Dexamethasone influences human clock gene expression in bronchial epithelium and peripheral blood mononuclear cells in vitro

We determined whether human peripheral blood mononuclear cells (PBMCs) could be used to analyze clock genes by studying their mRNA expressions in human bronchial epithelium (BEAS-2B) and PBMCs following stimulation by the glucocorticoid homologue dexamethasone (DEX) in vitro. PBMCs were obtained at 10:00 h from two diurnally active (∼07:00 to 23:00 h) healthy volunteers and were evaluated for hPer1 mRNA expression following DEX stimulation in vitro using real time-PCR analysis. DEX stimulation of human BEAS-2B cells and PBMCs in vitro led to a remarkable increase of hPer1 mRNA. The glucocorticoid rapidly affected the expression of hPer1 mRNA in PBMCs, suggesting that human PBMCs may be a useful surrogate marker for the investigation of drug effects on clock genes.     

Association of major histocompatibility complex class I haplotypes with disease progression after simian immunodeficiency virus challenge in burmese rhesus macaques

Nonhuman primate AIDS models are essential for the analysis of AIDS pathogenesis and the evaluation of vaccine efficacy. Multiple studies on human immunodeficiency virus and simian immunodeficiency virus (SIV) infection have indicated the association of major histocompatibility complex class I (MHC-I) genotypes with rapid or slow AIDS progression. The accumulation of macaque groups that share not only a single MHC-I allele but also an MHC-I haplotype consisting of multiple polymorphic MHC-I loci would greatly contribute to the progress of AIDS research. Here, we investigated SIVmac239 infections in four groups of Burmese rhesus macaques sharing individual MHC-I haplotypes, referred to as A, E, B, and J. Out of 20 macaques belonging to A(+) (n = 6), E(+) (n = 6), B(+) (n = 4), and J(+) (n = 4) groups, 18 showed persistent viremia. Fifteen of them developed AIDS in 0.5 to 4 years, with the remaining three at 1 or 2 years under observation. A(+) animals, including two controllers, showed slower disease progression, whereas J(+) animals exhibited rapid progression. E(+) and B(+) animals showed intermediate plasma viral loads and survival periods. Gag-specific CD8(+) T-cell responses were efficiently induced in A(+) animals, while Nef-specific CD8(+) T-cell responses were in A(+), E(+), and B(+) animals. Multiple comparisons among these groups revealed significant differences in survival periods, peripheral CD4(+) T-cell decline, and SIV-specific CD4(+) T-cell polyfunctionality in the chronic phase. This study indicates the association of MHC-I haplotypes with AIDS progression and presents an AIDS model facilitating the analysis of virus-host immune interaction.     

tfdA-like genes in 2,4-dichlorophenoxyacetic acid-degrading bacteria belonging to the Bradyrhizobium-Agromonas-Nitrobacter-Afipia cluster in alpha-Proteobacteria

The 2,4-dichlorophenoxyacetate (2,4-D)/alpha-ketoglutarate dioxygenase gene (tfdA) homolog designated tfdAalpha was cloned and characterized from 2,4-D-degrading bacterial strain RD5-C2. This Japanese upland soil isolate belongs to the Bradyrhizobium-Agromonas-Nitrobacter-Afipia cluster in the alpha subdivision of the class Proteobacteria on the basis of its 16S ribosomal DNA sequence. Sequence analysis showed 56 to 60% identity of tfdAalpha to representative tfdA genes. A MalE-TfdAalpha fusion protein expressed in Escherichia coli exhibited about 10 times greater activity for phenoxyacetate than 2,4-D in an alpha-ketoglutarate- and Fe(II)-dependent reaction. The deduced amino acid sequence of TfdAalpha revealed a conserved His-X-Asp-X(146)-His-X(14)-Arg motif characteristic of the active site of group II alpha-ketoglutarate-dependent dioxygenases. The tfdAalpha genes were also detected in 2,4-D-degrading alpha-Proteobacteria previously isolated from pristine environments in Hawaii and in Saskatchewan, Canada (Y. Kamagata, R. R. Fulthorpe, K. Tamura, H. Takami, L. J. Forney, and J. M. Tiedje, Appl. Environ. Microbiol. 63:2266-2272, 1997). These findings indicate that the tfdA genes in beta- and gamma-Proteobacteria and the tfdAalpha genes in alpha-Proteobacteria arose by divergent evolution from a common ancestor.     

