Visfatin induces sickness responses in the brain

Background/Objective

Visfatin, also known as nicotiamide phosphoribosyltransferase or pre-B cell colony enhancing factor, is a pro-inflammatory cytokine whose serum level is increased in sepsis and cancer as well as in obesity. Here we report a pro-inflammatory role of visfatin in the brain, to mediate sickness responses including anorexia, hyperthermia and hypoactivity.

Methodology

Rats were intracerebroventricularly (ICV) injected with visfatin, and changes in food intake, body weight, body temperature and locomotor activity were monitored. Real-time PCR was applied to determine the expressions of pro-inflammatory cytokines, proopiomelanocortin (POMC) and prostaglandin-synthesizing enzymes in their brain. To determine the roles of cyclooxygenase (COX) and melanocortin in the visfatin action, rats were ICV-injected with visfatin with or without SHU9119, a melanocortin receptor antagonist, or indomethacin, a COX inhibitor, and their sickness behaviors were evaluated.

Principal Findings

Administration of visfatin decreased food intake, body weight and locomotor activity and increased body temperature. Visfatin evoked significant increases in the levels of pro-inflammatory cytokines, prostaglandin-synthesizing enzymes and POMC, an anorexigenic neuropeptide. Indomethacin attenuated the effects of visfatin on hyperthermia and hypoactivity, but not anorexia. Further, SHU9119 blocked visfatin-induced anorexia but did not affect hyperthermia or hypoactivity.

Conclusions

Visfatin induced sickness responses via regulation of COX and the melanocortin pathway in the brain.     

In vivo post-translational modifications of recombinant mussel adhesive protein in insect cells

Mussel adhesive proteins (MAPs) have been suggested as promising bioadhesives for diverse application fields, including medical uses. Previously, we successfully constructed and produced a new type of functional recombinant MAP, fp-151, in a prokaryotic Escherichia coli expression system. Even though the E. coli-derived MAP showed several excellent features, such as high production yield and efficient purification, in vitro enzymatic modification is required to convert tyrosine residues to l-3,4-dihydroxyphenyl alanine (dopa) molecules for its adhesive ability, due to the intrinsic inability of E. coli to undergo post-translational modification. In this work, we produced a soluble recombinant MAP in insect Sf9 cells, which are widely used as an effective and convenient eukaryotic expression system for eukaryotic foreign proteins. Importantly, we found that insect-derived MAP contained converted dopa residues by in vivo post-translational modification. In addition, insect-derived MAP also had other post-translational modifications including phosphorylation of serine and hydroxylation of proline that originally occurred in some natural MAPs. To our knowledge, this is the first report on in vivo post-translational modifications of MAP containing dopa and other modified amino acid residues.     

Development of a Plasmid Display System using GAL4 DNA Binding Domain for the <Emphasis Type="Italic">in Vitro</Emphasis> Screening of Functional Proteins

A plasmid display system using GAL4 DNA binding domain (GAL4 DBD) was constructed to enrich the molecular diversity and in vitro selection of functional proteins. Model proteins used were enhanced green fluorescent protein (EGFP) and glutathione S-transferase (GST). The feasibility of this display system was examined using enrichment experiments of target protein from a model protein mixture and identifying the encoding genes by PCR, in which the model protein mixture includes GAL4 DBD/GST fusion protein, GAL4 DBD/EGFP fusion protein, and xylanase. Target proteins of GAL4 DBD/GST and GAL4 DBD/EGFP from the model protein mixture were efficiently isolated by the plasmid display, respectively. The results show that the display system is sufficiently sensitive to select a target protein from a protein mixture, and that it is possible to discover the functional proteins from large libraries using relatively simple approaches.     

Synthesis and biological activity of 3,4-dihydroquinazolines for selective T-type Ca2+ channel blockers

We have synthesized 3,4-dihydroquinazoline derivatives for the potent and selective T-type Ca(2+) channel blockers and evaluated for their inhibitory activities against two subtypes T-type Ca(2+) channels and N-type Ca(2+) channels. Among them, 5b (KYS05044, IC(50)=0.56+/-0.10 microM) was identified as potent T-type Ca(2+) channel blocker with in vitro selectivity profile at meaningful level (T/N-type, SI=>100).     

Synthesis and antifungal activity of noble 5-arylamino- and 6-arylthio-4,7-dioxobenzoselenazoles

5-Arylamino- and 6-arylthio-4,7-dioxobenzoselenazoles 4 and 5 were synthesized and tested for in vitro antifungal activity against Candida and Aspergillus species. 5-Arylamino-4,7-dioxobenzoselenazoles 4 showed, in general, more potent antifungal activity than 6-arylthio-4,7-dioxobenzoselenazoles 5. The results suggest that 5-arylamino-4,7-dioxobenzoselenazoles 4 would be potent antifungal agents.     

Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.     

Evidence that Na+-pumping occurs through the D-channel in Vitreoscilla cytochrome bo

The operon (cyo) encoding the Na(+)-pumping respiratory terminal oxidase (cytochrome bo) of the bacterium Vitreoscilla was transformed into Escherichia coli GV100, a deletion mutant of cytochrome bo. This was done for the wild type operon and five mutants in three conserved Cyo subunit I amino acids known to be crucial for H(+) transport in the E. coli enzyme, one near the nuclear center, one in the K-channel, and one in the D-channel. CO-binding, NADH and ubiquinol oxidase, and Na(+)-pumping activities were all substantially inhibited by each mutation. The wild type Vitreoscilla cytochrome bo can pump Na(+) against a concentration gradient, resulting in a transmembrane concentration differential of 2-3 orders of magnitude. It is proposed that Vitreoscilla cytochrome bo pumps four Na(+) through the D-channel to the exterior and transports four H(+) through the K-channel for the reduction of each O(2).     

Genistein, EGCG, and capsaicin inhibit adipocyte differentiation process via activating AMP-activated protein kinase

Phytochemicals such as soy isoflavone genistein have been reported to possess therapeutic effects for obesity, diabetes, and cardiovascular diseases. In the present study, the molecular basis of selective phytochemicals with emphasis on their ability to control intracellular signaling cascades of AMP-activated kinase (AMPK) responsible for the inhibition of adipogenesis was investigated. Recently, the evolutionarily conserved serine/threonine kinase, AMPK, emerges as a possible target molecule of anti-obesity. Hypothalamic AMPK was found to integrate nutritional and hormonal signals modulating feeding behavior and energy expenditure. We have investigated the effects of genistein, EGCG, and capsaicin on adipocyte differentiation in relation to AMPK activation in 3T3-L1 cells. Genistein (20-200muM) significantly inhibited the process of adipocyte differentiation and led to apoptosis of mature adipocytes. Genistein, EGCG, and capsaicin stimulated the intracellular ROS release, which activated AMPK rapidly. We suggest that AMPK is a novel and critical component of both inhibition of adipocyte differentiation and apoptosis of mature adipocytes by genistein or EGCG or capsaicin further implying AMPK as a prime target of obesity control.