Towards irreversible HIV inactivation: stable gp120 binding by nucleophilic antibodies

Conventional antibodies react with antigens reversibly. We report the formation of unusually stable complexes of HIV gp120 and nucleophilic antibodies raised by immunization with an electrophilic HIV gp120 analog (E-gp120). The stability of the complexes was evident from their very slow dissociation in a nondenaturing solvent (approximate t(1/2) 18.5 days) and their resistance to dissociation by a denaturant commonly employed to disrupt noncovalent protein-protein binding (sodium dodecyl sulfate). Kinetic studies indicated time-dependent and virtually complete progression of the antibody-gp120 complexes from the initial noncovalent state to a poorly dissociable state. The antibodies to E-gp120 displayed improved covalent reactivity with an electrophilic phosphonate probe compared to control antibodies, suggesting their enhanced nucleophilicity. One of the stably binding antibodies neutralized the infectivity of CCR5-dependent primary HIV strains belonging to clades B and C. These findings suggest the feasibility of raising antibodies capable of long-lasting inactivation of antigens by electrophilic immunization.     

Substrate Specificity of Streptococcal Unsaturated Glucuronyl Hydrolases for Sulfated Glycosaminoglycan

Unsaturated glucuronyl hydrolase (UGL) categorized into the glycoside hydrolase family 88 catalyzes the hydrolytic release of an unsaturated glucuronic acid from glycosaminoglycan disaccharides, which are produced from mammalian extracellular matrices through the β-elimination reaction of polysaccharide lyases. Here, we show enzyme characteristics of pathogenic streptococcal UGLs and structural determinants for the enzyme substrate specificity. The putative genes for UGL and phosphotransferase system for amino sugar, a component of glycosaminoglycans, are assembled into a cluster in the genome of pyogenic and hemolytic streptococci such as Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes, which produce extracellular hyaluronate lyase as a virulent factor. The UGLs of these three streptococci were overexpressed in Escherichia coli cells, purified, and characterized. Streptococcal UGLs degraded unsaturated hyaluronate and chondroitin disaccharides most efficiently at approximately pH 5.5 and 37 °C. Distinct from Bacillus sp. GL1 UGL, streptococcal UGLs preferred sulfated substrates. DNA microarray and Western blotting indicated that the enzyme was constitutively expressed in S. agalactiae cells, although the expression level increased in the presence of glycosaminoglycan. The crystal structure of S. agalactiae UGL (SagUGL) was determined at 1.75 Å resolution by x-ray crystallography. SagUGL adopts α₆/α₆-barrel structure as a basic scaffold similar to Bacillus UGL, but the arrangement of amino acid residues in the active site differs between the two. SagUGL Arg-236 was found to be one of the residues involved in its activity for the sulfated substrate through structural comparison and site-directed mutagenesis. This is the first report on the structure and function of streptococcal UGLs.Cell surface polysaccharides play an important role in linking neighboring cells and protecting cells against physicochemical stress such as osmotic pressure or invasion by pathogens. Glycosaminoglycans such as chondroitin, hyaluronan, and heparin are highly negatively charged polysaccharides with a repeating disaccharide unit consisting of an uronic acid residue (glucuronic or iduronic acid) and an amino sugar residue (glucosamine or galactosamine) (1), and they are widely present in mammalian cells as an extracellular matrix responsible for cell-to-cell association, cell signaling, and cell growth and differentiation (2). For example, in humans, glycosaminoglycans exist in tissues such as the eye, brain, liver, skin, and blood (3). Except for hyaluronan, glycosaminoglycans such as chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin sulfate, and heparan sulfate are often sulfated. Chondroitin consists of d-glucuronic acid (GlcA)² and N-acetyl-d-galactosamine (GalNAc) with a sulfate group(s) at position 4 or 6 or both (4). Hyaluronan, is composed of GlcA and N-acetyl-d-glucosamine (GlcNAc) (5).The adhesion of pathogenic bacteria to mammalian cells is regarded as a primary mechanism of bacterial infection, followed by secondary effects of the