Isolation of 2,4-Diacetylphloroglucinol from a Fluorescent Pseudomonad and Investigation of Physiological Parameters Influencing Its Production

Pseudomonas sp. strain F113 was isolated from the rhizosphere of sugar beets and shown to inhibit a range of plant pathogenic fungi by producing an antibioticlike compound. An antibiotic-negative mutant strain, F113G22, was generated by transposon mutagenesis. This mutant has lost the ability to inhibit both bacterial and fungal microorganisms on high-iron medium. The antibioticlike compound was subsequently identified as 2,4-diacetylphloroglucinol (DAPG), and a high-pressure liquid chromatographic assay was developed for to detect it quantitatively in growth culture media and soil. The growth temperature had a direct bearing on DAPG production by strain F113, with maximum production at 12°C. The iron concentration, pH, and oxygen had no influence on DAPG production by strain F113 under the assay conditions used. However, a low ratio of culture volume to surface area available to the microbe in the growth container was critical for optimum DAPG production. Different types of carbon sources influenced DAPG production by strain F113 to various degrees. For example, sucrose, fructose, and mannitol promoted high yields of DAPG by strain F113, whereas glucose and sorbose resulted in very poor DAPG production.     

3H]forskolin. Direct photoaffinity labeling of the erythrocyte D-glucose transporter

Irradiation of erythrocyte ghosts in the presence of [3H]forskolin resulted in a concentration-dependent, covalent incorporation of radiolabel into several of the major membrane protein bands. Most of the incorporation occurred in four regions of the gel. Peak 1 (216 kDa) was a sharp peak near the top of the gel in the region corresponding to spectrin. Peak 2 appeared to be associated with band 3 (89 kDa), while a third peak occurred around the position of band 4.2 (76 kDa). The fourth region of labeling was a broad area between 43-75 kDa which corresponds to the region of the glucose transporter. Forskolin labeling of this region was inhibited by cytochalasin B and D-glucose, but not L-glucose. Extraction of extrinsic membrane proteins resulted in a loss of radiolabeled protein from the 216- and 76-kDa regions. Treatment of membranes labeled with either cytochalasin B or forskolin with endo-beta-galactosidase resulted in identical shifts of the 43 to 75-kDa peaks to 42 kDa. Similarly, trypsinization of membranes photolabeled with either cytochalasin B or forskolin resulted in the generation of a 17-kDa radiolabeled fragment in both cases. Photoincorporation of [3H]cytochalasin B into the glucose transporter was blocked in a concentration-dependent manner by unlabeled forskolin.     

The endogenous Mus81-Eme1 complex resolves Holliday junctions by a nick and counternick mechanism

Functional studies strongly suggest that the Mus81-Eme1 complex resolves Holliday junctions (HJs) in fission yeast, but in vitro it preferentially cleaves flexible three-way branched structures that model replication forks or 3' flaps. Here we report that a nicked HJ is the preferred substrate of endogenous and recombinant Mus81-Eme1. Cleavage occurs specifically on the strand that opposes the nick, resulting in resolution of the structure into linear duplex products. Resolving cuts made by the endogenous Mus81-Eme1 complex on an intact HJ are quasi-simultaneous, indicating that Mus81-Eme1 resolves HJs by a nick and counternick mechanism, with a large rate enhancement of the second cut arising from the flexible nature of the nicked HJ intermediate. Recombinant Mus81-Eme1 is ineffective at making the first cut. We also report that HJs accumulate in a DNA polymerase alpha mutant that lacks Mus81, providing further evidence that the Mus81-Eme1 complex targets HJs in vivo.     

