Adjuvant Potential of Selegiline in Attenuating Organ Dysfunction in Septic Rats with Peritonitis

Selegiline, an anti-Parkinson drug, has antioxidant and anti-apoptotic effects. To explore the effect of selegiline on sepsis, we used a clinically relevant animal model of polymicrobial sepsis. Cecal ligation and puncture (CLP) or sham operation was performed in male rats under anesthesia. Three hours after surgery, animals were randomized to receive intravenously selegiline (3 mg/kg) or an equivalent volume of saline. The administration of CLP rats with selegiline (i) increased arterial blood pressure and vascular responsiveness to norepinephrine, (ii) reduced plasma liver and kidney dysfunction, (iii) attenuated metabolic acidosis, (iv) decreased neutrophil infiltration in liver and lung, and (v) improved survival rate (from 44% to 65%), compared to those in the CLP alone rats. The CLP-induced increases of plasma interleukin-6, organ superoxide levels, and liver inducible nitric oxide synthase and caspase-3 expressions were ameliorated by selegiline treatment. In addition, the histological changes in liver and lung were significantly attenuated in the selegiline -treated CLP group compared to those in the CLP group. The improvement of organ dysfunction and survival through reducing inflammation, oxidative stress and apoptosis in peritonitis-induced sepsis by selegiline has potential as an adjuvant agent for critical ill.     

Laser microirradiation of kinetochores in mitotic PtK2 cells

Irradiation of the kinetochore region of PtK₂ chromosomes by laser light of 532 nm was used to study the function of the kinetochore region in chromosome movement and to create artificial micronuclei in cells. When the sister kinetochores of a chromosome were irradiated at prometaphase, the affected chromosome detached from the spindle and exhibited no further directed movements for the duration of mitosis. The chromatids of the chromosome remained attached to one another until anaphase, at which point they separated. No poleward movement of the chromatids was observed, and at telophase they passively moved to one of the daughter cells and were enclosed in a micronucleus. The daughter cell containing the micronucleus was then isolated by micromanipulation and followed through subsequent mitoses. At the next mitosis, two chromosomes, each with two chromatids, condensed in the micronucleus. These chromosomes did not attach to the spindle and showed chromatid separation, but no poleward movements at anaphase. They were again enclosed in micronuclei at telophase. The third generation mitosis was similar to the second. Occasionally, both the irradiation-produced and naturally occurring micronuclei exhibited no chromosome condensation at mitosis. Feulgenstained monolayers of PtK₂ cells with naturally occurring micronuclei showed that some micronuclei stain positive for DNA and others do not. This finding raises questions about the fate of chromosomes in a micronucleus.     

Backbone dynamics of Escherichia coli thioesterase/protease I: evidence of a flexible active-site environment for a serine protease

Escherichia coli thioesterase/protease I (TEP-I) is a member of a novel subclass of the lipolytic enzymes with a distinctive GDSLS motif. In addition to possessing thioesterase and protease activities, TEP-I also exhibits arylesterase activity. We have determined the (15)N nuclear magnetic spin relaxation rates, R(1) and R(2), and the steady state (1)H-(15)N heteronuclear Overhauser effect, measured at both 11.74 T and 14.09 T, of (u-(15)N) TEP-I. These data were analyzed using model-free formalism (with axially symmetric rotational diffusion anisotropy) to extract the backbone dynamics of TEP-I. The results reveal that the core structure of the central beta-sheet and the long alpha-helices are rigid, while the binding pocket appears to be rather flexible. The rigid core serves as a scaffold to anchor the essential loops, which form the binding pocket. The most flexible residues display large amplitude fast (ps/ns time-scale) motion and lie on one stripe whose orientation is presumed to be the ligand-binding orientation. We also detected the presence of several residues displaying slow (microseconds/ms time-scale) conformational exchanging processes. These residues lie around the binding pocket and are oriented perpendicularly to the orientation of the flexible stripe. Two of the putative catalytic triads, Ser10 and His157, and their neighbors show motion on the microseconds/ms time-scale, suggesting that their slow motion may have a role in catalysis, in addition to their possible roles in ligand binding. The presence of a flexible substrate-binding pocket may also facilitate binding to a wide range of substrates and confer the versatile functional property of this protein.     

