Deciphering the Kinetic Binding Mechanism of Dimeric Ligands Using a Potent Plasma-stable Dimeric Inhibitor of Postsynaptic Density Protein-95 as an Example

Dimeric ligands can be potent inhibitors of protein-protein or enzyme-substrate interactions. They have increased affinity and specificity toward their targets due to their ability to bind two binding sites simultaneously and are therefore attractive in drug design. However, few studies have addressed the kinetic mechanism of interaction of such bivalent ligands. We have investigated the binding interaction of a recently identified potent plasma-stable dimeric pentapeptide and PDZ1–2 of postsynaptic density protein-95 (PSD-95) using protein engineering in combination with fluorescence polarization, isothermal titration calorimetry, and stopped-flow fluorimetry. We demonstrate that binding occurs via a two-step process, where an initial binding to either one of the two PDZ domains is followed by an intramolecular step, which produces the bidentate complex. We have determined all rate constants involved in the binding reaction and found evidence for a conformational transition of the complex. Our data demonstrate the importance of a slow dissociation for a successful dimeric ligand but also highlight the possibility of optimizing the intramolecular association rate. The results may therefore aid the design of dimeric inhibitors in general.     

Polymorphisms of interferon-inducible genes OAS-1 and MxA associated with SARS in the Vietnamese population

We hypothesized that host antiviral genes induced by type I interferons might affect the natural course of severe acute respiratory syndrome (SARS). We analyzed single nucleotide polymorphisms (SNPs) of 2',5'-oligoadenylate synthetase 1 (OAS-1), myxovirus resistance-A (MxA), and double-stranded RNA-dependent protein kinase in 44 Vietnamese SARS patients with 103 controls. The G-allele of non-synonymous A/G SNP in exon 3 of OAS-1 gene showed association with SARS (p=0.0090). The G-allele in exon 3 of OAS-1 and the one in exon 6 were in strong linkage disequilibrium and both of them were associated with SARS infection. The GG genotype and G-allele of G/T SNP at position -88 in the MxA gene promoter were found more frequently in hypoxemic group than in non-hypoxemic group of SARS (p=0.0195). Our findings suggest that polymorphisms of two IFN-inducible genes OAS-1 and MxA might affect susceptibility to the disease and progression of SARS at each level.     

Membrane topology of mouse stearoyl-CoA desaturase 1

Stearoyl-CoA desaturase (SCD) is an integral membrane protein anchored in the endoplasmic reticulum. It catalyzes the biosynthesis of monounsaturated fatty acids that are required for the synthesis of triglycerides, cholesteryl esters, and phospholipids. Four mouse isoforms of SCD (SCD1-4) and two human isoforms have been characterized. In the current study, we characterize the topology of the mouse SCD1 isoform. Hydropathy analysis of the 355-amino acid mouse SCD1 protein predicts that the protein contains four transmembrane domains (TMDs) and three loops connecting the membrane-spanning domains. To define the topology of the protein, recombinant SCD1 constructs containing epitope tags were transiently expressed in HeLa cells and analyzed by indirect immunofluorescence and cysteine derivatization. Our data provide evidence that the N and C termini of SCD1 are oriented toward the cytosol with four transmembrane domains separated by two very short hydrophilic loops in the ER lumen and one large hydrophilic loop in the cytosol. In addition, based on the previous observation that SCD is a thiol enzyme, we sought to investigate whether the cysteine residues were essential for enzyme activity through mutagenesis studies, and our data suggest that the cysteines in SCD are not catalytically essential.     

A transgenic plant cell‐suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles

We describe a novel strategy to produce vaccine antigens using a plant cell‐suspension culture system in lieu of the conventional bacterial or animal cell‐culture systems. We generated transgenic cell‐suspension cultures from Nicotiana benthamiana leaves carrying wild‐type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot‐and‐mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co‐expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large‐scale production of immunopeptide vaccines in a cost‐effective manner using a plant cell‐suspension culture system.     

Local Application of Ibandronate/Gelatin Sponge Improves Osteotomy Healing in Rabbits

Delayed healing or non-union of skeletal fractures are common clinical complications. Ibandronate is a highly potent anti-catabolic reagent used for treatment of osteopenia and fracture prevention. We hypothesized that local application of ibandronate after fracture fixation may improve and sustain callus formation and therefore prevent delayed healing or non-union. This study tested the effect of local application of an ibandronate/gelatin sponge composite on osteotomy healing. A right-side distal-femoral osteotomy was created surgically, with fixation using a k-wire, in forty adult male rabbits. The animals were divided into four groups of ten animals and treated by: (i) intravenous injection of normal saline (Control); (ii) local implantation of absorbable gelatin sponge (GS); (iii) local implantation of absorbable GS containing ibandronate (IB+GS), and (iv) intravenous injection of ibandronate (IB i.v.). At two and four weeks the affected femora were harvested for X-ray photography, computed tomography (CT), biomechanical testing and histopathology. At both time-points the results showed that the calluses in both the ibandronate-treated groups, but especially in the IB+GS group, were significantly larger than in the control and GS groups. At four weeks the cross sectional area (CSA) and mechanical test results of ultimate load and energy in the IB+GS group were significantly higher than in other groups. Histological procedures showed a significant reduction in osteoclast numbers in the IB+GS and IB i.v. groups at day 14. The results indicate that local application of an ibandronate/gelatin sponge biomaterial improved early osteotomy healing after surgical fixation and suggest that such treatment may be a valuable local therapy to enhance fracture repair and potentially prevent delayed or non-union.     

