Inactivation of the glycine transporter 1 gene discloses vital role of glial glycine uptake in glycinergic inhibition

The glycine transporter subtype 1 (GlyT1) is widely expressed in astroglial cells throughout the mammalian central nervous system and has been implicated in the regulation of N-methyl-D-aspartate (NMDA) receptor activity. Newborn mice deficient in GlyT1 are anatomically normal but show severe motor and respiratory deficits and die during the first postnatal day. In brainstem slices from GlyT1-deficient mice, in vitro respiratory activity is strikingly reduced but normalized by the glycine receptor (GlyR) antagonist strychnine. Conversely, glycine or the GlyT1 inhibitor sarcosine suppress respiratory activity in slices from wild-type mice. Thus, during early postnatal life, GlyT1 is essential for regulating glycine concentrations at inhibitory GlyRs, and GlyT1 deletion generates symptoms found in human glycine encephalopathy.     

The effects of high magnitude cyclic tensile load on cartilage matrix metabolism in cultured chondrocytes

Excessive mechanical load is thought to be responsible for the onset of osteoarthrosis (OA), but the mechanisms of cartilage destruction caused by mechanical loads remain unknown. In this study we applied a high magnitude cyclic tensile load to cultured chondrocytes using a Flexercell strain unit, which produces a change in cell morphology from a polygonal to spindle-like shape, and examined the protein level of cartilage matrixes and the gene expression of matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs) and proinflammatory cytokines such as IL-1beta and TNF-alpha. Toluidine blue staining, type II collagen immunostaining, and an assay of the incorporation of [35S]sulfate into proteoglycans revealed a decrease in the level of cartilage-specific matrixes in chondrocyte cultures subjected to high magnitude cyclic tensile load. PCR-Southern blot analysis showed that the high magnitude cyclic tensile load increased the mRNA level of MMP-1, MMP-3, MMP-9, IL-1beta, TNF-alpha and TIMP-1 in the cultured chondrocytes, while the mRNA level of MMP-2 and TIMP-2 was unchanged. Moreover, the induction of MMP-1, MMP-3 and MMP-9 mRNA expression was observed in the presence of cycloheximide, an inhibitor of protein synthesis. These findings suggest that excessive mechanical load directly changes the metabolism of cartilage by reducing the matrix components and causing a quantitative imbalance between MMPs and TIMPs.     

Current knowledge of dystrophin and dystrophin-associated proteins in the retina

Dramatical development of molecular genetics has been disclosing the molecular mechanism of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). DMD gene product, dystrophin, is a submembranous cytoskeletal protein and many dystrophin-associated proteins (DAPs) have been identified, such as utrophin, dystroglycans, sarcoglycans, syntrophins and dystrobrevins. Dystrophin and DAPs are very important proteins not only for skeletal, cardiac, or smooth muscles but also for peripheral and central nervous systems including the retina. The retina has been extensively examined to demonstrate that dystrophin and beta-dystroglycan localize at the photoreceptor terminal, and their deficiency produces the abnormal neurotransmission between photoreceptor cells and ON-bipolar cells. Dystrophin has seven isoforms in variable tissues, and the retina contains full-length dystrophin (Dp427), Dp260, and Dp71. Recent studies have demonstrated that Dp71 localizes in the inner limiting membrane (INL) and around the blood vessel, and Dp260 is expressed in the outer plexiform layer (OPL). beta-dystroglycan is also expressed in the same regions as well as dystrophin, but it remains unclear whether other DAPs are expressed in the retina or not. It is generally assumed that dystrophin functions to stabilize muscle fibers with DAPs by linking the sarcolemma to the basement membrane, but its function in the retina is totally unknown so far.     

Sgs1 helicase activity is required for mitotic but apparently not for meiotic functions

The SGS1 gene of Saccharomyces cerevisiae is a homologue for the Bloom's syndrome and Werner's syndrome genes. The disruption of the SGS1 gene resulted in very poor sporulation, and the majority of the cells were arrested at the mononucleated stage. The recombination frequency measured by a return-to-growth assay was reduced considerably in sgs1 disruptants. However, double-strand break formation, which is a key event in the initiation of meiotic DNA recombination, occurred; crossover and noncrossover products were observed in the disruptants, although the amounts of these products were slightly decreased compared with those in wild-type cells. The spores produced by sgs1 disruptants showed relatively high viability. The sgs1 spo13 double disruptants sporulated poorly, like the sgs1 disruptants, but spore viability was reduced much more than with either sgs1 or spo13 single disruptants. Disruption of the RED1 or RAD17 gene partially alleviated the poor-sporulation phenotype of sgs1 disruptants, indicating that portions of the population of sgs1 disruptants are blocked by the meiotic checkpoint. The poor sporulation of sgs1 disruptants was complemented with a mutated SGS1 gene encoding a protein lacking DNA helicase activity; however, the mutated gene could suppress neither the sensitivity of sgs1 disruptants to methyl methanesulfonate and hydroxyurea nor the mitotic hyperrecombination phenotype of sgs1 disruptants.     

Protective effect of beta-glucan against systemic Streptococcus pneumoniae infection in mice

The antimicrobial effect of soluble beta-1,3-D-glucan from Sclerotinia sclerotiorum (SSG) was examined in mice experimentally infected intraperitoneally (i.p.) with Streptococcus pneumoniae serotypes 4 and 6B. SSG was administered i.p. either 3 days before challenge or 3-48 h after challenge. The number of bacteria in blood samples and the mouse survival rates were recorded. Pre-challenge SSG administration protected dose-dependently against both S. pneumoniae type 4 and 6B infections. SSG injected 24 h post-challenge had a curative effect against type 6B but not type 4 pneumococcal infection. The data demonstrate that SSG administered systemically protects against pneumococcal infection in mice.     

