An estrogen responsive primary amphibian liver cell culture system

Amphibian hepatocytes have been prepared in both high yield and purity using a collagenase perfusion technique. The isolated cells attach efficiently in serum-free medium to collagen-coated culture dishes and subsequently form monolayers. These cultures can be maintained in an appropriate medium for over one week with minimal cell loss. The nuclear labelling index of cells exposed to [³H]thymidine indicates a very low level of cell growth. Twenty-four hour exposure to dexamethasone induces tyrosine aminotransferase activity throughout the culture period. Monolayers incorporate [³H]leucine linearly into acid-insoluble material with approx. 40% of all synthesis devoted to secreted protein. Polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate shows the majority of proteins present in whole serum are synthesized and secreted by the cultured hepatocytes. The absolute rate of protein secretion on the first day of culture is approx. 73 μg/day/mg cell protein which subsequently declines and plateaus at 30% of this level by the 4th–5th day of culture. However, when hepatocytes are cultured in the continued presence of insulin, the drop in protein secretion is completely inhibited.Cultures of hepatocytes isolated from female frogs and subsequently exposed to 17-B estradiol in culture, synthesize and secrete the egg-yolk protein precursor vitellogenin. The protein initially appears as a minor component in the medium 1–2 days after hormone addition. Its rate of synthesis, relative to other secreted proteins, increases with time so that it ultimately constitutes the majority of protein being exported after 6 days of treatment. Parallel with vitellogenin induction is an increase in rate of total protein secretion reaching a 2-fold increase at maximal stimulation.The results show that viable, monolayer cultures of amphibian hepatocytes can be prepared which retain the ability to respond directly to added estrogen by synthesizing vitellogenin.     

Enhancement in rats by the liver tumor promoter ethinyl estradiol of a serum factor(s) which is stimulatory for hepatocyte DNA synthesis

Fractionation of female rat serum or plasma on Sephadex G-200 revealed the presence of an activity stimulatory for hepatocyte DNA synthesis. Treatment of female rats with the liver tumor promoter ethinyl estradiol (EE) at 2.5 micrograms/day caused a 1.6 fold increase in the level of this activity at 24 hr in both serum and plasma. The stimulatory activity had a molecular weight of 135 kD, was sensitive to trypsin and heating and was not inhibited by the antiestrogen tamoxifen or antibody to epidermal growth factor (EGF). However, the pooled active fractions from EE-treated rats competed to a greater extent than comparable fractions from control rats for specific [125I]-EGF binding to rat liver membranes. These results demonstrate that treatment of female rats with EE, under conditions known to stimulate liver growth, caused an increase in level of a factor(s) stimulatory for hepatocyte DNA synthesis and whose activity may be mediated through the EGF receptor.     

Impedance of the chest wall during sustained respiratory muscle contraction

We measured total chest wall impedance (Zw), "pathway impedances" of the rib cage (Zrcpath), and diaphragm-abdomen (Zd-apath), and impedance of the belly wall including abdominal contents (Zbw+) in five subjects during sustained expiratory (change in average pleural pressure [Ppl] from relaxation = 10 and 20 cmH2O) and inspiratory (change in Ppl = -10 and -20 cmH2O) muscle contraction, using forced oscillatory techniques (0.5-4 Hz) we have previously reported for relaxation (J. Appl. Physiol. 66: 350-359, 1989). Chest wall configuration and mean lung volume were kept constant. Zw, Zrcpath, Zd-apath, and Zbw+ all increased greatly at each frequency during expiratory muscle contraction; increases were proportional to effort. Zw, Zrcpath, and Zd-apath increased greatly during inspiratory muscle contraction, but Zbw+ did not. Resistances and elastances calculated from each of the impedances showed the same changes during muscle contraction as the corresponding impedances. Each of the resistances decreased as frequency increased, independent of effort; elastances generally increased with frequency. These frequency dependencies were similar to those measured in relaxed or tetanized isolated muscle during sinusoidal stretching (P.M. Rack, J. Physiol. Lond. 183: 1-14, 1966). We conclude that during respiratory muscle contraction 1) chest wall impedance increases, 2) changes in regional chest wall impedances can be somewhat independent, depending on which muscles contract, and 3) increases in chest wall impedance are due, at least in part, to changes in the passive properties of the muscles themselves.     

Genetic Analysis of Suppressors of the Vea1 Mutation in Aspergillus Nidulans

Light-dependent conidiation in the filamentous ascomycete, Aspergillus nidulans, is contingent on the allelic state of the velvet (veA) gene. Light dependence is abolished by a mutation in this gene (veA1), which allows conidiation to occur in the absence of light. We have isolated and characterized six extragenic suppressors of veA1 that restore the light-dependent conidiation phenotype. Alleles of four genes, defined by complementation tests, were subjected to extensive genetic and phenotypic analysis. The results of light-dark shifting experiments and the phenotypes of double mutant combinations are consistent with the possibility that the expression of the light-dependent phenotype is regulated by specific interactions of the suppressor gene products with the velvet gene product and with each other.     

Mucolipidoses II and III variants with normal N-acetylglucosamine 1-phosphotransferase activity toward alpha-methylmannoside are due to nonallelic mutations.

