Effect of quebracho condensed tannin extract on fecal gas flux in steers.

Naturally occurring gaseous by-products of ruminant production-carbon dioxide (CO2 ), methane (CH4 ), and nitrous oxide (N2 O)-can negatively affect the environment. Along with enteric fermentation, manure on pasture is among the most significant contributors to non-CO2 emissions. Condensed tannins, a group of naturally occurring phenolic compounds, can alter the route of nutrient excretion and interact with microbes, suggesting they are a plausible feed additive for reducing excreta gas emissions. We evaluated how quebracho (Schinopsis balansae) tannin extract fed at 0, 15, 30, and 45 g kg-1 of dry matter (DM) within a roughage-based diet affected fecal gas emissions at multiple locations (College Station and Stephenville, TX) during two periods corresponding to winter and spring. During both periods, CO2 , CH4 , and N2 O fluxes were determined using the vented-static chamber methodology over 39 d, and cumulative emissions were calculated. A random coefficients model with animal nested within dietary treatment and period as the random factor was analyzed by location due to the presence of collinearity with soil parameters within periods. Daily CO2 flux was influenced by soil moisture and temperature (r = .34; P < .01), whereas CH4 and N2 O were associated with soil moisture. Cumulative gas production confirmed a dietary effect for CO2 and gross CO2 equivalent at the College Station site (P ≤ .001), demonstrating a linear reduction as quebracho inclusion increased. Variance partitioning indicated that dietary treatment and seasonal period likely influenced animal digestive and metabolic parameters. Within specific environments, quebracho supplementation may assist in reducing fecal gas emissions.

Nutritive value, fermentation characteristics, and in situ disappearance kinetics of sorghum silage treated with inoculants.

Fibrolytic enzymes and microbial inoculants have the potential to improve the value of sorghum feedstuff and feedstock. An experiment was conducted to determine nutritive value, ensiling characteristics, and in situ disappearance kinetics of 4 sorghum (Sorghum bicolor L.) silage varieties: Dairy Master BMR (DBMR; brown midrib; Richardson Seed, Vega, TX), PS 747 (PS; photoperiod sensitive; Pogue Seed, Kenedy, TX), Silo 700D (S700D; conventional forage type; Richardson Seed), and MMR 381/73 (MMR; conventional forage type; Richardson Seed) pretreated with fibrolytic enzyme (xylanase plus cellulase, XC; 50:50 mixture of Cellulase Plus and Xylanase Plus; Dyadic, Juniper, FL) or microbial [Promote ASB (Lactobacillus buchneri and Lactobacillus plantarum); Cargill Animal Nutrition, Indianapolis, IN; PRO] inoculants. The greatest yield was for cultivar PS and the least for MMR. Neutral detergent fiber (NDF) concentration was least for XC-treated silage, and acid detergent fiber (ADF) concentration was least for XC- and PRO-treated silage. When silage was treated with XC, concentrations of NDF concentrations decreased, on average, 4.81% across all cultivars and ADF concentrations decreased, on average, 3.23% in all cultivars except MMR. Inoculant PRO reduced the NDF concentration of DBMR by 6.47%. The ADF concentrations of DBMR and PS treated with PRO were decreased by 3.25%. Treating sorghum silage with XC or PRO reduced the NDF and ADF fractions, which increased cell wall degradability. In vitro true digestibility was greatest for PRO-treated DBMR, whereas acid detergent lignin was least for PRO-treated DBMR. Aerobic stability was not improved by PRO; however, aerobic stability of XC-treated MMR was 63 h greater than that of the control. Acetate concentrations were greatest for XC-treated MMR, which explains the 63-h improvement in aerobic stability due to the inhibition of fungi. However, inoculant PRO did not improve yeast and mold counts or aerobic stability of sorghum silage compared with the control, which may be due to the lesser acetate concentrations, especially of PRO-treated S700D silage. Generally, in situ disappearance kinetics were improved with the application of XC and PRO, and XC had the greatest effect on silage with greater NDF and ADF concentrations.

Nutritive value, fermentation characteristics, and in situ disappearance kinetics of ensiled warm-season legumes and bahiagrass.

