Role of side-edge site of sphingomyelinase from Bacillus cereus

Bacillus cereus sphingomyelinase (Bc-SMase) belongs to the Mg(2+)-dependent neutral sphingomyelinase (nSMase) which hydrolyzes sphingomyelin (SM) to produce phosphocholine and ceramide, and acts as an extracellular hemolysin. Bc-SMase has two metal ion-binding sites in a long horizontal cleft across the molecule, with one Mg(2+) in the central region of the cleft and one divalent metal ion at the side-edge of the cleft. The role of the Mg(2+) at the side-edge of the long horizontal cleft in Bc-SMase remains unresolved. The replacement of Asn-57, Glu-99, and Asp-100 located in close proximity to Mg(2+) at the side-edge with alanine resulted in a striking reduction in binding to and hydrolysis of sphingomyelin in membranes of sheep erythrocytes or SM-liposomes but that of Phe-55 did not. However, the replacement of these residues had little effect on the enzymatic activity. N57A, E99A, and D100A contained 2 mol of Mg(2+) per mol of protein, and the wild type and F55A contained 3 mol. A crystal analysis showed that N57A with Mg(2+) had no metal ion at the side-edge. These results indicate that the Mg(2+) at the side-edge of Bc-SMase plays an important role in the binding to membranes.     

Intake of dissolved organic matter from deep seawater inhibits atherosclerosis progression

Dissolved organic matter (DOM) in seawater can be defined as the fraction of organic matter that passes through a filter of sub micron pore size. In this study, we have examined the effect of DOM of deep seawater (DSW) from Pacific Ocean on platelet aggregation and atherosclerosis progression. DSW was passed through a series of filters and then through an Octadecyl C18 filter; the retained substance in ethanol was designated as C18 extractable DOM (C18-DOM). Our studies showed that C18-DOM treatment inhibited platelet aggregation, P-selectin expression and activity of COX-1 significantly. C18-DOM increased the expression of anti-atherogenic molecule namely heme oxygenase-1 in endothelial cells and all these data showed that C18-DOM is exhibiting aspirin-like effects. Moreover our in vivo studies showed that C18-DOM feeding slowed remarkably the progression of atherosclerosis. Our study demonstrated a novel biological effect of oceanic DOM, which has several important implications, including a possible therapeutic strategy for atherosclerosis.     

Assignment of the Gene for Porcine Insulin-Like Growth Factor Binding Protein 1 to Chromosome 18 and Detection of Polymorphisms in Intron 2 by PCR–RFLP

We have obtained a partial cDNA and three BAC clones for the porcine insulin-like growth factor binding protein 1 gene (IGFBP-1). Results of fluorescence in situ and radiation hybrid (RH) mapping assigned this gene to porcine chromosome (SSC) 18q24-qter. We found two types of polymerase chain reaction–restriction-fragment-length polymorphisms (PCR–RFLP) in intron 2 by using FokI and AluI.     

Adipose triglyceride lipase regulates basal lipolysis and lipid droplet size in adipocytes

In adipocytes, lipid droplet (LD) size reflects a balance of triglyceride synthesis (lipogenesis) and hydrolysis (lipolysis). Perilipin A (Peri A) is the most abundant phosphoprotein on the surface of adipocyte LDs and has a crucial role in lipid storage and lipolysis. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major rate-determining enzymes for lipolysis in adipocytes. Each of these proteins (Peri A, ATGL, and HSL) has been demonstrated to regulate lipid storage and release in the adipocyte. However, in the absence of protein kinase A (PKA) stimulation (basal state), the lipases (ATGL and HSL) are located mainly in the cytoplasm, and their contribution to basal rates of lipolysis and influence on LD size are poorly understood. In this study, we utilize an adenoviral system to knockdown or overexpress ATGL and HSL in an engineered model system of adipocytes in the presence or absence of Peri A. We are able to demonstrate in our experimental model system that in the basal state, LD size, triglyceride storage, and fatty acid release are mainly influenced by the expression of ATGL. These results demonstrate for the first time the relative contributions of ATGL, HSL, and Peri A on determination of LD size in the absence of PKA stimulation.     

Ceiling culture-derived proliferative adipocytes retain high adipogenic potential suitable for use as a vehicle for gene transduction therapy

Adipose tissue is expected to provide a source of proliferative cells for regenerative medicine and cell-transplantation therapies using gene transfer manipulation. We have recently identified ceiling culture-derived proliferative adipocytes (ccdPAs) from the mature adipocyte fraction as cells suitable as a therapeutic gene vehicle because of their stable proliferative capacity. In this study, we examined the capability of adipogenic differentiation of the ccdPAs compared with stromal vascular fraction (SVF)-derived progenitor cells (adipose-derived stem cells, ASCs) with regard to their multipotential ability to be converted to another lineage and therefore their potential to be used for regenerative medicine research. After in vitro passaging, the surface antigen profile and the basal levels of adipogenic marker genes of the ccdPAs were not obviously different from those of the ASCs. However, the ccdPAs showed increased lipid-droplet accumulation accompanied with higher adipogenic marker gene expression after stimulation of differentiation compared with the ASCs. The higher adipogenic potential of the ccdPAs than the ASCs from the SVF was maintained for 42 days in culture. Furthermore, the difference in the adipogenic response was enhanced after partial stimulation without indomethacin. These results indicate that the ccdPAs retain a high adipogenic potential even after in vitro passaging, thus suggesting the commitment of ccdPAs to stable mature adipocytes after autotransplantation, indicating that they may have potential for use in regenerative and gene-manipulated medicine.     

