Crystal structure of unautoprocessed precursor of subtilisin from a hyperthermophilic archaeon: evidence for Ca2+-induced folding

The crystal structure of an active site mutant of pro-Tk-subtilisin (pro-S324A) from the hyperthermophilic archaeon Thermococcus kodakaraensis was determined at 2.3 A resolution. The overall structure of this protein is similar to those of bacterial subtilisin-propeptide complexes, except that the peptide bond linking the propeptide and mature domain contacts with the active site, and the mature domain contains six Ca2+ binding sites. The Ca-1 site is conserved in bacterial subtilisins but is formed prior to autoprocessing, unlike the corresponding sites of bacterial subtilisins. All other Ca2+-binding sites are unique in the pro-S324A structure and are located at the surface loops. Four of them apparently contribute to the stability of the central alphabetaalpha substructure of the mature domain. The CD spectra, 1-anilino-8-naphthalenesulfonic acid fluorescence spectra, and sensitivities to chymotryptic digestion of this protein indicate that the conformation of pro-S324A is changed from an unstable molten globule-like structure to a stable native one upon Ca2+ binding. Another active site mutant, pro-S324C, was shown to be autoprocessed to form a propeptide-mature domain complex in the presence of Ca2+. The CD spectra of this protein indicate that the structure of pro-S324C is changed upon Ca2+ binding like pro-S324A but is not seriously changed upon subsequent autoprocessing. These results suggest that the maturation process of Tk-subtilisin is different from that of bacterial subtilisins in terms of the requirement of Ca2+ for folding of the mature domain and completion of the folding process prior to autoprocessing.     

Regulation of cyclic longitudinal mechanical stretch on proliferation of human bone marrow mesenchymal stem cells

Mechanical stimulation is critical to both physiological and pathological states of living cells. Although a great deal of research has been done on biological and biochemical regulation of the behavior of bone marrow mesenchymal stem cells (MSCs), the influence of biomechanical factors on their behavior is still not fully documented. In this study, we investigated the modulation of mechanical stretch magnitude, frequency, and duration on the human marrow mesenchymal stem cells (hMSCs) proliferation by an in vitro model system using a mechanical stretch loading apparatus, and optimized the stretch regime for the proliferation of hMSCs. We applied 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrasodium bromide (MTT) assay to estimate the overall proliferative effects of the stretch on hMSCs. We found that fibronectin coating increased adhesion to silicone chamber surface, however, it did not show significant effect on proliferation of hMSCs. A frequency of 1 Hz was more effective in stimulating hMSCs proliferation. At 1 Hz, 5% strain for 15, 30, 60 min, the significant increase of hMSCs proliferation was observed. Proliferation was enhanced at 1 Hz, 10% strain for 15, 30 min, while decreased for 60 min. At 1 Hz, 15% strain, 15 min stretch resulted in the decrease of proliferation, and 30 min and 60 min stretch showed an increased proliferation. Long time (12 and 24 h) strain application blocked the proliferation. These results indicate that mechanical stretch plays an important role in hMSCs growth and proliferation; an appropriate mechanical stretch regime could be a novel approach to promoting proliferation of hMSCs in vitro.     

Redox imbalance induces remodeling of glucose metabolism in Rhipicephalus microplus embryonic cell line

Carbohydrate metabolism not only functions in supplying cellular energy but also has an important role in maintaining physiological homeostasis and in preventing oxidative damage caused by reactive oxygen species. Previously, we showed that arthropod embryonic cell lines have high tolerance to H₂O₂ exposure. Here, we describe that Rhipicephalus microplus tick embryonic cell line (BME26) employs an adaptive glucose metabolism mechanism that confers tolerance to hydrogen peroxide at concentrations too high for other organisms. This adaptive mechanism sustained by glucose metabolism remodeling promotes cell survival and redox balance in BME26 cell line after millimolar H₂O₂ exposure. The present work shows that this tick cell line could tolerate high H₂O₂ concentrations by initiating a carbohydrate-related adaptive response. We demonstrate that gluconeogenesis was induced as a compensation strategy that involved, among other molecules, the metabolic enzymes NADP-ICDH, G6PDH, and PEPCK. We also found that this phenomenon was coupled to glycogen accumulation and glucose uptake, supporting the pentose phosphate pathway to sustain NADPH production and leading to cell survival and proliferation. Our findings suggest that the described response is not atypical, being also observed in cancer cells, which highlights the importance of this model to all proliferative cells. We propose that these results will be useful in generating basic biological information to support the development of new strategies for disease treatment and parasite control.     

Phylogenetic position of Rickettsia tsutsugamushi and the relationship among its antigenic variants by analyses of 16S rRNA gene sequences

Abstract The 16S rRNA gene sequences of Rickettsia tsutsugamushi and Rickettsia sibirica were determined by PCR and DNA sequencing. Phylogenetic analysis revealed that R. sibirica is positioned in a cluster of the genus Rickettsia with a similarity value of 98.1–99.6%, whereas R. tsutsugamushi is located apart from the cluster with a similarity value of 90.2–90.6%. This evidence suggests that R. tsutsugamushi should be excluded taxonomically from the genus Rickettsia . The phylogenetic classification of six antigenic variants in R. tsutsugamushi moderately reflected their antigenic relationship known in closely and distantly related strains.     

