Isolation and characterization of a new cellulosome-producing Clostridium thermocellum strain

The anaerobic thermophilic bacterium, Clostridium thermocellum, is a potent cellulolytic microorganism that produces large extracellular multienzyme complexes called cellulosomes. To isolate C. thermocellum organisms that possess effective cellulose-degrading ability, new thermophilic cellulolytic strains were screened from more than 800 samples obtained mainly from agriculture residues in Thailand using microcrystalline cellulose as a carbon source. A new strain, C. thermocellum S14, having high cellulose-degrading ability was isolated from bagasse paper sludge. Cellulosomes prepared from S14 demonstrated faster degradation of microcrystalline cellulose, and 3.4- and 5.6-fold greater Avicelase activity than those from C. thermocellum ATCC27405 and JW20 (ATCC31449), respectively. Scanning electron microscopic analysis showed that S14 had unique cell surface features with few protuberances in contrast to the type strains. In addition, the cellulosome of S14 was resistant to inhibition by cellobiose that is a major end product of cellulose hydrolysis. Saccharification tests conducted using rice straw soaked with sodium hydroxide indicated the cellulosome of S14 released approximately 1.5-fold more total sugars compared to that of ATCC27405. This newly isolated S14 strain has the potential as an enzyme resource for effective lignocellulose degradation.     

Is the Gut the Major Source of Virus in Early Simian Immunodeficiency Virus Infection?

The acute phases of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection are characterized by rapid and profound depletion of CD4⁺ T cells from the guts of infected individuals. The large number of CD4⁺ T cells in the gut (a large fraction of which are activated and express the HIV/SIV coreceptor CCR5), the high level of infection of these cells, and the temporal coincidence of this CD4⁺ T-cell depletion with the peak of virus in plasma in acute infection suggest that the intestinal mucosa may be the major source of virus driving the peak viral load. Here, we used data on CD4⁺ T-cell proportions in the lamina propria of the rectums of SIV-infected rhesus macaques (which progress to AIDS) and sooty mangabeys (which do not progress) to show that in both species, the depletion of CD4⁺ T cells from this mucosal site and its maximum loss rate are often observed several days before the peak in viral load, with few CD4⁺ T cells remaining in the rectum by the time of peak viral load. In contrast, the maximum loss rate of CD4⁺ T cells from bronchoalveolar lavage specimens and lymph nodes coincides with the peak in virus. Analysis of the kinetics of depletion suggests that, in both rhesus macaques and sooty mangabeys, CD4⁺ T cells in the intestinal mucosa are a highly susceptible population for infection but not a major source of plasma virus in acute SIV infection.The acute phase of human immunodeficiency virus (HIV) infection is characterized by moderate CD4⁺ T-cell depletion in blood, followed by a transient partial restoration of CD4⁺ T-cell numbers and eventually by a slow long-term CD4⁺ T-cell decline in the chronic phase that lasts for several years. Studies of CD4⁺ T-cell depletion in mucosal sites, often conducted with simian immunodeficiency virus (SIV)-infected macaques, have demonstrated that mucosal CD4⁺ T-cell depletion is more rapid and profound (3, 10, 13, 19, 21). The severe depletion of cells in the gut in early infection is thought to be driven in part by the phenotype of the cells present, which are predominantly CCR5⁺ and in general more activated than their circulating counterparts. As such, these mucosal CD4⁺ T cells are highly susceptible to productive infection with the dominant CCR5-tropic strains of HIV and SIV present in early infection (20). The rapid depletion of CD4⁺ T cells at mucosal sites is accompanied by relatively high numbers of infected cells (10, 13) and is temporally associated with the peak viral load in plasma, suggesting that the infection of mucosal CD4⁺ T cells may be responsible for the majority of virus replication occurring during acute infection (10, 15, 21, 22).The size of the CD4⁺ T-cell pool in the gut is a matter of some controversy, with estimates ranging from ∼5 to 50% of the total body pool of these cells (reviewed in reference 5). Regardless of the precise numbers, the gut (and particularly the mucosal lamina propria) contains a significant proportion of the body CD4⁺ CCR5⁺ memory T cells, which are depleted very early in infection. However, whether CD4⁺ T cells in the gut are merely a target of early infection or whether they are a major driver of early viral growth and peak viral loads in acute infection is unclear. Here we use a combination of experimental data and modeling to demonstrate that the gut is unlikely to be a major source of virus production in acute SIV infection.     

