Chondroitinase-mediated degradation of rare 3-O-sulfated glucuronic acid in functional oversulfated chondroitin sulfate K and E

Chondroitin sulfate K (CS-K) from king crab cartilage rich in rare 3-O-sulfated glucuronic acid (GlcUA(3S)) displayed neuritogenic activity and affinity toward various growth factors like CS-E from squid cartilage. CS-K-mediated neuritogenesis of mouse hippocampal neurons in culture was abolished by digestion with chondroitinase (CSase) ABC, indicating the possible involvement of GlcUA(3S). However, identification of GlcUA(3S) in CS chains by conventional high performance liquid chromatography has been hampered by its CSase ABC-mediated degradation. To investigate the degradation process, an authentic CS-E tetrasaccharide, Delta4,5HexUA-GalNAc(4S)-GlcUA(3S)-GalNAc(4S), was digested with CSase ABC, and the end product was identified as GalNAc(4S) by electrospray ionization mass spectrometry (ESI-MS). Putative GalNAc(6S) and GalNAc(4S,6S), derived presumably from GlcUA(3S)-GalNAc(6S) and GlcUA(3S)-GalNAc(4S,6S), respectively, were also detected by ESI-MS in the CSase ABC digest of a CS-E oligosaccharide fraction resistant to CSases AC-I and AC-II. Intermediates during the CSase ABC-mediated degradation of Delta4,5HexUA(3S)-GalNAc(4S) to GalNAc(4S) were identified through ESI-MS of a partial CSase ABC digest of a CS-K tetrasaccharide, GlcUA(3S)-GalNAc(4S)-GlcUA(3S)-GalNAc(4S), and the conceivable mechanism behind the degradation of the GlcUA(3S) moiety was elucidated. Although a fucose branch was also identified in CS-K, defucosylated CS-K exhibited greater neuritogenic activity than the native CS-K, excluding the possibility of the involvement of fucose in the activity. Rather, (3S)-containing disaccharides are likely involved. These findings will enable us to detect GlcUA(3S)-containing disaccharides in CS chains to better understand CS-mediated biological processes.     

Activation of histidine decarboxylase through post-translational cleavage by caspase-9 in a mouse mastocytoma P-815

L-Histidine decarboxylase (HDC) is the rate-limiting enzyme for histamine synthesis in mammals. Although accumulating evidence has indicated the post-translational processing of HDC, it remains unknown what kinds of proteases are involved. We investigated the processing of HDC in a mouse mastocytoma, P-815, using a lentiviral expression system. HDC was expressed as a 74-kDa precursor form, which is cleaved to yield the 55- and 60-kDa forms upon treatment with butyrate. Alanine-scanning mutations revealed that two tandem aspartate residues (Asp(517)-Asp(518), Asp(550)-Asp(551)) are critical for the processing. Treatment with butyrate caused an increase in the enzyme activity of the cells expressing the wild type HDC, but not in the cells expressing the processing-incompetent mutant. An increase in histamine synthesis by butyrate was accompanied by formation of the 55- and 60-kDa form of HDC. In addition, the in vitro translated 74-kDa form of HDC was found to undergo a limited cleavage by purified human caspase-9, whereas the alanine-substituted mutants were not. Processing and enzymatic activation of HDC in P-815 cells was enhanced in the presence of a Zn(2+) chelator, TPEN. Although treatment with butyrate and TPEN drastically augmented the protease activity of caspase-3, and -9, no apoptotic cell death was observed. Both enzymatic activation and processing of HDC were completely suppressed by a pan-caspase inhibitor, partially but significantly by a specific inhibitor for caspase-9, but not by a caspase-3 inhibitor. These results suggest that, in P-815 cells, histamine synthesis is augmented through the post-translational cleavage of HDC, which is mediated by caspase-9.     