Normal N-acetylglucosamine 1-phosphotransferase activity toward mono- and oligosaccharide acceptor substrates was detected in cultured skin fibroblasts from mucolipidoses II and III patients who were designated as variants (one of four mucolipidosis II and three out of six mucolipidosis III patients examined). The activity toward natural lysosomal protein acceptors was absent or deficient in cell preparations from all patients with classical as well as variant forms of mucolipidoses II and III. Complementation analysis, using fused and cocultivated mutant fibroblast combinations, revealed that, while cell lines with variant mucolipidosis III constituted a complementation group distinct from that of classical forms of mucolipidoses II and III, the variant mucolipidosis II cell line belonged to the same complementation group as did the classical forms. In contrast to the mutant enzyme from variant mucolipidosis III patients that failed to recognize lysosomal proteins as the specific acceptor substrates, the activity toward alpha-methylmannoside in the variant mucolipidosis II patient could be inhibited by exogenous lysosomal enzyme preparations (bovine beta-glucuronidase and human hexosaminidase A). These findings suggest that N-acetylglucosamine 1-phosphotransferase is composed of at least two distinct polypeptides: (1) a recognition subunit that is defective in the mucolipidosis III variants and (2) a catalytic subunit that is deficient or altered in the classical forms of mucolipidoses II and III as well as in the mucolipidosis II variant.     

Differential expression of cholesterol hydroxylases in Alzheimer's disease

Cholesterol is eliminated from neurons by oxidization, which generates oxysterols. Cholesterol oxidation is mediated by the enzymes cholesterol 24-hydroxylase (CYP46A1) and cholesterol 27-hydroxylase (CYP27A1). Immunocytochemical studies show that CYP46A1 and CYP27A1 are expressed in neurons and some astrocytes in the normal brain, and CYP27A1 is present in oligodendrocytes. In Alzheimer's disease (AD), CYP46A1 shows prominent expression in astrocytes and around amyloid plaques, whereas CYP27A1 expression decreases in neurons and is not apparent around amyloid plaques but increases in oligodendrocytes. Although previous studies have examined the effects of synthetic oxysterols on the processing of amyloid precursor protein (APP), the actions of the naturally occurring oxysterols have yet to be examined. To understand the role of cholesterol oxidation in AD, we compared the effects of 24(S)- and 27-hydroxycholesterol on the processing of APP and analyzed the cell-specific expression patterns of the two cholesterol hydroxylases in the human brain. Both oxysterols inhibited production of Abeta in neurons, but 24(S)-hydroxycholesterol was approximately 1000-fold more potent than 27-hydroxycholesterol. The IC(50) of 24(S)-hydroxycholesterol for inhibiting Abeta secretion was approximately 1 nm. Both oxysterols induced ABCA1 expression with IC(50) values similar to that for inhibition of A beta secretion, suggesting the involvement of liver X receptor. Oxysterols also inhibited protein kinase C activity and APP secretion following stimulation of protein kinase C. The selective expression of CYP46A1 around neuritic plaques and the potent inhibition of APP processing in neurons by 24(S)-hydroxycholesterol suggests that CYP46A1 affects the pathophysiology of AD and provides insight into how polymorphisms in the CYP46A1 gene might influence the pathophysiology of this prevalent disease.     

Structure-based design, synthesis, and antimicrobial activity of purine derived SAH/MTA nucleosidase inhibitors

The structure-based design, synthesis, and biological activity of novel inhibitors of S-adenosyl homocysteine/methylthioadenosine (SAH/MTA) nucleosidase are described. Using 6-substituted purine and deaza purines as the core scaffolds, a systematic and structure guided series of modifications provided low nM inhibitors with broad-spectrum antimicrobial activity.     

Rabies in an American bison from North Dakota

In North Dakota (USA) during April 1998, a ranched female bison (Bison bison) was found dead. At gross necropsy, there was profound hair loss and consolidated lung lobes. Intracytoplasmic neuronal inclusions suggestive of Negri bodies were observed in the brain stem and hippocampus, and a diagnosis of rabies was confirmed by the fluorescent antibody test. Antigenic typing demonstrated the occurrence of a rabies virus variant associated with skunks from the upper midwestern USA. This case of a rabid bison was one of only four such instances recorded from the USA over the past 40 yr, and is the first case report of rabies in a bison that reports clinical, pathologic, and antigenic findings. Although rabies in bison is rare, veterinarians and wildlife managers that work closely with such non-traditional species are reminded of the dangers that zoonoses such as rabies present.     

Therapeutic DNA vaccine induces broad T cell responses in the gut and sustained protection from viral rebound and AIDS in SIV-infected rhesus macaques

Immunotherapies that induce durable immune control of chronic HIV infection may eliminate the need for life-long dependence on drugs. We investigated a DNA vaccine formulated with a novel genetic adjuvant that stimulates immune responses in the blood and gut for the ability to improve therapy in rhesus macaques chronically infected with SIV. Using the SIV-macaque model for AIDS, we show that epidermal co-delivery of plasmids expressing SIV Gag, RT, Nef and Env, and the mucosal adjuvant, heat-labile E. coli enterotoxin (LT), during antiretroviral therapy (ART) induced a substantial 2-4-log fold reduction in mean virus burden in both the gut and blood when compared to unvaccinated controls and provided durable protection from viral rebound and disease progression after the drug was discontinued. This effect was associated with significant increases in IFN-γ T cell responses in both the blood and gut and SIV-specific CD8+ T cells with dual TNF-α and cytolytic effector functions in the blood. Importantly, a broader specificity in the T cell response seen in the gut, but not the blood, significantly correlated with a reduction in virus production in mucosal tissues and a lower virus burden in plasma. We conclude that immunizing with vaccines that induce immune responses in mucosal gut tissue could reduce residual viral reservoirs during drug therapy and improve long-term treatment of HIV infection in humans.