A cell-based reporter assay for the identification of protein kinase C activators and inhibitors

Transmission of extra cellular signals across biological membranes results in the generation of lipid metabolites which in turn influence specific cellular events such as cell growth or differentiation. Many of these lipid messengers can activate protein kinase C (PKC) isozymes of which one function is to perpetuate the extracellular signals to the nucleus by phosphorylating other targets proteins. We have engineered mammalian cell lines to identify and evaluate activators and inhibitors of PKC-dependent and independent signal transduction pathways. The A31 mouse fibroblast cell line, has been stably transfected with a construct containing a triplet repeat of the TPA response element (TRE) upstream of a thymidine kinase promoter fused to the human growth hormone (hGH) gene. A31 cells containing this reporter construct exhibit significant increases in hGH secretion following stimulation by phorbol esters or other mitogens. The levels of hGH secretion are modulated in this system using different pharmacological agents. We demonstrate that this assay can be used to identify specific and general inhibitors as well as activators of the signal transduction pathway mediated by PKC isozymes. (Mol Cell Biochem141: 129–134, 1994)     

Modeling an immune response to influenza A virus infection in alveolar epithelial cells

Influenza A viruses (IAV) have been the cause of several influenza pandemics in history and are a significant threat for the next global pandemic. Hospitalized influenza patients often have excess interferon production and a dysregulated immune response to the IAV infection. Obtaining a better understanding of the mechanisms of IAV infection that induce these harmful effects would help drug developers and health professionals create more effective treatments for IAV infection and improve patient outcomes. IAV stimulates viral sensors and receptors expressed by alveolar epithelial cells, like RIG-I and toll-like receptor 3 (TLR3). These two pathways coordinate with one another to induce expression of type III interferons to combat the infection. Presented here is a queuing theory-based model of these pathways that was designed to analyze the timing and amount of interferons produced in response to IAV single stranded RNA and double-stranded RNA detection. The model accurately represents biological data showing the necessary coordination of the RIG-I and TLR3 pathways for effective interferon production. This model can serve as the framework for future studies of IAV infection and identify new targets for potential treatments.     

Volatile oils of mature and juvenile leaves of Juniperus horizontalis: Chemosystematic significance

Comparison of 39 terpenoids between young (juvenile) foliage and mature (adult) foliage from naturally growing plants of Juniperus horizontalis revealed no significant differences. Canonical variate analysis of the terpenoids of J. scopulorum and J. virginiana along with the mature foliage of J. horizontalis and co-plotting juvenile foliage showed a slight loosening of the J. horizontalis group but not enough to blur taxonomic distinctions. These results stand in sharp contrast with the previous work on J. scopulorum and appear to be due to the indeterminant growth pattern seen in J. horizontalis.     

Influence of plasmids carrying the lexA gene on DNA repair and related processes in Escherichia coli K-12

Summary Plasmid pLC44-14 from the Clarke and Carbon collection has been shown to carry the lexA gene. The presence of lexA was demonstrated by complementation of tsl mutants which lie close to lexA on the E. coli K-12 linkage map and are probably in the lexA gene, and by crossing the dominant lexA mutation on to pLC44-14 to produce a recombinant plasmid, pSEl, which gave the host cell the properties of a lexA mutant. The lexA gene has been cloned on to pBR322 (Little, 1980). pJL21, which carries the lexA ⁺ gene, rendered the host cell moderately sensitive to UV light, greatly reduced the extent of Weigle reactivation and mutagenesis of UV-irradiated phage , and inhibited induction of protein X by either UV light or nalidixic acid. A similar plasmid carrying a mutant lexA3 allele produced extreme sensitivity to UV light, reduced recombinant production 10 to 50-fold following Hfr x F^– conjugation crosses, and otherwise mimicked the effects of pJL21. Introduction of an amber mutation into the lexA gene carried by the plasmid greatly reduced the UV-sensitivity of the host, thereby indicating that the extreme sensitivity was due to the mutant lexA gene product. These properties of strains with lexA plasmids are thought to originate from high levels of the lexA protein in the cell due to a large plasmid copy number. This protein, which appears from other studies to regulate negatively the recA gene, may inhibit expression of recA or other DNA repair genes when present in excess amounts in the cell.     

Wood construction more strongly shapes deadwood microbial communities than spatial location over 5 years of decay

Diverse communities of fungi and bacteria in deadwood mediate wood decay. While rates of decomposition vary greatly among woody species and spatially distinct habitats, the relative importance of these factors in structuring microbial communities and whether these shift over time remains largely unknown. We characterized fungal and bacterial diversity within pieces of deadwood that experienced 6.3–98.8% mass loss while decaying in common garden ‘rotplots’ in a temperate oak-hickory forest in the Ozark Highlands, MO, USA. Communities were isolated from 21 woody species that had been decomposing for 1–5 years in spatially distinct habitats at the landscape scale (top and bottom of watersheds) and within stems (top and bottom of stems). Microbial community structure varied more strongly with wood traits than with spatial locations, mirroring the relative role of these factors on decay rates on the same pieces of wood even after 5 years. Co-occurring fungal and bacterial communities persistently influenced one another independently from their shared environmental conditions. However, the relative influence of wood construction versus spatial locations differed between fungi and bacteria, suggesting that life history characteristics of these clades structure diversity differently across space and time in decomposing wood.     