The structure of the integrin αIIbβ3 transmembrane complex explains integrin transmembrane signalling

Heterodimeric integrin adhesion receptors regulate cell migration, survival and differentiation in metazoa by communicating signals bi‐directionally across the plasma membrane. Protein engineering and mutagenesis studies have suggested that the dissociation of a complex formed by the single‐pass transmembrane (TM) segments of the α and β subunits is central to these signalling events. Here, we report the structure of the integrin αIIbβ3 TM complex, structure‐based site‐directed mutagenesis and lipid embedding estimates to reveal the structural event that underlies the transition from associated to dissociated states, that is, TM signalling. The complex is stabilized by glycine‐packing mediated TM helix crossing within the extracellular membrane leaflet, and by unique hydrophobic and electrostatic bridges in the intracellular leaflet that mediate an unusual, asymmetric association of the 24‐ and 29‐residue αIIb and β3 TM helices. The structurally unique, highly conserved integrin αIIbβ3 TM complex rationalizes bi‐directional signalling and represents the first structure of a heterodimeric TM receptor complex.     

Caveolin-1-dependent and -independent membrane domains

Lipid rafts defined as cholesterol- and sphingomyelin-rich domains have been isolated from different cell types that vary greatly in their lipid profiles. Here, we investigated the contribution of the structural protein caveolin-1 (Cav1) to the overall lipid composition and domain abundance in mouse embryonic fibroblasts (MEFs) from wild-type (WT) or Cav1-deficient (Cav1^−/−) animals. Our findings show that Cav1 expression had no effect on free (membrane-associated) cholesterol levels. However, Cav1^−/−-deficient cells did have a higher proportion of sphingomyelin, decreased abundance of unsaturated phospholipids, and a trend toward shorter fatty acid chains in phosphatidylcholine. We isolated detergent-resistant membranes (DRMs), nondetergent raft domains (NDR), and cholesterol oxidase (CO)-sensitive domains and assessed the abundance of ordered domains in intact cells using the fluorescent dye Laurdan. Despite differences in phospholipid composition, we found that cholesterol levels in DRMs, NDR, and CO-sensitive domains were similar in both cell types. The data suggest that Cav1 is not required to target cholesterol to lipid rafts and that CO does not specifically oxidize caveolar cholesterol. In contrast, the abundance of ordered domains in adherent cells is reduced in Cav1^−/− compared with WT MEFs, suggesting that cell architecture is critical in maintaining Cav1-induced lipid rafts.     

Hydrologic regulation of gross methyl chloride and methyl bromide uptake from Alaskan Arctic tundra

The Arctic tundra has been shown to be a potentially significant regional sink for methyl chloride (CH₃Cl) and methyl bromide (CH₃Br), although prior field studies were spatially and temporally limited, and did not include gross flux measurements. Here we compare net and gross CH₃Cl and CH₃Br fluxes in the northern coastal plain and continental interior. As expected, both regions were net sinks for CH₃Cl and CH₃Br. Gross uptake rates (−793 nmol CH₃Cl m⁻² day⁻¹ and −20.3 nmol CH₃Br m⁻² day⁻¹) were 20–240% greater than net fluxes, suggesting that the Arctic is an even greater sink than previously believed. Hydrology was the principal regulator of methyl halide flux, with an overall trend towards increasing methyl halide uptake with decreasing soil moisture. Water table depth was one of the best predictors of net and gross uptake, with uptake increasing proportionately with water table depth. In drier areas, gross uptake was very high, averaging −1201 nmol CH₃Cl m⁻² day⁻¹ and −34.9 nmol CH₃Br m⁻² day⁻¹; in flooded areas, gross uptake was significantly lower, averaging −61 nmol CH₃Cl m⁻² day⁻¹ and −2.3 nmol CH₃Br m⁻² day⁻¹. Net and gross uptake was greater in the continental interior than in the northern coastal plain, presumably due to drier inland conditions. Within certain microtopographic features (low‐ and high‐centered polygons), uptake rates were positively correlated with soil temperature, indicating that temperature played a secondary role in methyl halide uptake. Incubations suggested that the inverse relationship between water content and methyl halide uptake was the result of mass transfer limitation in saturated soils, rather than because of reduced microbial activity under anaerobic conditions. These findings have potential regional significance, as the Arctic is expected to become warmer and drier due to anthropogenic climate forcing, potentially enhancing the Arctic sink for CH₃Cl and CH₃Br.     

