Reduced sensitivity of inducible nitric oxide synthase-deficient mice to chronic colitis.

BACKGROUND: Overproduction of nitric oxide by the inducible form of nitric oxide synthase (iNOS) has been implicated in colitis. Different authors have postulated both toxic and protective effects of nitric oxide (NO) in the pathophysiology of active inflammation. The objective of this study was to examine the role of iNOS in experimental chronic colitis using iNOS-deficient mice. METHODS: For induction of colitis, mice received three cycles of 2% of dextran sodium sulfate (DSS) (M.W. 40,000) treatment in drinking water. The degree of colonic inflammation, leukocyte infiltration, and the expression of cell adhesion molecules were determined. INOS expression and nitrotyrosine were also determined by immunohistochemistry. RESULTS: After DSS treatment, a moderate colitis with marked cell infiltration was observed. Intense expression of iNOS was observed on infiltrating cells as well as on the colonic mucosal epithelium in these animals. In the iNOS-deficient mice, tissue damage was significantly diminished. No iNOS or nitrotyrosine staining was found in iNOS-deficient mice. The number of infiltrating cells and the expression of mucosal adressin cell adhesion molecule-1 were significantly attenuated in the DSS-treated colon of iNOS-deficient mice. CONCLUSION: Induction of iNOS seems to act as a critical toxic effector molecule in the pathogenesis of chronic colonic inflammation.     

Novel methods to determine the epitopes on the asparagine-linked oligosaccharides of glycoproteins

Oligosaccharides obtained from glycoproteins by hydrazinolysis followed by N-acetylation were covalently coupled to an amino-bonded thin-layer plate or to a poly-L-lysine coating on the microtiter plate. Bound oligosaccharides can be directly detected by immunostaining or by enzyme-linked immunosorbent assay (ELISA) using a mouse monoclonal antibody. These methods are simple and require small amounts of oligosaccharides and antibodies. The methods were successfully applied to determine the epitope of F48-60, a monoclonal antibody which reacts with the N-linked sugar chains of nonspecific cross-reacting antigen 2, but not with those of carcinoembryonic antigen. The results revealed that the antibody recognizes the Gal beta 1----3GlcNAc beta 1----group.     

Enzymatic removal of O6-ethylguanine from mitochondrial DNA in rat tissues exposed to N-ethyl-N-nitrosourea in vivo

DNA repair is essential for maintaining the integrity of the genetic material, and a number of DNA repair mechanisms have been fairly well characterized for the nuclear DNA of eukaryotic cells as well as prokaryotes. However, little is known about DNA repair in mitochondria. Using highly sensitive immunoanalytical methods to detect specific DNA alkylation products, we found active removal of O6-ethyl-2'-deoxyguanosine (O6-EtdGuo) from rat liver mitochondrial DNA after pulse-exposure to N-ethyl-N-nitrosourea in vivo. In the kidney, O6-EtdGuo was removed from mitochondrial DNA with moderate efficiency, but nearly no removal was observed from the DNA of brain mitochondria. Among the rat tissues examined, the kinetics of O6-EtdGuo elimination from mitochondrial DNA was very similar to the kinetics of removal from nuclear DNA. O4-Ethyl-2'-deoxythymidine, another premutagenic DNA ethylation product, was stable in both mitochondrial and nuclear DNA of rat liver.     

The effects of subminimal inhibitory concentrations of beta-lactam antibiotics against Clostridium perfringens.

