Up-regulation of vascular endothelial growth factor in response to glucose deprivation

Vascular endothelial growth factor (VEGF), also known as a vascular permeability factor (VPF), is an endothelial specific mitogen and is a potent inducer of angiogenesis. Recently it has been reported that hypoxia induces VEGF mRNA expression in various cells. Since both oxygen and glucose are required for efficient production of energy, we examined the effect of glucose deprivation on VEGF mRNA expression and VEGF protein production in U-937 (a human monocytic cell line) cells. Both the mRNA expression and secretion of VEGF increased after exposure to low glucose. Addition of L-glucose, the L-stereoisomer of D-glucose, did not prevent the up-regulation of VEGF expression. The conditioned medium from glucose-deprived cells, followed by supplementation with glucose, did not up-regulate VEGF mRNA expression in U-937 cells. The low glucose-induced VEGF mRNA expression returned to the control level after supplementation with D-glucose. Furthermore, oligomycin, a mitochondrial ATP synthase inhibitor, increased VEGF protein production. The results suggest that the up-regulation of VEGF mRNA in U-937 cells in response to glucose deprivation is not mediated by autocrine factors from the cells nor is the osmotic change of the medium mediated by the deficiency of glucose metabolism in the cells. Our results also suggest that the intracellular ATP depletion due to glucose deprivation may be one of the causes for increased VEGF mRNA expression. We speculate that local hypoglycemia may act as an essential trigger for angiogenesis through the VEGF gene expression.     

Visualization of the stop of microtubule depolymerization that occurs at the high-density region of microtubule-associated protein 2 (MAP2).

Individual microtubules (MTs) repeat alternating phases of polymerization and depolymerization, a process known as dynamic instability. Microtubule-associated proteins (MAPs) regulate the dynamic instability by increasing the rescue frequency. To explore the influence of MAP2 on in vitro MT dynamics, we correlated the distribution of MAP2 on individual MTs with the dynamic phase changes of the same MTs. MAP2 was modified selectively on its projection region by X-rhodamine iodoacetamide without altering the MT-binding activity. When the labeled MAP2 was added to MTs, the fluorescence was distributed along almost the entire length of individual MTs. However, the inhomogeneity of the distribution gradually became obvious due to the fluorescence bleaching, and the MTs appeared to consist of rapidly bleached portions (RBPs) and slowly bleached portions (SBPs), which were distributed randomly along the MT. By measuring the duration of fluorescence bleaching, the density of MAP2 in SBP was estimated to be approximately 2.5 times higher than the RBP. The average tubulin:MAP2 ratio in SBP was calculated to be 16. When the MT dynamics were observed by dark-field microscopy after determining the MAP2 distribution, rescues were always found to occur only at the SBPs. MTs also displayed intermittent shortening by repeated depolymerization phases separated by pause phases. In these cases, depolymerization phases stopped only at the SBPs. Not every SBP stopped depolymerization, but depolymerization always stopped at an SBP. Taken together, we suggest that there is a minimum density of MAP2 that is necessary to stop depolymerization.     

Photosensitized Oxygenation of α-Pyran Derived from β-Ionone

Photosensitized oxygenation of α-pyran(l) (2,5,5,8a-tetramethyl-6,7,8,8a-tetrahydro-5H- l-benzopyran), in methanol using rose bengal, rapidly formed a stable peroxide (2).

The peroxide (2) gave 6,6-dimethyl-8-undecene-2,7,10-trione(11) at 140°C in xylene, and 8(or 9)-methoxy or hydroxy derivatives of 6,6-dimethyl-undecane-2,7,10-trione (12a or b) by hydrochloric acid.

These triketones (11 and 12a) were also obtained by a photo-reaction from the peroxide(2).     

Can reproductive traits help to explain the coexistence of mud crabs Panopeus (Decapoda: Panopeidae)? A case of two sympatric species inhabiting an impacted mangrove area of Southern Brazil

Coexistence among species is commonly related to niche divergence. However, congenerics usually are very similar in their microhabitat selection and food consumption. Thus, divergent life history strategies may represent the mechanism that allows sympatry in related species. Here, we describe and compare reproductive features in two sympatric mud crabs Panopeus americanus and P. occidentalis in an impacted mangrove area in Southern Brazil. As these species are ecologically similar, we hypothesize that these species diverge in their reproductive traits, which could explain their coexistence. Crabs were collected every two months from September 2004 to July 2006. Reproductive features such as number and size of ovigerous females, breeding season, fecundity, reproductive output, and embryo volume were assessed. Panopeus americanus produced embryos during the entire sampled period, while P. occidentalis produced only between September and March. Panopeus americanus produced more embryos considering the size of the species, had significantly lower embryo volume, and higher reproductive output than P. occidentalis. These data permit to classify P. americanus as an r-strategist and P. occidentalis as a K-strategist regarding their reproductive traits. In conclusion, our results support the hypothesis that divergent reproductive features may allow coexistence of these mud crabs.     

