Nuclear DNA content of Vitis vinifera cultivars and ploidy level analyses of somatic embryo-derived plants obtained from anther culture

Flow cytometry was employed to determine the ploidy level of Vitis vinifera L. somatic embryo-derived plants obtained from anther culture. Only one among the 41 analysed plants (2.4%) presented somaclonal variation (tetraploidy); the other plants were diploid. No significant differences (P≤0.05) were detected between diploid and parental field plants. No haploid or aneuploid plants were observed. The nuclear DNA content of nine V. vinifera cultivars was also estimated using flow cytometry. A non-significant variation was found among the cultivars, with DNA content ranging from 1.17 pg/2C (cv. ‘Tinta Barroca’ and ‘Viosinho’) to 1.26 pg/2C (cv. ‘Cabernet Sauvignon’). These results and previous studies on other Vitis species suggest that Vitis genome is stable with regard to nuclear DNA content.     

Oxidation of methionine residues in recombinant human interleukin-1 receptor antagonist: implications of conformational stability on protein oxidation kinetics

Oxidation of methionine residues is involved in several biochemical processes and in degradation of therapeutic proteins. The relationship between conformational stability and methionine oxidation in recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was investigated to document how thermodynamics of unfolding affect methionine oxidation in proteins. Conformational stability of rhIL-1ra was monitored by equilibrium urea denaturation, and thermodynamic parameters of unfolding (DeltaGH2O, m, and Cm) were estimated at different temperatures. Methionine oxidation induced by hydrogen peroxide at varying temperatures was monitored during "coincubation" of rhIL-1ra with peptides mimicking specific regions of the reactive methionine residues in the protein. The coincubation study allowed estimation of oxidation rates in protein and peptide at each temperature from which normalized oxidation rate constants and activation energies were calculated. The rate constants for buried Met-11 in the protein were lower than for methionine in the peptide with an associated increase in activation energy. The rate constants and activation energy of solvent exposed methionines in protein and peptide were similar. The results showed that conformational stability, monitored using the Cm value, has an effect on oxidation rates of buried methionines. The rate constant of buried Met-11 correlated well with the Cm value but not DeltaGH2O. No correlation was observed for the oxidation rates of solvent-exposed methionines with any thermodynamic parameters of unfolding. The findings presented have implications in protein engineering, in design of accelerated stability studies for protein formulation development, and in understanding disease conditions involving protein oxidation.     

Affinities of Shiga toxins 1 and 2 for univalent and oligovalent Pk-trisaccharide analogs measured by electrospray ionization mass spectrometry

The binding stoichiometry and affinities of the Shiga toxins, Stx1 and Stx2, for a series of uni- and oligovalent analogs of the Pk-trisaccharide were measured using the direct electrospray ionization mass spectrometry (ES-MS) assay. Importantly, it is shown that, for a given ligand, Stx1 and Stx2 exhibit similar affinities. The binding data suggest a high degree of similarity in the spatial arrangement and structural characteristics of the Pk binding sites in Stx1 and Stx2. The results confirm that both toxins recognize the alpha-D-Galp(1-->4)-beta-D-Galp(1-->4)-beta-D-Glcp carbohydrate motif of the cell surface glycolipid Gb3. This, taken together with the results of the chemical mapping study, suggests that the nature of the Pk binding interactions with Stx1 and Stx2 are similar. The affinities of Stx1-B(5) and Stx2 for the multivalent ligands reveals that site 2 of Stx2, which shares the same spatial arrangement as site 2 in Stx1, is the primary Pk binding site and that site 1 of Stx1 and of Stx2 can also participate in Pk binding.     

