The L1 major capsid protein of human papillomavirus type 11 recombinant virus-like particles interacts with heparin and cell-surface glycosaminoglycans on human keratinocytes

The L1 major capsid protein of human papillomavirus (HPV) type 11, a 55-kDa polypeptide, forms particulate structures resembling native virus with an average particle diameter of 50-60 nm when expressed in the yeast Saccharomyces cerevisiae. We show in this report that these virus-like particles (VLPs) interact with heparin and with cell-surface glycosaminoglycans (GAGs) resembling heparin on keratinocytes and Chinese hamster ovary cells. The binding of VLPs to heparin is shown to exhibit an affinity comparable to that of other identified heparin-binding proteins. Immobilized heparin chromatography and surface plasmon resonance were used to show that this interaction can be specifically inhibited by free heparin and dextran sulfate and that the effectiveness of the inhibitor is related to its molecular weight and charge density. Sequence comparison of nine human L1 types revealed a conserved region of the carboxyl terminus containing clustered basic amino acids that bear resemblance to proposed heparin-binding motifs in unrelated proteins. Specific enzymatic cleavage of this region eliminated binding to both immobilized heparin and human keratinocyte (HaCaT) cells. Removal of heparan sulfate GAGs on keratinocytes by treatment with heparinase or heparitinase resulted in an 80-90% reduction of VLP binding, whereas treatment of cells with laminin, a substrate for alpha6 integrin receptors, provided minimal inhibition. Cells treated with chlorate or substituted beta-D-xylosides, resulting in undersulfation or secretion of GAG chains, also showed a reduced affinity for VLPs. Similarly, binding of VLPs to a Chinese hamster ovary cell mutant deficient in GAG synthesis was shown to be only 10% that observed for wild type cells. This report establishes for the first time that the carboxyl-terminal portion of HPV L1 interacts with heparin, and that this region appears to be crucial for interaction with the cell surface.     

Pdx1 and Ngn3 overexpression enhances pancreatic differentiation of mouse ES cell-derived endoderm population

In order to define the molecular mechanisms regulating the specification and differentiation of pancreatic β-islet cells, we investigated the effect of upregulating Pdx1 and Ngn3 during the differentiation of the β-islet-like cells from murine embryonic stem (ES) cell-derived activin induced-endoderm. Induced overexpression of Pdx1 resulted in a significant upregulation of insulin (Ins1 and Ins2), and other pancreas-related genes. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we induced the overexpression express of Ngn3 together with Pdx1. This combination dramatically increased the level and timing of maximal Ins1 mRNA expression to approximately 100% of that found in the βTC6 insulinoma cell line. Insulin protein and C-peptide expression was confirmed by immunohistochemistry staining. These inductive effects were restricted to c-kit(+) endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions after the specification of pancreatic endoderm. Although insulin secretion was stimulated by various insulin secretagogues, these cells had only limited glucose response. Microarray analysis was used to evaluate the expression of a broad spectrum of pancreatic endocrine cell-related genes as well as genes associated with glucose responses. Taken together, these findings demonstrate the utility of manipulating Pdx1 and Ngn3 expression in a stage-specific manner as an important new strategy for the efficient generation of functionally immature insulin-producing β-islet cells from ES cells.     

Nematotoxicity of Marasmius oreades agglutinin (MOA) depends on glycolipid binding and cysteine protease activity

Fruiting body lectins have been proposed to act as effector proteins in the defense of fungi against parasites and predators. The Marasmius oreades agglutinin (MOA) is a Galα1,3Gal/GalNAc-specific lectin from the fairy ring mushroom that consists of an N-terminal ricin B-type lectin domain and a C-terminal dimerization domain. The latter domain shows structural similarity to catalytically active proteins, suggesting that, in addition to its carbohydrate-binding activity, MOA has an enzymatic function. Here, we demonstrate toxicity of MOA toward the model nematode Caenorhabditis elegans. This toxicity depends on binding of MOA to glycosphingolipids of the worm via its lectin domain. We show further that MOA has cysteine protease activity and demonstrate a critical role of this catalytic function in MOA-mediated nematotoxicity. The proteolytic activity of MOA was dependent on high Ca(2+) concentrations and favored by slightly alkaline pH, suggesting that these conditions trigger activation of the toxin at the target location. Our results suggest that MOA is a fungal toxin with intriguing similarities to bacterial binary toxins and has a protective function against fungivorous soil nematodes.     

