Modules in the photoreceptor RGS9-1.Gbeta 5L GTPase-accelerating protein complex control effector coupling, GTPase acceleration, protein folding, and stability

RGS (regulators of G protein signaling) proteins regulate G protein signaling by accelerating GTP hydrolysis, but little is known about regulation of GTPase-accelerating protein (GAP) activities or roles of domains and subunits outside the catalytic cores. RGS9-1 is the GAP required for rapid recovery of light responses in vertebrate photoreceptors and the only mammalian RGS protein with a defined physiological function. It belongs to an RGS subfamily whose members have multiple domains, including G(gamma)-like domains that bind G(beta)(5) proteins. Members of this subfamily play important roles in neuronal signaling. Within the GAP complex organized around the RGS domain of RGS9-1, we have identified a functional role for the G(gamma)-like-G(beta)(5L) complex in regulation of GAP activity by an effector subunit, cGMP phosphodiesterase gamma and in protein folding and stability of RGS9-1. The C-terminal domain of RGS9-1 also plays a major role in conferring effector stimulation. The sequence of the RGS domain determines whether the sign of the effector effect will be positive or negative. These roles were observed in vitro using full-length proteins or fragments for RGS9-1, RGS7, G(beta)(5S), and G(beta)(5L). The dependence of RGS9-1 on G(beta)(5) co-expression for folding, stability, and function has been confirmed in vivo using transgenic Xenopus laevis. These results reveal how multiple domains and regulatory polypeptides work together to fine tune G(talpha) inactivation.     

Nuclear death in living Tetrahymena: the case of the haploid nuclei

During sexual conjugation in Tetrahymena the micronucleus divides meiotically, producing four haploid nuclei. While one of these nuclei divides mitotically to yield two genetically identical gametic pronuclei, a stationary pronucleus and a migratory pronucleus, the remaining three haploid nuclei degenerate and disappear. Typically, they migrate to the posterior end of the cell where they remain as residual bodies until they disappear. In the present study we asked whether degenerating haploid nuclei share any properties with apoptotic nuclei. Specifically, we wondered whether they would be stained by "apofluor", a combination of vital fluorescent indicators that differentially stains apoptotic nuclei in living cells. "Apofluor" includes acridine orange, which becomes trapped in acidic compartments and stains lysosomal bodies a brilliant orange-red, and Hoechst 33342, which binds to DNA and stains nuclei bright blue. With this dye combination, while ordinary nuclei stain blue, the apoptotic macronucleus stains first blue-green, then yellow, and finally orange. The progression in color is presumed to be due to the accumulation of protons in the apoptotic nucleus compartment. We found that three of the four post-meiotic haploid nuclei, those that are eliminated, were stained differentially green, then yellow, and then come to be indistinguishable from the orange lysosomal bodies. Differential staining can occur even while the nuclei are located at the anterior ends of the cells, and before the "viable" nucleus divides to form pronuclei. These results indicate that haploid nuclei in the process of degradation are differentially stained in living cells by "apofluor", and that the differential staining occurs early in the elimination process. Further, since the degenerating haploid nuclei are stained by "apofluor" it is likely that they are degraded by a mechanism similar to the elimination of the apoptotic macronucleus.     

Prokaryotic expression of woodchuck cytotoxic T lymphocyte antigen 4 (wCTLA-4) and preparation of polyclonal antibody to wCTLA-4

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an inhibitory T cell receptor predominately expressed on activated T cells and plays an important role in regulation of specific T cell responses to viral infection. The woodchuck model is an informative animal model for hepatitis B virus (HBV) infection. In this study, the extracellular region of woodchuck CTLA-4 (wCTLA-4) was cloned and the fusion protein of GST-wCTLA-4 was expressed and purified. Polyclonal antibody against GST-wCTLA-4 (anti-GST-wCTLA-4) was prepared. The full length wCTLA-4 protein expressed in transfected baby hamster kidney cells was detected by anti-GST-wCTLA-4 in western blot analysis and immunofluorescence staining. Anti-GST-wCTLA-4 provides a useful tool to study the role of CTLA-4 in T-cell response in the woodchuck model. Further, the blocking of CTLA-4 with anti-GST-wCTLA-4, as a novel therapy approach for chronic hepatitis B virus infection, could be studied in woodchuck model now.     

Inhibition of Hepatitis B Virus cccDNA by siRNA in Transgenic Mice

The elimination of viral covalently closed circular DNA (cccDNA) from the nucleus of infected hepatocytes is an obstacle to achieving sustained viral clearance during antiviral therapy of chronic hepatitis B virus (HBV) infection. The aim of our study was to determine whether treatment with siRNA is able to suppress viral cccDNA amplification using a HBV-transgenic mice model. The experimental results revealed that siRNAs can serve as efficient alternative anti-HBV agents, because they showed better inhibitory effect on viral replication and antigen expression in transgenic mice. More importantly, the siRNA markedly inhibited HBV cccDNA amplification.     