Physiological basis for novel drug therapies used to treat the inflammatory bowel diseases I. Pathophysiological basis and prospects for probiotic therapy in inflammatory bowel disease

Mechanisms underlying the conditioning influence of the intestinal flora on mucosal homeostasis, including development and function of immune responses, are attracting increasing scientific scrutiny. The intestinal flora is a positive asset to host defense, but some of its components may, in genetically susceptible hosts, become a risk factor for development of inflammatory bowel disease (IBD). It follows that strategies to enhance assets or offset microbial liabilities represent a therapeutic option; therein lies the rationale for manipulation of the flora in IBD. In addition, the diversity of regulatory signalling among the flora and host epithelum, lymphoid tissue, and neuromuscular apparatus is an untapped reservoir from which novel therapeutics may be mined. Moreover, the capacity to engineer food-grade or commensal bacteria to deliver therapeutic molecules to the intestinal mucosa promises to extend the scope of microbial manipulation for the benefit of mankind.     

Grape seed extract proanthocyanidins downregulate HIV-1 entry coreceptors,CCR2b,CCR3 and CCR5 gene expression by normal peripheral blood mononuclear cells

Flavonoids and related polyphenols, in addition to their cardioprotective, anti-tumor, anti-inflammatory, anti-carcinogenic and anti-allergic activities, also possess promising anti-HIV effects. Recent studies documented that the beta-chemokine receptors, CCR2b, CCR3 and CCR5, and the alpha-chemokine receptors, CXCR1, CXCR2 and CXCR4 serve as entry coreceptors for HIV-1. Although flavonoids and polyphenolic compounds elicit anti-HIV effects such as inhibition of HIV-1 expression and virus replication, the molecular mechanisms underlying these effects remain to be clearly elucidated. We hypothesize that flavonoids exert their anti-HIV effects, possibly by interfering at the HIV co-receptor level. We investigated the effect of flavonoid constituents of a proprietary grape seed extract (GSE) on the expression of HIV-1 coentry receptors by immunocompetent mononuclear leukocytes. Our results showed that GSE significantly downregulated the expression of the HIV-1 entry co-receptors, CCR2b, CCR3 and CCR5 in normal PBMC in a dose dependent manner. Further, GSE treated cultures showed significantly lower number of CCR3 positive cells as quantitated by flow cytometry analysis which supports RT-PCR gene expression data. Investigations of the mechanisms underlying the anti-HIV-1 effects of grape seed extracts may help to identify promising natural products useful in the prevention and/or amelioration of HIV-1 infection.     

Depth-Dependent Differences in Community Structure of the Human Colonic Microbiota in Health

Objective

The aims of this study were to develop techniques for spatial microbial assessment in humans and to establish colonic luminal and mucosal spatial ecology, encompassing longitudinal and cross-sectional axes.

Design

A microbiological protected specimen brush was used in conjunction with a biopsy forceps to sample the colon in nine healthy volunteers undergoing colonoscopy. Terminal Restriction Fragment Length Polymorphism analysis was used to determine the major variables in the spatial organization of the colonic microbiota.

Results

Protected Specimen Brush sampling retrieved region-specific, uncontaminated samples that were enriched for bacterial DNA and depleted in human DNA when compared to biopsy samples. Terminal Restriction Fragment Length Polymorphism analysis revealed a segmentation of bacterial communities between the luminal brush and biopsy-associated ecological niches with little variability across the longitudinal axis of the colon and reduced diversity in brush samples.

Conclusion

These results support the concept of a microbiota with little longitudinal variability but with some degree of segregation between luminal and mucosal communities.     

Coupling of the nucleus and cytoplasm: role of the LINC complex

The nuclear envelope defines the barrier between the nucleus and cytoplasm and features inner and outer membranes separated by a perinuclear space (PNS). The inner nuclear membrane contains specific integral proteins that include Sun1 and Sun2. Although the outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum, it is nevertheless enriched in several integral membrane proteins, including nesprin 2 Giant (nesp2G), an 800-kD protein featuring an NH(2)-terminal actin-binding domain. A recent study (Padmakumar, V.C., T. Libotte, W. Lu, H. Zaim, S. Abraham, A.A. Noegel, J. Gotzmann, R. Foisner, and I. Karakesisoglou. 2005. J. Cell Sci. 118:3419-3430) has shown that localization of nesp2G to the ONM is dependent upon an interaction with Sun1. In this study, we confirm and extend these results by demonstrating that both Sun1 and Sun2 contribute to nesp2G localization. Codepletion of both of these proteins in HeLa cells leads to the loss of ONM-associated nesp2G, as does overexpression of the Sun1 lumenal domain. Both treatments result in the expansion of the PNS. These data, together with those of Padmakumar et al. (2005), support a model in which Sun proteins tether nesprins in the ONM via interactions spanning the PNS. In this way, Sun proteins and nesprins form a complex that links the nucleoskeleton and cytoskeleton (the LINC complex).     