Complete genome sequence and updated annotation of Desulfovibrio alaskensis G20

Desulfovibrio alaskensis G20 (formerly Desulfovibrio desulfuricans G20) is a Gram-negative mesophilic sulfate-reducing bacterium (SRB), known to corrode ferrous metals and to reduce toxic radionuclides and metals such as uranium and chromium to sparingly soluble and less toxic forms. We present the 3.7-Mb genome sequence to provide insights into its physiology.     

Increased Osteoclastogenesis in Mice Lacking the Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1

Alterations in bone remodeling are a major public health issue, as therapeutic options for widespread bone disorders such as osteoporosis and tumor-induced osteolysis are still limited. Therefore, a detailed understanding of the regulatory mechanism governing bone cell differentiation in health and disease are of utmost clinical importance. Here we report a novel function of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a member of the immunoglobulin superfamily involved in inflammation and tumorigenesis, in the physiologic regulation of bone remodeling. Assessing the expression of all members of the murine Ceacam family in bone tissue and marrow, we found CEACAM1 and CEACAM10 to be differentially expressed in both bone-forming osteoblasts and bone-resorbing osteoclasts. While Ceacam10-deficient mice displayed no alteration in structural bone parameters, static histomorphometry demonstrated a reduced trabecular bone mass in mice lacking CEACAM1. Furthermore, cellular and dynamic histomorphometry revealed an increased osteoclast formation in Ceacam1-deficient mice, while osteoblast parameters and the bone formation rate remained unchanged. In line with these findings, we detected accelerated osteoclastogenesis in Ceacam1-deficient bone marrow cells, while osteoblast differentiation, as determined by mineralization and alkaline phosphatase assays, was not affected. Therefore, our results provide in vivo and in vitro evidence for a physiologic role of CEACAM1 in the regulation of osteoclastogenesis.     

Bacterial fingerprinting by flow cytometry: bacterial species discrimination.

BACKGROUND: A flow cytometric measurement (FCM) technique has been developed to size DNA fragments. Individual fragments of a restriction digest of genomic DNA, stained with an intercalating dye, are passed through an ultrasensitive cytometer. The measured fluorescence intensity from each fragment is proportional to the fragment length. METHODS: The isolation of bacterial genomic DNA and digestion by restriction enzymes were performed inside an agarose plug. Rare cutting enzymes were employed to produce a manageable number of DNA fragments. Electroelution was used to move the DNA fragments from the agarose plug into a solution containing polyamines to protect the DNA from shear-induced breakage. The DNA was stained with the bisintercalating dye thiazole orange homodimer and introduced into our ultrasensitive flow cytometer. A histogram of the fluorescence intensities (fingerprint) was constructed. RESULTS: Gram-positive Bacillus globigii and gram-negative bacteria Escherichia coli and Erwinia herbicola were distinguished by the fingerprint pattern of restriction fragments of their genomic DNA. DNA sizes determined by FCM are in good agreement with pulsed-field gel electrophoresis (PFGE) analysis. Flow cytometry requires only picogram quantities of purified DNA and takes less than 10 min for data collection and analysis. When the total sample preparation time is included, the analysis times for PFGE and FCM are similar ( approximately 3 days). CONCLUSIONS: FCM is an attractive technique for the identification of bacterial species. It is more sensitive and potentially much faster than PFGE.     

