Hepatocyte Growth Factor Induces Transglutaminase Activity That Negatively Regulates the Growth Signal in Primary Cultured Hepatocytes

Transglutaminase (TGase) activity increased 2.5-fold at 6 h after treatment of rat hepatocytes with 117 nMhepatocyte growth factor (HGF). In the same manner, putrescine incorporation into proteins of cells occurred in HGF-treated cells but did not in those pretreated with monodansylcadaverine (MDC), a TGase inhibitor, even in the presence of HGF. These results suggest that HGF-induced TGase was active and catalyzed some cross-linkage reaction. Cycloheximide completely blocked the increase in TGase activity induced by HGF, suggesting that HGF stimulatedde novosynthesis of TGase within 6 h. Both [³⁵S]methionine incorporation and Northern blotting analyses supported this possibility. Pretreatment of cells with MDC additionally increased HGF-induced DNA synthesis and the ratio of cells in S-phase. Similarly, TGase antisense oligonucleotide inhibitedde novosynthesis of TGase, resulting in increase in the ratio of S-phase cells in the presence of HGF. Analyses of cross-linking of HGF to the receptor indicated that the antisense oligonucleotide inhibited the downregulation of HGF receptor subsequent to HGF-addition. These results provide the first evidence for inducibility ofde novosynthesis of TGase by HGF and suggest that TGase negatively regulates the growth signal of HGF through the downregulation of receptor.     

Attachment and invasion of host cells by Toxoplasma gondii

Recent studies indicate that Toxoplasma gondii attachment is mediated via a parasite ligand-host cell receptor interaction. Lloyd Kosper and Jose Mineo here survey factors involved in the attachment to and penetration and invasion of host cells by T. gondii.     

Model-based definition of population heterogeneity and its effects on metabolism in sporulating Bacillus subtilis

The soil bacterium Bacillus subtilis forms dormant, robust spores as a tactic to ensure survival under conditions of starvation. However, the sporulating culture includes sporulating and non-sporulating cells, because a portion of the cell population initiates sporulation in wild-type strain. We anticipated that the population effect must be considered carefully to analyse samples yielding population heterogeneity. We first built a mathematical model and simulated for signal transduction of the sporulation cue to see what mechanisms are responsible for generating the heterogeneity. The simulated results were confirmed experimentally, where heterogeneity is primarily modulated by negative feedback circuits, resulting in generation of a bistable response within the sporulating culture. We also confirmed that mutants relevant to negative feedback yield either sporulating or non-sporulating subpopulations. To see the effect of molecular mechanism between sporulating and non-sporulating cells in distinct manner, metabolome analysis was conducted using the above mutants. The metabolic profiles exhibited distinct characteristics with time regardless of whether sporulation was initiated or not. In addition, several distinct characteristics of metabolites were observed between strains, which was inconsistent with previously reported data. The results imply that careful consideration must be made in the interpretation of data obtained from cells yielding population heterogeneity.     

Absence of increased alpha1-microglobulin in IgA nephropathy proteinuria

To search for biomarkers of IgA nephropathy, protein profiles of urine samples from patients with IgA nephropathy and normal volunteers were compared using two-dimensional DIGE. Most of the 172 spots identified in the urine were serum proteins, and their amounts in IgA nephropathy urine were much higher than those in normal urine; this can be explained as proteinuria caused by glomerular dysfunction. However, only alpha(1)-microglobulin, also one of the major serum proteins, in IgA nephropathy urine was not higher in amount than that in normal urine. We confirmed using ELISA analysis that the amounts of transferrin and albumin in IgA nephropathy and diabetic nephropathy urine were much higher than those in normal urine, whereas the amount of alpha(1)-microglobulin in IgA nephropathy urine was not higher than that in normal urine and was much lower than that in diabetic nephropathy urine. Approximately 50% of alpha(1)-microglobulin forms a complex with IgA in serum. These results suggest that alpha(1)-microglobulin in IgA nephropathy urine is a characteristic protein and might be a biomarker for IgA nephropathy and that alpha(1)-microglobulin might have a relationship with IgA nephropathy pathology.     

