Macrophage ubiquitin-specific protease 2 modifies insulin sensitivity in obese mice.

We previously reported that ubiquitin-specific protease (USP) 2 in macrophages down-regulates genes associated with metabolic diseases, suggesting a putative anti-diabetic role for USP2 in macrophages. In this study, we evaluate this role at both cellular and individual levels. Isolated macrophages forcibly expressing Usp2a, a longer splicing variant of USP2, failed to modulate the insulin sensitivity of 3T3-L1 adipocytes. Similarly, macrophage-selective overexpression of Usp2a in mice (Usp2a transgenic mice) had a negligible effect on insulin sensitivity relative to wild type littermates following a three-month high-fat diet. However, Usp2a transgenic mice exhibited fewer M1 macrophages in their mesenteric adipose tissue. Following a six-month high-fat diet, Usp2a transgenic mice exhibited a retarded progression of insulin resistance in their skeletal muscle and liver, and an improvement in insulin sensitivity at an individual level. Although conditioned media from Usp2a-overexpressing macrophages did not directly affect the insulin sensitivity of C2C12 myotubes compared to media from control macrophages, they did increase the insulin sensitivity of C2C12 cells after subsequent conditioning with 3T3-L1 cells. These results indicate that macrophage USP2A hampers obesity-elicited insulin resistance via an adipocyte-dependent mechanism.

Piebald mutation on a C57BL/6J background.

The classic piebald mutation in the endothelin receptor type B (Ednrb) gene was found on rolling Nagoya genetic background (PROD-s/s) mice with white coat spotting. To examine whether genetic background influenced the phenotype in the piebald mutant mice, we generated a congenic strain (B6.PROD-s/s), produced by repeated backcrosses to the C57BL/6J (B6) strain. Although B6.PROD-s/s mice showed white coat spotting, 7% of B6.PROD-s/s mice died between 2 and 5 weeks after birth due to megacolon. The PROD-s/s, s/s and Japanese fancy mouse 1 (JF1) strains, which also have piebald mutations on different genetic backgrounds with B6, showed only pigmentation defects without megacolon. In expression analyses, rectums of B6.PROD-s/s with megacolon mice showed ~5% of the level of Ednrb gene expression versus B6 mice. In histological analyses, aganglionosis was detected in the rectum of megacolon animals. The aganglionic rectum was thought to lead to severe constipation and intestinal blockage, resulting in megacolon. We also observed an abnormal intestinal flora, including a marked increase in Bacteroidaceae and Erysipelotrichaceae and a marked decrease in Lactobacillus and Clostridiales, likely inducing endotoxin production and a failure of the mucosal barrier system, leading ultimately to death. These results indicate that the genetic background plays a key role in the development of enteric ganglion neurons, controlled by the Ednrb gene, and that B6 has modifier gene (s) regarding aganglionosis.

Reassessment of the in vivo functions of DNA polymerase I and RNase H in bacterial cell growth.

A major factor in removing RNA primers during the processing of Okazaki fragments is DNA polymerase I (Pol I). Pol I is thought to remove the RNA primers and to fill the resulting gaps simultaneously. RNase H, encoded by rnh genes, is another factor in removing the RNA primers, and there is disagreement with respect to the essentiality of both the polA and rnh genes. In a previous study, we looked for the synthetic lethality of paralogs in Bacillus subtilis and detected several essential doublet paralogs, including the polA ypcP pair. YpcP consists of only the 5'-3' exonuclease domain. In the current study, we first confirmed that the polA genes of both Escherichia coli and B. subtilis could be completely deleted. We found that the 5'-3' exonuclease activity encoded by either polA or ypcP xni was required for the growth of B. subtilis and E. coli. Also, the 5'-3' exonuclease activity of Pol I was indispensable in the cyanobacterium Synechococcus elongatus. These results suggest that a 5'-3' exonuclease activity is essential in these organisms. Our success in constructing a B. subtilis strain that lacked all RNase H genes indicates that the enzymatic activity is dispensable, at least in the wild type. Increasing the 5'-3' exonuclease activity partially compensated for a defective phenotype of an RNase H-deficient mutant, suggesting cooperative functions for the two enzyme systems. Our search for the distribution of the 5'-3' exonuclease domain among 250 bacterial genomes resulted in the finding that all eubacteria, but not archaea, possess this domain.

The putative ABC transporter YheH/YheI is involved in the signalling pathway that activates KinA during sporulation initiation.

The primary kinases that control the supply of phosphate to the phosphorelay are KinA and KinB, although it is not yet known what type of signal(s) activates these kinases. Our systematic study of protein-protein interactions using yeast two-hybrid analysis revealed an interaction between KinA and YheH. YheH with the preceding gene product YheI is categorized as an ABC transporter. Overexpression of yheH/yheI in the kinB mutant resulted in a reduced sporulation efficiency. Moreover, reporter assays using Spo0A approximately P dependent promoters revealed that the deficiency in sporulation is probably due to a failure in the activation of Spo0A. Our results further suggest that the N-terminal region of YheH may play an important role in sensing the signal to be delivered to the C-terminally bound KinA.