Hepatitis B Virus (HBV) Surface Antigen Interacts with and Promotes Cyclophilin A Secretion: Possible Link to Pathogenesis of HBV Infection

Cyclophilin A (CypA), predominantly located intracellularly, is a multifunctional protein. We previously reported decreased CypA levels in hepatocytes of transgenic mice expressing hepatitis B virus (HBV) surface antigen (HBsAg). In this study, we found that expression of HBV small surface protein (SHBs) in human hepatoma cell lines specifically triggered CypA secretion, whereas SHBs added extracellularly to culture medium did not. Moreover, CypA secretion was not promoted by the expression of a secretion deficient SHBs mutant, suggesting a close association between secretion of CypA and SHBs. Interaction between CypA and SHBs was observed by using coimmunoprecipitation and glutathione S-transferase pull-down assays. Hydrodynamic injection of the SHBs expression construct into C57BL/6J mice resulted in increased serum CypA levels and ALT/AST levels, as well as the infiltration of inflammatory cells surrounding SHBs-positive hepatocytes. The inflammatory response and serum ALT/AST level were reduced when the chemotactic effect of CypA was inhibited by cyclosporine and anti-CD147 antibody. Furthermore, higher serum CypA levels were detected in chronic hepatitis B patients than in healthy individuals. In HBV patients who had received liver transplantation, serum CypA levels declined dramatically after the loss of HBsAg as a consequence of liver transplantation. Taken together, these results indicate that expression and secretion of SHBs can promote CypA secretion, which may contribute to the pathogenesis of HBV infection.Hepatitis B virus (HBV) infects more than 350 million people worldwide and is a major cause of chronic viral hepatitis and hepatocellular carcinoma (25). Three morphologically distinct forms of viral particles exist in the sera of HBV-infected patients, namely, the 22-nm-diameter spherical particles, tubular particles, and 42-nm-diameter virions (19). Strikingly, the subviral particles (spheres and tubules), containing only viral envelop glycoproteins and host-derived lipids, typically outnumber the virions by a factor of 1,000- to 10,000-fold (6, 11). There are three HBV envelop glycoproteins collectively known as HBV surface antigen (HBsAg), including the large (LHBs), middle (MHBs), and small (SHBs) surface proteins. Among them, SHBs is the most abundant viral envelop protein in virion and subviral particles. Although excess HBsAg subviral particles have been suggested to sequester the neutralizing antibody against HBV and contribute to a state of immune tolerance, thereby enabling the survival of infectious virions and leading to persistent infections (6, 27), the biological and pathological significance of the overproduction of HBsAg subviral particles still remains elusive.HBsAg has been proved extremely effective in inducing protective antibodies (anti-HBs) and thus has been used as the prophylactic vaccine. Thus far, most studies on HBsAg have focused on the development of hepatitis B vaccines (41), identification of HBsAg-interacting membrane proteins as potential host HBV receptors (9, 13), and characterization of the impact of naturally occurring HBsAg mutations on its antigenicity (12, 43). However, specific interactions between HBsAg and host intracellular factors have not been extensively studied.To address this issue, SHBs-secreting cell lines and lineages of HBV transgenic mice persistently expressing HBsAg were used in our previous studies (28, 34, 44). We found that the level of cyclophilin A (CypA) decreased markedly in the livers of HBsAg transgenic mice but increased significantly in their sera (44). CypA is a multifunctional cellular protein. It is the major binding protein for the immunosuppressive drug cyclosporine (Cs) (14) and exhibits peptidyl-prolyl cis-trans isomerase activity (35). Recently, it was found CypA played important roles in regulating inflammatory responses and viral infections. Regarding these newly recognized physiological functions, CypA was speculated to be involved in HBV infection. In the present study, the mechanism and clinical implications of elevated secretion of CypA induced by SHBs were explored in detail, including studies in cell cultures, hydrodynamic injected mouse models, and chronic hepatitis B patients. Our findings indicate that expression and secretion of SHBs can trigger the secretion of CypA, which may induce liver inflammation and contribute to the pathogenesis of HBV infection.     