Biosynthesis of lactosamine in bovine mammary gland

Lactosamine (beta-D-Galp-(1-->4)-D-GlcN) was isolated from bovine milk sampled after intravenous infusion of glucosamine through the jugular vein of a lactating cow. The chemical structure was established by 2D NMR spectroscopy and electrospray ionisation mass spectrometry (ESIMS). Selected ion monitoring liquid chromatography-mass spectrometry (SIMLC-MS) of the perbenzoylated carbohydrate fraction showed the presence of the novel disaccharide in the milk sample after infusion, but not in the control bovine milk sample. The results showed the uptake of glucosamine in bovine mammary gland, and also indicated that a part of glucosamine was metabolised to the product lactosamine.     

Subunit Interacting Surfaces of Human Hemoglobin in Solution: Localization of the α–β Subunit Interacting Surfaces on the α-Chain by a Comprehensive Synthetic Strategy

By using synthetic overlapping peptides encompassing the entire -chain of adult human hemoglobin (HbA), we have mapped on the -chain the regions responsible for its binding to the -chain in solution. These binding surfaces were, in general, in good agreement with those expected from the crystal structure (peptides 81–95, 101–115, 111–125, and 131–141). However, we observed some significant differences in the levels of binding found here in solution and those expected from the crystal structure. Peptide 31–45, which in the crystal had the highest number of contact residues of all the -chain peptides, did not bind the -chain in solution. Similarly, peptide 91–105, with seven contact residues in the crystal, showed low binding with the -chain in solution. On the other hand, peptides 41–55 and 121–135 possessed much higher binding activity in solution than would be expected from their contribution to subunit association in the crystal. In fact, peptide 121–135 had the highest binding activity of the -chain peptides. These studies and our previous findings, which localized on the -chain the regions that bind to the -chain in solution, have shown that the regions of subunit association in solution are close to, but not identical with, those in the crystal. The approach should be quite useful for mapping subunit association in oligomeric proteins and could even be applied to proteins that are isolated only in traces or whose three-dimensional structure is not yet known.     

Running inhibits osteoporosis induced by protein-deficient (PD) food intake

Running at 0.7 km/h for 10 min every day inhibited development of osteoporosis caused by protein deficient (PD) food intake. Urine alkaline phosphatase (ALP), a marker of bone formation osteoporosis, was not elevated in rats fed PD, when the osteoporosis was inhibited by running. Estrogen supplementation increased bone-breaking energy (BBE), but did not increase bone mineral density (BMD), and did not decrease urinary ALP levels.     

Fluorescent detection of α-aminoadipic and γ-glutamic semialdehydes in oxidized proteins

The oxidative modification of proteins is believed to play a critical role in the etiology and/or progression of several diseases. α-Aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) residues represent major oxidized amino acids generated in oxidized proteins. This paper describes a novel procedure for the specific and sensitive determination of AAS and GGS after their reductive amination with sodium cyanoborohydride and p-aminobenzoic acid, a fluorescence reagent, to their corresponding derivatives, followed by a high-performance liquid chromatography (HPLC) analysis. This fluorescent labeling of protein-associated aldehyde moieties is a simple and accurate technique that may be widely used to reveal increased levels of oxidatively modified proteins with reactive oxygen species during aging and disease.     

Incidence of symptomatic vertebral fractures in women of childbearing age newly treated with high-dose glucocorticoid

