Evaluation of phage display system and leech-derived tryptase inhibitor as a tool for understanding the serine proteinase specificities

A small combinatorial library of LDTI mutants (5.2 x 10(4)) restricted to the P1-P4' positions of the reactive site was displayed on the pCANTAB 5E phagemid, and LDTI fusion phages were produced and selected for potent neutrophil elastase and plasmin inhibitors. Strong fusion phage binders were analyzed by ELISA on enzyme-coated microtiter plates and the positive phages had their DNA sequenced. The LDTI variants: 29E (K8A, I9A, L10F, and K11F) and 19E (K8A, K11Q, and P12Y) for elastase and 2Pl (K11W and P12N), 8Pl (I9V, K11W, and P12E), and 10Pl (I9T, K11L, and P12L) for plasmin were produced with a Saccharomyces cerevisiae expression system. New strong elastase and plasmin inhibitors were 29E and 2Pl, respectively. LDTI-29E was a potent and specific neutrophil elastase inhibitor K(i) =0.5 nM), affecting no other tested enzymes. LDTI-2Pl was the strongest plasmin inhibitor ( K(i) =1.7nM) in the LDTI mutant library. This approach allowed selection of new specific serine proteinase inhibitors for neutrophil elastase and plasmin (a thrombin inhibitor variant was previously described), from a unique template molecule, LDTI, a Kazal type one domain inhibitor, by only 2-4 amino acid replacements. Our data validate this small LDTI combinatorial library as a tool to generate specific serine proteinase inhibitors suitable for drug design and enzyme-inhibitor interaction studies.     

Molecular and functional characterization of a novel splice variant of ANKHD1 that lacks the KH domain and its role in cell survival and apoptosis

Multiple ankyrin repeat motif-containing proteins play an important role in protein-protein interactions. ANKHD1 proteins are known to possess multiple ankyrin repeat domains and a single KH domain with no known function. Using yeast two-hybrid system analysis, we identified a novel splice variant of ANKHD1. This splice variant of ANKHD1, which we designated as HIV-1 Vpr-binding ankyrin repeat protein (VBARP), does not contain the signature KH domain, and codes for only a single ankyrin repeat motif. We characterized VBARP by molecular and functional analysis, revealing that VBARP is ubiquitously expressed in different tissues as well as cell lines of different lineage. In addition, blast searches indicated that orthologs and homologs to VBARP exist in different phyla, suggesting that VBARP might be evolutionarily conserved, and thus may be involved in basic cellular function(s). Furthermore, biochemical analysis revealed the presence of two VBARP isoforms coding for 69 and 49 kDa polypeptides, respectively, that are primarily localized in the cytoplasm. Functional analysis using short interfering RNA approaches indicate that this gene product is essential for cell survival through its regulation of caspases. Taken together, these results indicate that VBARP is a novel splice variant of ANKHD1 and may play a role in cellular apoptosis (antiapoptotic) and cell survival pathway(s).     

Expression of clock genes in human subcutaneous and visceral adipose tissues

Disrupted circadian rhythms are associated with obesity and metabolic alterations, but little is known about the participation of peripheral circadian clock machinery in these processes. The aim of the present study was to analyze RNA expression of clock genes in subcutaneous (SAT) and visceral (VAT) adipose tissues of male and female subjects in AM (morning) and PM (afternoon) periods, and its interactions with body mass index (BMI). Ninety-one subjects (41 ± 11 yrs of age) presenting a wide range of BMI (21.4 to 48.6 kg/m(2)) were included. SAT and VAT biopsies were obtained from patients undergoing abdominal surgeries. Clock genes expressions were evaluated by qRT-PCR. The only clock gene that showed higher expression (p 相似文献   

Towards a semen proteome of the dengue vector mosquito: protein identification and potential functions

Background

No commercially licensed vaccine or treatment is available for dengue fever, a potentially lethal infection that impacts millions of lives annually. New tools that target mosquito control may reduce vector populations and break the cycle of dengue transmission. Male mosquito seminal fluid proteins (Sfps) are one such target since these proteins, in aggregate, modulate the reproduction and feeding patterns of the dengue vector, Aedes aegypti. As an initial step in identifying new targets for dengue vector control, we sought to identify the suite of proteins that comprise the Ae. aegypti ejaculate and determine which are transferred to females during mating.