This study determined the nutritive value, ensiling characteristics, and in situ disappearance kinetics of bahiagrass (Paspalum notatum Flügge 'Tifton 9'), perennial peanut (Arachis glabrata Benth. 'Florigraze'), annual peanut [Arachis hypogaea (L.) 'FL MDR 98'], cowpea [Vigna unguiculata (L.) Walp. 'Iron clay'], and pigeonpea [Cajanus cajan (L.) Millsp. 'GA-2']. All forages were harvested at maturity stages that optimized dry matter (DM) yield and nutritive value. After harvest, forages were wilted to 45% DM, and 4 replicate bales of each legume and 8 bales of bahiagrass were wrapped in polyethylene and ensiled for 180 d. After each bale was opened, the forage was thoroughly mixed, and representative subsamples were taken for laboratory analysis and in situ incubation. Wilting and ensiling decreased the rumen-undegradable protein, water-soluble carbohydrate, crude protein (CP), and in vitro true digestibility (IVTD) of bahiagrass, perennial peanut, and cowpea, and increased their neutral detergent fiber (NDF) concentrations. Among haylages, CP concentration was greatest for annual peanut, followed by perennial peanut and cowpea, and least for bahiagrass. In contrast, NDF concentration was greater in bahiagrass than in legumes. Pigeonpea had the greatest NDF concentration among legumes and lowest IVTD of all haylages. All haylages were aerobically stable for at least 84 h, but pH was lower in perennial peanut and cowpea than in pigeonpea. Ammonia-N concentrations tended to be greater in legume haylages than in bahiagrass haylage. Butyrate concentration was greater in annual and perennial peanut than in bahiagrass. Total VFA concentration was greater in annual and perennial peanut and cowpea haylages than in bahiagrass haylage. Undegradable DM fractions were greater and extent of DM degradation was lower in bahiagrass and pigeonpea than in other haylages but lag time and degradation rates did not differ. Annual and perennial peanut and cowpea haylages were as aerobically stable and had greater CP, IVTD, and extent of degradation than did bahiagrass haylage; therefore, they are promising forages for dairy cow diets in the southeastern United States.

Intake, digestibility, and nitrogen retention by sheep supplemented with warm-season legume haylages or soybean meal.

The high cost of commercial supplements necessitates evaluation of alternatives for ruminant livestock fed poor quality warm-season grasses. This study determined how supplementing bahiagrass haylage (Paspalum notatum Flügge cv. Tifton 9) with soybean [Glycine max (L.) Merr.] meal or warm-season legume haylages affected the performance of lambs. Forty-two Dorper x Katadhin lambs (27.5 +/- 5 kg) were fed for ad libitum intake of bahiagrass haylage (67.8% NDF, 9.6% CP) alone (control) or supplemented with soybean meal (18.8% NDF, 51.4% CP) or haylages of annual peanut [Arachis hypogaea (L.) cv. Florida MDR98; 39.6% NDF, 18.7% CP], cowpea [Vigna unguiculata (L.) Walp. cv. Iron clay; 44.1% NDF, 16.0% CP], perennial peanut (Arachis glabrata Benth. cv. Florigraze; 40.0% NDF, 15.8% CP), or pigeonpea [Cajanus cajan (L.) Millsp. cv. GA-2; 65.0% NDF, 13.7% CP]. Haylages were harvested at the optimal maturity for maximizing yield and nutritive value, wilted to 45% DM, baled, wrapped in polyethylene plastic, and ensiled for 180 d. Legumes were fed at 50% of the dietary DM, and soybean meal was fed at 8% of the dietary DM to match the average CP concentration (12.8%) of legume haylage-supplemented diets. Lambs were fed each diet for a 14-d adaptation period and a 7-d data collection period. Each diet was fed to 7 lambs in period 1 and 4 lambs in period 2. Pigeonpea haylage supplementation decreased (P < 0.01) DM and OM intake and digestibility vs. controls. Other legume haylages increased (P < 0.05) DM and OM intake vs. controls; however, only soybean meal supplementation increased (P = 0.01) DM digestibility. All supplements decreased (P = 0.05) NDF digestibility. Except for pigeonpea haylage, all supplements increased (P < 0.01) N intake, digestibility, and retention, and the responses were greatest (P = 0.04) with soybean meal supplementation. Microbial N synthesis was reduced (P = 0.02) by pigeonpea haylage supplementation, but unaffected (P = 0.05) by other supplements. Efficiency of microbial protein synthesis was unaffected (P = 0.05) by diet. Ruminal ammonia concentration was increased (P = 0.01) by all supplements, but only soybean meal and annual peanut haylage increased (P < 0.03) plasma urea-N concentrations. Perennial peanut, annual peanut, and cowpea haylages are promising protein supplements for growing lambs.