The sino-atrial node as an indicator of oscillatory processes in the cardiovascular system

The spectrogram of heart rate in denervated (vagotomy + propranolol) and artificially ventilated cats always contains the true respiratory peak and 1-3 resonance ones pacing at intervals equivalent to frequency of breathing. Hypothermic decrease of heart rate periodically draws the splitting of respiratory peaks and generation of supplementary rate-dependent peaks reflecting the interference of heart and breathing rhythms. The functional base for detection of mentioned peaks is myogenic reaction of sino-atrial node to its extension by fluctuations of venous inflow.     

The lipopeptide antibiotic A54145 biosynthetic gene cluster from Streptomyces fradiae

Ca(2+)-dependent cyclic lipodepsipeptides are an emerging class of antibiotics for the treatment of infections caused by Gram-positive pathogens. These compounds are synthesized by nonribosomal peptide synthetase (NRPS) complexes encoded by large gene clusters. The gene cluster encoding biosynthetic pathway enzymes for the Streptomyces fradiae A54145 NRP was cloned from a cosmid library and characterized. Four NRPS-encoding genes, responsible for subunits of the synthetase, as well as genes for accessory functions such as acylation, methylation and hydroxylation, were identified by sequence analysis in a 127 kb region of DNA that appears to be located subterminally in the bacterial chromosome. Deduced epimerase domain-encoding sequences within the NRPS genes indicated a D: -stereochemistry for Glu, Lys and Asn residues, as observed for positionally analogous residues in two related compounds, daptomycin, and the calcium-dependent antibiotic (CDA) produced by Streptomyces roseosporus and Streptomyces coelicolor, respectively. A comparison of the structure and the biosynthetic gene cluster of A54145 with those of the related peptides showed many similarities. This information may contribute to the design of experiments to address both fundamental and applied questions in lipopeptide biosynthesis, engineering and drug development.     

The involvement of hydrogen peroxide in UV-B-inhibited pollen germination and tube growth of Paeonia suffruticosa and Paulownia tomentosa in vitro

Previous studies have shown that UV-B could affect pollen germination and tube growth. However, the mechanism of response of pollen to UV-B has not been clear. The purpose of this study was to investigate the role of hydrogen peroxide (H₂O₂) in the UV-B-induced reduction of in vitro pollen germination and tube growth of Paeonia suffruticosa Andr. and Paulownia tomentosa Steud. Exposure of pollen of the two species to 0.4 and 0.8 W m⁻² UV-B radiation for 3 h resulted in not only the reduction of pollen germination and tube growth, but also the H₂O₂ production in pollen grain and tube. Also, exogenous H₂O₂ inhibited pollen germination and tube growth of the two species in a dose-dependence manner. Two scavengers of H₂O₂, ascorbic acid and catalase, largely prevented not only the H₂O₂ generation, but also the reduction of pollen germination and tube growth induced by UV-B radiation in the two species. These results indicate that H₂O₂ is involved in the UV-B-inhibited pollen germination and tube growth.     

Isolation and characterization of a novel thermostable non-specific lipid transfer protein-like antimicrobial protein from motherwort (Leonurus japonicus Houtt) seeds

In screening for potent antimicrobial proteins from plant seeds, a novel heat-stable antimicrobial protein, designated LJAMP2, was purified from seeds of the motherwort (Leonurus japonicus Houtt), a medicine herb, with a procedure involving cation exchange chromatography on a CM FF column, and reverse phase HPLCs on C8 column and C18 column. LJAMP2 exhibited a molecular mass of 6.2 kDa determined. Automated Edman degradation determined the partial N-terminal sequence of LJAMP2 to be NH2-AIGCNTVASKMAPCLPYVTGKGPLGGCCGGVKGLIDAARTTPDRQAVCNCLKTLAKSYSG, which displays homology with plant non-specific lipid transfer proteins (nsLTPs). In vitro bioassays showed that LJAMP2 inhibits the growth of a variety of microbes, including filamentous fungi, bacteria and yeast. The growth of three phytopathogenic fungi, Alternaria brassicae, Botrytis maydis, and Rhizoctonia cerealis, are inhibited at 7.5 μM of LJAMP2, whereas Bacillus subtilis is about 15 μM. The IC₅₀ of LJAMP2 for Aspergillus niger, B. maydis, Fusarium oxysporum, Penicillium digitatum and Saccharomyces cerevisiae are 5.5, 6.1, 9.3, 40.0, and 76.0 μM, respectively.     

