin synthesis, nor was the phosphoribosylpyrophosphate content of untreated erythroleukemic cells significantly different from that of cultures exposed to dimethylsulfoxide for 96 hours. However, the rate of the early steps of de novo purine biosynthesis as measured by the incorporation of [1-14C] glycine and [1-14C] formate into formyglycinamide ribonucleotide, was significantly lower in differentiating cell cultures. The addition of glutamine or ammonia increased glycine incorporation of control cultures, but failed to do so in treated cultures. In the course of the normal development of erythrocytes in vivo, phosphoribosylpyrophosphate synthetase activity is preserved, while the capacity to synthesize purines de novo is lost, as is the activity of the phosphoribosyl-l-amine synthesizing enzymes. Our present study suggests that the rate of de novo purine biosynthesis in this erythroleukemic cell line is not limited by the availability of phosphoribosylphrophosphate, but rather by a decrease in the phosphoribosyl-l-amine synthesizing enzymes. These findings provide further evidence that during dimethylsulfoxide-stimulated erythroid maturation, the same regulatory mechanisms are operative as in normal cellular development, and that ammonia-dependent purine biosynthesis is subject to the same regulatory mechanisms as is glutamine-dependent biosynthesis.


Turnover at nicotinamide adenine dinucleotide in cultures of human cells.

The rate of turnover of nicotinamide adenine dinucleotide (NAD) in the human cell line, D98/AH2, has been estimated by measuring the rates of entry into and exit from NAD molecules of 14C-adenine. In one set of experiments, cells were labeled by growth in medium containing 14C-adenine for six hours and then shifted to medium without labeled adenine. The loss of 14C-adenine from the adenine nucleotide and pyridine nucleotide pools was measured, and the data were analyzed using an analytical treatment which corrects for the relatively slow turnover of precursor pools. The loss of 14C-adenine from the NAD pool and from the precursor ATP pool could be related to the absolute rate of NAD breakdown. Under the experimental conditions used, the rate of NAD turnover ranged from 83,000 to 126,000 molecules per second per cell. In a complementary experiment cells were grown in the presence of unlabeled adenine, then shifted into medium containing 14C-adenine and the rate of entry of 14C-adenine into adenine and pyridine nucleotides was measured. The data were treated using a similar analysis to relate the rate of entry of 14C-adenine into NAD and the precursor ATP pools to the absolute turnover rate of NAD. This analysis gave a value for NAD turnover of 78,000 molecules per second per cell in excellent agreement with results from the pulse-chase experiments. The results from both types of experiment indicate that within D98/AH2 cells the half-life of an intact NAD molecule is 60 +/- 18 minutes. Thus, in a human D98/AH2 cell growing with a generation time of 24 hours, NAD is turning over at twice the rate found in Escherichia coli with a generation time of half an hour.


Development of a clonal myogenic cell line with unusual biochemical properties.

The morphological, ultrastructural, biochemical and electrophysiological properties of B104-F, a clonal cell line derived from a nitrosoethylurea-induced neoplasm in a rat, were studied as a function of the growth phase of the culture. Cells in exponentially growing cultures are mononucleate and produce action potentials when stimulated electrically. Stationary phase cultures contain three types of cells: cells of the first type are mononucleate and have long processes containing microfilaments and many parallel microtubules; cells of the second type are mononucleate but contain no microtubules and few microfilaments; and cells of the third type have ultrastructural features typical of multinucleate, striated myotubes. Multinucleate cells generate action potentials with both sodium and calcium components and are depolarized by acetylcholine. The acetylcholine response is blocked by d-tubocurarine. The specific activity of creatine phosphokinase is nine times higher in stationary phase cultures than in exponentially growing ones while the myokinase specific activity is unchanged. The gamma-aminobutyric acid content of the cells is 3.5- to 26-fold higher in stationary phase than in exponentially growing cultures, depending on the degree of fusion of the culture. The properties of B104-F are discussed in relation to the properties of developing skeletal muscle and of central nervous system cell lines.


Establishment in culture of a multihormone-secreting cell strain derived from the MtT/F4 rat pituitary tumor.

A clonal cell strain F4C1 has been established from the transplantable rat pituitary tumor MtT/F4 and has been maintained in continuous culture for two years. The cells grow with a population doubling time of 48 hours; the karyotype with a modal number of 39 chromosomes includes a pair of large metacentric marker chromosomes. F4C1 cells in culture produce growth hormone and prolactin but not adrenocorticotropin in contrast to the MtT/F4 tumor which secretes all three hormones in the host rat. The cloned cells lack specific receptors for thyrotropin-releasing hormone and do not respond to this agent with increased prolactin or decreased growth hormone production. Treatment with hydrocortisone results in a small increase in growth hormone and a small decrease in prolactin production. Tumors generated in rats from injected F4C1 cells secrete prolactin and growth hormone but not adrenocorticotropin. The results suggest that growth hormone and prolactin are produced by a single cell type in the MtT/F4 tumor.


Increased susceptibility of murine teratocarcinoma cells to Simian virus 40 and polyoma virus following treatment with 5-bromodeoxyuridine.

Cultures of the multipotential stem cell, embryonal carcinoma (EC), of a murine teratocarcinoma were treated with 5-bromodeoxyuridine (BrdU). Within 2-4 days at concentrations of 1-50 mugm/ml of BrdU, there was a marked change in the morphology of cells observed by light and electron microscopy. A comparison of the growth potential showed that for up to four days the BrdU-treated cultures were similar to untreated cultures. When these BrdU-treated cells were infected with Simian virus 40 (SV40) and polyoma virus (Py), there was an increase in susceptibility of the treated cells. The untreated embryonal carcinoma cells were refractory. These results suggest that BrdU modifies the embryonal carcinoma cells to allow infection with two DNA viruses.


Cellular responsiveness to stimulation in vitro: increased responsiveness to colony stimulating factor of bone marrow colony-forming cells treated with surface-active agents and cyclic 3'5' AMP.

Addition of low concentrations (10 ng/ml) of saponin or Tween 80 to stimulated cultures of normal mouse bone marrow in agar increased the number of granulocyte-macrophage colonies which developed. Addition of cyclic AMP or dibutyryl cyclic AMP in low concentration (10(-8) to 10(-10) M) also enhanced colony numbers although concentrations above 10(-5) M were inhibitory. enhancement was found when marrow cells were pre-treated with these agents and cultured in their absence. The agents did not stimulate colony development in the absence of colony-stimulating factor and enhancement of colony number occurred only in cultures containing a concentration of colony-stimulating factor which was sub-optimal in terms of maximum colony development. There was no indication of increased colony-stimulating factor production by treated marrow cells under the experimental conditions used to show colony enhancement. It was concluded that the agents caused an increased responsiveness of colony-forming cells to colony-stimulating factor.