EmPLiCS: an empirical approach for structure-based design of natural peptide drugs

The computer implementation of a peptide drug-design strategy has been developed. The system is named EmPLiCS (Empirical Peptide Ligand Construction System) according to the strategy of the system, which searches for peptide-ligand structures by referring to empirical rules that are derived from known protein 3D structures. The system was tested on several known peptide-protein complexes. The results demonstrated the ability of this system to detect key residues of peptides that are crucial for interaction with their specific proteins. The system also showed the ability to detect the main chain trace of these peptides. Some of the main chain atoms were detected even though the complete primary structures were not reproduced, suggesting that main chain structure is important in peptide-protein recognition. The results of the present study demonstrated that the empirical rules-based system can generate significant information for use in the design of natural peptide drugs.     

Studies on ATPase(GTPase) intrinsic to E. coli ribosomes

(1) Escherichia coli 70S ribosomes showed intrinsic ATPase and GTPase activities, although they were much lower than those of rat liver ribosomes. The latter activity was higher than the former one. (2) The ATPase activity was inhibited by GTP and GMP-P(NH)P, and the GTPase activity was inhibited by ATP and AMP-P(NH)P, indicating a close relationship between the two enzymes. (3) Elongation components alone or in combination enhanced the ATPase activity, indicating the possible correlation of ribosomal ATPase with elongational components. (4) Vanadate at the concentrations that did not inhibit the GTPase activities of EF-Tu and EF-G, depressed the poly(U)-dependent polyphe synthesis, suggesting that ribosomal ATPase (GTPase) participates in peptide elongation by inducing positive conformational changes of ribosomes required for the attachment of elongational components.     

MAPK upstream kinase (MUK)-binding inhibitory protein, a negative regulator of MUK/dual leucine zipper-bearing kinase/leucine zipper protein kinase

Mitogen-activated protein kinase upstream kinase/dual leucine zipper-bearing kinase/leucine-zipper protein kinase (MUK/DLK/ZPK) is a MAPKKK class protein kinase that induces JNK/SAPK activation. We report here a protein named MBIP that binds to MUK/DLK/ZPK. MUK-binding inhibitory protein (MBIP) contains two tandemly orientated leucine-zipper-like motifs with a cluster of basic amino acids located between the two motifs. MBIP interacts with one of the two leucine-zipper-like motifs of MUK/DLK/ZPK and inhibits the activity of MUK/DLK/ZPK to induce JNK/SAPK activation. Notably, no similar effect was observed with another JNK/SAPK-inducing MAPKKK, COT/Tpl-2, showing the specificity of MBIP action. Furthermore, the overexpression of MBIP partially inhibits the activation of JNK by 0.3 m sorbitol in 293T cells. Taken together, these observations indicate that MBIP can function as a regulator of MUK/DLK/ZPK, a finding that may provide a clue to understanding the molecular mechanism of JNK/SAPK activation by hyperosmotic stress.     

Amelioration of experimental autoimmune uveoretinitis by pretreatment with a pathogenic peptide in liposome and anti-CD40 ligand monoclonal antibody

We have defined a peptide K2 (ADKDVVVLTSSRTGGV) that corresponds to residues 201-216 of bovine interphotoreceptor retinoid-binding protein and induces experimental autoimmune uveoretinitis (EAU)4 in H-2Ak-carrying mice (H-2Ak mice). In this study, we attempted to ameliorate EAU in the H-2Ak mice without nonspecific suppression of T cell responses. Preceding s.c. administration of liposomes including K2 (liposomal K2) specifically inhibited subsequent generation of T cell response to K2. The same result was obtained with a combination of OVA323-339 peptide and the OVA-specific TCR-transgenic T cells. It was suggested that the inhibition was mainly attributed to peripheral anergy induction of T cells specific for the peptide Ag, although specific cell death might also be involved in the inhibition. Pretreatment with liposomal K2 also considerably abolished IFN-gamma production but not IL-4 production. The specific inhibitory effect of the pretreatment with liposomal peptide was augmented by a simultaneous administration of anti-CD40 ligand (anti-CD40L) mAb. Moreover, it was shown that the pretreatment with liposomal K2 reduced both the incidence and severity of the subsequent K2-induced EAU, and the simultaneous administration of anti-CD40L mAb augmented this preventive effect by liposomal K2. Our findings demonstrate that the s.c. administration of liposomal pathogenic peptide and anti-CD40L mAb can be applied to preventing autoimmune diseases without detrimental nonspecific suppression of T cell responses.     

Immunocytochemistry of collagenase expression in transitional cell carcinoma of the bladder

OBJECTIVE: To evaluate the usefulness of collagenase immunocytochemistry as well as its immunohistochemistry in assessing the correlation with prognostic factors in transitional cell carcinoma (TCC) of the urinary bladder. STUDY DESIGN: We investigated the expression of collagenase in catheterized urine and histologic specimens from 38 patients with TCC and 20 cases with benign lesions of the urinary tract. RESULTS: Thirteen (34.2%) and 17 (44.7%) patients with TCC showed positive expression of collagenase on cytologic and histologic specimens, respectively, whereas in no cases with benign lesions was such expression found (P < .01). Invasive and nonpapillary TCC had higher positive rates than noninvasive and papillary TCC. Grade 3 TCC was positive at a higher rate than was grade 2, whereas there were no positive cases with grade 1. Collagenase expression did not correlate significantly with stage. CONCLUSION: Collagenase expression in urinary TCC correlated well with tumor growth pattern, pathologic grade and invasiveness of the carcinoma; all are known to be prognostic factors. The application of collagenase immunostaining to urinary cytology is very useful for assessing prognosis in TCC.