A high field NMR study of the products ensuing from konjak glucomannan C(6)-oxidation followed by enzymatic C(5)-epimerization

Konjak glucomannan (KGM) is a water-soluble linear copolymer of (1-->4) linked beta-D-mannopyranosyl and beta-D-glucopyranosyl units. It has been selectively C6-oxidized by a 2,2,6,6-tetramethylpiperidin-1-oxy mediated reaction to obtain the corresponding uronan. Oxidized KGM has been treated with three different C-5 epimerases, AlgE4, AlgE6, and AlgE1, to obtain uronans with a various content of alpha-L-gulopyranuronate residues, namely, KGME4, KGME6, and KGME1. By use of 1D selective and 2D NMR techniques, a full assignment of the high field (600 MHz) NMR spectra of the purified native KGM and of the oxidized and epimerized derivatives has been obtained. Since in the anomeric region of the (1)H NMR spectrum of native KGM, diads sensitivity is present, the glucose-glucose, glucose-mannose, mannose-mannose, and mannose-glucose distribution has been obtained. In the (13)C spectrum of oxidized KGM, due to the presence of triad sensitivity on the C-4 resonance of glucuronic and mannuronic units, a better sequential investigation has been possible. As a result the average length of mannuronic blocks, N(M) is obtained. When AlgE4, AlgE6, and AlgE1 enzymes are used for the epimerization of oxidized KGM, the reaction products differ significantly both in the proportion and in the distribution of the mannuronic and guluronic residues. In epimerized KGM derivatives, a careful deconvolution of (1)H spectra allows the measurement of the degree of epimerization. In the case of KGME1 and KGME6, the average blocks length, N(G), of the guluronic blocks introduced in the polysaccharidic chain with the epimerization has also been calculated. Due to the shortness of mannuronic blocks in the oxidized KGM before the epimerization, N(G) in the epimerized compounds is also very low.     

Jaw transformation with gain of symmetry after Dlx5/Dlx6 inactivation: Mirror of the past?

In modern vertebrates upper and lower jaws are morphologically different. Both develop from the mandibular arch, which is colonized mostly by Hox-free neural crest cells. Here we show that simultaneous inactivation of the murine homeobox genes Dlx5 and Dlx6 results in the transformation of the lower jaw into an upper jaw and in symmetry of the snout. This is the first homeotic-like transformation found in this Hox-free region after gene inactivation. A suggestive parallel comes from the paleontological record, which shows that in primitive vertebrates both jaws are essentially mirror images of each other. Our finding supports the notion that Dlx genes are homeotic genes associated with morphological novelty in the vertebrate lineage.     

Mga2p processing by hypoxia and unsaturated fatty acids in Saccharomyces cerevisiae: impact on LORE-dependent gene expression

In Saccharomyces cerevisiae, OLE1 encodes a Δ9 fatty acid desaturase, an enzyme that plays a critical role in maintaining the correct ratio of saturated to monounsaturated fatty acids in the cell membrane. Previous studies have demonstrated that (i) OLE1 expression is repressed by unsaturated fatty acids (UFAs) and induced by low oxygen tension, (ii) a component of this regulation is mediated through the same low oxygen response element (LORE) in the OLE1 promoter, and (iii) Mga2p is involved in LORE-dependent hypoxic induction of OLE1. We now report that LORE-CYC1 basal promoter-lacZ fusion reporter assays demonstrate that UFAs repress the reporter expression under hypoxic conditions in a dose-dependent manner via LORE. Electrophoretic mobility shift assays show that UFAs repress the hypoxia-induced complex formation with LORE. Studies with a construct encoding a truncated form of Mga2p support the hypothesis that both hypoxia and UFA signals affect the processing of Mga2p and the UFA repression of OLE1 hypoxic induction is mediated through Mga2p. Data from Western blot assays provide evidence that under normoxic conditions, Mga2p processing produces approximately equimolar levels of the membrane-bound and processed forms and is unaffected by UFAs. Hypoxic induction of OLE1, however, is associated with increased processing of the protein, resulting in an approximately fivefold increase in the soluble active form that is counteracted by exposure of the cells to unsaturated fatty acids. Data from this study suggest that the Mga2p-LORE interaction plays an important role in OLE1 expression under both normoxic and hypoxic conditions.     

HIV protease inhibitors are potent anti-angiogenic molecules and promote regression of Kaposi sarcoma

Treatment with HIV-1 protease inhibitors (PI) is associated with a reduced incidence or regression of Kaposi sarcoma (KS). Here we show that systemic administration of the PIs indinavir or saquinavir to nude mice blocks the development and induces regression of angioproliferative KS-like lesions promoted by primary human KS cells, basic fibroblast growth factor (bFGF), or bFGF and vascular endothelial growth factor (VEGF) combined. These PIs also block bFGF or VEGF-induced angiogenesis in the chorioallantoic membrane assay with a potency similar to paclitaxel (Taxol). These effects are mediated by the inhibition of endothelial- and KS-cell invasion and of matrix metalloproteinase-2 proteolytic activation by PIs at concentrations present in plasma of treated individuals. As PIs also inhibit the in vivo growth and invasion of an angiogenic tumor-cell line, these data indicate that PIs are potent anti-angiogenic and anti-tumor molecules that might be used in treating non-HIV KS and in other HIV-associated tumors.     

The alpha1b-adrenergic receptor subtype: molecular properties and physiological implications

The aim of this review is to summarize some of the main findings from our laboratory as well as from others concerning the biochemical, molecular, and functional properties of the alpha1b-adrenergic receptor. Experimental and computational mutagenesis of the alpha1b-adrenergic receptor have been instrumental in elucidating some of the molecular mechanisms underlying receptor activation and receptor coupling to Gq. The knockout mouse model lacking the alpha1b-adrenergic receptor has highlighted the potential implication of this receptor subtype in variety of functions including the regulation of blood pressure, glucose homeostasis, and the rewarding response to drugs of abuse.