Intake, digestibility, and nitrogen retention by sheep supplemented with warm-season legume hays or soybean meal.

The increasing cost of feed supplements necessitates evaluation of alternatives for ruminant livestock grazing poor quality warm-season grasses. This study determined how supplementing bahiagrass hay (Paspalum notatum Flügge cv. Pensacola) with soybean [Glycine max (L.) Merr.] meal or warm-season legume hays affected intake, digestibility, and N utilization by lambs. Dorper x Katadhin crossbred lambs (30.6 +/- 5.5 kg; n = 42) were fed bahiagrass hay (73.8% NDF, 8.1% CP) for ad libitum intake and supplemented with nothing (control), soybean meal, or hays of annual peanut [Arachis hypogaea (L.) cv. Florida MDR98; 46.2% NDF, 14.7% CP], cowpea [Vigna unguiculata (L.) Walp. cv. Iron clay; 62.2% NDF, 11.7% CP], perennial peanut (Arachis glabrata Benth. cv. Florigraze; 43.3% NDF, 15.2% CP), pigeonpea [Cajanus cajan (L.) Millsp. cv. GA-2; 78.6% NDF, 12.2% CP], or soybean (cv. Pioneer 97B52; 59.0% NDF, 13.5% CP). Legume hays were supplemented at 50% of total diet DM, and soybean meal was supplemented at a level (4.25% of diet DM) that matched the average dietary CP content (10.8%) of the legume hay-supplemented diets. The cowpea, pigeonpea, and soybean were harvested at respective maturities that maximized DM yield and nutritive value, and the peanuts were first cuttings. Diets were fed to 6 lambs per treatment for 2 consecutive 21-d periods. Supplementation with hays of annual and perennial peanut, cowpea, and soybean increased (P < 0.01) DMI vs. control, but apparent DM digestibility was only increased (P = 0.03) by supplementation with annual or perennial peanut hay. Compared with the control, N intake, digestibility, and retention were increased (P < 0.01) by supplementation with legume hay or soybean meal. Responses were greatest when annual or perennial peanut hays were fed. Ruminal ammonia concentration was increased (P < 0.01) by all legume hay supplements vs. the control. Microbial N synthesis and ruminally degraded OM were increased (P = 0.03) by perennial and annual peanut hay supplementation, but efficiency of microbial synthesis was not different (P = 0.52) among diets. Unlike other supplements, annual and perennial peanut hays increased DM and N intake and digestibility and improved microbial N synthesis; therefore, they were the best supplements for the bahiagrass hay under the conditions of this study.

Genetic and functional diversity of human immunodeficiency virus type 1 subtype B Nef primary isolates.

We have characterized the functional integrity of seven primary Nef isolates: five from a long-term nonprogressing human immunodeficiency virus (HIV)-infected individual and one each from two patients with AIDS. One of the seven Nefs was defective for CD4 downregulation, two others were defective for PAK-2 activation, and one Nef was defective for PAK-2 activation and major histocompatibility complex (MHC) class I downregulation. Five of the Nefs were tested and found to be functional for the enhancement of virus particle infectivity. The structural basis for each of the functional defects has been analyzed by constructing a consensus nef, followed by mutational analysis of the variant amino acid residues. Mutations A29V and F193I were deleterious to CD4 downregulation and PAK-2 activation, respectively, while S189R rendered Nef defective for both MHC class I downregulation and PAK-2 activation. A search of the literature identified HIVs from five patients with Nefs predominantly mutated at F193 and from one patient with Nefs predominantly mutated at A29. A29 is highly conserved in all HIV subtypes except for subtype E. F193 is conserved in subtype B (and possibly in the closely related subtype D), but none of the other HIV group M subtypes. Our results suggest that functional distinctions may exist between HIV subtypes.