Molecular cloning of N-methylputrescine oxidase from tobacco

Nicotine biosynthesis in Nicotiana species requires an oxidative deamination of N-methylputrescine, catalyzed by N-methylputrescine oxidase (MPO). In a screen for tobacco genes that were down-regulated in a tobacco mutant with altered regulation of nicotine biosynthesis, we identified two homologous MPO cDNAs which encode diamine oxidases of a particular subclass. Tobacco MPO genes were expressed specifically in the root, and up-regulated by jasmonate treatment. Recombinant MPO protein expressed in Escherichia coli formed a homodimer and deaminated N-methylputrescine more efficiently than symmetrical diamines. These results indicate that MPO evolved from general diamine oxidases to function effectively in nicotine biosynthesis.     

Action of ascorbic acid on a myosin molecule derived from carp

The influence of L-ascorbic acid at 40 degrees C incubation on the subfragment-1 and rod regions, prepared by chymotryptic digestion of myosin, and myosin was investigated by SDS-polyacrylamide gel electrophoresis and transmission electron microscopy respectively. It was observed that L-ascorbic acid acted more readily on the subfragment-1 region of myosin. Further, circular dichroism measurement indicated that L-ascorbic acid did not affect the structure of myosin. These results suggest that L-ascorbic acid acts more readily on the myosin subfragment-1 region and promotes the gelation of myosin without producing a conformational change in this protein.     

Bile acid modulates transepithelial permeability via the generation of reactive oxygen species in the Caco-2 cell line

The barrier functions in epithelial and endothelial cells seem to be very important for maintaining normal biological homeostasis. However, it is unclear whether or how bile acids affect the epithelial barrier. We examined the bile acid-induced disruption of the epithelial barrier. We measured the transepithelial electrical resistance (TEER) of Caco-2 cells as a marker of disruption of the epithelial barrier. Reactive oxygen species (ROS) generation was also measured. Cholic acid (CA) decreased the TEER and increased intracellular ROS generation. PLA2 (phospholipase A2), COX (cyclooxygenase), PKC (protein kinase), ERK 1/2 (extracellular signal-regulated kinase 1/2), PI 3 K (phosphatidylinositol 3-kinase), p38 MAPK (p38 mitogen-activated protein kinase), MLCK (myosin light-chain kinase), NADH dehydrogenase, and XO (xanthine oxidase) inhibitors or ROS scavengers prevented the CA-induced TEER decrease. PLA2, COX, PKC, NADH dehydrogenase, and XO inhibitors prevented the CA-induced ROS generation but not ERK 1/2, PI 3 K, p38 MAPK, and MLCK inhibitors. If the cells were treated with ROS generators such as superoxide dismutase, the TEER decreased. ERK 1/2, PI 3 K, p38 MAPK, and MLCK inhibitors prevent these ROS generators from inducing the TEER decrease. These results suggest that ROS play an important role. In addition, PLA2, COX, PKC, NADH dehydrogenase, and XO are located upstream of the ROS generation, but ERK 1/2, PI 3 K, p38 MAPK, and MLCK are downstream during the signaling of CA-induced TEER alterations.     

Glucosamine induces autophagy via an mTOR-independent pathway

Autophagy is a cellular process that nonspecifically degrades cytosolic components and is involved in many cellular responses. We found that amino sugars with a free amino group such as glucosamine, galactosamine and mannosamine induced autophagy via an mTOR-independent pathway. Glucosamine-induced autophagy at concentrations of at least 500 μM to over 40 mM. In the presence of 40 mM glucosamine, autophagy induction was initiated at 6 h and reached a plateau at 36 h. Glucosamine-induced autophagy could remove accumulated ubiquitin-conjugated proteins as well as 79-glutamine repeats. Therefore, orally administered glucosamine could contribute to the prevention of neurodegenerative diseases and promotion of antiaging effects.