Occurrence in chick embryo vitreous humor of a type IX collagen proteoglycan with an extraordinarily large chondroitin sulfate chain and short alpha 1 polypeptide

We have prepared a high buoyant density proteoglycan fraction from the vitreous humor of 13-day-old chick embryos. Using immunoblot analysis coupled with chondroitinase digestion, we demonstrate that the purified preparation is composed predominantly of type IX collagen-like chondroitin sulfate proteoglycan with an alpha 1(IX) chain Mr approximately 23,000 shorter than the known alpha 1 in cartilage type IX. Also different from cartilage type IX is the size of the chondroitin sulfate chain attached to the alpha 2(IX) polypeptide; its Mr is approximately 350,000 indicating that it is approximately 10 times larger in vitreous humor than in cartilage. Examination of vitreous bodies at different developmental stages indicates that a transition occurs in the size of alpha 1(IX) in a well defined temporal pattern; at about stage 31, a cartilage-type alpha 1(IX) of Mr 84,000 is the predominant species, whereas at stage 36 and thereafter, a Mr 61,000 species appears with a concomitant disappearance of the Mr 84,000 species. Immunostaining for type IX collagen followed by electron microscopic observation of 13-day-old chick embryo vitreous humor reveals a regular D-periodic arrangement of vitreous type IX collagen proteoglycan along thin fibrils. It seems possible that the chondroitin sulfate chains of extraordinarily high viscosity and high molecular weight may extend away from the fibrils, thus contributing to structural as well as functional properties of this unique matrix.     

Identification of the phospholipid-binding site of human beta(2)-glycoprotein I domain V by heteronuclear magnetic resonance

To understand the mechanism of the interaction between human beta(2)-glycoprotein I (beta(2)-GPI) and negatively charged phospholipids, we determined the three-dimensional solution structure of the fifth domain of beta(2)-GPI by heteronuclear multidimensional NMR. The results showed that the molecule is composed of well-defined four anti-parallel beta-strands and two short alpha-helices, as well as a long highly flexible loop. Backbone dynamic analysis demonstrated significant mobility of the flexible loop on a subnanosecond time scale. Structural modeling of the nicked fifth domain, in which the Lys317-Thr318 peptide bond was specifically cleaved, revealed the importance of this long C-terminal loop for the interaction between beta(2)-GPI and negatively charged phospholipids. A titration experiment with the anionic surfactant SDS showed that this highly mobile loop, as well as the short beta-hairpin between betaC and betaD strands, which is rich in positively charged residues, specifically interact with the surfactant. The mobile loop, together with the surrounding positively charged residues, probably construct the binding site for negatively charged phospholipids such as cardiolipin.     

A novel membrane-anchored cytochrome c-550 of alkaliphilic Bacillus clarkii K24-1U: expression, molecular features and properties of redox potential

A membrane-anchored cytochrome c-550, which is highly expressed in obligately alkaliphilic Bacillus clarkii K24-1U, was purified and characterized. The protein contained a conspicuous sequence of Gly²²-Asn³⁴, in comparison with the other Bacillus small cytochromes c. Analytical data indicated that the original and lipase-treated intermediate forms of cytochrome c-550 bind to fatty acids of C₁₅, C₁₆ and C₁₇ chain lengths and C₁₅ chain length, respectively, and it was considered that these fatty acids are bound to glycerol–Cys¹⁸. Since there was a possibility that the presence of a diacylglycerol anchor contributed to the formation of dimeric states of this protein (20 and 17 kDa in SDS-PAGE), a C18M (Cys¹⁸ → Met)-cytochrome c-550 was constructed. The molecular mass of the C18M-cytochrome c-550 was determined as 15 and 10 kDa in SDS-PAGE and 23 kDa in blue native PAGE. The C18M-cytochrome c-550 bound with or without Triton X-100 formed a tetramer as the original cytochrome c-550 bound with Triton X-100, as determined by gel filtration. The midpoint redox potential of cytochrome c-550 as determined by redox titration was +83 mV, while that determined by cyclic voltammetric measurement was +7 mV. The above results indicate that cytochrome c-550 is a novel cytochrome c.     

Identification of diterpene biosynthetic gene clusters and functional analysis of labdane-related diterpene cyclases in Phomopsis amygdali

Two diterpene biosynthesis gene clusters in the fusicoccin-producing fungus, Phomopsis amygdali, were identified by genome walking from PaGGS1 and PaGGS4 which encode the geranylgeranyl diphosphate (GGDP) synthases. The diterpene cyclase-like genes, PaDC1 and PaDC2, were respectively located proximal to PaGGS1 and PaGGS4. The amino acid sequences of these two enzymes were similar to those of fungal labdane-related diterpene cyclases. Recombinant PaDC1 converted GGDP mainly into phyllocladan-16 alpha-ol via (+)-copalyl diphosphate (CDP) and trace amounts of several labdane-related hydrocarbons which had been identified from the P. amygdali F6 mycelia. Since phyllocladan-16 alpha-ol had not been identified in P. amygdali F6 mycelia, we isolated phyllocladan-16 alpha-ol from the mycelia. Recombinant PaDC2 converted GGDP into (+)-CDP. Furthermore, we isolated the novel diterpenoid, phyllocladan-11 alpha,16 alpha,18-triol, which is a possible metabolite of phyllocladan-16 alpha-ol in the mycelia. We propose that genome walking offers a useful strategy for the discovery of novel natural products in fungi.     