The interaction of macrophages and bacteria: a comparative study of the induction of tumoricidal activity and of reactive nitrogen intermediates

The abilities of various bacteria to induce in a pure population of bone marrow-derived mononuclear phagocytes (BMM phi) tumoricidal activity and/or the generation of reactive nitrogen intermediates (RNI) were comparatively assessed. Interaction of BMM phi with bacteria led to expression of these functional activities, indicating that the organisms were recognized as foreign. As the majority of bacteria elicited in BMM phi either tumoricidal activity (that is maintained for days) or the production of RNI, measured by the release of nitrite (that is short-lived), it appears that the two functions are under separate control. However, both functions are inhibited or even abrogated by arginase or the L-arginine analogue, NG-monomethyl-L-arginine, suggesting that their expression is dependent on L-arginine.     

Auxins,ABA and gibberellin-like activity in abscising and non-abscising flowers and pods of Vicia faba L.

Abscission probability varies among floral positions within inflorescences of Vicia faba L. Flowers from proximal positions have a greater chance to develop into mature pods than flowers from more distal positions which normally abscise either as older flowers or as young pods. In three field experiments with the indeterminate single stem variety Herz-Freya, changes in the contents of extractable auxins, abscisic acid (ABA) and gibberellins in flowers and pods during their development, and their possible influence on abscission were investigated.Inflorescences at different positions along the stem were divided into the two proximal and the remaining fruits. The content of all three hormones was at a low level during flower development, increased greatly in parallel with dry matter accumulation in the young pods, and then decreased to maturity. The first hormone to increase in the fruits was auxin and this took place when abscission from the distal positions began. ABA and gibberellins at this time were still at a low level. This ontogenic course of hormone production was very similar in fruits of both positions within an inflorescence, but in flowers and young pods from proximal positions, auxin content in most inflorescences was greater than in those from the abscising distal positions. No such positional differences were observed with ABA and gibberellins. Decapitation of the plants reduced flower and pod drop from the remaining reproductive nodes. Although decapitation resulted in less abscission among distal flowers and young pods from these nodes, it did not affect the ontogenic course of auxin and ABA production in these fruits.     

Late neogene biostratigraphy and paleoceanography of DSDP site 310 Central North Pacific and correlation with the Southwest Pacific

Late Neogene planktonic foraminifera have been examined at Site 310 in the Central North Pacific and their stratigraphic ranges and frequencies are presented here. Blow's (1969) zonation developed for tropical regions has been applied where applicable. Where tropical index taxa are rare or absent in this temperate region, Globorotalia crassaformis, and the evolutionary bioseries G. conoidea — G. conomiozea and G. puncticula — G. inflata have been found useful for zonal subdivisions. A correlation between stratigraphic ranges and frequency distributions of these species at Site 310 in the Central North Pacific, and Site 284 in the Southwest Pacific indicates that these species are relatively consistent biostratigraphic markers in temperate regions of both the North and South Pacific Oceans. An informal zonation for temperate latitudes of the Southwest Pacific has been established by Kennett (1973) and a similar zonal subdivision can be made at Site 310.Paleoclimatic/paleoceanographic interpretations based on coiling ratios, percent abundance, and phenotypic variations of Neogloboquadrina pachyderma indicate four major cold events during early, middle, and late Pliocene, and early Pleistocene. Faunal correlations of these events with similar events elsewhere in the Northeast and Southwest Pacific which have been paleomagnetically dated indicate the following approximate ages for these cold events: 4.7 Ma, 3.0 Ma, 2.6–1.8 Ma. and 1.2 Ma. Faunal assemblages have been divided into three groups representing cool, intermediate, and warmer water assemblages. Cool water assemblages are dominated by >60% N. pachyderma; intermediate temperature faunas are dominated by species of Globigerina and Globigerinita and contain between 20% and 30% N. pachyderma. Warmer water assemblages are dominated by species of Globorotalia and contain <10% N. pachyderma. Frequency oscillations within these groups, in addition to paleotemperature parameters evident in N. pachyderma, afford refined paleoclimatic/paleoceanographic interpretations.