Background: The treatment and prevention of glucocorticoid (GC)-induced osteoporosis have been controversial in premenopausal women during their childbearing years.Objective: This study assessed the incidence and risk factors for symptomatic vertebral fracture in women of childbearing age newly treated with high-dose GC.Methods: An observational cohort study was conducted at the rheumatic center of Shimoshizu National Hospital in Chiba, Japan, from 1986 to 2006. The prevalence of symptomatic vertebral fractures, as determined by x-rays, was assessed in premenopausal (aged <50 years) women with collagen vascular disease newly treated with high-dose GC (≥20 mg/d prednisolone equivalent) compared with their counterparts who did not receive GC. Differences in the incidences of vertebral fractures were compared between groups by the Kaplan-Meier method and evaluated by the log-rank test. Hazard ratios (HRs) with 95% CIs were estimated using the Cox proportional hazards regression model.Results: A total of 373 women were assessed: 292 patients in the high-dose GC treatment group (mean [SD] initial age, 32.4 [8.2] years; initial dose, 43.8 [14.9] mg/d; follow-up time, 124.2 [75.4] months) and 81 patients in the non-GC control group (initial age, 39.3 [7.8] years; follow-up time, 106.5 [79.7] months). Symptomatic vertebral fractures occurred more frequently in the high-dose GC group (11.3%) than in the non-GC group (1.2%). Using the Cox model, the adjusted HR for the high-dose GC group was 13.96 (95% CI, 1.87–104.22) relative to the non-GC group. In the high-dose GC group, Kaplan-Meier analyses revealed that the incidence of fractures in women in their forties was significantly higher in comparison with those in their twenties (P < 0.001) and thirties (P < 0.05), and that the incidence of fractures in those who consumed alcohol (>80 g/wk of pure alcohol) was significantly higher than in those who did not (P < 0.05). The Cox model also revealed that the risk was independently higher with every 10-year increment of initial age (HR = 2.27; 95% CI, 1.46–3.53), with every GC dose increase (HR = 2.28; 95% CI, 1.58–3.31), and with each 1-gram decrease of cumulative GC dose (HR = 0.95; 95% CI, 0.93–0.98).Conclusions: In this study, high-dose GC use was associated with a significantly high prevalence of symptomatic vertebral fractures in premenopausal women with collagen vascular disease during their childbearing years. However, the fracture risk was relatively low in women of childbearing age, especially those in their twenties and thirties during the early years of treatment.     

Lipopolysaccharide Decreases Single Immunoglobulin Interleukin-1 Receptor-related Molecule (SIGIRR) Expression by Suppressing Specificity Protein 1 (Sp1) via the Toll-like Receptor 4 (TLR4)-p38 Pathway in Monocytes and Neutrophils

Single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) is one of the immunoglobulin-like membrane proteins that is crucial for negative regulation of toll-like receptor 4 (TLR4) and interleukin-1 receptor. Despite the importance of understanding its expression and function, knowledge is limited on the regulatory mechanism in the epithelial tissues, such as the liver, lung, and gut, where its predominant expression is originally described. Here, we found expression of SIGIRR in non-epithelial innate immune cells, including primary peripheral blood monocytes, polymorphonuclear neutrophils, monocytic RAW264 cells, and neutrophilic-differentiated HL-60 cells. Consistent with previous findings in epithelial tissues, SIGIRR gene and protein expression were also down-regulated by LPS treatment in a time-dependent manner in primary blood monocytes and polymorphonuclear neutrophils. A reduction was also observed in RAW264 and differentiated HL-60 cells. Notably, exogenous introduction of the dominant negative form of TLR4 and siRNA of p38 resulted in inhibition of LPS-induced SIGIRR down-regulation, whereas treatment with p38 activator anisomycin showed a dose-dependent decrease in SIGIRR expression, suggesting TLR4-p38 signal as a critical pathway for LPS-induced SIGIRR down-regulation. Finally, reporter gene and chromatin immunoprecipitation assays demonstrated that Sp1 is a key factor that directly binds to the proximal promoter of SIGIRR gene and consequently regulates basal SIGIRR expression, which is negatively regulated by the LPS-dependent TLR4-p38 pathway. In summary, the data precisely demonstrate how LPS down-regulates SIGIRR expression and provide a role of LPS signal that counteracts Sp1-dependent basal promoter activation of SIGIRR gene via TLR4-p38 pathway in non-epithelial innate immune cells.     

Identification of genes that function in the TNF-alpha-mediated apoptotic pathway using randomized hybrid ribozyme libraries

Now that the sequences of many genomes are available, methods are required for the rapid identification of functional genes. We describe here a simple system for the isolation of genes that function in the tumor necrosis factor-alpha (TNF-alpha)-mediated pathway of apoptosis, using RNA helicase-associated ribozyme libraries with randomized substrate-binding arms. Because target-site accessibility considerably limits the effective use of intracellular ribozymes, the effectiveness of a conventional ribozyme library has been low. To overcome this obstacle, we attached to ribozymes an RNA motif (poly(A)-tail) able to interact with endogenous RNA helicase(s) so that the resulting helicase-attached, hybrid ribozymes can more easily attack target sites regardless of their secondary or tertiary structures. When the phenotype of cells changes upon introduction of a ribozyme library, genes responsible for these changes may be identified by sequencing the active ribozyme clones. In the case of TNF-alpha-mediated apoptosis, when a ribozyme library was introduced into MCF-7 cells, surviving clones were completely or partially resistant to TNF-alpha-induced apoptosis. We identified many pro-apoptotic genes and partial sequences of previously uncharacterized genes using this method. Our gene discovery system should be generally applicable to the identification of functional genes in various systems.