Bioinformatics in molecular immunology laboratories demonstrated: Modeling an anti-CMV scFv antibody

A scFv (single chain variable fragment) antibody clone from anti-CMV (anti-cucumber mosaic virus) was successfully constructed from immunized mouse and the DNA sequence was submitted to GenBank (AY337618 and AY337619). The expression of a 32 kDa recombinant antibody in bacteria was verified using ELISA (enzyme-linked immunoassay) and western blot. However, elucidation of specific anti-CMV scFv function requires detailed and time consuming immuno-assays. Alternatively, useful functional information on anti-CMV scFV antibody can be obtained using available Bioinformatics tools and techniques without performing tedious assays. Here, we use the commonly used Bioinformatics tools and databases such as BLAST (basic local alignment search tool), GenBank, PDB (protein databank), KABAT numbering, SWISS-MODEL and Insight II to gain specific functional insights into anti-CMV scFv.     

Morphological variation of perch Perca fluviatilis in humic lakes: the effect of predator density,competition and prey abundance

Between and within‐lake variations in morphology of perch Perca fluviatilis were studied in four humic lakes in eastern Finland. Perca fluviatilis were more streamlined and smaller headed in a lake with the highest abundance of cyprinids, but lowest abundance of predators (Lake Tuopanjärvi), indicating adaptation to planktivorous feeding and low predator density. Highest bodied fish were found from a lake with the lowest cyprinid but highest predator abundance (Lake Koppelojärvi), which conversely indicates adaptation to more effective predator avoidance. Furthermore, the length of the paired fins was longest in Lake Kinnasjärvi and Lake Tuopanjärvi, where the abundance of benthic macroinvertebrates was lowest, suggesting selection for more effective benthivory. Clear morphological differences of P. fluviatilis between habitats were found only in Lake Kinnasjärvi, whereas in Lake Koppelojärvi and Lake Tuopanjärvi only the length of the paired fins differed and in Lake Harkkojärvi no differences were found. Taken together, these results suggest that inter and intrapopulation morphological differences are probably highly dependent on different biotic factors (i.e. predation risk, resource availability and competition). Spatial and temporal variations in these factors may have a great effect on body morphology of P. fluviatilis.     

Cholesterol-induced caveolin targeting to lipid droplets in adipocytes: a role for caveolar endocytosis

We have investigated the targeting of caveolin to lipid bodies in adipocytes that express high levels of caveolins and contain well-developed lipid droplets. We observed that the lipid droplets isolated from adipocytes of caveolin-1 knock out mice contained dramatically reduced levels of cholesterol, indicating that caveolin is required for maintaining the cholesterol content of this organelle. Analysis of caveolin distribution by cell fractionation and fluorescent light microscopy in 3T3-L1 adipocytes indicated that addition of cholesterol rapidly stimulated translocation of caveolin to lipid droplets. The cholesterol-induced trafficking of caveolins to lipid droplets was shown to be dynamin- and protein kinase C (PKC)-dependent and modulated by src tyrosine kinase activation, suggesting a role for caveolar endocytosis in this novel trafficking pathway. Consistent with this, caveolae budding was stimulated by cholesterol addition. The present data identify lipid droplets as potential target organelles for caveolar endocytosis and demonstrate a role for caveolin-1 in the maintenance of free cholesterol levels in adipocyte lipid droplets.     

Angiostatin formation involves disulfide bond reduction and proteolysis in kringle 5 of plasmin

Plasmin is processed in the conditioned medium of HT1080 fibrosarcoma cells producing fragments with the domain structures of the angiogenesis inhibitor, angiostatin, and microplasmin. Angiostatin consists of kringle domains 1-4 and part of kringle 5, while microplasmin consists of the remainder of kringle 5 and the serine proteinase domain. Our findings indicate that formation of angiostatin/microplasmin involves reduction of plasmin by a plasmin reductase followed by proteolysis of the reduced enzyme. We present evidence that the Cys461-Cys540 and Cys511-Cys535 disulfide bonds in kringle 5 of plasmin were reduced by plasmin reductase. Plasmin reductase activity was secreted by HT1080 and Chinese hamster ovary cells and the human mammary carcinoma cell lines MCF-7, MDA231, and BT20 but not by the monocyte/macrophage cell line THP-1. Neither primary foreskin fibroblasts, blood monocyte/macrophages, nor macrovascular or microvascular endothelial cells secreted detectable plasmin reductase. In contrast, cultured bovine and rat vascular smooth muscle cells secreted small but reproducible levels of plasmin reductase. Reduction of the kringle 5 disulfide bonds triggered cleavage at either Arg529-Lys530 or two other positions C-terminal of Cys461 in kringle 5 by a serine proteinase. Plasmin autoproteolysis could account for the cleavage, although another proteinase was mostly responsible in HT1080 conditioned medium. Three serine proteinases with apparent Mr of 70, 50, and 39 were purified from HT1080 conditioned medium, one or more of which could contribute to proteolysis of reduced plasmin.