infectious process. Polysaccharides, including the glycosaminoglycans that form part of the cell surface matrix, are typical targets for microbial pathogens that invade host cells, and many specific interactions between pathogens and these polysaccharides have been described (6). Glycosaminoglycans in the extracellular matrix are also degraded enzymatically by hydrolases and lyases (1). Generally, hydrolases cleave the glycoside bonds between the glycosyl oxygen and the anomeric carbon atom through the addition of water and play an important role in glycosaminoglycan metabolism in mammals (7). On the other hand, bacterial pathogens invading host cells degrade glycosaminoglycans through the action of lyases. Bacterial polysaccharide lyases recognize the uronic acid residue in polysaccharides, cleave the glycoside bonds through the β-elimination reaction without water addition, and produce unsaturated saccharides with the unsaturated uronic acid residue having a CC double bond at the nonreducing terminus (8).Streptococci such as group B Streptococcus agalactiae, group nonassigned Streptococcus pneumoniae, and group A Streptococcus pyogenes are typical pyogenic and hemolytic pathogens causing severe infections (e.g. pneumonia, bacteremia, sinusitis, or meningitis) (9–11). In S. pneumoniae, hyaluronate lyase, neuraminidases, autolysin, choline-binding protein A, and pneumococcal surface protein A are suggested to function as cell surface virulent factors (12). Hyaluronate lyase degrades the extracellular matrix component hyaluronan in mammalian cells through the β-elimination reaction and releases unsaturated disaccharide, indicating that the enzyme produced by pathogenic bacteria functions as a spreading factor (13). Because hyaluronate lyase is commonly produced by the three pyogenic and hemolytic streptococci (14–16), the structure and function of their enzymes have been intensively studied (17, 18). Groups A, B, C, and G streptococci also produce hyaluronate lyase (19), suggesting that the enzyme is ubiquitously present in pathogenic streptococci. Streptococcal hyaluronate lyase can also act on sulfated and nonsulfated chondroitin (20). The metabolism of the resultant unsaturated disaccharides in streptococci, however, remains to be clarified.Unsaturated glucuronyl hydrolase (UGL), a member of the glycoside hydrolase family 88 in the CAZY data base (21), acts on unsaturated oligosaccharides having an unsaturated GlcA (ΔGlcA) with β-glycoside bond, such as ΔGlcA-GalNAc produced by chondroitin lyase and ΔGlcA-GlcNAc produced by hyaluronate lyase (22) (Fig. 1A). We have first identified the UGL-coding gene in Bacillus sp. GL1 (23) and clarified the structure and function of the enzyme by x-ray crystallography (24–27). The enzyme reaction generates ΔGlcA and the leaving saccharide. ΔGlcA is spontaneously converted to 4-deoxy-1-threo-5-hexosulose-uronate (Fig. 1A) because the ringed form of ΔGlcA has not been obtained because of keto-enole equilibrium (23, 28). In contrast with general glycoside hydrolases with retention or inversion catalytic mechanism of an anomeric configuration, UGL uniquely triggers hydrolysis of vinyl ether groups in unsaturated saccharides but not of the glycoside bond (26) (Fig. 1B). This article deals with the characteristics of streptococcal UGLs by using recombinant enzymes, gene expression in S. agalactiae cells by DNA microarray, and structural determinants of S. agalactiae UGL for substrate specificity by x-ray crystallography and site-directed mutagenesis.Open in a separate windowFIGURE 1.UGL reaction. A, degradation scheme of Δ6S by UGL. B, catalytic reaction mechanism of UGL. C, structures of unsaturated oligosaccharides. ΔGellan, unsaturated gellan tetrasaccharide; ΔHA, unsaturated hyaluronan disaccharide; Δ0S, unsaturated chondroitin disaccharide; Δ2′S, unsaturated chondroitin disaccharide sulfated at C-2 position of ΔGlcA residue; Δ2′S4S, unsaturated chondroitin disaccharide sulfated at C-2 position of ΔGlcA residue and C-4 position of GalNAc residue; Δ2′S6S, unsaturated chondroitin disaccharide sulfated at C-2 position of ΔGlcA residue and C-6 position of GalNAc residue; Δ4S6S, unsaturated chondroitin disaccharide sulfated at C-4 and C-6 positions of GalNAc residue; Δ2′S4S6S, unsaturated chondroitin disaccharide sulfated at C-2 position of ΔGlcA residue and C-4 and C-6 positions of GalNAc residue.     

