Anorectic actions of prolactin-releasing peptide are mediated by corticotropin-releasing hormone receptors

Prolactin-releasing peptide (PrRP) reduces food intake and body weight and modifies body temperature when administered centrally in rats, suggesting a role in energy homeostasis. However, the mediators of PrRP's actions are unknown. The present study, therefore, first examined the possible involvement of the anorectic neuropeptides corticotropin-releasing hormone (CRH) and the melanocortins (e.g., alpha-melanocyte-stimulating hormone) in PrRP's effects on food intake and core body temperature and, second, determined if PrRP affects energy expenditure by measuring oxygen consumption (Vo2). Intracerebroventricular injection of PrRP (4 nmol) to 24-h-fasted male Sprague-Dawley rats decreased food intake and modified body temperature. Blockade of central CRH receptors by intracerebroventricular coadministration of the CRH receptor antagonist astressin (20 microg) reversed the PrRP-induced reduction in feeding. However, astressin's effect on PrRP-induced changes in body temperature was complicated because the antagonist itself caused a slight rise in body temperature. In contrast, intracerebroventricular coadministration of the melanocortin receptor-3/4 antagonist SHU-9119 (0.1 nmol) had no effect on any of PrRP's actions. Finally, intracerebroventricular injection of PrRP (4 nmol) caused a significantly greater Vo2 over a 3-h test period compared with vehicle-treated rats. These results show that the anorectic actions of PrRP are mediated by central CRH receptors but not by melanocortin receptors-3/4 and that PrRP can modify Vo2.     

The role of erythromycin C-12 hydroxylase, EryK, as a substitute for PikC hydroxylase in pikromycin biosynthesis

The substrate flexibility of the erythromycin C-12 hydroxylase from Saccharopolyspora erythraea, EryK, was investigated to test its potential for the generation of novel polyketide structures. We have shown that EryK can accept the substrates of PikC from Streptomyces venezuelae which is responsible for the hydroxylation of YC-17 and narbomycin. In a S. venezuelae pikC deletion mutant, EryK could catalyze the hydroxylation of YC-17 and narbomycin to generate methymycin/neomethymycin and pikromycin, respectively. Molecular modeling of the enzyme-substrate complex suggested the possible interaction of EryK with alternative substrates. The results indicate that EryK is flexible toward some alternative polyketides and can be useful for structural diversification of macrolides by post-polyketide synthase hydroxylation.     

Expression of functional recombinant mussel adhesive protein Mgfp-5 in Escherichia coli

Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine. However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed. Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the first time. Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in Escherichia coli and was highly purified using affinity chromatography. The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance. Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments.     

Growth promotion of red pepper plug seedlings and the production of gibberellins by Bacillus cereus, Bacillus macroides and Bacillus pumilus

The growth of red pepper plug seedlings was promoted by Bacillus cereus MJ-1, B. macroides CJ-29, and B. pumilus CJ-69 isolated from the rhizosphere. Gibberellins (GAs), a well-known plant growth-promoting hormone, were detected in the culture broth of their rhizobacteria. Among the GAs, the contents of GA1, GA3, GA4, and GA7, physiologically active GAs, were comparatively higher than those of others, suggesting that the growth promoting effect was originated from the GAs. This isthe first report on the production of GA5, GA8, GA34, GA44, and GA53 by bacteria.     

In vivo monitoring of intracellular expression of human interleukin-2 using green fluorescent protein fusion partner in Pichia pastoris

The use of Pichia pastoris for protein production was simplified by creation of fusion proteins containing green fluorescent protein (GFP) and the product of interest. Human interleukin-2 (hIL-2) was used as a model product: GFP enabled clear identification of fusion protein expression and, more importantly, the quantification of hIL-2. Although GFP fusions for bioprocess monitoring have been demonstrated in other hosts, this is its first use in P. pastoris.     

New preparation of PM2 phage DNA and an endonuclease assay for a single-strand break

PM2 is a bacteriophage which has closed circular double-stranded DNA as a genome, which is the sole source for endonuclease assay for a single strand break in the fmol range. Therefore, it is important to isolate PM2 DNA with low control nicks for the endonuclease assay. Usually, the isolation method of phage DNA is to use ultracentrifugation which takes at least 4 days. In this report, a fast and effective method which takes only 2 days was developed to purify DNA using polyethylene glycol (PEG) 8000 and the yields of phage DNA isolated by these two methods were compared. The method using PEG 8000 increased the yield of PM2 DNA from 31.2% to 45.2%, and decreased the nick from 17.1% to 13.1%. Recently, the complete PM2 DNA genome sequence of 10,079 bp was published. The exact number of nucleotides of PM2 DNA is important for the correct enzyme assay which measures nicks generated by an endonuclease. The correct calculation of endonuclease activity of rpS3 for nick-circle assay was performed to measure single-strand breaks in this report. This revised version was published online in June 2006 with corrections to the Cover Date.     

Implication of two glutathione S-transferases in the optimal metabolism of m-toluate by Sphingomonas yanoikuyae B1

A putative glutathione S-transferase (GST) gene (bphK) was identified in the meta-cleavage operon for the degradation of m-toluate by Sphingomonas yanoikuyae B1. Disruption of bphK resulted in the loss of GST activity against 1-chloro-2,4-dinitrobenzene and a much increased lag time of the mutant strain MB3 (bphK::Km) following subculture into m-toluate medium. In contrast, an increased lag time was not observed when MB3 was grown on biphenyl or m-xylene and MB3 showed normal growth on m-toluate when complemented with a subclone containing the bphK gene only. Furthermore, an additional GST activity was detected in MB3. The induction timing of this second GST activity coincided with the beginning of the exponential growth phase of MB3 on m-toluate, reached maximal activity within three hours, and then dropped sharply to the basal level. Thus, it is apparent that BphK and/or the second GST are necessary for optimal growth of B1 on m-toluate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Solution structure of a novel disintegrin, salmosin, from Agkistrondon halys venom

Disintegrins are potent inhibitors of both platelet aggregation and integrin-dependent cell adhesion. A new disintegrin, salmosin, isolated from the venom of the Korean snake Agkistrodon halys brevicaudus, has been characterized by mass spectrometry and NMR spectroscopy, and its in vitro biological activity has been assessed. The IC(50) value of the purified salmosin was determined to be 2.2 nM in an assay for the inhibition of glycoprotein IIb-IIIa/fibrinogen interaction. Salmosin also inhibited the bovine capillary endothelial cell proliferation induced by bFGF in a dose-dependent manner. The NMR solution structures were well converged with a root-mean-square deviation of 0.76 A for backbone atoms among the 20 lowest energy structures, except for the arginylglycylaspartic acid (RGD) loop. The structure revealed that the conserved RGD motif with an unusual finger shape is distal from the rigid core of the C-terminal domain. Furthermore, even though the RGD motif did not interact with the hydrophobic core of the protein, it was stabilized by a network of molecular contacts through a small antiparallel beta-sheet comprising residues of Ile46-Ala50 and Asp54-Tyr58. Last, the electrostatic charge distribution on the surface of salmosin differs dramatically from that of other disintegrin proteins in that there is a cluster of negatively charged residues in close proximity to the RGD loop.     

Synthesis of carbocyclic 1-[4-(hydroxymethyl)cyclopent-2-enyl]-1,2,4-triazole-3-carboxamide and its derivatives

The synthesis of carbocyclic 1-[4-(hydroxymethyl)cyclopent-2-enyl]-1,2,4-triazole-3-carboxamide (6a) and its derivatives was achieved from triol 10 in excellent overall yield. This route involves a Pd(0)-catalyzed coupling reaction as a key step.