The effects of subminimal inhibitory concentrations (sub-MIC) of four beta-lactam antibiotics [penicillin-G (PCG), ampicillin (AMP), cephaloridine (CER), cephalothin (CET)] were tested against Clostridium perfringens type A PB6K, after determining the minimum inhibitory concentrations (MIC) of 29 different Clostridium strains. The majority of the strains were sensitive to all beta-lactam antibiotics. Morphological changes, such as filamentous development and lysis, occurred at concentrations considerably lower than the MIC of CER and CET in C. perfringens. Clear cooperation of AMP and CER with rabbit polymorphonuclear leucocytes (PMNL) against C. perfringens was observed. The filamentous bacteria produced as a result of exposure to sub-MIC of each antibiotic, were phagocytosed easily. The ratios between the drug concentrations (microg/ml) at which the morphological changes began to occur, the minimum antibiotic concentrations (MAC), and the MIC values (microg/ml), were calculated. A large ratio indicated a wide range of effective concentrations below the MIC value for the antibiotics.     

HGF triggers activation of the COX-2 gene in rat gastric epithelial cells: action mediated through the ERK2 signaling pathway.

Although it is established that growth factors and prostaglandins function in the maintenance of gastric mucosal integrity and in the healing of gastric mucosal injury and ulceration, the regulatory relationship between growth factors and prostaglandins in the gastric mucosa is not well characterized. Therefore, we investigated whether hepatocyte growth factor (HGF) affects expression of COX-2 (the inducible form of the prostaglandin synthesizing enzyme, cyclooxygenase) in gastric epithelial cells and whether this action is mediated through the MAP (ERK) kinase signaling pathway. In RGM1 cells (an epithelial cell line derived from normal rat gastric mucosa), HGF caused an increase in COX-2 mRNA and protein by 236% and 175%, respectively (both P<0.05). This induction of COX-2 expression was abolished by pretreatment with the MAPK kinase (MEK) inhibitor PD98059. HGF also triggered a 13-fold increase in c-Met/HGF receptor phosphorylation (P<0.005) and increased ERK2 activity by 684% (P<0.01). Pretreatment with PD98059 abolished the HGF-induced increase in ERK2 activity, but not c-Met/HGF receptor phosphorylation. The specific inhibitor of p38 MAP kinase, SB203580, had no effect on HGF-induced COX-2 expression. Thus, HGF triggers activation of the COX-2 gene in gastric epithelial cells through phosphorylation of c-Met/HGF receptor and activation of the ERK2 signaling pathway.-Jones, M. K., Sasaki, E., Halter, F., Pai, R., Nakamura, T., Arakawa, T., Kuroki, T., Tarnawski, A. S. HGF triggers activation of the COX-2 gene in rat gastric epithelial cells: action mediated through the ERK2 signaling pathway.     

Taxonomic diversity within the Japanese green lacewing, Chrysoperla carnea (Neuroptera: Chrysopidae), identified by courtship song analyses and crossing tests

The green lacewing, Chrysoperla carnea (Stephens), is an important natural enemy of various crop pests, especially aphids. In the Japanese fauna, there are two types of larval forms, A and B, characterized by different head capsule markings. The Type A form is distributed throughout Japan, but the Type B form has a more limited distribution. Adults use abdominal vibration as a communication signal (courtship song) during mating. We analyzed oscillograms of these songs among several Japanese populations of C. carnea. The courtship songs of types A and B are distinctly different from one another. We then performed crossing tests between the two types. Copulation between same-type pairings was much more likely than between different-type pairings. We also analyzed courtship songs of European C. carnea sensu stricti, introduced to Japan as a natural enemy of crop pests. The song of these introduced green lacewings appeared to differ from either type of Japanese C. carnea. The two types of C. carnea are likely to be different species, and also distinct from C. carnea sensu stricti of Europe.     

Reshuffling of the Bacillus subtilis 168 genome by multifold inversion

The genome of Bacillus subtilis 168 was modified to yield a genome vector for the cloning of DNA several Mb in size. Unlike contemporary plasmid-based vectors, this 4.2 Mb genome vector requires specific in vivo handling protocols because of its large size. Inversion mutagenesis, a method to modify local genome structure without gain or loss of genes, was applied intensively to the B. subtilis genome; this technique made possible both exchange and translocation of designated regions of the genome. This method not only reshuffles the genome of B. subtilis, but can provide insight into the biologic principles underlying genome plasticity.     

