Increased response of liver microsomal delta 6-desaturase activity to dietary methionine in rats

The effects of dietary casein level (5-40%) on the liver microsomal phospholipid profile, delta 6-desaturase activity and related variables were investigated in rats to examine whether the dietary protein level affected the delta 6-desaturase activity through an alteration of the liver microsomal phospholipid profile. The effects of supplementing a 10% casein diet with certain amino acids were also investigated. The concentration of hepatic S-adenosylmethionine (SAM), the ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) and the delta 6-desaturase activity in liver microsomes, and the ratio of arachidonate to linoleate of microsomal PC increased with increasing dietary casein level. There were significant correlations between the dietary methionine content and hepatic SAM concentration, hepatic SAM concentration and microsomal PE concentration, and microsomal PE concentration and delta 6-desaturase activity. Supplementation of the 10% casein diet with methionine significantly increased the hepatic SAM concentration, PC/PE ratio, delta 6-desaturase activity, and arachidonate/linoleate ratio, whereas cystine supplementation had no or little effect on these variables. These increases induced by methionine were significantly suppressed by additional glycine. The results obtained here, together with those in our previous report, suggest that quantity and type of dietary protein might affect the delta 6-desaturase activity through an alteration of the liver microsomal profile of phospholipids, especially PE, and that the alteration of phospholipid profile might be mediated by a hepatic SAM concentration that reflects the dietary methionine level.     

Differential level in co-down-modulation of CD4 and CXCR4 primed by HIV-1 gp120 in response to phorbol ester, PMA, among HIV-1 isolates

HIV-1 enters cells through interacting with cell surface molecules such as CD4 and chemokine receptors. We generated recombinant soluble gp120s derived from T-cell line-tropic (T-tropic) and macrophage-tropic (M-tropic) HIV-1 strains using a baculovirus expression system and investigated the association of CD4-gp120 complex with the chemokine receptor and/or other surface molecule(s). For monitoring the co-down-modulations of the CD4-gp120 complex, a cytoplasmic domain deletion mutant (tailless CD4), which is not capable of undergoing down-modulation by itself in response to phorbol ester PMA, was used. Our studies revealed both cell-type and HIV-1 strain-specific differences. We found that T-tropic gp120s were capable of priming co-down-modulation with tailless CD4 by interacting with CXCR4, whereas M-tropic SF162 gp120 could not after PMA treatment even in the presence of CCR5. Among the T-tropic HIV-1 envelopes, IIIB gp120 was the most potent. Furthermore, the ability of gp120 to prime the PMA induced co-down-modulation of tailless CD4 appeared to be dependent on the concentration of the principal coreceptor CXCR4. Nevertheless, the observation that IIIB gp120 strongly primed tailless CD4 co-down-modulation on human osteosarcoma HOS cells that express undetectable levels of surface CXCR4 raised the possibility that membrane component(s) other than those recently identified can be involved in down-modulation of the CD4/gp120 complexes.     

Immunohistochemical Localization of Arginase II and Other Enzymes of Arginine Metabolism in Rat Kidney and Liver

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were collocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.     

Organic matter processing by chironomid larvae (Diptera: Chironomidae)

In laboratory experiments, we used fluorescent dye markers to investigate processing of organic matter by larvae of Psectrocladius limbatellus (Holm.) (Diptera: Chironomidae). 59% of the organic matter used was incorporated into tubes, 39% was present in faecal pellets (both after 24 h), and 2% was found in the larval gut at the end of experiments. Ingested matter passed through the gut rapidly, resulting in the gut being emptied more than 20 times each day. Further 24-h experiments using dye-marked faecal pellets showed that 6% of pellets produced were re-ingested and 12% were incorporated into tubes. There was no preference for conditioned faecal pellets as food over those that had recently been egested and tubes also provided a food reserve on which larvae feed. Chironomid larvae recycle organic matter resulting in its mineralization and their ‘engineering’ has a dramatic effect on the substratum. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation of 48 genetic markers appropriate for high throughput genotyping of inbred rat strains by B1 repetitive sequence-representational difference analysis