HIV-1 Accessory Protein Vpu Internalizes Cell-surface BST-2/Tetherin through Transmembrane Interactions Leading to Lysosomes

Bone marrow stromal antigen 2 (BST-2, also known as tetherin) is a recently identified interferon-inducible host restriction factor that can block the production of enveloped viruses by trapping virus particles at the cell surface. This antiviral effect is counteracted by the human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein U (Vpu). Here we show that HIV-1 Vpu physically interacts with BST-2 through their mutual transmembrane domains and leads to the degradation of this host factor via a lysosomal, not proteasomal, pathway. The degradation is partially controlled by a cellular protein, β-transducin repeat-containing protein (βTrCP), which is known to be required for the Vpu-induced degradation of CD4. Importantly, targeting of BST-2 by Vpu occurs at the plasma membrane followed by the active internalization of this host protein by Vpu independently of constitutive endocytosis. Thus, the primary site of action of Vpu is the plasma membrane, where Vpu targets and internalizes cell-surface BST-2 through transmembrane interactions, leading to lysosomal degradation, partially in a βTrCP-dependent manner. Also, we propose the following configuration of BST-2 in tethering virions to the cell surface; each of the dimerized BST-2 molecules acts as a bridge between viral and cell membranes.     

Essential role of p38 MAPK in caspase-independent, iPLA2-dependent cell death under hypoxia/low glucose conditions

The mechanisms of cell death induced by hypoxia or ischemia are not yet fully understood. We have previously demonstrated that cell death induced by hypoxia occurs independently of caspases, and is mediated by phospholipase A₂ (PLA₂).Here, we show that p38 mitogen-activated protein kinase is activated under hypoxia. A selective inhibitor of p38 or decrease in the p38alpha protein level prevents hypoxia-induced cell death. The p38 inhibitor abolishes PLA₂ activation by hypoxia, indicating that p38 acts upstream of PLA₂. The antioxidant N-acetyl-cysteine inhibits activation of p38 and cell death induced by hypoxia, indicating that reactive oxygen species (ROS) are responsible for p38 activation. These results demonstrate that the ROS/p38/PLA₂ signaling axis has a crucial role in caspase-independent cell death induced by hypoxia.     

Lupeol reduces triglyceride and cholesterol synthesis in human hepatoma cells

Lupeol suppressed triglyceride and cholesterol secretion from HepG2-Lipo human hepatoma cells in a dose-dependent manner. Quantitative real-time RT-PCR analysis demonstrated that lupeol inhibited the expressions of sterol regulatory element-binding protein-1c and -2, fatty acid synthase, 3-hydroxy-3-methylglutaryl-Coenzyme A synthetase-1, and farnesyl-diphosphate farnesyl transferase-1, which are required for lipid synthesis in HepG2-Lipo cells. Furthermore, lupeol markedly inhibited apolipoproteinB-100 and microsomal triglyceride transfer protein in cells at the mRNA level. These results suggest that lupeol lowers lipid secretion from HepG2-Lipo cells by attenuating synthesis within the cells.     

Toll-Like Receptor 4 +3725 G/C Polymorphism, Helicobacter pylori Seropositivity, and the Risk of Gastric Atrophy and Gastric Cancer in Japanese

