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Biomedical subjects

J Muller

Publications and source records attributed to J Muller.

At least 91 records · Page 5Linked to original sources
PubMed1995

Impairment of the melanogenic pathway in B16 melanoma cells transfected with class I H-2 genes.

Transfection of class I H-2Kb or H-2Kd into cells of a pigmented subclone of B16-F10 BL6 (termed BL6-8) results in the loss of melanin production. In contrast, transfected BL6-8 cells expressing H-2Dd, H-2Ld, class I H-21Ak and/or the neor genes maintained their pigmented phenotype. Melanogenesis was also inhibited in cells which expressed the endogenous H-2Kb, but not the endogenous H-2Db, gene. In order to identify the specific defects in the melanogenic pathway responsible for the absence of melanin production, factors known to be related to the regulation of pigment formation were evaluated in H-2K-expressing cells. These studies showed that: (1) transfection of BL6-8 cells with the H-2Kb or H-2Kd, but not with the H-2Dd, H-2Ld or H-21Ak, genes was associated with complete inhibition of tyrosinase activity; (2) alpha-melanocyte-stimulating hormone (MSH) and theophylline (an inhibitor of cAMP phosphodiesterase) failed to stimulate tyrosinase activity in H-2K-positive cells, whereas tyrosinase activities in untransfected, or H-2DdH-2Ld, neor or H-21Ak-transfected cells were dramatically increased by those agents; (3) treatment with MSH had no effect on cAMP levels in H-2K-positive cells but stimulated cAMP levels more than 100-fold in H-2K-negative cells; (4) in contrast to MSH, forskolin, a stimulator of adenylate cyclase, was able to stimulate cAMP levels in all cell lines tested, but in H-2Kb-positive cells the levels of forskolin-induced cAMP were significantly less than those elicited in H-2Kb-negative cells; (5) electron microscopy showed that H-2K-positive cells lacked mature melanosomes; (6) Northern blot analyses showed that H-2K-positive cells lacked mRNA for tyrosinase or for the MSH receptor. Taken together, expression of the endogenous or transfected H-2K gene in BL6 melanoma cells results in down-regulation of the entire melanogenic pathway, including the inhibition of tyrosinase and MSH receptor gene expression, cAMP responses and melanosomal biogenesis.

Animals↗
PubMed1995

Comparative analysis of melanins and melanosomes produced by various coat color mutants.

Pigmentation in the skin, hair, and eyes of animals is influenced by a number of genes that modulate the activity of melanocytes, the intervention of enzymatic controls at different stages of the melanogenic process, and the physico-chemical properties of the final pigment. The results of combined phenotypic, ultrastructural, biochemical, and chemical analyses of hairs of a variety of defined genotypes on a common genetic background performed in this study are consistent with the view that pigmentation of dark to black hairs results from the incorporation of eumelanin pigments whereas that of yellow hairs results from the incorporation of eu- and pheomelanins. It is also clear that relatively minor differences in melanin content can have dramatic effects on visible hair color. A good correlation was found for expression of (and enzyme activities associated with) TRP1 and TRP2 with eumelanin synthesis and eumelanosome production.

Animals↗
PubMed1994

Susceptibility of the Sf9 insect cell line to infection with adventitious viruses.

Sf9, the insect cell line commonly used for gene expression by recombinant baculovirus (BV), can be infected by St. Louis encephalitis (SLE) virus, a flavivirus, resulting in a persistent, productive, and cytopathic infection, while retaining the ability to be infected with a recombinant baculovirus (rBV). We now demonstrate using double immunofluorescence that single cells are dually infected with SLE virus and rBV. Fourteen additional viruses including additional flaviviruses, other arbovirus classes, vesicular stomatitis virus (VSV), and herpes simplex virus, type 1 (HSV-1) failed to produce a cytopathic effect (CPE) in Sf9 cells. Plaque assays indicated infectious virus was present for several weeks post-inoculation for Yellow fever (YF), Dengue types 1 and 2 (DEN-1 and DEN-2), Gumbo limbo (GL), Eastern equine encephalomyelitis virus (EEE), Western equine encephalomyelitis virus (WEE), HSV-1, and VSV viruses. For HSV-1, GL, EEE, WEE and VSV, but not for YF, DEN-1 or DEN-2 viruses, this could be attributed solely to survival in the Sf9 cell culture media. Of the 14 viruses tested, only HSV-1 could be detected after 2 weeks in serum-free media. The data indicate that several viruses which are pathogenic for humans are stable for long periods of time at 27 degrees C in the serum-containing media used for cultivation of Sf9 cells. YF, DEN-1 and DEN-2 viruses may replicate in Sf9 cells at extremely low levels. This suggests that adventitious agents which do not produce obvious CPE or interfere with rBV infection or recombinant protein expression could contaminate Sf9 cell cultures or media.