Core fucose and bisecting GlcNAc, the direct modifiers of the N-glycan core: their functions and target proteins

Among the various posttranslational modification reactions, glycosylation is the most common, and nearly 50% of all known proteins are thought to be glycosylated. In particular, most of the molecules involved in cell–cell communication are glycosylated, and glycosylation is thus implicated in many physiological and pathological events, including cell growth, cell–cell adhesion, and tumor metastasis. As many of the glycosyltransferases are cloned, it is becoming possible to alter the oligosaccharide structures artificially and examine the effects. Among the glycosyltransferases involved in the biosynthesis of N-glycan branching, this review will focus on the function of Fut8 and N-acetylglucosaminyltransferase III, which directly modify the N-glycan core. It is suggested that these two glycosyltransferases are involved in the conformation and the function of the modified proteins including cell-surface receptors and adhesion molecules.     

Mannose binding lectin and lung collectins interact with Toll-like receptor 4 and MD-2 by different mechanisms

Background

We have previously shown that lung collectins, surfactant protein A (SP-A) and surfactant protein D, interact with Toll-like receptor (TLR) 2, TLR4, or MD-2. Bindings of lung collectins to TLR2 and TLR4/MD-2 result in the alterations of signaling through these receptors, suggesting the immunomodulatory functions of lung collectins. Mannose binding lectin (MBL) is another collectin molecule which has structural homology to SP-A. The interaction between MBL and TLRs has not yet been determined.

Methods

We prepared recombinant MBL, and analyzed its bindings to recombinant soluble forms of TLR4 (sTLR4) and MD-2.

Results

MBL bound to sTLR4 and MD-2. The interactions were Ca²⁺-dependent and inhibited by mannose or monoclonal antibody against the carbohydrate-recognition domain of MBL. Treatment of sTLR4 or MD-2 by peptide N-glycosidase F significantly decreased the binding of MBL. SP-A bound to deglycosylated sTLR4, and this property did not change in chimeric molecules of SP-A/MBL in which Glu¹⁹⁵–Phe²²⁸ or Thr¹⁷⁴–Gly¹⁹⁴ of SP-A were replaced with the corresponding MBL sequences.

General Significance

These results suggested that MBL binds to TLR4 and MD-2 through the carbohydrate-recognition domain, and that oligosaccharide moieties of TLR4 and MD-2 are important for recognition by MBL. Since our previous studies indicated that lung collectins bind to the peptide portions of TLRs, MBL and lung collectins interact with TLRs by different mechanisms. These direct interactions between MBL and TLR4 or MD-2 suggest that MBL may modulate cellular responses by altering signals through TLRs.     

Analyses of non-leucine-rich repeat (non-LRR) regions intervening between LRRs in proteins

Background

Many proteins have LRR (leucine-rich repeat) units interrupted by non-LRRs which we call IR (non-LRR island region).

Methods

We identified proteins containing LRR@IRs (LRRs having IR) by using a new method and then analyzed their natures and distributions.

Results

LRR@IR proteins were found in over two hundred proteins from prokaryotes and from eukaryotes. These are divided into twenty-one different protein families. The IRs occur one to four times in LRR regions and range in length from 5 to 11,265 residues. The IR lengths in Fungi adenylate cyclases (acys) range from 5 to 116 residues; there are 22 LRR repeats. The IRs in Leishmania proteophosphoglycans (ppgs) vary from 105 to 11,265 residues. These results indicate that the IRs evolved rapidly. A group of LRR@IR proteins—LRRC17, chondroadherin-like protein, ppgs, and four Pseudomonas proteins—have a super motif consisting of an LRR block and its adjacent LRR@IR region. This indicates that the entire super motif experienced duplication. The sequence analysis of IRs offers functional similarity in some LRR@IR protein families.

General significance

This study suggests that various IRs and super motifs provide a great variety of structures and functions for LRRs.