Mapping of genetic suppressors, modifiers, and quantitative trait loci (QTLs) requires genetic markers that can be efficiently and inexpensively genotyped for a large number of individuals. To isolate rat genetic markers suitable for this purpose, representational difference analysis (RDA) was performed with amplicons prepared by PCR with the B1 repetitive sequence used as the primer (B1-amplicons). In total, 48 polymorphic DNA fragments were isolated by five series of RDA, subtracting the B1-amplicons prepared from an ACI/N (ACI) rat from those prepared from BUF/Nac (BUF), and vice versa. All the polymorphic fragments detected ``presence-or-absence' polymorphisms with B1-amplicons prepared from ACI, BUF, and their F₂ progeny, and each fragment was linkage mapped. Dot-blotting amplicons onto filters at a high density and hybridization of the filters with these B1-RDA markers made it possible to genotype a large number of rats simultaneously for multiple loci. These B1-RDA markers were polymorphic between two given inbred strains of rat at frequencies between 30% and 70%. This is the first report on the isolation of B1-RDA markers among inbred strains of rats. Received: 15 July 1998 / Accepted: 18 August 1998     

Epigenetic regulation of autophagy by the methyltransferase EZH2 through an MTOR-dependent pathway

Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the autophagy regulation machinery has been widely studied, the key epigenetic control of autophagy process still remains unknown. Here we report that the methyltransferase EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) epigenetically represses several negative regulators of the MTOR (mechanistic target of rapamycin [serine/threonine kinase]) pathway, such as TSC2, RHOA, DEPTOR, FKBP11, RGS16 and GPI. EZH2 was recruited to these genes promoters via MTA2 (metastasis associated 1 family, member 2), a component of the nucleosome remodeling and histone deacetylase (NuRD) complex. MTA2 was identified as a new chromatin binding protein whose association with chromatin facilitated the subsequent recruitment of EZH2 to silenced targeted genes, especially TSC2. Downregulation of TSC2 (tuberous sclerosis 2) by EZH2 elicited MTOR activation, which in turn modulated subsequent MTOR pathway-related events, including inhibition of autophagy. In human colorectal carcinoma (CRC) tissues, the expression of MTA2 and EZH2 correlated negatively with expression of TSC2, which reveals a novel link among epigenetic regulation, the MTOR pathway, autophagy induction, and tumorigenesis.     

Characterization of a broadly reactive monoclonal antibody against norovirus genogroups I and II: recognition of a novel conformational epitope

Norovirus, which belongs to the family Caliciviridae, is one of the major causes of nonbacterial acute gastroenteritis in the world. The main human noroviruses are of genogroup I (GI) and genogroup II (GII), which were subdivided further into at least 15 and 18 genotypes (GI/1 to GI/15 and GII/1 to GII/18), respectively. The development of immunological diagnosis for norovirus had been hindered by the antigen specificity of the polyclonal antibody. Therefore, several laboratories have produced broadly reactive monoclonal antibodies, which recognize the linear GI and GII cross-reactive epitopes or the conformational GI-specific epitope. In this study, we characterized the novel monoclonal antibody 14-1 (MAb14-1) for further development of the rapid immunochromatography test. Our results demonstrated that MAb14-1 could recognize 15 recombinant virus-like particles (GI/1, 4, 8, and 11 and GII/1 to 7 and 12 to 15) and showed weak affinity to the virus-like particle of GI/3. This recognition range is the broadest of the existing monoclonal antibodies. The epitope for MAb14-1 was identified by fragment, sequence, structural, and mutational analyses. Both terminal antigenic regions (amino acid positions 418 to 426 and 526 to 534) on the C-terminal P1 domain formed the conformational epitope and were in the proximity of the insertion region (positions 427 to 525). These regions contained six amino acids responsible for antigenicity that were conserved among genogroup(s), genus, and Caliciviridae. This epitope mapping explained the broad reactivity and different titers among GI and GII. To our knowledge, we are the first group to identify the GI and GII cross-reactive monoclonal antibody, which recognizes the novel conformational epitope. From these data, MAb14-1 could be used further to develop immunochromatography.