Regulation and distribution of MAdCAM-1 in endothelial cells in vitro

Mucosal addressin cell adhesion molecule-1(MAdCAM-1) is a 60-kDa endothelial cell adhesion glycoprotein thatregulates lymphocyte trafficking to Peyer's patches and lymph nodes.Although it is widely agreed that MAdCAM-1 induction is involved inchronic gut inflammation, few studies have investigated regulation ofMAdCAM-1 expression. We used two endothelial lines [bEND.3 (brain) and SVEC (high endothelium)] to study the signal paths that regulate MAdCAM-1 expression in response to tumor necrosis factor (TNF)- using RT-PCR, blotting, adhesion, and immunofluorescence. TNF- induced both MAdCAM-1 mRNA and protein in a dose- and time-dependent manner. This induction was tyrosine kinase (TK), p42/44, p38mitogen-activated protein kinase (MAPK), and nuclear factor(NF)-B/poly-ADP ribose polymerase (PARP) dependent. Because MAdCAM-1is regulated via MAPKs, we examined mitogen/extracellularsignal-regulated kinase (MEK)-1/2 activation in SVEC. We found thatMEK-1/2 is activated by TNF- within minutes and is dependent on TKand p42/44 MAPKs. Similarly, TNF- activated NF-B through TK,p42/44, p38 MAPKs, and PARP pathways in SVEC cells. MAdCAM-1 was alsoshown to be frequently distributed to endothelial junctions both invitro and in vivo. Cytokines like TNF- stimulate MAdCAM-1 inhigh endothelium via TK, p38, p42/22 MAPKs, and NF-B/PARP.MAdCAM-1 expression requires NF-B translocation through both directp42/44 and indirect p38 MAPK pathways in high endothelial cells.

     

Crystal structures of 3-isopropylmalate dehydrogenases with mutations at the C-terminus: crystallographic analyses of structure-stability relationships

Thermal stability of the Thermus thermophilus isopropylmalate dehydrogenase enzyme was substantially lost upon the deletion of three residues from the C-terminus. However, the stability was partly recovered by the addition of two, four and seven amino acid residues (called HD177, HD708 and HD711, respectively) to the C-terminal region of the truncated enzyme. Three structures of these mutant enzymes were determined by an X-ray diffraction method. All protein crystals belong to space group P2(1) and their structures were solved by a standard molecular replacement method where the original dimer structure of the A172L mutant was used as a search model. Thermal stability of these mutant enzymes is discussed based on the 3D structure with special attention to the width of the active-site groove and the minor groove, distortion of beta-sheet pillar structure and size of cavity in the domain-domain interface around the C-terminus. Our previous studies revealed that the thermal stability of isopropylmalate dehydrogenase increases when the active-site cleft is closed (the closed form). In the present study it is shown that the active-site cleft can be regulated by open-close movement of the minor groove located at the opposite side to the active-site groove on the same subunit, through a paperclip-like motion.     

Wnt4 expression in the differentiating gonad of the frog Rana rugosa

Wnt4, a member of the Wnt family, is known to influence the sex-determination cascade. In mice having a targeted deletion of Wnt4, masculinization occurs in XX pups. Therefore, in addition to Sry, Wnt4 is also involved in sex determination in mice. In humans, a chromosomal duplication of the WNT4 causes feminization of XY-individuals. Thus, for better understanding of the mechanism of sex determination in vertebrates, it is necessary to examine the expression of Wnt4 at early gonadal development stages in non-mammalians. We first isolated the Wnt4 cDNA from the tetsis of the frog Rana rugosa. R. rugosa Wnt4 had a high similarity (>86%) at the amino acid level with zebra fish, chicken, mouse, and human Wnt4s. We next employed RT-PCR analysis to examine whether Wnt4 was expressed in a sexually dimorphic fashion at early stages of gonadal development in R. rugosa. Wnt4 was transcribed first in the embryos at the late gastrula stage, and its expression was maintained until the indifferent gonad differentiated into a testis or an ovary. Wnt4 expression in the differentiating gonad appeared in a non-sexually dimorphic pattern. The results, taken together, suggest that Wnt4 is highly conserved through evolution, and that its expression in the indifferent gonad takes place with no sexual dimorphism. Thus, Wnt4 is not a key factor to initiate the development of a testis or an ovary from the indifferent gonads in R. rugosa. However, this gene probably forms part of a gonadal-development pathway in this species.     

Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures

Random mutagenesis coupled with screening of the active enzyme at a low temperature was applied to isolate cold-adapted mutants of a thermophilic enzyme. Four mutant enzymes with enhanced specific activities (up to 4.1-fold at 40 degrees C) at a moderate temperature were isolated from randomly mutated Thermus thermophilus 3-isopropylmalate dehydrogenase. Kinetic analysis revealed two types of cold-adapted mutants, i.e. k(cat)-improved and K(m)-improved types. The k(cat)-improved mutants showed less temperature-dependent catalytic properties, resulting in improvement of k(cat) (up to 7.5-fold at 40 degrees C) at lower temperatures with increased K(m) values mainly for NAD. The K(m)-improved enzyme showed higher affinities toward the substrate and the coenzyme without significant change in k(cat) at the temperatures investigated (30-70 degrees C). In k(cat)-improved mutants, replacement of a residue was found near the binding pocket for the adenine portion of NAD. Two of the mutants retained thermal stability indistinguishable from the wild-type enzyme. Extreme thermal stability of the thermophilic enzyme is not necessarily decreased to improve the catalytic function at lower temperatures. The present strategy provides a powerful tool for obtaining active mutant enzymes at lower temperatures. The results also indicate that it is possible to obtain cold-adapted mutant enzymes with high thermal stability.     

