Induction of umuC gene expression by nitrogen dioxide in Salmonella typhimurium

Gaseous nitrogen dioxide (NO2) was found to induce umuC gene expression in Salmonella typhimurium carrying the umuC-lacZ fusion plasmid. The induction level of the umu operon responsible for inducible mutagenesis was measured by the level of beta-galactosidase in the cell, encoded by the fusion gene. NO2 gas was bubbled into bacterial suspensions at 10, 30 and 90 microliters/l for 30 min at a flow rate of 100 ml/min. Expression of the umuC gene varied with the concentration, flow rate and bubbling time of the NO2 gas. Although NO2 gas induces SOS functions, mutagenesis due to it was not detectable in Salmonella typhimurium TA100 and TA102. Nitric oxide gas (NO) did not induce any umuC gene expression.     

Alginate degradation by bacteria isolated from the gut of sea urchins and abalones

Degradation of alginate and its constituents, polymannuronate (polyM) and polyguluronate (polyG), by gut bacteria isolated from sea urchins and abalones in the northern part of Japan, were investigated. Bacterial counts in the guts of sea urchin S. intermedius, were 10⁵ to 10⁸ CFU/g, and in abalone H. discus hannai, counts ranged from 10⁶ to 10⁹ CFU/g. More than 80% of total 600 isolates were found to have alginolytic activity. The alginolytic bacteria were predominantly fermentative, but some differences were observed in their substrate specificity as well as between the flora in the gut of sea urchins and the abalones. Seventy percent of the alginolytic bacteria from the sea urchins showed no degrading preference for polyM or polyG blocks, and were able to degrade both the substrates simultaneously. Most of the alginolytic bacteria (96.6%) from sea urchins belonged to the genus Vibrio. The majority of alginolytic bacteria (68.0% on average) from abalones only degraded polyG and they were predominantly non-motile fermenters. From these results, it appeared that a different type of association exists between alginolytic gut microflora and the marine algal feeders with respect to the level of contribution by bacteria to the host's digestion of alginate. Correspondence to: T. Sawabe     

In Vivo Messenger RNA Introduction into the Central Nervous System Using Polyplex Nanomicelle

Messenger RNA (mRNA) introduction is a promising approach to produce therapeutic proteins and peptides without any risk of insertion mutagenesis into the host genome. However, it is difficult to introduce mRNA in vivo mainly because of the instability of mRNA under physiological conditions and its strong immunogenicity through the recognition by Toll-like receptors (TLRs). We used a novel carrier based on self-assembly of a polyethylene glycol (PEG)-polyamino acid block copolymer, polyplex nanomicelle, to administer mRNA into the central nervous system (CNS). The nanomicelle with 50 nm in diameter has a core-shell structure with mRNA-containing inner core surrounded by PEG layer, providing the high stability and stealth property to the nanomicelle. The functional polyamino acids possessing the capacity of pH-responsive membrane destabilization allows smooth endosomal escape of the nanomicelle into the cytoplasm. After introduction into CNS, the nanomicelle successfully provided the sustained protein expression in the cerebrospinal fluid for almost a week. Immune responses after mRNA administration into CNS were effectively suppressed by the use of the nanomicelle compared with naked mRNA introduction. In vitro analyses using specific TLR-expressing HEK293 cells confirmed that the nanomicelle inclusion prevented mRNA from the recognition by TLRs. Thus, the polyplex nanomicelle is a promising system that simultaneously resolved the two major problems of in vivo mRNA introduction, the instability and immunogenicity, opening the door to various new therapeutic strategies using mRNA.     

Design and synthesis of novel opioid ligands with an azabicyclo[2.2.2]octane skeleton having a 7-amide side chain and their pharmacologies

Several derivatives with an azabicyclo[2.2.2]octane skeleton having a 7-amide side chain were synthesized. Compounds that had an electron-donating group exhibited high affinity for the μ opioid receptor while those with a bulky substituent at the 8-nitrogen atom had low affinities for all receptor types. High affinities and selectivities for the κ receptor resulted from the introduction of the longer amide side chain at the 7α-position. Our studies indicate that the orientation of the amide side chain at the 7-position within the azabicyclo[2.2.2]octane skeleton is related to selectivity for the κ receptor.     

A Computational Module Assembled from Different Protease Family Motifs Identifies PI PLC from Bacillus cereus as a Putative Prolyl Peptidase with a Serine Protease Scaffold

Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.     

Complete rectal prolapse in wild anubis baboons (Papio anubis)

This report describes two cases of rectal prolapse in wild anubis baboons (Papio anubis), with one spontaneous resolution. Both occurred after individuals consumed low‐water, high‐fibre dried maize during provisioning prior to capture, while one also experienced distress during capture.