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Biomedical subjects

R W Jansen

Publications and source records attributed to R W Jansen.

81 records · Page 5Linked to original sources
PubMed1987

Genomic heterogeneity among human and nonhuman strains of hepatitis A virus.

Cloned cDNA probes derived from the P1 and P2 regions of the genome of HM175 virus, a reference strain of human hepatitis A virus (HAV), failed to hybridize under standard stringency criteria with RNA from PA21 and PA33 viruses, two epizootiologically related HAV strains recovered from naturally infected New World owl monkeys. Hybridization of these probes to PA21 RNA was only evident under reduced stringency conditions. However, cDNA representing the 5' nontranslated region of the HM175 genome hybridized equally to HM175 and PA21 RNA under standard stringency conditions, while a probe derived from the 3' 1,400 bases of the genome yielded a reduced hybridization signal with PA21 RNA. In contrast, no differences could be discerned between HM175 virus and three other HAV strains of human origin (GR8, LV374, and MS1) in any region of the genome, unless increased stringency conditions were used. These results suggest that PA21 and PA33 are unique among HAV isolates and may represent a virus native to the owl monkey. Despite extremely poor homology within the P1 region, which encodes capsid polypeptides, monoclonal antibody analysis confirmed that the immunodominant neutralization epitopes of HAV were highly conserved between HM175 and PA21 viruses. Furthermore, experimental challenge of the owl monkey with successive PA33 and HM175 inocula confirmed a high but incomplete degree of cross-protection. Only one of six monkeys previously infected with PA33 developed recurrent hepatitis 28 days after intravenous HM175 challenge, while none of six monkeys previously infected with HM175 had demonstrable hepatitis following PA33 challenge. These data provide molecular evidence for the existence of HAV strains unique to nonhuman primate species and indicate that strict conservation of antigenic function may accompany substantial genetic divergence in HAV.

Animals↗
PubMed1987

Antihypertensive treatment and postprandial blood pressure reduction in the elderly.

Recently it has been demonstrated that blood pressure in the elderly decreases after a meal. To evaluate the influence of antihypertensive treatment on postprandial blood pressure reduction in the elderly, the effects of a breakfast (405 kcal) on blood pressure and heart rate were studied in 15 healthy normotensive elderly subjects (mean age 79.5 +/- 6.0 years), in 10 healthy hypertensive elderly subjects (mean age 80.2 +/- 5.7 years) and in 22 hypertensive elderly subjects (mean age 71.4 +/- 5.0 years) treated with antihypertensive medication (diuretics, beta-blockers, vasodilators). In the three groups there was a fall of mean arterial blood pressure of 9.3 +/- 1.9%, 13.8 +/- 1.9% and 7.9 +/- 1.3%, respectively, at 40 min after the start of the breakfast. In all three groups heart rate increased significantly. It is concluded that antihypertensive treatment with the regimens used in this study does not induce an additional fall of blood pressure postprandially. However, the influence of eating should be avoided in the assessment of antihypertensive drug effects in the elderly.

Adrenergic beta-Antagonists↗
PubMed1986

A complex noncoordinate regulation of alpha-lactalbumin and 25 K beta-casein by corticosterone, prolactin, and insulin in long term cultures of normal rat mammary cells.