Methodology and Principal Findings

Using a stable-isotope labeling method coupled with proteomics to distinguish male- and female-derived proteins, we identified Sfps and sperm proteins transferred from males to females. Sfps were distinguished from sperm proteins by comparing the transferred proteins to sperm-enriched samples derived from testes and seminal vesicles. We identified 93 male-derived Sfps and 52 predicted sperm proteins that are transferred to females during mating. The Sfp protein classes we detected suggest roles in protein activation/inactivation, sperm utilization, and ecdysteroidogenesis. We also discovered that several predicted membrane-bound and intracellular proteins are transferred to females in the seminal fluids, supporting the hypothesis that Ae. aegypti Sfps are released from the accessory gland cells through apocrine secretion, as occurs in mammals. Many of the Ae. aegypti predicted sperm proteins were homologous to Drosophila melanogaster sperm proteins, suggesting conservation of their sperm-related function across Diptera.

Conclusion and Significance

This is the first study to directly identify Sfps transferred from male Ae. aegypti to females. Our data lay the groundwork for future functional analyses to identify individual seminal proteins that may trigger female post-mating changes (e.g., in feeding patterns and egg production). Therefore, identification of these proteins may lead to new approaches for manipulating the reproductive output and vectorial capacity of Ae. aegypti.     

应用红外相机技术研究秦岭观音山自然保护区内野猪的行为和丰富度

2009年7月,在陕西观音山自然保护区凉风垭小区域（中高海拔）和西沟小区域（低海拔）安装18台红外相机,2009年8月至2013年4月共收集野猪照片1 195张。定义9种野猪行为,分别为站立、走动、跑动、采食、饮水、修饰、发情、拱土、坐着休息,并逐一比对照片中野猪的行为,统计各种行为所占的比例;引入月相对丰富度和时间段相对丰富度两个指数分别研究野猪的年活动规律和日活动规律;利用一个种群估测模型探讨野猪密度的年际变化。结果表明：（1）春季野猪以走动、采食和站立为主,分别占总行为次数的36%、25.6%和17.4%;夏季野猪以走动、站立、采食和跑动为主,分别占总行为次数的35.7%、23.6%、17%和16.5%;秋季野猪以采食、走动和发情为主,分别占总行为次数的50.3%、19.3%和17.8%;冬季野猪以采食、走动和站立为主,分别占总行为次数的53.7%、26.7%和11.9%。（2）野猪在8月、9月和12月活动较为频繁;全年日活动高峰出现在午后14:00-16:00,低谷出现在22:00-04:00,四季显示活动规律不同。（3）2009-2012年野猪密度呈逐年上升趋势。这些研究结果有助于了解野猪的行为活动和种群动态,并采取针对性的措施对野猪进行有效管理。     

A MIV-150/Zinc Acetate Gel Inhibits SHIV-RT Infection in Macaque Vaginal Explants