The effects of standard care counseling or telephone/in-person counseling on beliefs, knowledge, and behavior related to mammography screening.

PURPOSE/OBJECTIVES: To determine the most effective methods of increasing mammography adherence while also considering ease of intervention delivery in evolving healthcare systems. DESIGN: Experimental. SETTING: Women from a health maintenance organization and a large general medicine practice. SAMPLE: Women 50-85 years of age who had not had breast cancer and did not have a mammogram within the last 15 months. METHODS: Once consent and baseline information were obtained, women were randomized to receive in-person, telephone, or no mammography counseling. MAIN RESEARCH VARIABLES: Mammography adherence, perception of susceptibility to breast cancer, and benefits, barriers to, and knowledge of mammography. FINDINGS: Compared to standard care, telephone counseling was more than twice as effective at increasing mammography adherence, whereas in-person counseling resulted in almost three times the mammography adherence postintervention. Both telephone and in-person counseling are successful in changing perceived susceptibility, knowledge, barriers, and benefits. CONCLUSION: Both telephone and in-person counseling interventions were successful in changing beliefs, which, in turn, increased mammography adherence. IMPLICATIONS FOR NURSING PRACTICE: Interventions based on altering beliefs are effective for increasing mammography adherence.

Lentivirus Nef specifically activates Pak2.

Nef proteins from human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) have been found to associate with an active cellular serine/threonine kinase designated Nef-associated kinase (Nak). The exact identity of Nak remains controversial, with two recent studies indicating that Nak may be either Pak1 or Pak2. In this study, we investigated the hypothesis that such discrepancies arise from the use of different Nef alleles or different cell types by individual investigators. We first confirm that Pak2 but not Pak1 is cleaved by caspase 3 in vitro and then demonstrate that Nak is caspase 3 sensitive, regardless of Nef allele or cell type used. We tested nef alleles from three lentiviruses (HIV-1 SF2, HIV-1 NL4-3, and SIVmac239) and used multiple cell lines of myeloid, lymphoid, and nonhematopoietic origin to evaluate the identity of Nak. We demonstrate that ectopically expressed Pak2 can substitute for Nak, while ectopically expressed Pak1 cannot. We then show that Nef specifically mediates the robust activation of ectopically expressed Pak2, directly demonstrating that Nef regulates Pak2 activity and does not merely associate with activated Pak2. We report that most of the active Pak2 is found bound to Nef, although a fraction is not. In contrast, only a small amount of Nef is found associated with Pak2. We conclude that Nak is Pak2 and that Nef specifically mediates Pak2 activation in a low-abundance complex. These results will facilitate both the elucidation of the role of Nef in pathogenesis and the development of specific inhibitors of this highly conserved function of Nef.

Colorectal cancer screening beliefs. Focus groups with first-degree relatives.

OBJECTIVES: The purpose of this paper is to describe the perceived benefits and barriers to colorectal cancer screening reported by first-degree relatives of colorectal cancer patients. MATERIALS AND METHODS: In this study, the authors used focus groups to identify perceived benefits and barriers to colorectal cancer screening among parents and children of colorectal cancer patients. Four focus groups were conducted with relatives of colorectal cancer patients seen at two university medical centers in the Midwest. The groups ranged in size from five to eight members each and were stratified by gender. RESULTS: Four benefits of colorectal cancer screening were identified by participants: finding colorectal cancer early, decreasing the chances of dying from colorectal cancer, freedom from worry about colorectal cancer, and reassurance that one was cancer-free. Four main barriers were identified that applied to all four types of colorectal cancer screening or to colorectal cancer screening in general. These included inadequate public awareness of colorectal cancer, inconsistent recommendations from healthcare providers, concerns about the efficacy of screening tests, and embarrassment. Barriers unique to each screening test also were identified. CONCLUSIONS: Understanding individual beliefs about the benefits and barriers to colorectal cancer screening will allow clinicians and researchers to develop effective interventions to increase screening. Results from the focus groups have been used to develop an instrument to measure benefits and barriers to colorectal cancer screening, which now needs to be tested with more culturally and socioeconomically diverse groups.

Lentiviral and murine retroviral transduction of T cells for expression of human CD40 ligand.