Efficacy of oral vaccine against bacterial coldwater disease in ayu Plecoglossus altivelis

The development of a practical vaccination method against bacterial coldwater disease (BCWD) in ayu Plecoglossus altivelis and the efficacy of oral administration of formalin-killed cells (FKCs) of Flavobacterium psychrophilum was investigated. The FKC was administrated at a dose of 0.1-0.2 g kg(-1) body weight to juvenile ayu (0.5 g body weight) every day for 2 wk or on 5 days over 2 wk. Experimental immersion challenge at 3 and 7 wk after vaccination showed significantly higher survival rates than the controls. The results show the effectiveness of oral vaccination against BCWD in ayu.     

The initial step of the thermal unfolding of 3-isopropylmalate dehydrogenase detected by the temperature-jump Laue method

A temperature-jump (T-jump) time-resolved X-ray crystallographic technique using the Laue method was developed to detect small, localized structural changes of proteins in crystals exposed to a temperature increase induced by laser irradiation. In a chimeric protein between thermophilic and mesophilic 3-isopropylmalate dehydrogenases (2T2M6T), the initial structural change upon T-jump to a denaturing temperature (approximately 90 degrees C) was found to be localized at a region which includes a beta-turn and a loop located between the two domains of the enzyme. A mutant, 2T2M6T-E110P/S111G/S113E, having amino acid replacements in this beta-turn region with the corresponding residues of the thermophilic enzyme, showed greater stability than the original chimera (increase of T:(m) by approximately 10 degrees C) and no T-jump-induced structural change in this region was detected by our method. These results indicate that thermal unfolding of the original chimeric enzyme, 2T2M6T, is triggered in this beta-turn region.     

Genetic variation of the cytochrome b gene in the rosy bitterling, Rhodeus ocellatus (Cyprinidae) in Japan

According to conventional views, the rosy bitterling, Rhodeus ocellatus, comprises two subspecies, R. ocellatus kurumeus and R. ocellatus ocellatus, the former being native to Japan whereas the latter was introduced into Japan from China during World War II. To examine the genetic structure of Japanese R. ocellatus, part of the mitochondrial cytochrome b gene from 48 individuals collected from various locations in Japan was sequenced. Three major mitochondrial lineages were found. Based on historical evidence, two of these represent R. ocellatus ocellatus and the third R. ocellatus kurumeus. The existence of two distinct lineages of R. ocellatus ocellatus in Japan suggests at least two colonizations. Some local populations comprised purely R. ocellatus kurumeus, but those from Kashima and Ogori included both subspecies. Because the proportion of R. ocellatus ocellatus in Kashima increased from 1994 to 1995, invasion by R. ocellatus ocellatus into R. ocellatus kurumeus habitats is apparently in progress. Received: April 30, 1999 / Revised: March 22, 2000 / Accepted: December 20, 2000     

Parkin-independent mitophagy via Drp1-mediated outer membrane severing and inner membrane ubiquitination

Here, we report that acute reduction in mitochondrial translation fidelity (MTF) causes ubiquitination of the inner mitochondrial membrane (IMM) proteins, including TRAP1 and CPOX, which occurs selectively in mitochondria with a severed outer mitochondrial membrane (OMM). Ubiquitinated IMM recruits the autophagy machinery. Inhibiting autophagy leads to increased accumulation of mitochondria with severed OMM and ubiquitinated IMM. This process occurs downstream of the accumulation of cytochrome c/CPOX in a subset of mitochondria heterogeneously distributed throughout the cell (“mosaic distribution”). Formation of mosaic mitochondria, OMM severing, and IMM ubiquitination require active mitochondrial translation and mitochondrial fission, but not the proapoptotic proteins Bax and Bak. In contrast, in Parkin-overexpressing cells, MTF reduction does not lead to the severing of the OMM or IMM ubiquitination, but it does induce Drp1-independent ubiquitination of the OMM. Furthermore, high–cytochrome c/CPOX mitochondria are preferentially targeted by Parkin, indicating that in the context of reduced MTF, they are mitophagy intermediates regardless of Parkin expression. In sum, Parkin-deficient cells adapt to mitochondrial proteotoxicity through a Drp1-mediated mechanism that involves the severing of the OMM and autophagy targeting ubiquitinated IMM proteins.     

Overexpression of genes of an extreme thermophile Thermus thermophilus, in Escherichia coli cells.

The 3-isopropylmalate dehydrogenase gene from an extreme thermophile, Thermus thermophilus, was not expressed in Escherichia coli unless a palindromic structure around the ribosome binding site was eliminated or a leader open reading frame was introduced into the upstream flanking region of the gene. This report suggests a way to increase the expression of this gene, with a high G+C content, in E. coli.