To extend our observations that single or repeated application of a gel containing the NNRTI MIV-150 (M) and zinc acetate dihydrate (ZA) in carrageenan (CG) (MZC) inhibits vaginal transmission of simian/human immunodeficiency virus (SHIV)-RT in macaques, we evaluated safety and anti-SHIV-RT activity of MZC and related gel formulations ex vivo in macaque mucosal explants. In addition, safety was further evaluated in human ectocervical explants. The gels did not induce mucosal toxicity. A single ex vivo exposure to diluted MZC (1∶30, 1∶100) and MC (1∶30, the only dilution tested), but not to ZC gel, up to 4 days prior to viral challenge, significantly inhibited SHIV-RT infection in macaque vaginal mucosa. MZC''s activity was not affected by seminal plasma. The antiviral activity of unformulated MIV-150 was not enhanced in the presence of ZA, suggesting that the antiviral activity of MZC was mediated predominantly by MIV-150. In vivo administration of MZC and CG significantly inhibited ex vivo SHIV-RT infection (51–62% inhibition relative to baselines) of vaginal (but not cervical) mucosa collected 24 h post last gel exposure, indicating barrier effect of CG. Although the inhibitory effect of MZC (65–74%) did not significantly differ from CG (32–45%), it was within the range of protection (∼75%) against vaginal SHIV-RT challenge 24 h after gel dosing. Overall, the data suggest that evaluation of candidate microbicides in macaque explants can inform macaque efficacy and clinical studies design. The data support advancing MZC gel for clinical evaluation.     

A novel allele of the Arabidopsis thaliana MACPF protein CAD1 results in deregulated immune signaling

Immune recognition in plants is governed by two major classes of receptors: pattern recognition receptors (PRRs) and nucleotide-binding leucine-rich repeat receptors (NLRs). Located at the cell surface, PRRs bind extracellular ligands originating from microbes (indicative of “non-self”) or damaged plant cells (indicative of “infected-self”), and trigger signaling cascades to protect against infection. Located intracellularly, NLRs sense pathogen-induced physiological changes and trigger localized cell death and systemic resistance. Immune responses are under tight regulation in order to maintain homeostasis and promote plant health. In a forward-genetic screen to identify regulators of PRR-mediated immune signaling, we identified a novel allele of the membrane-attack complex and perforin (MACPF)-motif containing protein CONSTITUTIVE ACTIVE DEFENSE 1 (CAD1) resulting from a missense mutation in a conserved N-terminal cysteine. We show that cad1-5 mutants display deregulated immune signaling and symptoms of autoimmunity dependent on the lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), suggesting that CAD1 integrity is monitored by the plant immune system. We further demonstrate that CAD1 localizes to both the cytosol and plasma membrane using confocal microscopy and subcellular fractionation. Our results offer new insights into immune homeostasis and provide tools to further decipher the intriguing role of MACPF proteins in plants.     

Variation in growth rate,carbon assimilation,and photosynthetic efficiency in response to nitrogen source and concentration in phytoplankton isolated from upper San Francisco Bay

Six species of phytoplankton recently isolated from upper San Francisco Bay were tested for their sensitivity to growth inhibition by ammonium (NH₄⁺), and for differences in growth rates according to inorganic nitrogen (N) growth source. The quantum yield of photosystem II (F_v/F_m) was a sensitive indicator of NH₄⁺ toxicity, manifested by a suppression of F_v/F_m in a dose‐dependent manner. Two chlorophytes were the least sensitive to NH₄⁺ inhibition, at concentrations of >3,000 μmoles NH₄⁺ · L^?1, followed by two estuarine diatoms that were sensitive at concentrations >1,000 μmoles NH₄⁺ · L^?1, followed lastly by two freshwater diatoms that were sensitive at concentrations between 200 and 500 μmoles NH₄⁺ · L^?1. At non‐inhibiting concentrations of NH₄⁺, the freshwater diatom species grew fastest, followed by the estuarine diatoms, while the chlorophytes grew slowest. Variations in growth rates with N source did not follow taxonomic divisions. Of the two chlorophytes, one grew significantly faster on nitrate (NO₃^?), whereas the other grew significantly faster on NH₄⁺. All four diatoms tested grew faster on NH₄⁺ compared with NO₃^?. We showed that in cases where growth rates were faster on NH₄⁺ than they were on NO₃^?, the difference was not larger for chlorophytes compared with diatoms. This holds true for comparisons across a number of culture investigations suggesting that diatoms as a group will not be at a competitive disadvantage under natural conditions when NH₄⁺ dominates the total N pool and they will also not have a growth advantage when NO₃^? is dominant, as long as N concentrations are sufficient.