Efficient transduction of primary human T cells is an important goal toward treating a number of genetic defects. Patient T cells could be harvested by leukapheresis, transduced, and returned to the donor. A wide range of secreted or cell surface therapeutic proteins may be delivered in this way. The ability to produce antibodies is the consequence of interactions between T cells and B cells and lack of expression of CD40 ligand (CD40L) on T cells causes X-linked hyper-IgM syndrome (XHIM). We are investigating delivery of a normal CD40 ligand to treat this disorder. We tested promoters driving the expression of either reporter genes such as enhanced green fluorescent protein (eGFP) or human CDC40L. Using murine retroviruses, the best able to drive gene expression in T cells was the cytomegalovirus (CMV) promoter enhancer element; however, transduction efficiency was low. To achieve efficient, high-level gene expression we tested lentiviral gene delivery vectors. At a low multiplicity of infection (MOI) (0.5-2) a large fraction of target cells was transduced by lentiviral vectors (40-93%), and the strength of gene expression was high, as determined by flow cytometric analysis. We monitored the expression of eGFP or human CD40L on T cell lines and untransformed primary human T cells from normal and CD40L-deficient patients. We achieved efficient gene expression without an extended exposure to virus, and without the need for selection. These results are encouraging for efficient lentivirus-mediated transduction of refractory human cells to achieve therapeutic gene delivery.

The impact of age and race on mammography practices.

Mortality from breast cancer has recently begun to decline, primarily because of increased use of mammography screening. Although initial mammography utilization rates for women over 50 in the general population are high, compliance with regular, repeat mammograms is quite low. Both initial and repeat mammography utilization rates are much lower for older women and minority women. The study purpose was to identify age and racial differences in mammography beliefs of women for whom cost had been eliminated. Effects of age and race on perceived benefits and barriers to mammography were examined. Differences in reasons for not getting a mammogram were explored. Data were collected via self-report surveys from 817 noncompliant women at baseline and 1 to 2 months after an intervention to increase mammography compliance. An interaction effect on total barriers with race and age was demonstrated. Controlling for education and income, younger Caucasians and older African Americans had the highest total barriers scores. Racial and age differences on individual barrier and benefit items also were found. Results can be used to develop interventions targeted to address different barrier patterns for women of different age and racial groups.

Diversity of expressed cytochrome P450 genes in the adult Mediterranean Fruit Fly, Ceratitis capitata.

The cytochrome P450s comprise a superfamily of mostly microsomal haemoproteins which play a dominant role in the metabolism of a variety of endogenous and foreign compounds. The use of a degenerate PCR primer targeted to the haem-binding decapeptide unique to the cytochrome P450 superfamily resulted in the identification of 14 novel cytochrome P450s in the Mediterranean Fruit Fly, Ceratitis capitata. Analysis of the relative frequency of individual isoforms within the pool of isolated sequences suggests that the CYP4 and CYP6 P450 families contain the most highly expressed isoforms in adult C. capitata. Phylogenetic analyses of the conceptual amino acid translations of PCR-amplified cDNAs provides evidence that one of isolated sequences may represent a new P450 family.

Induction by alkaloids and phenobarbital of Family 4 Cytochrome P450s in Drosophila: evidence for involvement in host plant utilization.

In vertebrates, cytochrome P450s of the CYP2 and CYP3 families play a dominant role in drug metabolism, while in insects members of the CYP6 and CYP28 families have been implicated in metabolism of insecticides and toxic natural plant compounds. A degenerate 3' RACE strategy resulted in the identification of fifteen novel P450s from an alkaloid-resistant species of Drosophila. The strong (17.4-fold) and highly specific induction of a single gene (CYP4D10) by the toxic isoquinoline alkaloids of a commonly utilized host-plant (saguaro cactus) provides the first indication that members of the CYP4 family in insects may play an important role in the maintenance of specific insect-host plant relationships. Strong barbiturate inducibility of CYP4D10 and two other D. mettleri P450 sequences of the CYP4 family was also observed, suggesting a pattern of xenobiotic responsiveness more similar to those of several vertebrate drug-metabolizing enzymes than to putative vertebrate CYP4 homologs.

Tailoring interventions for health behavior change in breast cancer screening.