Animals↗
PubMed1993

Rapid purification of rabbit reticulocyte lipoxygenase for electron paramagnetic spectroscopy characterization of the non-heme iron.

An efficient three-step purification technique has been developed for the reticulocyte 15-lipoxygenase from rabbit. Ammonium sulfate fractionated reticulocyte lysate was purified by size exclusion chromatography and preparative scale isoelectric focusing. The entire procedure was complete in less than eight hours and was carried out in batches which typically yielded 10 mg of purified enzyme. The identity and purity of the enzyme were evaluated by N-terminal sequencing, sodium dodecylsulfate polyacrylamide gel electrophoresis and specific activity determinations. The enzyme contained approximately one g-atom iron per mole of protein. The iron was present in an electron paramagnetic spectroscopy (EPR) silent, presumably high spin iron(II), form in the isolated enzyme. Treatment with one equivalent of 13-hydroperoxy-9(Z),11(E)-octadecadienoic acid resulted in the appearance of an EPR signal around g6.

Animals↗
PubMed1993

St Louis encephalitis virus establishes a productive, cytopathic and persistent infection of Sf9 cells.

The Sf9 cell line, commonly used for gene expression by recombinant baculovirus, has been productively infected by St Louis encephalitis (SLE) virus, a flavivirus. SLE viral infection produced a c.p.e. in the Sf9 cells characterized by giant cells and the presence of 10-fold fewer cells in the infected cultures after the first week of infection compared with uninoculated control cultures. Infected Sf9 cells expressed SLE viral antigens, and intracellular virus particles were observed by electron microscopy. Titres of cell-associated SLE virus rose slightly over an 8 week period, whereas titres of cell-free virus remained stable, suggesting that SLE virus establishes a productive and persistent infection of Sf9 cells. The SLE virus produced by the Sf9 cells could be neutralized by SLE virus-immune mouse ascitic fluid, and no evidence of escape mutants was detected. Sf9 cells persistently infected with SLE virus could be superinfected with a recombinant baculovirus and expressed recombinant antigen. The successful infection of Sf9 cells by SLE virus represents the first report of production of c.p.e. by SLE virus in insect cells under routine cell culture conditions and of the infection of Sf9 cells by a human pathogen.

Animals↗
PubMed1993

Mortality from lung cancer in Ontario uranium miners.

Mortality from lung cancer was greater in Ontario uranium miners than in the general male population of Ontario (observed = 152, expected = 67.6, standardised mortality ratio 225, 95% confidence interval 191-264). Part of the excess of lung cancer may be because the proportion of men who are smokers or have smoked is greater in uranium miners than in Ontario men. Smoking does not explain the whole excess. Mortality from lung cancer in Ontario uranium miners is clearly related to exposure to short lived radon progeny. The excess relative risk of lung cancer from the same degree of exposure to short lived radon progeny is greatest five to 14 years after exposure and less subsequently. It is greater in men under the age of 55 years and less in older men. Part of the excess of lung cancer mortality in Ontario uranium miners is probably also due to exposure to arsenic that occurred earlier in gold mines. In Ontario uranium miners, the lung cancer mortality from exposure to arsenic increases as the intensity of exposure to short lived radon progeny increases. This finding is consistent with the hypothesis that the risk of lung cancer from exposure to arsenic is enhanced by exposure to other carcinogens. In Ontario uranium miners, the proportion of lung cancers that are small cell carcinomas is greater than in the general population. The proportion of small cell carcinomas is especially great five to 14 years after exposure to short lived radon progeny and in men who die from lung cancer at younger ages.

Adult↗
PubMed1993

Mortality from stomach cancer in Ontario miners.