Meteorological factors are associated with hemorrhagic fever with renal syndrome in Jiaonan County,China, 2006–2011

This study examined the effect of meteorological factors on the occurrence of hemorrhagic fever with renal syndrome (HFRS) using a generalized additive model with penalized smoothing splines in Jiaonan, China, from 2006 to 2011. The dose–response relationship was first examined, and then the association between daily meteorological variables and HFRS occurrence was investigated according to the dose–response curves. There were two linear segments in the temperature–HFRS relationship curve. When daily temperature was lower than 17 °C, a positive association was found [with excessive risk (ER) for 1 °C increase on the current day being 2.56 %, 95 % confidence interval (CI): 0.36 % to 4.80 %]. An inverse association was found when daily temperature was higher than 17 °C [ER for 1 °C increase on the current day was ?12.82 % (95 % CI: ?17.51 % to ?7.85 %)]. Inverse associations were observed for relative humidity [ER for 1 % increase on lag day 4 was ?1.21 % (95 % CI: ?1.63 % to ?0.79 %)] and rainfall [ER for 1 mm increase on lag day 1 was ?2.20 % (95 % CI: ?3.56 % to ?0.82 %)]. Meteorological factors might be important predictor of HFRS epidemics in Jiaonan County.     

Serum testosterone,progesterone, and estradiol concentrations and sexual maturation in spotted seals (Phoca largha)

Spotted seals (Phoca largha) are ice-breeding phocid found in eight different breeding colonies all over the world. They exhibit a seasonal breeding pattern, with annual and synchronous cycles; however, little is known about their reproductive endocrinology. In this study, we measured serum testosterone, progesterone, and 17β-estradiol concentrations in captive spotted seals (simple number: female n = 68; male n = 89) throughout a full reproductive cycle. Males that were older than 4 years had significant testosterone fluctuations and were, therefore, classified as sexually mature. These animals show significant seasonal changes in testosterone levels, with average peak concentrations of 10.81 ± 9.57 nmol/L (±SD) from November to February, compared with mean concentrations of 1.42 ± 3.09 nmol/L throughout the remainder of the year. Females that reported a significant variation in progesterone concentrations and were older than 4 years were considered to be sexually mature. In these females, progesterone levels increased in February, remained elevated for 7 months with a mean value of 37.39 ± 17.03 nmol/L, and then dropped to 0.74 ± 0.54 nmol/L. Serum 17β-estradiol levels were also found to be significantly increased in January, remained so for 8 months (15.80 ± 14.15 ng/L), and then declined after August (7.77 ± 6.78 ng/L). In seals, mating typically occurs in February and March, 1 month after the observed peaks in testosterone and estradiol concentrations and corresponding to the increase in progesterone. A moderate positive correlation between testosterone and progesterone concentrations in sexually mature males was also observed (Spearman rho, r = 0.63, P < 0.01). In sexually immature females, progesterone and estradiol concentrations were found to be significantly lower than those in mature females. Finally, the observed patterns of estradiol and progesterone in sexually mature females suggest that embryonic diapause or successful implantation occurs in August.     

Structural insights into the T6SS effector protein Tse3 and the Tse3–Tsi3 complex from Pseudomonas aeruginosa reveal a calcium‐dependent membrane‐binding mechanism

The opportunistic pathogen Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to deliver the muramidase Tse3 into the periplasm of rival bacteria to degrade their peptidoglycan (PG). Concomitantly, P. aeruginosa uses the periplasm‐localized immunity protein Tsi3 to prevent potential self‐intoxication caused by Tse3, and thus gains an edge over rival bacteria in fierce niche competition. Here, we report the crystal structures of Tse3 and the Tse3–Tsi3 complex. Tse3 contains an annexin repeat‐like fold at the N‐terminus and a G‐type lysozyme fold at the C‐terminus. One loop in the N‐terminal domain (Loop 12) and one helix (α9) from the C‐terminal domain together anchor Tse3 and the Tse3–Tsi3 complex to membrane in a calcium‐dependent manner in vitro, and this membrane‐binding ability is essential for Tse3's activity. In the C‐terminal domain, a Y‐shaped groove present on the surface likely serves as the PG binding site. Two calcium‐binding motifs are also observed in the groove and these are necessary for Tse3 activity. In the Tse3–Tsi3 structure, three loops of Tsi3 insert into the substrate‐binding groove of Tse3, and three calcium ions present at the interface of the complex are indispensable for the formation of the Tse3–Tsi3 complex.