PURPOSE: Health professionals continue to seek strategies to help individuals increase health-promoting behaviors and decrease those behaviors related to unhealthy lifestyles. Various health behavior theories currently guide interventions that focus on health behavior changes. Of these, a tailored approach uses individual data on beliefs and behavior to guide the content and delivery of interventions. The purpose of this paper is to explain the usefulness of theories in tailoring health promotion messages to increase health-protecting behaviors. OVERVIEW: A brief overview of current popular theories that focus on individual behavior is presented, followed by illustrations of a tailored intervention approach for breast cancer screening. Applications of the tailored intervention approach in ongoing studies to increase breast cancer screening are described. CLINICAL IMPLICATIONS: Clinical implications of the ongoing projects suggest practical and widespread utility of tailoring health-promoting messages to increase healthy lifestyles. The tailored approach may be used by a multidisciplinary team to focus on the individual's unique factors to impact behavior change rather than to deliver the same standard message to all individuals.

Molecular Determinants of Nef Function.

The human immunodeficiency virus (HIV), the causative agent of the acquired immune deficiency syndrome (AIDS), in addition to encoding for the gag, pol and env structural genes common to all retroviruses also encodes six accessory genes: tat, rev, nef, vpr, vpu and vif. These accessory genes are responsible for the regulation of HIV replication. Recent advances in our understanding of the function(s) of these genes have illustrated the complex interplay between HIV, the infected cell and the host. In addition, identification of cellular proteins interacting with accessory gene products have provided new tools to study cellular processes. The topic of this review, nef, has been shown in vitro to induce the cell surface downregulation of CD4, the receptor for HIV, to enhance the infectivity of HIV particles and to associate with at least one cellular serine/threonine kinase. In vivo, Nef is essential for the efficient virus replication responsible for disease progression. In this review, several prominent aspects of Nef function are discussed including its effect on CD4 trafficking, on signaling pathways and on virus infectivity enhancement. Copyright 1997 S. Karger AG, Basel

The amphotropic murine leukemia virus receptor gene encodes a 71-kilodalton protein that is induced by phosphate depletion.

The amphotropic murine leukemia virus (MuLV) can infect cells from a number of mammals, including humans, via its specific receptor. Basic knowledge of amphotropic MuLV receptor expression is likely to be useful in the development and improvement of gene therapy protocols based on amphotropic-pseudotyped vectors. To investigate the expression of the human receptor for the amphotropic MuLV (GLVR-2, newly termed Pit2), we determined its mRNA levels in several cell lines and found them to vary significantly. Induction of increased levels of mRNA after removal of phosphate from the media was observed in two osteosarcoma cell lines. The increase in GLVR-2 mRNA resulted in a concomitant rise in the levels of a 71-kDa protein specifically recognized by affinity-purified antibodies against GLVR-2. Using these antibodies, we were able to confirm the intracellular topology of the large hydrophilic domain between the proposed sixth and seventh transmembrane domains of the GLVR-2 protein. This assignment is in agreement with the fourth extracellular loop being outside the cell, consistent with the proposal that the fourth extracellular loop of GLVR-2 contains the envelope binding site.

Development and validation of a Structured Telephone Interview for Dementia Assessment (STIDA): the NIMH Genetics Initiative.

As part of the NIMH Genetics Initiative Alzheimer's Disease (AD) Study Group, a brief structured telephone interview to distinguish individuals with normal cognitive functioning from those with changes in cognition and daily functioning suggestive of early AD was developed. The Structured Telephone Interview for Dementia Assessment (STIDA), yields a dementia score between 0 and 81 (higher scores indicating greater impairment). Subscales corresponding to the subscales of the Clinical Dementia Rating Scale (CDR) can be derived. The STIDA performed well as a screening instrument for mildly demented individuals. When a score of 10 or more (based on informant interview and subject testing) was used to identify mildly impaired individuals, the STIDA had a sensitivity of .93 and a specificity of .92 for a clinician-derived CDR of 0.5 or more. The STIDA was also capable of accurately assessing the level of dementia. STIDA-derived CDR ratings agreed with clinician-derived CDR scores in 23 of 28 cases, corresponding to an unweighted kappa of.71 and a weighted kappa of.81. A much-abbreviated short STIDA that could be administered directly to the subject was able to detect possible impairment with a sensitivity of .93 and a specificity of.77. These results suggest that the short STIDA provides a sensitive and fairly specific telephone screen for dementia, and that the full STIDA, consisting of an interview with a knowledgeable informant and subject testing, approximates the success of a face-to-face clinical interview, and provides reliable and valid screening and staging of dementia over the telephone.

