Role of peripheral and central opioid activity in analgesia induced by restraint stress

Rats subjected to prolonged restraint showed an increase in tail flick latency which outlasted the period of restraint by 15 min. This restraint could be blocked but not reversed by 1 mg/kg of naltrexone hydrochloride given subcutaneously. Naltrexone methobromide, administered subcutaneously in doses of 10 or 25 mg/kg, did not block the analgesia indicating that peripheral opioid receptors were probably not involved. Naltrexone hydrochloride was shown to have no effect on brain tryptophan uptake in restrained rats, a neurochemical event which had previously been shown to be critical to restraint-induced analgesia.     

Localization and functional analysis of transposon mutations in regulatory genes of the TOL catabolic pathway.

Mutant derivatives of the TOL plasmid pWW0-161, containing Tn5 insertions in the xylS and xylR regulatory genes of the catabolic pathway, have been identified and characterized. The two genes are located together on a 1.5- to 3.0-kilobase segment of TOL, just downstream of genes of the enzymes of the meta-cleavage pathway. As predicted by a current model for regulation of the TOL catabolic pathway, benzyl alcohol dehydrogenase, a representative enzyme of the upper (hydrocarbon leads to carboxylic acid) pathway, was induced by m-methylbenzyl alcohol in xylS mutant bacteria but not in a xylR mutant, whereas catechol 2,3-oxygenase, a representative enzyme of the lower (meta-cleavage) pathway, was induced by m-toluate in a xylR mutant but not in the xylS mutants. Unexpectedly, however, catechol 2,3-oxygenase was not induced by m-methylbenzyl alcohol in xylS mutants but was induced by benzyl alcohol and benzoate. These results indicate that expression of the TOL plasmid-encoded catabolic pathway is regulated by at least three control elements, two of which (the products of the xylS and xylR genes) interact in the induction of the lower pathway by methylated hydrocarbons and alcohols and one of which responds only to nonmethylated substrates.     

The use of PCR to monitor the population abundance of six human intestinal bacterial species in an in vitro semicontinuous culture system

Abstract Six PCR primer sets complementary to the 16S rDNAs (rRNA genes) were developed and shown to be specific for the following anaerobic bacteria: Clostridium clostridiiforme, C. perfringens, C. leptum, Bacteroides vulgatus, B. distasonis , and B. thetaiotaomicron , respectively. These primers were used for PCR to detect and monitor the bacteria in a semicontinuous culture system designed to mimic intestinal microflora in the human gastrointestinal tract. Except for C. perfringens , the five species of Bacteroides and Clostridia present in the in vitro culture system were detected by the PCR, and the titers varied from 10⁻² to 10⁻⁶ dilutions. The role of azo dye reduction by these bacterial species in the system was examined and discussed.     

Mapping the interactions between a RUN domain from DENND5/Rab6IP1 and sorting nexin 1

Eukaryotic cells have developed a diverse repertoire of Rab GTPases to regulate vesicle trafficking pathways. Together with their effector proteins, Rabs mediate various aspects of vesicle formation, tethering, docking and fusion, but details of the biological roles elicited by effectors are largely unknown. Human Rab6 is involved in the trafficking of vesicles at the level of Golgi via interactions with numerous effector proteins. We have previously determined the crystal structure of Rab6 in complex with DENND5, alternatively called Rab6IP1, which comprises two RUN domains (RUN1 and RUN2) separated by a PLAT domain. The structure of Rab6/RUN1-PLAT (Rab6/R1P) revealed the molecular basis for Golgi recruitment of DENND5 via the RUN1 domain, but the functional role of the RUN2 domain has not been well characterized. Here we show that a soluble DENND5 construct encompassing the RUN2 domain binds to the N-terminal region of sorting nexin 1 by surface plasmon resonance analyses.     

GSK3β-Dependent Phosphorylation Alters DNA Binding,Transactivity and Half-Life of the Transcription Factor USF2

The upstream stimulatory factor 2 (USF2) is a regulator of important cellular processes and is supposed to have also a role during tumor development. However, the knowledge about the mechanisms that control the function of USF2 is limited. The data of the current study show that USF2 function is regulated by phosphorylation and identified GSK3β as an USF2-phosphorylating kinase. The phosphorylation sites within USF2 could be mapped to serine 155 and threonine 230. In silico analyses of the 3-dimensional structure revealed that phosphorylation of USF2 by GSK3β converts it to a more open conformation which may influence transactivity, DNA binding and target gene expression. Indeed, experiments with GSK-3β-deficient cells revealed that USF2 transactivity, DNA binding and target gene expression were reduced upon lack of GSK3β. Further, experiments with USF2 variants mimicking GSK3β phosphorylated USF2 in GSK3β-deficient cells showed that phosphorylation of USF2 by GSK3β did not affect cell proliferation but increased cell migration. Together, this study reports a new mechanism by which USF2 may contribute to cancerogenesis.     

Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5B

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature.