Prenatal phthalate exposure and 8-isoprostane among Mexican-American children with high prevalence of obesity.

Oxidative stress has been linked to many obesity-related conditions among children including cardiovascular disease, diabetes mellitus and hypertension. Exposure to environmental chemicals such as phthalates, ubiquitously found in humans, may also generate reactive oxygen species and subsequent oxidative stress. We examined longitudinal changes of 8-isoprostane urinary concentrations, a validated biomarker of oxidative stress, and associations with maternal prenatal urinary concentrations of phthalate metabolites for 258 children at 5, 9 and 14 years of age participating in a birth cohort residing in an agricultural area in California. Phthalates are endocrine disruptors, and in utero exposure has been also linked to altered lipid metabolism, as well as adverse birth and neurodevelopmental outcomes. We found that median creatinine-corrected 8-isoprostane concentrations remained constant across all age groups and did not differ by sex. Total cholesterol, systolic and diastolic blood pressure were positively associated with 8-isoprostane in 14-year-old children. No associations were observed between 8-isoprostane and body mass index (BMI), BMI Z-score or waist circumference at any age. Concentrations of three metabolites of high molecular weight phthalates measured at 13 weeks of gestation (monobenzyl, monocarboxyoctyl and monocarboxynonyl phthalates) were negatively associated with 8-isoprostane concentrations among 9-year olds. However, at 14 years of age, isoprostane concentrations were positively associated with two other metabolites (mono(2-ethylhexyl) and mono(2-ethyl-5-carboxypentyl) phthalates) measured in early pregnancy. Longitudinal data on 8-isoprostane in this pediatric population with a high prevalence of obesity provides new insight on certain potential cardiometabolic risks of prenatal exposure to phthalates.

Use of Lactobacillus acidophilus and Lactobacillus casei for a potential probiotic legume-based fermented product using pigeon pea (Cajanus cajan).

The aim of the present study was to evaluate the use of pigeon pea (Cajanus cajan) as an appropriate substrate in the production of a legume-based fermented product with Lactobacillus acidophilus ATCC 314 or Lactobacillus casei ATCC 393 and then to ascertain the effects of the addition of ingredients such as powdered milk and banana or strawberry sauce. The products were analyzed for viable cell counts, pH, and sensory attributes during product manufacture and throughout the refrigerated storage period at 3, 7, 14, 21, and 28 days. Nine types of products were produced. At the end of the storage period, the viability of L. acidophilus was above 7 log CFU/g in the presence of milk and 20% sucrose fruit sauce. For products with L. casei, the lack of ingredients such as milk caused no significant loss in viability; however, a high concentration of sucrose in the fruit sauce was an important factor in maintaining a high L. casei population. L. casei had high viability and good sensory attributes. Both strains could be considered suitable for a pigeon pea-based fermented potential probiotic product and a low-cost protein source.

Effect of oat's soluble fibre (ß-glucan) as a fat replacer on physical, chemical, microbiological and sensory properties of low-fat beef patties.

This study evaluated the effect of adding oat fibre source of ß-glucan (13.45%) on physical, chemical, microbiological and sensory traits of low-fat (<10%) beef patties as compared to 20% fat control patties. Significant (p<0.05) improvements in cooking yield (74.19%), and retentions of fat (79.74%) and moisture (48.41%) of low-fat patties were attributed to the water binding ability of ß-glucan. Because of larger water retentions moisture contents of raw and cooked low-fat patties were higher (p<0.05) than those of the control patties. Cholesterol content was similar across formulations. Low-fat and control beef patties remained stable in microbiological quality during 60days frozen storage. Low-fat patties were found to be of lower degree of likeness in the taste but juicer than control (p<0.05). Besides appearance, tenderness and colour were not affected by the addition of oat's soluble fibre. Oat fibre can be used successfully as a fat substitute in low-fat beef patties.

The H subunit (Vma13p) of the yeast V-ATPase inhibits the ATPase activity of cytosolic V1 complexes.