Oral administration of a zinc complex improves type 2 diabetes and metabolic syndromes

Previously, we reported that intraperitoneal injections of the Zn(II) complex (Zn(alx)(2)) with allixin, which is isolated from dry garlic, with a Zn(O(4)) coordination environment, exhibited high anti-diabetic effects in obesity-linked type 2 diabetic KKA(y) mice. However, this complex exhibited low activity when administered orally. To improve the effect of Zn(alx)(2), we prepared a novel Zn(II) complex with the allixin-derivative bis(1,6-dimethyl-3-hydroxy-5-methoxy-2-pentyl-1,4-dihydropyridine-4- thionato)Zn(II), abbreviated as Zn(II)-thioallixin-N-methyl (Zn(tanm)(2)), having a Zn(S(2)O(2)) coordination environment; this complex has extremely high in vitro insulin-like activity. Because Zn was extensively absorbed from the gastrointestinal tract when Zn(tanm)(2) was orally administered, its anti-diabetic effects were examined in KKA(y) mice. Daily oral administrations of Zn(tanm)(2) for 4 weeks in KKA(y) mice significantly improved hyperglycemia, glucose intolerance, insulin resistance, hyperleptinemia, obesity, and hypertension. Interestingly, Zn(tanm)(2) increased depressed plasma adiponectin levels in the mice. Here, we propose that Zn(tanm)(2) will be an orally active therapeutic for obesity-linked type 2 diabetes and metabolic syndromes.     

Six strains of human immunodeficiency virus type 1 isolated in Japan and their molecular phylogeny

Summary Five strains of human immunodeficiency virus type 1 (HIV-1) were isolated from five Japanese hemophilia patients. Two isolates, HIV1[GUN-1] and HIV-1[GUN-2], were from brother patients with hemophilia B and the other three isolates, HIV-I[GUN-3], HIV-1[GUN-4], and HIV1[GUN-5], were from hemophilia A patients. Another HIV-1 strain, HIV-1[GUN-6], was isolated from a Canadian male homosexual with AIDS. The restriction endonuclease cleavage maps of the proviral genomes of these six HIV-1 strains revealed that they were apparently different from each other. The phylogenetic trees constructed using restriction maps and nucleotide sequences were quite similar, indicating that phylogenetic analyses of Japanese HIV-1 isolates can be done using restriction maps of the proviruses. Phylogenetic analyses showed that they were more closely related to HIV-1s which had been reported to be isolated from homosexual patients in the United States than those isolated from African patients. In particular, GUN-1 and GUN-2 isolates were on the branch of a San Francisco isolate, ARV2, while GUN-5 and GUN-6 isolates were on the branch of HTLV-III_B-related isolates.Offprint requests to: H. Hoshino     

Early divergence of Fc epsilon receptor I signals for receptor up-regulation and internalization from degranulation, cytokine production, and survival

Mast cells play a critical role in IgE-dependent immediate hypersensitivity. Monomeric IgE binding to its high affinity receptor (FcepsilonRI) results in a number of biological outcomes in mouse mast cells, including increased surface expression of FcepsilonRI and enhanced survival. IgE molecules display heterogeneity in inducing cytokine production; highly cytokinergic IgEs cause extensive FcepsilonRI aggregation, leading to potent enhancement of survival and other activation events, whereas poorly cytokinergic IgEs can do so less efficiently. In this study, we demonstrate that IgE-induced receptor up-regulation is not sensitive to monovalent hapten, which can prevent receptor aggregation induced by IgE, whereas other activation events such as receptor internalization, degranulation, IL-6 production, and survival are sensitive to monovalent hapten. IgE-induced receptor up-regulation is also unique in that no Src family kinases, Syk, or Btk are required for it. By contrast, highly cytokinergic IgE-induced receptor internalization is dependent on Lyn, but not other Src family kinases, Syk, or Btk, whereas degranulation, IL-6 production, and survival require Syk. Weak to moderate stimulation with IgE plus anti-IgE or IgE plus Ag enhances survival, while stronger signals are required for degranulation and IL-6 production. Collectively, signals emanated from IgE-bound FcepsilonRI for receptor up-regulation and internalization are shown to diverge at the receptor or receptor-proximal levels from those for other biological outcomes.