Neuronal regulation by which microglia enhance the production of neurotrophic factors for GABAergic, catecholaminergic, and cholinergic neurons

A phenomenon-in which microglia are activated in axotomized rat facial nucleus suggests that a certain neuronal stimulus triggers the activation of microglia. However, how the microglial characteristics are regulated by this neuronal stimulus has not previously been determined. In this study, therefore, the regulation of microglial properties by neurons was characterized in vitro from a neurotrophic perspective. To evaluate the neurotrophic effects of microglia stimulated with neurons, the effects of conditioned medium (CM) of microglia stimulated with neuronal CM (NCM) were assessed in neuronal cultures. The amounts of tyrosine hydroxylase (TH) in neuronal culture exposed to CM of microglia stimulated with NCM was much more than those in neurons exposed to CM of control microglia, suggesting that neuronal stimulus enhances the production of neurotrophic factors for catecholaminergic neurons in microglia. Therefore, the neurotrophic effects of CM of microglia stimulated with NCM were analyzed in detail. The immunocytochemical and biochemical experiments revealed that the CM of microglia stimulated with NCM enhances the survival/maturation of GABAergic and catecholaminergic neurons. The levels of choline acetyltransferase specific to cholinergic neurons also significantly increased in response to stimulation with the same microglial CM. These results allowed us to investigate the production of neurotrophic factors in the CM of microglia stimulated with NCM. The results indicated that NCM induces nerve growth factor (NGF), and enhances neurotrophin-4/5 (NT-4/5), transforming growth factor beta1 (TGFbeta1), glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), interleukin-3 (IL-3), and IL-10 in microglia. The promoted neurotrophic effects of CM of microglia stimulated with NCM were significantly abrogated by deprivation of neurotrophic factors by means of an immunoprecipitation method. Taken together, neuronal stimulus was found to activate microglia to produce more neurotrophic factors as above, thereby changing microglia into more neurotrophic cells.     

Delta-opioid receptor activation before ischemia reduces gap junction permeability in ischemic myocardium by PKC-epsilon-mediated phosphorylation of connexin 43

The aim of this study was to examine the hypothesis that delta-opioid receptor activation before ischemia suppresses gap junction (GJ) permeability by PKC-mediated connexin 43 (Cx43) modulation, which contributes to infarct size limitation afforded by the delta-opioid receptor activation. A delta-opioid receptor agonist, [D-Ala(2),D-Leu(5)]-enkephalin acetate (DADLE, 300 nM), was used in place of preconditioning (PC) ischemia to trigger PC mechanisms in rat hearts. GJ permeability during ischemia, which was assessed by Lucifer yellow, was reduced by DADLE to 47% of the control level, and this effect of DADLE was almost abolished by a PKC-epsilon inhibitor [PKC-epsilon translocation inhibitory peptide (PKC-epsilon-TIP)] but was not affected by a PKC-delta inhibitor (rottlerin). After DADLE infusion, PKC-epsilon, but not PKC-delta, was coimmunoprecipitated with Cx43, and the level of phosphorylation of Cx43 at a PKC-dependent site (Ser(368)) was significantly elevated during ischemia. DADLE reduced infarct size after 35 min of ischemia followed by 2 h of reperfusion by 69%, and PKC-epsilon-TIP and rottlerin eliminated 48% and 63%, respectively, of the infarct size-limiting effect of DADLE. Infusion of a GJ blocker, heptanol, before reperfusion reduced infarct size by 36%, and this protection was not enhanced by preischemic infusion of rottlerin + DADLE, which allows PKC-epsilon activation by DADLE. These results suggest that phosphorylation of Cx43 by PKC-epsilon plays a crucial role in delta-opioid-induced suppression of GJ permeability in ischemic myocardium and that this modulation of the GJ is possibly an adjunct mechanism of infarct size limitation afforded by preischemic delta-opioid receptor activation.     

Effect of <Emphasis Type="Italic">poxB</Emphasis> gene knockout on metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis> based on growth characteristics and enzyme activities

The effect of poxB gene knockout on metabolism in Escherichia coli was investigated in the present paper based on the growth characteristics and the activities of the enzymes involved in the central metabolic pathways. The absence of pyruvate oxidase reduced the glucose uptake rate and cell growth rate, and increased O₂ consumption and CO₂ evolution. The enzyme assay results showed that although glucokinase activity increased, the flux through glycolysis was reduced due to the down-regulation of the other glycolytic enzymes such as 6-phosphofructosekinase and fructose bisphosphate aldolase in the poxB mutant. TCA cycle enzymes such as citrate synthase and malate dehydrogenase were repressed in the poxB mutant when the cells were cultivated in LB medium. The pyruvate oxidase mutation also resulted in the activation of glucose-6-phosphate dehydrogenase and acetyl-CoA synthetase. All these results suggest that pyruvate oxidase is not only a stationary-phase enzyme as previously known, and that the removal of the poxB gene affects the central metabolism at the enzyme level in E. coli.     