V-ATPases are composed of a peripheral complex containing the ATP-binding sites, the V(1) sector, attached to a membrane complex containing the proton pore, the V(o) sector. In vivo, free, inactive V(1) and V(o) sectors exist in dynamic equilibrium with fully assembled, active V(1) V(o) complexes, and this equilibrium can be perturbed by changes in carbon source. Free V(1) complexes were isolated from the cytosol of wild-type yeast cells and mutant strains lacking V(o) subunit c (Vma3p) or V(1) subunit H (Vma13p). V(1) complexes from wild-type or vma3Delta mutant cells were very similar, and contained all previously identified yeast V(1) subunits except subunit C (Vma5p). These V(1) complexes hydrolyzed CaATP but not MgATP, and CaATP hydrolysis rapidly decelerated with time. V(1) complexes from vma13Delta cells contained all V(1) subunits except C and H, and had markedly different catalytic properties. The initial rate of CaATP hydrolysis was maintained for much longer. The complexes also hydrolyzed MgATP, but showed a rapid deceleration in hydrolysis. These results indicate that the H subunit plays an important role in silencing unproductive ATP hydrolysis by cytosolic V(1) complexes, but suggest that other mechanisms, such as product inhibition, may also play a role in silencing in vivo.

Assembly and regulation of the yeast vacuolar H(+)-ATPase.

The yeast vacuolar H(+)-ATPase (V-ATPase) consists of a complex of peripheral subunits containing the ATP binding sites, termed the V(1) sector, attached to a complex of membrane subunits containing the proton pore, termed the V(o) sector. Interaction between the V(1) and V(o) sectors is essential for ATP-driven proton transport, and this interaction is manipulated in vivo as a means of regulating V-ATPase activity. When yeast (Saccharomyces cerevisiae) cells are deprived of glucose for as little as 5 min, up to 75% of the assembled V-ATPase complexes are disassembled into cytoplasmic V(1) sectors and membrane-bound V(o) sectors. Remarkably, this disassembly is completely reversible. Restoration of glucose to the growth medium results in quantitative reassembly of the disassembled complexes in as little as 5 min, even in the absence of any new protein synthesis. Cells also appear to regulate the extent of V(1)V(o) assembly on a long-term basis. Yeast cells grown for extended periods in a poor carbon source contain a high proportion of free V(1) and V(o) sectors, and these sectors remain poised for reassembly when growth conditions improve. Parallel experiments on the Manduca sexta V-ATPase suggest that reversible disassembly may be a general regulatory mechanism for V-ATPases. These results imply that V-ATPases are surprisingly dynamic structures, and their unique 'regulated instability' raises a number of interesting physiological and structural questions. How are extracellular conditions such as carbon source communicated to V-ATPase complexes present on intracellular membranes? How are such major structural changes in the V-ATPase generated and how are V(1) sectors 'silenced' in vivo to prevent unproductive hydrolysis of cytoplasmic ATP by the dissociated enzyme? We are addressing these questions using a combination of genetic and biochemical approaches.

Reversible association between the V1 and V0 domains of yeast vacuolar H+-ATPase is an unconventional glucose-induced effect.

The yeast vacuolar H+-ATPase (V-ATPase) is a multisubunit complex responsible for organelle acidification. The enzyme is structurally organized into two major domains: a peripheral domain (V1), containing the ATP binding sites, and an integral membrane domain (V0), forming the proton pore. Dissociation of the V1 and V0 domains inhibits ATP-driven proton pumping, and extracellular glucose concentrations regulate V-ATPase activity in vivo by regulating the extent of association between the V1 and V0 domains. To examine the mechanism of this response, we quantitated the extent of V-ATPase assembly in a variety of mutants with known effects on other glucose-responsive processes. Glucose effects on V-ATPase assembly did not involve the Ras-cyclic AMP pathway, Snf1p, protein kinase C, or the general stress response protein Rts1p. Accumulation of glucose 6-phosphate was insufficient to maintain or induce assembly of the V-ATPase, suggesting that further glucose metabolism is required. A transient decrease in ATP concentration with glucose deprivation occurs quickly enough to help trigger disassembly of the V-ATPase, but increases in cellular ATP concentrations with glucose readdition cannot account for reassembly. Disassembly was inhibited in two mutant enzymes lacking ATPase and proton pumping activities or in the presence of the specific V-ATPase inhibitor, concanamycin A. We propose that glucose effects on V-ATPase assembly occur by a novel mechanism that requires glucose metabolism beyond formation of glucose 6-phosphate and generates a signal that can be sensed efficiently only by a catalytically competent V-ATPase.