Structural and functional correlates between HIV-1 and SIV Nef isolates.

The nef genes of HIV-1 and SIV encode 27-34 kDa myristoylated proteins which have been shown to induce cell surface CD4 downregulation and bind to a cellular protein kinase. To identify regions of Nef important for function, structure-function correlates of HIVSF2 nef (Nef) and SIVmm239open nef (SNef) were sought by constructing Nef/SNef hybrids. Metabolic labeling with 35[S]methionine/cysteine demonstrated similar amounts of 35[S] incorporation into all but one hybrid, SNeftll, in which the C-terminus of SNef was replaced by that of Nef. The weak protein expression of SNeftll was attributable to its short half-life of approximately 45 min. Nef, SNef, and SHSNef, a hybrid containing the internal sequences of Nef and the N- and C-terminal sequences from SNef, downregulated CD4 in human CEM cells. Only Nef and SHSNef downregulated CD4 in mouse AKR1-G1 cells. Nef, SNef, and SHSNef also effectively bound phosphoproteins of MW = 62,000 and 78,000 in CEM cells. Two additional hybrids, in which the Nef sequences of SHSNef were replaced with additional SNef sequences, were essentially ineffective in both assays. Thus, in two different assays of Nef function, swapping the SIV and HIV internal nef sequences were shown to be greatly deleterious to Nef function while SHSNef remained functional.

Biochemical properties and cellular localization of the Drosophila DG1 cGMP-dependent protein kinase.

The protein encoded by the Drosophila cGMP-dependent protein kinase gene, DG1, was expressed in Sf9 cells. cGMP (10 microM) stimulated histone H2B phosphorylation by the DG1 protein kinase 20-fold. Maximal activity was observed at 40-50 mM Mg2+. The concentrations of cGMP, cAMP, cIMP, 8-bromo-cGMP, and 8-bromo-cAMP that gave 50% activation were 0.19 +/- 0.06, 11.7 +/- 2.8, 5.3 +/- 1.5, 0.04 +/- 0. 01, and 0.62 +/- 0.06 microM, respectively. cGMP activation was cooperative with a Hill coefficient (nH) of 1.28 +/- 0.10, whereas activation by cAMP was not cooperative. DG1 kinase expressed in Sf9 cells was found to be a dimer with an amino-terminal dimerization domain. It also autophosphorylated in a reaction stimulated by cGMP and cAMP. Immunoadsorbed DG1 protein from fly extracts was also capable of autophosphorylation, and this assay was used to quantitate the DG1 kinase in extracts from heads and bodies of adults and whole embryos. Activity was highest in heads of either sex and male bodies, intermediate in female bodies, and lowest in embryos. These results were in accord with DG1 mRNA abundance. Tissue distribution of the DG1 kinase was investigated by immunohistochemistry. In embryos, specific immunoreactivity was observed in large cells scattered along the anterior-posterior axis at stage 13. Prominent staining of adult heads was restricted to the proximal level of the lamina cortex.

Carbohydrate fluxes into alginate biosynthesis in Azotobacter vinelandii NCIB 8789: NMR investigations of the triose pools.

The metabolite flux of carbohydrates through primary catabolism and into hexose resynthesis has been investigated for alginic acid biosynthesis in Azotobacter vinelandii. To do these studies, we fed the microorganism a variety of 13C-labeled glucose precursors, including [U-13C6]glucose. The incorporations of the precursors were determined by 1D 13C-NMR, 2D 13C-DQF-COSY, and inverse triple-quantum correlation experiments. The results clearly show that the entire catabolism of hexose is through the Entner-Doudoroff (E-D) pathway and that the triose pools are in equilibrium. Reentry into gluconeogenesis prior to alginate synthesis occurs totally from the glyceraldehyde 3-phosphate generated by the E-D pathway. The obligatory intermediacy of triose intermediates in alginate biosynthesis was proved. The experiments and results presented in this paper constitute a new method for distinguishing the E-D pathway from glycolysis in bacteria.