Genetic portrait of Lisboa immigrant population from Cabo Verde with mitochondrial DNA analysis

Journal of Genetics -     

Production of agarase from a novel <Emphasis Type=Italic>Micrococcus</Emphasis> sp. GNUM-08124 strain isolated from the East Sea of Korea

The agar degrading bacterial strain GNUM-08124 was isolated from Enteromorpha compressa collected in the East Sea of Korea by using a selective artificial sea water (ASW) agar plate containing agar as the sole carbon source. GNUM-08124 grows to produce a circular, smooth, yellow-colored, and raised colony. Its ability to hydrolyze agar was confirmed by staining the ASW agar plate with Lugol’s solution. In liquid culture, the cell density (A₆₀₀) increased exponentially and reached a maximum level on the third day of cultivation. The specific agarase activity also increased in proportion to the cell density and reached maximum agarolytic activity on the third day. The 16S rRNA sequence of GNUM-08124 showed a close relationship to Micrococcus luteus (99.65%) and Micrococcus endophyticus (99.15%), which led us to assign it to the genus Micrococcus. Physiological studies indicated that optimal growth conditions were between 30 and 40°C, pH 4 and 7, using media containing between 5 and 10% NaCl (w/v), respectively. The GNUM-08124 strain was a grampositive, urease-positive, and catalase-positive bacterium. It could not hydrolyze gelatin, cellulose, xylan, or starch, but fermented a broader range of substrates, including Dglucose, D-galactose, D-fructose, D-lactose, D-trehalose, D-mannitol, D-melibiose, D-raffinose, D-xylose, methyl-α-D-glucopyranoside, N-acetyl-glucosamine, and xylitol, than those fermented by M. luteus or M. endophyticus, suggesting GNUM-08124 is a novel agar hydrolyzing microorganism belonging to Genus Micrococcus. Micrococcus sp. GNUM-08124 showed the highest agarase activity when it was cultured in ASW-YP medium supplemented with 0.4% glucose, but demonstrated lower activity in rich media (LB or TSB), in spite of superior cell growth, implying that agarase production is tightly regulated in an agar-dependent manner and repressed in rich conditions.     

Tea epigallocatechin-3-gallate increases 8-isoprostane level and induces caudal regression in developing rat embryos

Tea is the most common beverage after water. Concerns have been raised about the safety of tea during pregnancy, especially for embryo development. We aimed at studying the effects of active tea components on developing embryos by in vitro rat embryo culture. Rat embryos during early organogenesis were cultivated in serum supplemented with one of the tea catechins. Developmental hallmarks and malformations (Mal) in the developing embryos were compared and evaluated by a standard morphological scoring system. The embryotoxicity of each tea catechin was classified according to the European Center for the Validation of Alternative Methods. Cell viability was assessed by supervital dye staining, apoptosis by TUNEL assay, and peroxidation by the 8-isoprostane EIA method. We found that (+)-catechin had the least effect on developing embryos (Mal(50)=715.1 mg/L; IC50(Mal)=435 mg/L), whereas (-)-epigallocatechin gallate had the most adverse effect (Mal(50)=54.2 mg/L; IC50(Mal)=45.8 mg/L). The major malformation in affected embryos included caudal retardation with abnormal axial flexion and delayed hind-limb formation. All catechins were classified as nonembryotoxic except (-)-epigallocatechin gallate, which was classified as weakly embryotoxic. With (-)-epigallocatechin gallate, increased numbers of nonviable and apoptotic cells in the malformed embryos were associated with increased embryo 8-isoprostane.     

内蒙古多伦县土地沙漠化景观格局变化特征

利用景观分析软件Fragstats 3.3和内蒙古多伦县1960、1975、1987、1995、2000、2005年6期遥感影像解译结果,分析了1960—2005年间土地沙漠化景观格局变化.结果表明:1960—1995年间研究区土地沙漠化面积呈先增后减趋势,总沙漠化面积增加212.70km2,沙漠化斑块数先增多后减少,景观多样性和均匀性上升,轻度、中度、重度沙漠化斑块形状趋向简单;1995—2005年间研究区沙漠化斑块数大幅增加,景观多样性和均匀性下降,轻度、中度、重度沙漠化斑块形状趋向复杂;严重沙漠化斑块形状在1960年以后一直趋向复杂化.整个景观在研究期间表现为完整-破碎-完整-破碎的趋势,由轻度到严重沙漠化,其景观类型的破碎化程度逐渐降低.