Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-L-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95)

A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the alpha-(1-->2) linkage of 2'-fucosyllactose, and a gene encoding 1,2-alpha-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal alpha-(1-->2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2'-fucosyllactose was determined to be inversion by using (1)H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).     

An autocrine/paracrine loop linking keratin 14 aggregates to tumor necrosis factor alpha-mediated cytotoxicity in a keratinocyte model of epidermolysis bullosa simplex

Epidermolysis bullosa simplex (EBS) is a blistering cutaneous disease featuring protein aggregates. Here we investigate the molecular mechanisms linking protein aggregates to cell death in a cellular model of EBS in which HaCaT keratinocytes are transfected with plasmids expressing various mutant forms of keratin 14 (K14). In HaCaT cells, mutant K14 was found to form ubiquitinated protein aggregates that suppressed 20 S proteasome function instead of being degraded by 20 S proteasome. Keratinocytes with mutant K14-induced phosphorylation of the stress-activated kinase c-Jun, as well as up-regulation of unfolding protein Bip, indicates induction of endoplasmic reticulum stress. HaCaT cells were susceptible to apoptosis by activation of caspases-3, and -8, but not caspase-9 or -12. Tumor necrosis factor-alpha (TNFalpha) in the culture medium was increased in keratinocytes with mutant K14 compared with wild K14, and the addition of neutralizing anti-TNFalpha antibody to the culture medium rescued keratinocytes from cell death. Thus, TNFalpha release and the subsequent activation of the TNFalpha receptor by an autocrine/paracrine pathway links protein aggregates to cell death in this keratinocyte EBS cellular model. Furthermore, mutation in K14 reduced its affinity to TNFalpha receptor-associated death domain (TRADD), suggesting that the susceptibility of keratinocytes to caspase-8-mediated apoptosis is increased in mutated K14 because of impairment of the cytoprotective mechanism mediated by K14-TRADD interaction.     

C protein-induced myocarditis and subsequent dilated cardiomyopathy: rescue from death and prevention of dilated cardiomyopathy by chemokine receptor DNA therapy

Severe experimental autoimmune myocarditis and subsequent dilated cardiomyopathy (DCM) were successfully produced in Lewis rats by immunization with recombinant cardiac C protein. Seventy-five percent of immunized rats died between days 15 and 49 postimmunization, and all of the survived rats showed typical DCM characterized by the presence of ventricular dilatation and extensive fibrosis. Immunopathological and chemokine analysis during the acute phase revealed that there were marked macrophage infiltration with myocyte necrosis and up-regulation of MCP-1 and IFN-gamma-inducible protein-10 (IP-10). Based on these findings, we prepared plasmid DNAs encoding the binding site of CCR2 and CXCR3, which are receptors for MCP-1 and IP-10, respectively. The culture supernatant of cells transfected with these DNAs inhibited the migration of T cells and macrophages induced by MCP-1 and IP-10. Remarkably, administration of the DNAs to C protein-immunized rats prevented the disease progression and rescued animals from death. The present study has demonstrated for the first time that gene therapy targeting the chemokine receptor could be a powerful tool for the control of experimental autoimmune myocarditis and DCM.     

Induction of Fos Immunoreactivity in Oxytocin Neurons in the Paraventricular Nucleus After Female Odor Exposure in Male Rats: Effects of Sexual Experience

1. We examined whether oxytocin (OT) neurons in the paraventricular nucleus of the hypothalamus (PVN) were activated by estrus female odor and sexual contact in sexually naïve and experienced Long-Evans rats. 2. Male rats were not presented to anesthetized estrus females (control) or presented to the females without (exposure to the female odor without sexual contact) or with direct contact (exposure to the female odor with sexual contact). 3. Exposure to the female odor with sexual contact significantly increased OT neurons with Fos-ir in both males. Exposure to the female odor without contact increased OT neurons with Fos-immunoreactive cells (Fos-ir) in sexually experienced males but not in naïve males, suggesting that the female odor without sexual contact activated the oxytocinergic neuronal system in the PVN in the experienced males. 4. Therefore, exposure to the estrus female odor itself may exert different effects on sexually naïve and experienced males.     