Bifidobacterium breve with α-Linolenic Acid and Linoleic Acid Alters Fatty Acid Metabolism in the Maternal Separation Model of Irritable Bowel Syndrome

The aim of this study was to compare the impact of dietary supplementation with a Bifidobacterium breve strain together with linoleic acid & α-linolenic acid, for 7 weeks, on colonic sensitivity and fatty acid metabolism in rats. Maternally separated and non-maternally separated Sprague Dawley rats (n = 15) were orally gavaged with either B. breve DPC6330 (10⁹ microorganisms/day) alone or in combination with 0.5% (w/w) linoleic acid & 0.5% (w/w) α-linolenic acid, daily for 7 weeks and compared with trehalose and bovine serum albumin. Tissue fatty acid composition was assessed by gas-liquid chromatography and visceral hypersensitivity was assessed by colorectal distension. Significant differences in the fatty acid profiles of the non-separated controls and maternally separated controls were observed for α-linolenic acid and arachidonic acid in the liver, oleic acid and eicosenoic acid (c11) in adipose tissue, and for palmitoleic acid and docosahexaenoic acid in serum (p<0.05). Administration of B. breve DPC6330 to MS rats significantly increased palmitoleic acid, arachidonic acid and docosahexaenoic acid in the liver, eicosenoic acid (c11) in adipose tissue and palmitoleic acid in the prefrontal cortex (p<0.05), whereas feeding B. breve DPC6330 to non separated rats significantly increased eicosapentaenoic acid and docosapentaenoic acid in serum (p<0.05) compared with the NS un-supplemented controls. Administration of B. breve DPC6330 in combination with linoleic acid and α-linolenic acid to maternally separated rats significantly increased docosapentaenoic acid in the serum (p<0.01) and α-linolenic acid in adipose tissue (p<0.001), whereas feeding B. breve DPC6330 with fatty acid supplementation to non-separated rats significantly increased liver and serum docosapentaenoic acid (p<0.05), and α-linolenic acid in adipose tissue (p<0.001). B. breve DPC6330 influenced host fatty acid metabolism. Administration of B. breve DPC6330 to maternally separated rats significantly modified the palmitoleic acid, arachidonic acid and docosahexaenoic acid contents in tissues. The effect was not observed in non-separated animals.     

Polyphosphoinositide inclusion in artificial lipid bilayer vesicles promotes divalent cation-dependent membrane fusion.

Recent studies suggest that phosphoinositide kinases may participate in intracellular trafficking or exocytotic events. Because both of these events ultimately require fusion of biological membranes, the susceptibility of membranes containing polyphosphoinositides (PPIs) to divalent cation-induced fusion was investigated. Results of these investigations indicated that artificial liposomes containing PPI or phosphatidic acid required lower Ca2+ concentrations for induction of membrane fusion than similar vesicles containing phosphatidylserine, phosphatidylinositol, or phosphatidylcholine. This trend was first observed in liposomes composed solely of one type of phospholipid. In addition, however, liposomes designed to mimic the phospholipid composition of the endofacial leaflet of plasma membranes (i.e., liposomes composed of combinations of PPI, phosphatidylethanolamine, and phosphatidylcholine) also required lower Ca2+ concentrations for induction of aggregation and fusion. Liposomes containing PPI and phosphatidic acid also had increased sensitivity to Mg(2+)-induced fusion, an observation that is particularly intriguing given the intracellular concentration of Mg2+ ions. Moreover, the fusogenic effects of Ca2+ and Mg2+ were additive in vesicles containing phosphatidylinositol bisphosphate. These data suggest that enzymatic modification of the PPI content of intracellular membranes could be an important mechanism of fusion regulation.