The Yeast Oxysterol Binding Protein Kes1 Maintains Sphingolipid Levels

The oxysterol binding protein family are amphitropic proteins that bind oxysterols, sterols, and possibly phosphoinositides, in a conserved binding pocket. The Saccharomyces cerevisiae oxysterol binding protein family member Kes1 (also known as Osh4) also binds phosphoinositides on a distinct surface of the protein from the conserved binding pocket. In this study, we determine that the oxysterol binding protein family member Kes1 is required to maintain the ratio of complex sphingolipids and levels of ceramide, sphingosine-phosphate and sphingosine. This inability to maintain normal sphingolipid homeostasis resulted in misdistribution of Pma1, a protein that requires normal sphingolipid synthesis to occur to partition into membrane rafts at the Golgi for its trafficking to the plasma membrane.     

Constant light alters serum hormone levels related to thyroid function in male CD-1 mice

ABSTRACT

Disruptions to the circadian rhythm can lead to altered metabolism. Modification of thyroid function may be a reason why circadian misalignment may contribute to future metabolic disorders. We investigated whether circadian disruption through constant light (LL) can lead to variations in hormone levels associated with thyroid function. Mice were exposed to LL or a 12:12 Light:Dark (LD) cycle for 6 weeks; then glucose tolerance and thyroid hormone levels were measured at ZT 6 and ZT 18. There was day/night variation in glucose tolerance, but LL had no effect. LL reduced TSH, increased fT4, and abolished day/night variation in fT3 and leptin. These findings illustrate that LL alters thyroid-related hormones, providing evidence of a link between circadian disruption and thyroid function.     

Female parity, male aggression, and the Challenge Hypothesis in wild chimpanzees

The Challenge Hypothesis proposes that testosterone mediates aggression during periods of heightened conflict between males, especially episodes that have important fitness consequences. Considerable evidence from seasonally breeding species provides support for this hypothesis, but few data exist in animals that mate year-round. We tested predictions generated by the Challenge Hypothesis in chimpanzees, a non-seasonally breeding primate, through a study of individuals living in an exceptionally large community at Ngogo, Kibale National Park, Uganda. Results indicated that dominance rank had no influence on testosterone levels. Instead of rank influencing testosterone production, additional analyses revealed an important role for reproductive competition. Male chimpanzees displayed more aggression when they were in the same party as parous estrous females than when reproductively active females were unavailable. Male chimpanzees competed more intensely for mating opportunities with parous females than with nulliparas, and as a consequence, males displayed more aggression around the former than the latter. When males accompanied parous estrous females, their urinary testosterone concentrations were significantly higher than baseline concentrations. In contrast, urinary testosterone concentrations did not exceed baseline when males associated with nulliparous estrous females. These differences in testosterone levels could not be attributed to mating per se because males copulated equally often with parous and nulliparous females. Furthermore, variation in testosterone concentrations were not due to males gathering together in large parties, as their levels in these situations did not exceed baseline. Taken together, these findings, derived from a relatively large sample of males and estrous females, replicate those from a prior study and furnish additional support for the Challenge Hypothesis. Our results suggest that the Challenge Hypothesis is likely to be broadly applicable to chimpanzees and increase our understanding of the physiological costs to males who compete for estrous females.     

Chronic senolytic treatment alleviates established vasomotor dysfunction in aged or atherosclerotic mice

While reports suggest a single dose of senolytics may improve vasomotor function, the structural and functional impact of long‐term senolytic treatment is unknown. To determine whether long‐term senolytic treatment improves vasomotor function, vascular stiffness, and intimal plaque size and composition in aged or hypercholesterolemic mice with established disease. Senolytic treatment (intermittent treatment with Dasatinib + Quercetin via oral gavage) resulted in significant reductions in senescent cell markers (TAF⁺ cells) in the medial layer of aorta from aged and hypercholesterolemic mice, but not in intimal atherosclerotic plaques. While senolytic treatment significantly improved vasomotor function (isolated organ chamber baths) in both groups of mice, this was due to increases in nitric oxide bioavailability in aged mice and increases in sensitivity to NO donors in hypercholesterolemic mice. Genetic clearance of senescent cells in aged normocholesterolemic INK‐ATTAC mice phenocopied changes elicited by D+Q. Senolytics tended to reduce aortic calcification (alizarin red) and osteogenic signaling (qRT–PCR, immunohistochemistry) in aged mice, but both were significantly reduced by senolytic treatment in hypercholesterolemic mice. Intimal plaque fibrosis (picrosirius red) was not changed appreciably by chronic senolytic treatment. This is the first study to demonstrate that chronic clearance of senescent cells improves established vascular phenotypes associated with aging and chronic hypercholesterolemia, and may be a viable therapeutic intervention to reduce morbidity and mortality from cardiovascular diseases.