RU486 inhibits induction of aromatase by dexamethasone via glucocorticoid receptor in cultured human skin fibroblasts

Effects of RU486 on the induction of aromatase by dexamethasone via glucocorticoid receptor were determined using cultured human skin fibroblasts. Competition of [3H]dexamethasone binding to the cytosol receptor was 7 times stronger with RU486 than with dexamethasone. The order of the strength of competition was RU486 greater than dexamethasone greater than betamethasone greater than prednisolone greater than hydrocortisone. RU486 abolished a specific 8.6 S [3H]dexamethasone binding peak in the cytosol, determined using a sucrose density gradient analysis. Dexamethasone markedly induced aromatase and this event was strongly suppressed by RU486, in a dose-dependent manner, in the cultured skin fibroblasts. A linear correlation between the strength of competition and the induction of aromatase of various glucocorticoids was observed. RU486 non-competitively inhibited aromatase induction by dexamethasone determined from a double reciprocal plot of aromatase activity, with respect to [3H]androstenedione concentration in the presence of RU486. These results show that RU486 is a peripheral noncompetitive antiglucocorticoid on aromatase induction by glucocorticoid in human skin fibroblasts and that aromatase induction is a good marker for the biological function of glucocorticoid receptor in human skin fibroblasts.     

Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.     

Systematic search for single nucleotide polymorphisms in the insulin gene: evidence for a high frequency of -23T-->A in Japanese subjects

It has recently been shown that the A/A genotype at g.-23 of the insulin gene correlates with impaired insulin secretion in response to body weight gain in subjects of European descent. To examine whether there are single nucleotide polymorphisms (SNPs) in the insulin gene associated with type 2 diabetes, all exons with their flanking sequences for 113 Japanese type 2 diabetic patients and 99 nondiabetic control subjects were analyzed using PCR direct sequencing. We have only found g.-23T --> A, 806G --> C, 1128T --> C, and 1141A --> C, which have previously been reported in alpha (A-C-C-C) and beta (T-G-T-A) alleles. The allele frequency of -23T --> A in control Japanese subjects was 97.4%, whereas that in Europeans is about 30%. The A/A genotype was found in 94 of 99 Japanese subjects (94.9%) and the allele frequencies of 806G --> C, 1128T --> C, and 1141A --> C were all 96.5%. The estimated haplotype frequencies were (A-C-C-C) (96.0%), (T-G-T-A) (2.0%), (A-G-T-A) (1.5%), and (T-C-C-C) (0.5%). No association of these SNPs or haplotypes with type 2 diabetes was evident. Thus, the A/A genotype at the g.-23 of insulin gene was generally high in Japanese subjects, which could account for the fact that they typically secrete lower levels of insulin.     

Translesion DNA synthesis catalyzed by human pol eta and pol kappa across 1,N6-ethenodeoxyadenosine

1,N(6)-Ethenodeoxyadenosine, a DNA adduct generated by exogenous and endogenous sources, severely blocks DNA synthesis and induces miscoding events in human cells. To probe the mechanism for in vivo translesion DNA synthesis across this adduct, in vitro primer extension studies were conducted using newly identified human DNA polymerases (pol) eta and kappa, which have been shown to catalyze translesion DNA synthesis past several DNA lesions. Steady-state kinetic analyses and analysis of translesion products have revealed that the synthesis is >100-fold more efficient with pol eta than with pol kappa and that both error-free and error-prone syntheses are observed with these enzymes. The miscoding events include both base substitution and frameshift mutations. These results suggest that both polymerases, particularly pol eta, may contribute to the translesion DNA synthesis events observed for 1,N(6)-ethenodeoxyadenosine in human cells.     

Asialoglycoprotein receptor deficiency in mice lacking the major receptor subunit. Its obligate requirement for the stable expression of oligomeric receptor

The asialoglycoprotein receptor is an abundant hetero-oligomeric endocytic receptor that is predominantly expressed on the sinusoidal surface of the hepatocytes. A number of physiological and pathophysiological functions have been ascribed to this hepatic lectin (HL), the removal of desialylated serum glycoproteins and apoptotic cells, clearance of lipoproteins, and the sites of entry for hepatotropic viruses. The assembly of two homologous subunits, HL-1 and HL-2, is required to form functional, high affinity receptors on the cell surface. However, the importance of the individual subunits for receptor transport to the cell surface is controversial. We have previously generated HL-2-deficient mice and showed that the expression of HL-1 was significantly reduced, and the functional activity as the asialoglycoprotein receptor was virtually eliminated. However, we failed to detect phenotypic abnormalities. To explore the significance of the major HL-1 subunit for receptor expression and function in vivo, we have disrupted the HL-1 gene in mice. Homozygous HL-1-deficient animals are superficially normal. HL-2 expression in the liver is virtually abrogated, indicating that HL-1 is strictly required for the stable expression of HL-2. Although these mice are almost unable to clear asialo-orosomucoid, a high affinity ligand for asialoglycoprotein receptor, they do not accumulate desialylated glycoproteins or lipoproteins in the plasma.