An excess of mortality from stomach cancer has been found in Ontario gold miners (observed (obs) 104, standardised mortality ratio (SMR) 152, 95% confidence interval (95% CI) 125-185) and no excess of stomach cancer could be detected in other miners in Ontario (obs 74, SMR 102, 95% CI 80-128). The excess of stomach cancer appeared five to 19 years after the miners began gold mining in Ontario. In that interval, similar patterns of excess mortality from stomach cancer were found in miners born in north America (obs 14, SMR 268, CI 147-450) and in miners born outside north America (obs 12, SMR 280, 95% CI 145-489). Twenty or more years after the miners began mining gold, an excess of mortality from stomach cancer was found in gold miners born outside of north American (obs 41, SMR 160, 95% CI 115-218) but not in gold miners born in north America (obs 37, SMR 113, 95% CI 80-156). The excess of stomach cancer in gold miners under the age of 60 (obs 45, SMR 167, 95% CI 122-223) seems larger than the excess in gold miners between the ages of 60 and 74 (obs 59, SMR 143, 95% CI 109-184). Exposures to arsenic, chromium, mineral fibre, diesel emissions, and aluminium powder were considered as possible explanations of the excess of stomach cancer in Ontario gold miners. Exposure to diesel emissions and aluminium powder was rejected as gold miners and uranium miners were exposed to both agents but an excess of stomach cancer was noted only in gold miners. The association between the excess of stomach cancer and the time since the miner began mining gold suggested that duration of exposure to dust in gold mines ought to be weighted according to the time since the exposure to dust occurred and that an appropriate time weighting function would be one in the interval five to 19 years after each year of exposure to dust and zero otherwise. A statistically significant association between the relative risk of mortality from stomach cancer and the time weighted duration of exposure to dust in gold mines was found in miners under the age of 60. Time weighted indices of exposure to chromium and arsenic were formed for each gold miner by time weighting the product of the duration of exposure to dust in a gold mine and the percentages of arsenic and chromium in rocks in that gold mine. Exposure to mineral fibre was measured in terms of the time weighted duration of employment in those gold mines that contain mineral fibre. A statistically significant association between the excess of stomach cancer in gold miners under the age of 60 and the time weighted index of exposure to chromium occurred and not association was found between the excess of stomach cancer and either the time weighted duration of employment in mines containing mineral fibre. The excess of stomach cancer in gold miners under the age of 60 was better associated with the time weighted index of exposure to chromium than to the time weighted duration of exposure to dust in gold mines. Although the number of cases of gastric cancer that were classified according to the system of Lauren was small, the data suggest that for miners under the age of 60, exposure to chromium is associated with the development of the intestinal rather than the diffuse type of gastric cancer.

Adult↗
PubMed1993

Vasopressin- and cAMP-induced changes in ultrastructure of isolated perfused inner medullary collecting ducts.

Studies were performed to correlate arginine vasopressin (AVP)-induced changes in epithelial ultrastructure with changes in osmotic water permeability in isolated perfused rat terminal inner medullary collecting ducts (tIMCD). The tubules were perfused in three time periods, i.e., a 40-min basal period, a 40-min period with 0.1 nM AVP in the bath, and a 60-min withdrawal period. In each phase, the osmotic water permeability (Pf) was measured, and the perfused tubules were fixed for electron microscopy. AVP caused a four- to eightfold increase in Pf and induced several ultrastructural changes as follows: increased cell height of IMCD cells, expansion of the intercellular spaces, formation of large vacuoles, and increased coated pit density in the apical plasma membrane [from 0.6 +/- 0.2 (n = 6) to 2.9 +/- 0.3 (n = 7) pits/100 microns membrane length]. During AVP withdrawal, Pf decreased toward the basal value in association with partial reversal of the ultrastructural changes including a decrease in coated pit density to 1.0 +/- 0.2 (n = 4). Stimulation with 8-bromoadenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) (0.1 mM) produced similar changes in Pf. Coated pit density increased to 2.1 +/- 0.4 (n = 4) after cAMP stimulation and after cAMP withdrawal decreased to 1.2 +/- 0.2 (n = 6). In contrast to stimulation with AVP, cAMP stimulation did not result in dilated intercellular spaces or formation of large vacuoles. The only ultrastructural feature that directly correlated with the water permeability was the density of coated pits in the apical membrane. Organelles involved in the endocytic pathway were studied with cationized ferritin or albumin-gold in the luminal perfusate. At the end of 40 min basal perfusion or AVP stimulation, luminal tracer was found almost exclusively in large multivesicular bodies (MVB). Tubules perfused with tracer during AVP withdrawal demonstrated rapid tracer accumulation in small vesicles and small MVB within 3-5 min, a time point corresponding to the rapid phase of Pf decrease. Later (30-60 min) the label was mainly confined to large MVB. Occasionally during AVP stimulation or withdrawal, small coated vesicles and smooth vesicles with coated extensions were noted to contain tracer. The data demonstrate AVP-mediated coated pit formation and cellular changes and show very rapid internalization of apical membrane after AVP withdrawal.

Animals↗