Rat <Emphasis Type="Italic">wct</Emphasis> mutation prevents differentiation of maturation-stage ameloblasts resulting in hypo-mineralization in incisor teeth

A recent study provided genetic and morphological evidence that rat autosomal-recessive mutation, whitish chalk-like teeth (wct), induced tooth enamel defects resembling those of human amelogenesis imperfecta (AI). The wct locus maps to a specific interval of rat chromosome 14 corresponding to human chromosome 4q21 where the ameloblastin and enamelin genes exist, although these genes are not included in the wct locus. The effect of the wct gene mutation on the enamel matrix synthesis and calcification remains to be elucidated. This study clarifies how the wct gene mutation influences the synthesis of enamel matrix and its calcification by immunocytochemistry for amelogenin, ameloblastin and enamelin, and by electron probe micro-analysis (EPMA). The immunoreactivity for enamel proteins such as amelogenin, ameloblastin, and enamelin in the ameloblasts in the homozygous teeth was the same as that in the heterozygous teeth from secretory to transitional stages, although the homozygous ameloblasts became detached from the enamel matrix in the transitional stage. The flattened ameloblasts in the maturation stage of the homozygous samples contained enamel proteins in their cytoplasm. Thus, the wct mutation was found to prevent the morphological transition of ameloblasts from secretory to maturation stages without disturbing the synthesis of enamel matrix proteins, resulting in the hypo-mineralization of incisor enamel and cyst formation between the enamel organ and matrix. This mutation also prevents the transfer of iron into the enamel.     

Allelic diversity in the merozoite surface protein 1 gene of Plasmodium falciparum on Palawan Island, the Philippines

Allelic diversity of the Plasmodium falciparum merozoite surface protein 1 gene (msp1) is mainly generated by meiotic recombination at the mosquito stage. We investigated recombination-based allelic diversity of msp1 in a P. falciparum population from Palawan Island, the Philippines, where malaria transmission is moderate. We identified the 5' recombinant types, 3' sequence types and msp1 haplotypes (unique combinations of 5' recombinant type and 3' sequence type), and compared them with those of P. falciparum from the Solomon Islands, where malaria transmission is high. The mean number of 5' recombinant types per patient in Palawan was 1.44, which is comparable to the number for the Solomon Islands (1.41). The Palawan parasite population had 15 msp1 haplotypes, whereas the Solomon Islands population had only 8 haplotypes. The Palawan population showed strong linkage disequilibrium between polymorphic blocks/sites within msp1, which is comparable to the results for the Solomon Islands. These findings support our hypothesis that the extent of allelic diversity of msp1 is determined not only by the transmission intensity but also by the number of msp1 alleles prevalent in the local parasite population and the extent of mixed-allele infections. Contribution of a high prevalence of the chloroquine (CQ)-sensitive allele of P. falciparum CQ resistance transporter (pfcrt) to the relatively high msp1 diversity in the Palawan population is discussed.     

Progress in the molecular biology of malaria and the immunology of nematode infections

Japan is one of a small number of countries to have successfully controlled or eliminated major parasitic diseases, including malaria, filariasis, schistosomiasis and enteric parasitoses. Of particular importance in this success was a close collaboration between primary research and public health efforts. Many Japanese researchers continue to study malaria, particularly the areas of genetics and immunology, and this should contribute to global parasite eradication strategies. Furthermore, studies of immunoregulation of nematode infection using the interleukin-18 pathway, most of which have been conducted in Japan, are helping to improve researchers' understanding of human immune mechanisms and host-parasite interactions.     

Problem-based learning is suitable for the curriculum of “Sleep disorders and disease” for students in dentistry

Sleep and Biological Rhythms -