Mutant PLP/DM20 Cannot Be Processed to Secrete PLP-Related Oligodendrocyte Differentiation/Survival Factor

Most of the mutations within the PLP gene result in degeneration of oligodendrocytes and this is believed to be caused by intracellular trafficking defects. Previous studies have demonstrated that cells expressing the wild type PLP gene release a factor promoting differentiation/survival of oligodendrocyte and that this factor is the C-terminal portion of the protein itself. In this study we asked how the naturally occurring mutations of the PLP gene (jimpy, jimpy msd, and rumpshaker) affect this activity. We developed a transient expression system for retroviral production and transduction that enabled the expression of mutant PLP/DM20 cDNAs in NIH3T3 cells. None of the NIH3T3 cells producing mutant PLP/DM20s secreted the PLP-related factor that increases the number of oligodendrocytes. Since it has been shown that rumpshaker DM20 can be transported to the cell surface, but its folding is incorrect, absence of secretion of this factor is more heavily attributable to incorrect protein folding than to the defect in the PLP/DM20 trafficking.     

A new factor from Bacillus mesentericus which promotes the growth of Bifidobacterium

It was reported previously that supernatants of cultures of Bacillus mesentericus TO-A promote the growth of Bifidobacterium species. In this study, a new growth-promoting factor, BM-1, was purified from the supernatant of such a culture and its chemical structure was determined. BM-1 was identified as 3,3-dihydroxyazetidine, and it promoted the growth of several strains of Bifidobacterium.     

Cholesteryl ester transfer protein deficiency causes slow egg embryonation of Schistosoma japonicum

It was investigated whether proteasome activity was implicated in susceptibility of human vascular smooth muscle cells (VSMCs) to Fas-mediated death. Human fetal aorta smooth muscle cells were treated with agonistic anti-Fas antibody (CH11) and proteasome inhibitors (MG115 or MG132) and then cell death was determined by morphology, viability, and DNA fragmentation. The present study reports that: (a) crosslinking of Fas receptor with anti-Fas antibody in the presence of proteasome inhibitor-induced death and DNA degradation in human VSMCs that were blocked by caspases inhibitor z-DEVD.fmk; (b) cotreatment with anti-Fas antibody and proteasome inhibitor activated caspase-3; (c) proteasome inhibitors did not influence expression of procaspase-8, procaspase-3, c-FLIP, and Bcl-2; and (d) proteasome inhibitors up-regulated Fas and FADD. The data indicate that proteasome activity is important in survival of VSMCs and provide the first evidence that proteasome is involved in Fas signal transduction. The present study proposes novel mechanism(s) by which VSMCs become susceptible to FasL.     

Characterization of multiple Chinese hamster carbonyl reductases

Carbonyl reductase (CR) is an enzyme which can catalyze the oxidoreduction of various carbonyl compounds in the presence of NAD(P)H. With the PCR method, using primers carrying the conserved nucleotide sequence among mammalian CRs, we isolated three different cDNAs (CHCR1, CHCR2 and CHCR3) which encode a unique carbonyl reductase from the Chinese hamster. The PCR products of CHCR1 and CHCR2 were clearly isolated with Bpu1102I, BspEI and XmaI restriction enzymes. The nucleotide-sequence of CHCR3 was completely different from those of CHCR1 and CHCR2. The predicted double-wound betaalphabetaalpha-structures of the CHCRs suggests the presence of a typical NADP(+)-binding motif and is similar to the corresponding region of 3alpha,20beta-hydroxysteroid dehydrogenase and mouse lung tetrameric carbonyl reductase. The deduced amino acid sequence of CHCR1 showed a high homology to CHCR2 (>96%) and the other mammalian CRs (>81%). However, CHCR3 showed a high homology to human CBR3 (>86%) and a relatively lower homology to the other CHCRs (<76%). Bacterial recombinant CHCRs showed typical carbonyl reductase activities towards 4-benzoylpyridine, 4-nitrobenzaldehyde and pyridine 4-carboxyaldehyde. These three CRs showed not only 3-keto reductase of steroids, but also 20-keto reductase. However, these CRs did not show any activity of 17-keto reductase activity. Both CHCR1 and CHCR2 have prostaglandin 9-keto reductase and 15-hydroxyprostaglandin dehydrogenase activities towards PGE(2) and PGF(2alpha) from the analyses of enzymatic reaction products. The results of Western blotting and RT-PCR suggest these CHCRs have a tissue-dependent-distribution in the Chinese hamster.     

Identification of an Intra- and Inter-specific Tear Protein Signal in Rodents

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