Characterization of a temperature-sensitive yeast vacuolar ATPase mutant with defects in actin distribution and bud morphology.

The 27-kDa E subunit, encoded by the VMA4 gene, is a peripheral membrane subunit of the yeast vacuolar H+-ATPase. We have randomly mutagenized the VMA4 gene in order to examine the structure and function of the 27-kDa subunit. Cells lacking a functional VMA4 gene are unable to grow at pH > 7 or in elevated concentrations of CaCl2. Plasmid-borne, mutagenized vma4 genes were screened for failure to complement these phenotypes. Mutants producing Vma4 proteins detectable by immunoblot were selected; one (vma4-1(ts)) is temperature conditional, exhibiting the Vma- phenotype only at elevated temperature (37 degreesC). Sequencing revealed that a single point mutation, D145G, was responsible for the phenotypes of the vma4-1(ts) allele. The unassembled 27-kDa subunit made in the vma4-1(ts) cells is rapidly degraded, particularly at 37 degreesC, but can be protected from degradation by prior assembly into the V-ATPase complex. In purified vacuolar vesicles from the mutant cells, the peripheral subunits are localized to the vacuolar membrane at decreased levels and a comparably decreased level of ATPase activity (14% of the activity in wild-type vesicles) is observed. When vma4-1(ts) mutant cells are shifted to pH 7.5 medium at 37 degrees C, the cells become enlarged and exhibit multiple large buds, elongated buds, and other abnormal morphologies, together with delocalization of actin and chitin, within 4 h. These phenotypes suggest connections between the vacuolar ATPase, bud morphology, and cytokinesis that had not been recognized previously.

After-the-fact strategies Mexican Americans use to prevent HIV and STDs.

Hispanics make up less than 10% of the U.S. population but account for 83,923 (17%) of all U.S AIDS cases and are disproportionately affected by sexually transmitted diseases (STDs). Nurses have a mandate to provide culturally competent care, but to do so, they must understand what their clients' needs are and how best to deliver nursing care. A key finding of this exploratory focus group study with newly immigrated Mexican Americans in North Carolina was that respondents were actively striving to prevent HIV or STDs. Their preventive practices for both HIV and STDs centered around a "gonorrhea model" of prevention and casual transmission. The emphasis was not on the biomedical model of transmission or prevention (condoms use). The findings also suggested that the level of counseling for prevention needs to be targeted differently for men than for women.

Determination of genotypes of hepatitis C virus in Venezuela by restriction fragment length polymorphism.

Hepatitis C virus genotypes in Venezuela were analyzed by restriction fragment length polymorphism in the 5' noncoding region. The absence of BstUI digestion was found to be a useful marker for genotype 2 specimens. From 122 serum samples, 66, 20, and 2.5% were classified as genotypes 1, 2, and 3, respectively; 0.8% were classified as genotype 4; and 10% appeared to be mixed infections.

Wild-type and mutant vacuolar membranes support pH-dependent reassembly of the yeast vacuolar H+-ATPase in vitro.

Treatment of the yeast vacuolar proton-translocating ATPase (H+-ATPase) with 300 mM KI in the presence of 5 mM MgATP results in a 90% inhibition of ATPase activity accompanied by removal of at least five of the peripheral subunits of the enzyme from the membrane. Functional reassembly of the enzyme, as indicated by reattachment of the peripheral subunits and a partial (30-70%) recovery of ATPase activity, could be achieved by dialysis of the stripped wild-type membranes to remove the KI and MgATP, but proved to be strongly pH-dependent, with optimal reassembly and recovery of activity occurring after dialysis at pH 5.5. Vacuolar membranes isolated from vma2Delta mutants, which lack one of the peripheral subunits of the enzyme, do not contain any of the peripheral subunits but are shown to contain assembled membrane (Vo) complexes. The vma2Delta mutant vacuoles are demonstrated to be competent for attachment of KI-stripped peripheral subunits and reactivation of ATPase activity. The results indicate that previously assembled Vo complexes are capable of inducing assembly of the peripheral subunits, both with each other and with the membrane subunits, and of activating the ATPase activity that resides in the peripheral subunits in a pH-dependent manner.