A Vaccine Targeted at CETP Alleviates High Fat and High Cholesterol Diet-Induced Atherosclerosis and Non-Alcoholic Steatohepatitis in Rabbit

Low HDL-C levels are associated with atherosclerosis and non-alcoholic steatohepatitis, and increased levels may reduce the risk of these diseases. Inhibition of cholesteryl ester transfer protein (CETP) activity is considered a promising strategy for increasing HDL-C levels. Since CETP is a self-antigen with low immunogenicity, we developed a novel CETP vaccine (Fc-CETP₆) to overcome the low immunogenicity of CETP and for long-term inhibition of CETP activity. The vaccine consists of a rabbit IgG Fc domain for antigen delivery to antigen-presenting cells fused to a linear array of 6 repeats of a CETP epitope to efficiently activate B cells. Rabbits were fed a high fat/cholesterol (HFC) diet to induce atherosclerosis and NASH, and immunized with Fc-CETP₆ vaccine. The Fc-CETP₆ vaccine successfully elicited anti-CETP antibodies and lowered plasma CETP activity. The levels of plasma HDL-C and ApoA-I were higher, and plasma ox-LDL lower, in the Fc-CETP₆-immunized rabbits as compared to the unimmunized HFC diet-fed rabbits. Pathological analyses revealed less lipid accumulation and inflammation in the aorta and liver of the Fc-CETP₆-immunized rabbits. These results show that the Fc-CETP₆ vaccine efficiently elicited antibodies against CETP and reduced susceptibility to both atherosclerosis and steatohepatitis induced by the HFC diet. Our findings suggest that the Fc-CETP₆ vaccine may improve atherosclerosis and NASH and has high potential for clinical use.     

Osteopontin as a positive regulator in the osteoclastogenesis of arthritis

We examined the role of osteopontin (OPN) in the osteoclastogenesis of arthritis using collagen-induced arthritis (CIA). Cells from arthritic joints of wild-type (OPN +/+) mice spontaneously developed bone-resorbing osteoclast-like cells (OCLs). The cultured cells showed an enhanced expression of receptor activator of nuclear factor kappaB ligand (RANKL) and a decreased expression of osteoprotegerin (OPG). The addition of OPG reduced the number of OCLs, indicating that the osteoclastogenesis depends on the RANK/RANKL/OPG system. The cells also produced OPN abundantly and anti-OPN neutralizing antibodies suppressed the development of OCLs. Moreover, the addition of OPN increased the expression of RANKL and augmented differentiation of OCLs from OPN-deficient (OPN -/-) cells. OPN, like the combination of 1alpha,25-dihydroxyvitamin D(3) and dexamethasone, also enhanced the RANKL expression and decreased OPG expression in a stromal cell line, ST2. These results suggest that OPN acts as a positive regulator in the osteoclastogenesis of arthritis through the RANK/RANKL/OPG system.     

Combined Interactions of Plant Homeodomain and Chromodomain Regulate NuA4 Activity at DNA Double-Strand Breaks

DNA double-strand breaks (DSBs) represent one of the most threatening lesions to the integrity of genomes. In yeast Saccharomyces cerevisiae, NuA4, a histone acetylation complex, is recruited to DSBs, wherein it acetylates histones H2A and H4, presumably relaxing the chromatin and allowing access to repair proteins. Two subunits of NuA4, Yng2 and Eaf3, can interact in vitro with methylated H3K4 and H3K36 via their plant homeodomain (PHD) and chromodomain. However, the roles of the two domains and how they interact in a combinatorial fashion are still poorly characterized. In this study, we generated mutations in the PHD and chromodomain that disrupt their interaction with methylated H3K4 and H3K36. We demonstrate that the combined mutations in both the PHD and chromodomain impair the NuA4 recruitment, reduce H4K12 acetylation at the DSB site, and confer sensitivity to bleomycin that induces DSBs. In addition, the double mutant cells are defective in DSB repair as judged by Southern blot and exhibit prolonged activation of phospho-S129 of H2A. Cells harboring the H3K4R, H3K4R, K36R, or set1Δ set2Δ mutant that disrupts H3K4 and H3K36 methylation also show very similar phenotypes to the PHD and chromodomain double mutant. Our results suggest that multivalent interactions between the PHD, chromodomain, and methylated H3K4 and H3K36 act in a combinatorial manner to recruit NuA4 and regulate the NuA4 activity at the DSB site.     

Intra blood-cerebrospinal fluid-barrier detection of hepatitis B virus

Hepatitis B, a major public health concern worldwide, is caused by hepatitis type B virus, a hepdnavirus that infects only human and certain nonhuman primates, and replicates strictly in hepatocytes. By using the techniques of slot and Southern blot DNA hybridization, and electron microscopy, the presence of HBV was identified in the cerebrospinal fluid of three affected individuals.