NMR characterization of intramolecular interaction of osteopontin, an intrinsically disordered protein with cryptic integrin-binding motifs

Osteopontin (OPN) is an integrin-binding protein found in a variety of tissues and physiological fluids and is involved in divergent biological processes such as migration, adhesion and signaling in integrin-independent as well as dependent manners. The adhesive activity of this protein is modulated upon cleavage by thrombin at the central part of the molecule, in the vicinity of the integrin-binding sequences. Although detailed structural characterization is crucial for further understanding of the regulatory mechanisms of the OPN functions, its intrinsically disordered property hampers in-depth conformational analyses. Here we report an NMR study of mouse OPN and its N-terminal thrombin-cleavage product to characterize intramolecular interaction of this molecule. Paramagnetic relaxation enhancement experiment revealed that OPN exhibits a long-range intramolecular interaction between the N- and C-terminal regions. Furthermore, our NMR data showed that anti-OPN antibody OPN1.2, whose reactivity is impaired by deletion or amino acid substitutions of the arginine-aspartate-glycine integrin-binding motif, binds the N-terminal side of the integrin-binding motifs suggesting the existence of intramolecular interaction. These data suggest that functional interactions of OPN with integrins and the other binding partners can be modulated by the intramolecular interactions.     

Chemotaxonomic study of Vibrio cholerae bio-serogroup Hakata on the basis of the sugar composition of the polysaccharide portion of its lipopolysaccharides

A chemotaxonomic study was carried out on 31 strains of non-O1 Vibrio cholerae bio-serogroup Hakata, isolated in Japan, which possesses the Inaba antigen C of O1 V. cholerae. On the basis of the compositional sugar pattern of the polysaccharide portion of their lipopolysaccharides, the 23 strains isolated from the environment were separated into two groups, one (20 strains) containing mannose, glucose, fructose, L-glycero-D-mannoheptose, glucosamine, perosamine, quinovosamine, and an unidentified amino sugar AS, and the other (3 strains) containing two additional sugars, galactose and a trace amount of galactosamine. All of the eight strains isolated from imported seafoods belonged to the former group.     

Revision of the Capsaspora genome using read mating information adjusts the view on premetazoan genome

The genome sequences of unicellular holozoans, the closest relatives to animals, are shedding light on the evolution of animal multicellularity, shaping the genetic contents of the putative premetazoans. However, the assembly quality of the genomes remains poor compared to the major model organisms such as human and fly. Improving the assembly is critical for precise comparative genomics studies and further molecular biological studies requiring accurate sequence information such as enhancer analysis and genome editing. In this report, we present a new strategy to improve the assembly by fully exploiting the information of Illumina mate-pair reads. By visualizing the distance and orientation of the mapped read pairs, we could highlight the regions where possible assembly errors exist in the genome sequence of Capsaspora, a lineage of unicellular holozoans. Manual modification of these errors repaired 590 assembly problems in total and reassembled 84 supercontigs into 55. Our telomere prediction analysis using the read pairs containing the pan-eukaryotic telomere-like sequence identified at least 13 chromosomes. The resulting new assembly posed us a re-annotation of 112 genes, including 15 putative receptor protein tyrosine kinases. Our strategy thus provides a useful approach for improving assemblies of draft genomes, and the new Capsaspora genome offers us an opportunity to adjust the view on the genome of the unicellular animal ancestor.     

Construction of multi-chimeric pyrroloquinoline quinone glucose dehydrogenase with improved enzymatic properties and application in glucose monitoring

A multi-chimeric enzyme was constructed by combining the protein regions responsible for the enzymatic properties of Escherichia coli and Acinetobacter calcoaceticus pyrroloquinoline quinone glucose dehydrogenase (PQQGDH). The constructed multi-chimeric PQQGDH showed increased co-factor binding stability, thermal stability, an alteration in substrate specificity and a 10-fold increase in the K _m value for glucose compared with the wild-type E. coli PQQGDH. The cumulative effect of each introduced protein region on the improvement of enzymatic properties was observed. The application of the multi-chimeric PQQGDH in amperometric glucose sensor construction achieved an expanded dynamic range together with increased operational stability and narrower substrate specificity. The glucose sensor can measure glucose from 5 to 40 mM, suggesting its potential for the direct measurement of high blood-glucose levels in diabetic patients.     

Polymorphism and distribution of putative cell-surface adhesin-encoding ORFs among human fecal isolates of Bifidobacterium longum subsp. longum

The polymorphism of ORFs encoding putative cell-surface adhesins was investigated in Bifidobacterium longum subsp. longum. Firstly, we performed a PCR assay targeting 15 ORFs encoding putative adhesion proteins, which included 8 ORFs with a sortase targeting LPXTG motif, in 42 strains of different pulsotypes isolated from fecal samples from 12 human individuals. We found a variability in the presence of an ORF, BL0675, which encodes a putative fimbrial subunit protein. We sequenced ORFs corresponding to BL0675 in the 42 strains and adjacent ORFs corresponding to BL0674 and BL0676. The results indicated that ORFs corresponding to BL0675 were highly polymorphic with five variant types (i.e. A-, B-, C-, D-, and E-types). Meanwhile, BL0674 and BL0676, which encode an additional putative fimbrial subunit protein and a fimbrial-associated sortase-like protein, were highly conserved. Subsequent quantitative polymerase chain reaction (qPCR) assays targeting the variant types in 89 human fecal samples revealed that A-type was the most commonly distributed (74.2%), followed by B-type (59.6%), D-type (31.5%), E-type (32.6%) and C-type (5.6% prevalence). Since BL0675 is considered to be a fimbrial protein with glycoprotein-binding ability, the proteins encoded by the five variant types of BL0675 may have specific binding properties to various carbohydrate structures expressed on the human intestinal wall, thereby allowing B. longum to colonize the intestine in a host-specific manner.