The concentrations of PRL, corticosterone, and insulin required by long term cultures of normal rat mammary cells to produce alpha-lactalbumin (alpha LA) and the 25,000 mol wt beta-casein were evaluated with a variety of hormone ratios and concentrations. For these studies a double antibody RIA for beta-casein capable of measuring 0.5 ng beta-casein/100 microliter growth media was developed and used along with our previously reported RIA for alpha LA. PRL was active at physiological levels (0.05-0.15 micrograms/ml) and quantitatively stimulated beta-casein more than alpha LA, whereas physiological levels of corticosterone (0.05-0.15 micrograms/ml) quantitatively stimulated alpha LA more than beta-casein. The concentration of corticosterone greatly altered the magnitude of the cells' response to insulin and PRL for alpha LA output by cells from either virginal or midpregnant rats. Insulin also enhanced production of these milk proteins, but very little effect was measured in the physiological range. alpha LA was increased more by insulin than by PRL, and beta-casein was enhanced more by insulin than by corticosterone. Cells from midpregnant rats required less insulin to stimulate beta-casein production than to stimulate alpha LA. Cells from virginal rats required a supraphysiological insulin level to stimulate both beta-casein and alpha LA under these conditions. These cells generally require 5-6 weeks to achieve a steady-state rate of milk protein output. The complexities of our observations help explain some of the conflicting reports in the literature concerning which hormone is of prime importance for quantitatively increasing the synthesis of a particular milk protein, particularly since high hormone levels are often employed and time in culture varies considerably among reports. We conclude that lower levels of all these hormones can and should be used in vitro. Our messenger RNA (mRNA) studies using cloned complementary DNA probes for two rat casein mRNAs show that cells grown for 2 months with hormones contain significant amounts of both alpha- and beta-casein mRNAs. Simultaneous quantification of beta-casein mRNA levels and rates of beta-casein protein production in these long term cell cultures indicated that a substantial portion of their beta-casein protein production is regulated by the amount of its mRNA. This could be controlled by mRNA synthesis and/or mRNA degradation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

A simple method for clonal selection of hepatitis A virus based on recovery of virus from radioimmunofocus overlays.

Hepatitis A virus (HAV), a non-cytopathic picornavirus, has been quantitated in cell culture by autoradiographic detection of foci of viral replication developing beneath an agarose overlay following fixation and 'staining' of the cell sheet with radiolabelled antibody (radioimmunofocus assay). Using a modification of this basic technique, a clonal variant of HM-175 strain HAV was isolated from agarose overlying individual radioimmunofoci. Virus recovered from the agarose was amplified in small volume cultures of BS-C-1 cells and identified in supernatant culture fluids by cDNA-RNA hybridization. No virus was recovered from agarose which did not overlie a focus of viral replication. This method offers a simple, yet relatively rapid and certain means of selecting clonal variants of non-plaquing viruses such as hepatitis A virus.

Animals↗
PubMed1985

Combined immunoaffinity cDNA-RNA hybridization assay for detection of hepatitis A virus in clinical specimens.

To apply cDNA-RNA hybridization methods to the detection of hepatitis A virus (HAV) in clinical materials, we developed a two-step method in which a microtiter-based, solid-phase immunoadsorption procedure incorporating a monoclonal anti-HAV capture antibody was followed by direct blotting of virus eluates to nitrocellulose and hybridization with 32P-labeled recombinant HAV cDNA. This immunoaffinity hybridization method is simple and involves few sample manipulations, yet it retains high sensitivity (10- to 30-fold more than radioimmunoassay) and is capable of detecting approximately 1 X 10(5) to 2 X 10(5) genome copies of virus. The inclusion of the immunoaffinity step removes most contaminating proteins and thus facilitates subsequent immobilization of the virus for hybridization. It also permits positive hybridization signals to be related to specific antigens and adds a level of specificity to the hybridization procedure. When the method was applied to 23 fecal specimens collected from individuals during week 1 of symptoms due to hepatitis A, 13 specimens were found to be reproducibly positive for HAV RNA by immunoaffinity hybridization, whereas only 11 contained viral antigen detectable by radioimmunoassay.

Animals↗
PubMed1985

Infectious hepatitis A virus particles produced in cell culture consist of three distinct types with different buoyant densities in CsCl.