Background:  Toll-like receptor 4 (TLR4) Asp299Gly and Thr399Ile polymorphisms were reported to be a risk factor of gastric carcinoma or its precursors in Caucasian and Indian population, but these polymorphisms are absent in Japanese. We investigated the associations of TLR4 +3725 G/C polymorphism, another functional polymorphism of TLR4 , with risk of gastric cancer and gastric atrophy in Japanese.
Materials and Methods:  Study subjects were 583 histologically diagnosed gastric cancer patients and age- and sex-matched 1592 control outpatients, who visited Aichi Cancer Center Hospital from 2001 to 2005. Serum anti- H. pylori IgG antibody and pepsinogens were measured to evaluate H. pylori infection and gastric atrophy, respectively. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by a logistic model.
Results:  Among the seropositive subjects, the age- and sex-adjusted OR of gastric atrophy was 1.17 (95%CI: 0.91–1.50) for G/C , 1.20 (95%CI: 0.76–1.89) for C/C , and 1.18 (95%CI: 0.93–1.49) for G/C + C/C relative to G/G genotype. The age- and sex-adjusted OR of severe gastric atrophy among H. pylori seropositive subjects was 1.43 (95%CI: 0.99–2.06) for G/C , 1.47 (95%CI: 0.76–2.88) for C/C , and 1.43 (95%CI: 1.01–2.04) for G/C + C/C . The OR of gastric cancer compared with gastric atrophy controls was not statistically significant.
Conclusion:  Our study found that TLR4 +3725 G/C polymorphism was a risk factor of severe gastric atrophy in H. pylori seropositive Japanese. Our results underscored the significance of the variations in host innate immunity due to TLR4 polymorphism as genetic predispositions to gastric precancerous lesions in Eastern Asian populations with the same backgrounds.     

Does land-use change affect biodiversity dynamics at a macroecological scale? A case study of birds over the past 20 years in Japan

Because the effects of land-use change on biodiversity have primarily been examined at or below the regional scale, it remains unclear whether such effects scale up to the macroecological scale (i.e. nationwide or continental scale). In Japan, forests have become more mature since the cessation of most forestry efforts in the 1970s. At a nationwide scale, this forest maturation may lead to reductions in the abundance of species that depend on early successional forests (early successional species) and increases in the abundance of species that depend on mature forests (mature forest species). Japan has met its high demand for wood through imports from South-east Asia, resulting in deforestation there. Therefore, the abundance of mature forest species that migrate long distances to overwinter in South-east Asia may decrease. We examined changes in the range sizes of birds in Japan over the past 20 years using the living planet index (LPI). The LPI indicated that the range sizes of early successional species decreased. For mature forest species, the range sizes of long-distance migrants decreased, whereas those of short-distance migrants and residents increased. Our predictions were generally supported. Our results indicate that the effects of land-use change extend to the macroecological scale and that such changes in one country can affect the biodiversity dynamics in other countries. Forest maturation in Japan and concomitant deforestation in South-east Asia have been caused by internationally coupled socioeconomic processes. Therefore, biodiversity conservation at the macroecological scale must consider the role of land use, and such efforts will require both international and socioeconomic perspectives.     

Alfalfa fields promote high reproductive rate of an invasive predatory lady beetle

Invasive insect species often may attain high fecundity in agricultural habitats, thereby contributing to their establishment in new geographic regions and their displacement of similar native species. Such may be true for predatory lady beetles (Coleoptera: Coccinellidae) that have been introduced to North America in recent decades, raising concerns of adverse impact on native lady beetles. In northern Utah, Coccinella septempunctata L. first appeared in 1991, and is now predominant among lady beetles especially in alfalfa fields. We assessed the suitability of alfalfa fields as breeding habitat for females of C. septempunctata and the native, similarly sized Coccinella transversoguttata richardsoni Brown. The timing and amount of egg production differed significantly between C. septempunctata and C. transversoguttata as populations of aphids increased through spring and early summer. Reproduction by both species conformed to the egg window hypothesis, with populations of the predators producing most eggs before aphid numbers peaked. But consistently among fields and years, females of C. septempunctata produced more eggs, and did so earlier in the spring, than C. transversoguttata females even at low prey density. Furthermore, C. septempunctata females were more successful than females of C. transversoguttata in approaching their maximum body weights and reproductive output as measured in the laboratory under ideal conditions. The strong reproductive success of C. septempunctata may contribute to its displacement of C. transversoguttata in irrigated alfalfa in the generally arid Intermountain West of North America and to its establishment as an abundant species in this region of North America.