Evaluation of the membrane-spanning domain of ClC-2

The ClC family of chloride channels and transporters includes several members in which mutations have been associated with human disease. An understanding of the structure-function relationships of these proteins is essential for defining the molecular mechanisms underlying pathogenesis. To date, the X-ray crystal structures of prokaryotic ClC transporter proteins have been used to model the membrane domains of eukaryotic ClC channel-forming proteins. Clearly, the fidelity of these models must be evaluated empirically. In the present study, biochemical tools were used to define the membrane domain boundaries of the eukaryotic protein, ClC-2, a chloride channel mutated in cases of idiopathic epilepsy. The membrane domain boundaries of purified ClC-2 and accessible cysteine residues were determined after its functional reconstitution into proteoliposomes, labelling using a thiol reagent and proteolytic digestion. Subsequently, the lipid-embedded and soluble fragments generated by trypsin-mediated proteolysis were studied by MS and coverage of approx. 71% of the full-length protein was determined. Analysis of these results revealed that the membrane-delimited boundaries of the N- and C-termini of ClC-2 and the position of several extramembrane loops determined by these methods are largely similar to those predicted on the basis of the prokaryotic protein [ecClC (Escherichia coli ClC)] structures. These studies provide direct biochemical evidence supporting the relevance of the prokaryotic ClC protein structures towards understanding the structure of mammalian ClC channel-forming proteins.     

Disordered osteoclast formation in RAGE-deficient mouse establishes an essential role for RAGE in diabetes related bone loss

The mechanisms underlying diabetes-mediated bone loss are not well defined. It has been reported that the advanced glycation endproducts (AGEs) and receptor for AGEs (RAGEs) are involved in diabetic complications. Here, mice deficient in RAGE were used as a model for investigating the effects of RAGE on bone mass. We found that RAGE-/- mice have a significantly increased bone mass and bone biomechanical strength and a decreased number of osteoclasts compared to wild-type mice. The serum levels of IL-6 and bone breakdown marker pyridinoline were significantly decreased in RAGE-/- mice. RAGE-/- mice maintain bone mass following ovariectomy, whereas wild-type mice lose bone mass. Furthermore, osteoclast-like cells do express RAGE mRNA. Our data therefore indicate that RAGE serves as a positive factor to regulate the osteoclast formation, directly implicates a role for RAGE in diabetes-promoted bone destruction, and documents that the AGE-RAGE interaction may account for diabetes associated bone loss.     

Effects of Aluminum on Immune Functions of Cultured Splenic T and B Lymphocytes in Rats

The effects of Aluminum (Al) exposure on immune functions of cultured splenic T and B lymphocytes of rats were studied. The lymphocytes were isolated from spleen of healthy male Wistar rats weighing 110-120 g. The cultured cells in RPMI-1640 medium were exposed to 0 (control group), 0.035 (low-dose group), 0.07 (medial-dose group), and 0.14 (high-dose group) mg/mL Al(3+) as aluminum trichloride (AlCl(3)) in an incubator under 5% CO(2) at 37°C for 24 h. The T and B lymphocyte proliferation was measured with a tetrazolium dye colorimetric assay. The levels of interleukin (IL)-2, IL-6, and tumor necrosis factor (TNF)-α were determined by iodine [(125)I] IL-2, IL-6, and TNF-α radioimmunoassay kits, respectively. The proportions of CD3(+), CD4(+), and CD8(+) T lymphocytes were measured with a flow cytometer. The results showed that the T and B lymphocyte proliferation, the levels of IL-2, IL-6, TNF-α, the proportions of CD3(+) and CD4(+) T lymphocytes, and the ratio of CD4(+)/CD8(+) T lymphocytes were lowered by Al treatments, while the proportion of CD8(+) T lymphocytes was increased. These findings indicate that Al exposure can inhibit the immune functions of splenic T and B lymphocytes of rats in vitro.