Although hepatitis A virus (HAV) released by infected BS-C-1 cells banded predominantly at 1.325 g/cm3 (major component) in CsCl, smaller proportions of infectious virions banded at 1.42 g/cm3 (dense HAV particles) and at 1.27 g/cm3 (previously unrecognized light HAV particles). cDNA-RNA hybridization confirmed the banding of viral RNA at each density, and immune electron microscopy demonstrated apparently complete viral particles in each peak fraction. The ratio of the infectivity (radioimmunofocus assay) titer to the antigen (radioimmunoassay) titer of the major component was approximately 15-fold greater than that of dense HAV particles and 4-fold that of light HAV particles. After extraction with chloroform, the buoyant density of light and major component HAV particles remained unchanged, indicating that the lower density of the light particles was not due to association with lipids. Light particles also banded at a lower density (1.21 g/cm3) in metrizamide than did the major component (1.31 g/cm3). Dense HAV particles, detected by subsequent centrifugation in CsCl, were indistinguishable from the major component when first banded in metrizamide (1.31 g/cm3). However, dense HAV particles recovered from CsCl subsequently banded at 1.37 g/cm3 in metrizamide. Electrophoresis of virion RNA under denaturing conditions demonstrated that dense, major-component, and light HAV particles all contained RNA of similar length. Thus, infectious HAV particles released by BS-C-1 cells in vitro consist of three distinct types which band at substantially different densities in CsC1, suggesting different capsid structures with varied permeability to cesium or different degrees of hydration.

Animals↗
PubMed1981

Normal mammary cells in long term culture. I. development of hormone-dependent functional monolayer cultures and assay of alpha-lactalbumin production.

Mammary cells from normal tissue of virginal, pregnant, and lactating rats have been adapted to long term monolayer culture in plastic culture dishes with retention of hormone-responsive functional activity. The addition of PRL, insulin, and corticosterone resulted in an increase in the proportion of epithelial cells. the development of intracellular lipid droplets, and the ordered aggregations of these cells. In the presence of these hormones, the milk protein, alpha-lactalbumin (a-LA), was secreted into the growth medium at rates of 20-100 ng/mg cellular protein . 24 h. A double antibody RIA for a-LA capable of measuring 0.1 ng a-LA/100 microliter growth medium was developed in our laboratory for these studies. Both intracellular and extracellular a-LA fell below detectability within 2-3 weeks after hormone withdrawal. Intracellular a-LA accounted for less than 3% of the total a-LA accumulated in each culture in 24 h. the production rate of cells continuously given hormones increased 4- to 7-fold over a period of several months in culture, and their output was greater than 100-fold above that of cells not given hormones. These cells were obtained by overnight digestion and dispersion of tissue using selected batches of collagenase in the presence of 5% fetal calf serum. Plating densities of at least 3 X 10(4) cells/cm2 in Minimum Essential Medium supplemented with 14% fetal calf serum were required for optimal functional activity. Despite several months without added hormones, these cultures can retain their hormone responsiveness, since subsequent hormone addition resulted in detectable a-LA production beginning within 7-14 days. Our studies demonstrate for the first time that normal mammary cells can be maintained in a functional hormone-responsive state for extended periods in primary cell culture. These long term cell cultures provide a system with which the effects of these and other hormones on milk production and cell differentiation can be assessed under conditions which minimize the influence of the prior in vivo hormonal millieu. (Endocrinology 108: 573, 1981)

Animals↗
PubMed1981

Glucocorticoid regulation of alpha-lactalbumin production in long-term cultures of normal rat mammary cells.

Cultures of normal rat mammary cells have been developed which produce alpha-lactalbumin (a-LA) for several months in response to physiological levels of corticosterone whereas none is measureable without glucocorticoid. In contrast to recent studies in short-term organ cultures, these long-term cell cultures derived from virginal or mid-pregnant rats show no inhibition of a-LA production at high glucocorticoid concentrations. Comparable a-LA production was observed after a few months whether cells were initially cultured into media with various corticosterone levels or initially into high levels of corticosterone for 3 weeks prior to the shift to various lower doses.

Animals↗