An Enzymatic Platform for the Synthesis of Isoprenoid Precursors

The isoprenoid family of compounds is estimated to contain ∼65,000 unique structures including medicines, fragrances, and biofuels. Due to their structural complexity, many isoprenoids can only be obtained by extraction from natural sources, an inherently risky and costly process. Consequently, the biotechnology industry is attempting to genetically engineer microorganisms that can produce isoprenoid-based drugs and fuels on a commercial scale. Isoprenoid backbones are constructed from two, five-carbon building blocks, isopentenyl 5-pyrophosphate and dimethylallyl 5-pyrophosphate, which are end-products of either the mevalonate or non-mevalonate pathways. By linking the HMG-CoA reductase pathway (which produces mevalonate) to the mevalonate pathway, these building block can be synthesized enzymatically from acetate, ATP, NAD(P)H and CoA. Here, the enzymes in these pathways are used to produce pathway intermediates and end-products in single-pot reactions and in remarkably high yield, ∼85%. A strategy for the regio-specific incorporation of isotopes into isoprenoid backbones is developed and used to synthesize a series of isotopomers of diphosphomevalonate, the immediate end-product of the mevalonate pathway. The enzymatic system is shown to be robust and capable of producing quantities of product in aqueous solutions that meet or exceed the highest levels achieved using genetically engineered organisms in high-density fermentation.     

RhoA guanine exchange factor expression profile in arteries: evidence for a Rho kinase-dependent negative feedback in angiotensin II-dependent hypertension

Sustained overactivation of RhoA is a common component for the pathogenesis of several cardiovascular disorders, including hypertension. Although activity of Rho proteins depends on Rho exchange factors (Rho-GEFs), the identity of Rho-GEFs expressed in vascular smooth muscle cells (VSMC) and participating in the control of Rho protein activity and Rho-dependent functions remains unknown. To address this question, we analyzed by quantitative RT-PCR the expression profile of 28 RhoA-GEFs in arteries of normotensive (saline-treated) and hypertensive (ANG II-treated) rats. Sixteen RhoA-GEFs were downregulated in mesenteric arteries of hypertensive rats, among which nine are also downregulated in cultured VSMC stimulated by ANG II (100 nM, 48 h), suggesting a direct effect of ANG II. Inhibition of type 1 ANG II receptors (losartan, 1 μM) or Rho kinase (fasudil, 10 μM) prevented ANG II-induced RhoA-GEF downregulation. Functionally, ANG II-induced downregulation of RhoA-GEFs is associated with decreased Rho kinase activation in response to endothelin-1, norepinephrine, and U-46619. This work thus identifies a group of RhoA-GEFs that controls RhoA and RhoA-dependent functions in VSMC, and a negative feedback of RhoA/Rho kinase activity on the expression of these RhoA-GEFs that may play an adaptative role to limit RhoA/Rho kinase activation.     

Inhibitory effects of Spirulina in zymosan-induced arthritis in mice

The anti-inflammatory effect of microalgae Spirulina was studied in zymosan-induced arthritis in mice. Four days after the intra-articular injection of zymosan (15 mg/ml), Spirulina (100 and 400 mg/kg perorally) was administered to animals for 8 days. The mice were than killed and beta-glucuronidase was measured in the synovial fluid. Each knee joint was totally removed for histopathological studies. Spirulina significantly reduced the levels of beta-glucuronidase that had been increased by zymosan. Histopathological and ultrastructural studies showed inhibition of the inflammatory reaction, whereas no destruction of cartilage, well-preserved chondrocytes, and normal rough endoplasmic reticulum and mitochondria were seen. The anti-arthritic effect exerted by Spirulina as shown in this model may be at least partly due to the previously reported antiinflammatory and antioxidative properties of its constituent, phycocyanin. To our knowledge, this is the first report on the anti-inflammatory effect of Spirulina in an experimental model of arthritis.     

Structural and physiological features of sterols necessary to satisfy bulk membrane and sparking requirements in yeast sterol auxotrophs

A variety of sterols and stanols have been analyzed for their ability to satisfy bulk membrane and high-specificity (sparking) functions in three yeast sterol auxotrophs. While many sterols and stanols satisfied bulk membrane requirements, only those possessing a C-5,6 unsaturation or capable of being desaturated at C-5 fulfilled the high-specificity sparking requirement. Unsaturation of the A-ring or beta-saturation of a C-5,6 double bond rendered both sterol and stanol unsuitable for either function. The C-28 methyl group of ergosterol, while not required for growth, allowed for greater ease of desaturation at C-5 in vivo. As a result some sterols and stanols lacking the C-28 methyl were incapable of satisfying the sparking requirement while identical compounds possessing the C-28 methyl were able to fulfill the sparking function(s). These data are extended to hypothesize a role for the C-28 methyl group of ergosterol in yeast.