CO binding studies of nitric oxide synthase: effects of the substrate, inhibitors and tetrahydrobiopterin

The dissociation constant (K_d) for CO from neuronal nitric oxide synthase heme in the absence of the substrate and cofactor was less than 10⁻³ μM. In the presence of

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-Arg, it dramatically increased up to 1 μM. In the presence of inhibitors such as N^G-nitro-

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-arginine methyl ester and 7-nitroindazole (NI), the K_d value further increased up to more than 100 μM. Addition of the cofactor, 5,6,7,8-tetrahydrobiopterin (H₄B), increased the K_d value by 10-fold in the presence of

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-Arg, whereas it decreased the value to less than one 250th in the presence of NI. Addition of H₄B increased the recombination rate constant (k_on) for CO by more than two-fold in the presence of

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-Arg or N⁶-(1-iminoethyl)-

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-lysine, whereas it decreased the k_on value by three-fold in the presence of

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-thiocitrulline. Thus, the binding fashion of some of inhibitors, such as NI, may be different from that of

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-Arg with respect to the H₄B effect.     

The Large Isoform of Myelin-Associated Glycoprotein Is Scarcely Expressed in the Quaking Mouse Brain

Two polypeptide isoforms of myelin-associated glycoprotein (MAG) with molecular masses of 72 and 67 kDa are produced by alternative splicing of the exon 12 portion. Our previous work has demonstrated that in the quaking mouse brain this alternative splicing is lacking and that the mRNA coding the large MAG isoform (L-MAG) is scarcely expressed, whereas that of small MAG isoform (S-MAG) is overexpressed. In the present study, we prepared antisera specific to the S-MAG and L-MAG amino acid residues, respectively. Immunoblots showed that the L-MAG band was scarcely detectable in the quaking mouse brain, whereas the S-MAG band had an apparently higher molecular mass than in the normal control. Our immunohistochemical study also showed that L-MAG was scarcely stained in the quaking mouse brain. These results seemed to reflect a reduction in content of L-MAG mRNA and abnormal glycosylation in the quaking mouse brain.     

Antibiotics LL-Z1272 identified as novel inhibitors discriminating bacterial and mitochondrial quinol oxidases

To counter antibiotic-resistant bacteria, we screened the Kitasato Institute for Life Sciences Chemical Library with bacterial quinol oxidase, which does not exist in the mitochondrial respiratory chain. We identified five prenylphenols, LL-Z1272β, γ, δ, ? and ζ, as new inhibitors for the Escherichia coli cytochrome bd. We found that these compounds also inhibited the E. coli bo-type ubiquinol oxidase and trypanosome alternative oxidase, although these three oxidases are structurally unrelated. LL-Z1272β and ? (dechlorinated derivatives) were more active against cytochrome bd while LL-Z1272γ, δ, and ζ (chlorinated derivatives) were potent inhibitors of cytochrome bo and trypanosome alternative oxidase. Thus prenylphenols are useful for the selective inhibition of quinol oxidases and for understanding the molecular mechanisms of respiratory quinol oxidases as a probe for the quinol oxidation site. Since quinol oxidases are absent from mammalian mitochondria, LL-Z1272β and δ, which are less toxic to human cells, could be used as lead compounds for development of novel chemotherapeutic agents against pathogenic bacteria and African trypanosomiasis.     

Aluminium resistance requires resistance to acid stress: a case study with spinach that exudes oxalate rapidly when exposed to Al stress

Spinach is a vegetable with a high oxalate concentration in its tissues. Oxalate efflux from spinach (Spinacia oleracea L. cv. Quanneng) roots was rapidly stimulated (within 30 min) by aluminium (Al) treatment. The efflux was constant within 6 h, but increased with increasing Al concentration. The efflux was confined to the root tip (0-5 mm), which showed a 5-fold greater efflux than the root zone distal to the tip (5-10 mm). Oxalate efflux could not be triggered by treatment with the trivalent cation lanthanum or by phosphorus deficiency, indicating that the efflux was specific to the Al treatment. All this evidence suggested that spinach possesses Al-resistance mechanisms. However, spinach was found to be as sensitive to Al toxicity as the Al-sensitive wheat line ES8, which had no Al-dependent organic acids efflux. The Al accumulated in the apical 5 mm of the roots of spinach which was also similar to that in the Al-sensitive wheat after 24 h treatment with 50 microM AlCl(3), indicating a non-exclusion mechanism. In addition, root elongation in spinach was significantly inhibited at pH 4.5, compared with that at pH 6.5. Based on this evidence, it is concluded that the sensitivity to acid stress in spinach could mask the potential role for oxalate to protect the plant roots from Al toxicity.