Pigs expressing salivary phytase produce low-phosphorus manure.

To address the problem of manure-based environmental pollution in the pork industry, we have developed the phytase transgenic pig. The saliva of these pigs contains the enzyme phytase, which allows the pigs to digest the phosphorus in phytate, the most abundant source of phosphorus in the pig diet. Without this enzyme, phytate phosphorus passes undigested into manure to become the single most important manure pollutant of pork production. We show here that salivary phytase provides essentially complete digestion of dietary phytate phosphorus, relieves the requirement for inorganic phosphate supplements, and reduces fecal phosphorus output by up to 75%. These pigs offer a unique biological approach to the management of phosphorus nutrition and environmental pollution in the pork industry.

Purification, crystallization and preliminary X-ray analysis of the Escherichia coli glucose-1-phosphatase.

Encoded by the agp gene, Escherichia coli glucose-1-phosphatase hydrolyzes glucose-1-phosphate in the periplasmic space of the bacterium. It is a potential drug-design target because inositol phosphatases have been identified as important virulence determinants in several human and animal pathogens. The enzyme was isolated and purified to homogeneity from a strain of E. coli CU1867 (an appA-deficient mutant). Crystals were obtained overnight by the equilibrium vapour-diffusion method from a solution containing 10 mg ml(-1) enzyme, 1.2 M ammonium sulfate and 25% polyethylene glycol monomethyl ether 5000 in 0.1 M MES at pH 6.5. The crystals belong to space group R3, with unit-cell parameters a = b = 156.0, c = 92.2 A. The diffraction limit was 2.6 A at a rotating-anode X-ray source; a 2.7 A resolution data set has been collected using light mineral oil as a cryoprotectant. The data set was 95.2% complete, with an R(sym) of 0.058. There were two monomers of glucose-1-phosphatase in the asymmetric unit, which correspond to a V(M) of 2.36 A Da(-1) and 47.5% solvent content. Self-rotation analysis unambiguously shows a twofold non-crystallographic symmetry.

Two omeprazole-based Helicobacter pylori eradication regimens for the treatment of duodenal ulcer disease in general practice.

BACKGROUND: Helicobacter pylori is the main acquired factor in the pathogenesis of duodenal ulcer disease. METHODS: This multicentre study conducted in 32 general practice centres in the UK and Ireland was a double-blind, placebo-controlled, randomized, parallel-group comparison of triple therapy (n = 98: omeprazole 40 mg once daily and amoxycillin 1 g b.d. for 2 weeks, and metronidazole 400 mg t.d.s. for the first week) and dual therapy (n = 85: omeprazole 40 mg once daily and amoxycillin 1 g b.d. for 2 weeks, with placebo during the first week) for the eradication of H. pylori in patients with symptomatic duodenal ulcer disease. Patients who were successfully treated entered a follow-up phase for 12 months to assess symptomatic relapse and use of health-care resources. RESULTS: Eradication of H. pylori based on a second 13C-urea breath test was successful in 95% (95% confidence interval (CI) = 90-100%) of patients receiving omeprazole triple therapy and 53% (95% CI = 41-65%) of those receiving omeprazole dual therapy (P < 0.0001 between groups, all data available analysis). The all-patients-treated analysis gave eradication rates of 80 and 44% for omeprazole triple therapy and omeprazole dual therapy, respectively. Symptomatic relapse occurred in 16% (18/116) of the H. pylori-negative patients who entered the 12-month follow-up period, and there were significant reductions in the number of consultations, investigations and prescriptions relating to upper gastrointestinal symptoms compared with the 12 months prior to the eradication therapies (all P < 0.0001). The two treatment strategies were comparable in terms of the number of adverse events reported. CONCLUSIONS: Omeprazole triple therapy provides a highly effective treatment for the eradication of H. pylori infection in patients in general practice, with an adverse event profile similar to that seen with omeprazole dual therapy. The successful eradication of H. pylori with these omeprazole regimens results in a significant decrease in the use of health-care resources in the 12 months following treatment.

High levels of anti-human immunodeficiency virus type 1 (HIV-1) memory cytotoxic T-lymphocyte activity and low viral load are associated with lack of disease in HIV-1-infected long-term nonprogressors.

Lack of disease in long-term nonprogressors with human immunodeficiency virus type 1 (HIV-1) infection was strongly associated with very low copy numbers of HIV-1 DNA and RNA in peripheral blood mononuclear cells and plasma and the presence of high levels of anti-HIV-1 CD8+ memory cytotoxic T lymphocytes specific for Gag, Pol, and Env, compared with levels present in intermediate and advanced progressors. CD8+ memory cytotoxic T lymphocytes may have an important role in controlling HIV-1 replication and preventing disease in long-term nonprogressors.

Quantitation of human immunodeficiency virus type 1 DNA and RNA by a novel internally controlled PCR assay.

A novel internally controlled PCR (ICPCR) assay was developed to accurately quantitate human immunodeficiency virus type 1 (HIV-1) DNA and RNA in peripheral blood mononuclear cells and plasma. The ICPCR assay was sensitive and reproducible within a linear range of amplification of 10(0) to 10(3) copies for HIV-1 DNA and 10(1) to 10(4) copies for HIV-1 RNA. The assay detected HIV-1 RNA in plasma and peripheral blood mononuclear cells from all HIV-1 subjects regardless of disease stage. ICPCR was compared with a branched-DNA signal amplification assay for subjects beginning antiretroviral therapy. The reductions in plasma HIV-1 RNA in response to therapy were comparable with the two assays. The ICPCR assay should be useful in monitoring HIV-1 RNA levels both in natural history studies and in clinical trials of antiretroviral agents.

Enhanced expression of human immunodeficiency virus type 1 correlates with development of AIDS.

The progression to AIDS may be significantly related to the level of human immunodeficiency virus type 1 (HIV-1) replication. We have used quantitative cell culture and quantitative DNA and RNA PCR to measure viral load and expression in peripheral blood mononuclear cells obtained cross-sectionally and longitudinally from HIV-1-seropositive homosexual men enrolled in the Multicenter AIDS Cohort Study. Our results indicate that the number of circulating CD4+ T-lymphocytes producing HIV-1 increased as the total number of CD4+ T-cells declined. However, there was no correlation between the number of HIV-1-producing CD4+ cells and the duration of infection. Furthermore, the level of HIV-1 gag RNA increased as the disease progressed and CD4+ cell numbers declined. Subjects who remained asymptomatic with stable CD4+ cell counts, however, maintained a very low level of HIV-1 RNA expression during the entire period of follow-up (38-71 months). In contrast to viral RNA expression, the level of proviral DNA did not change significantly as the disease progressed. However, the level of proviral DNA was significantly higher in AIDS patients than in men who remained asymptomatic. Such increased levels of HIV-1 DNA were detected 34-68 months before the development of AIDS. These results support the role of HIV-1 RNA expression in the development of AIDS.

Low prevalence of HIV in high-risk seronegative homosexual men evidenced by virus culture and polymerase chain reaction.

OBJECTIVE: To assess the presence of covert HIV-1 infection. SETTING: High-risk seronegative homosexual men from the Pittsburgh portion of the Multicenter AIDS Cohort Study were examined for the presence of HIV-1 infection. PATIENTS, PARTICIPANTS: Ten men (group 1) were examined prospectively for the presence of HIV-1 in their freshly-obtained peripheral blood mononuclear cells (PBMC). Furthermore, cryopreserved PBMC from 26 men (group 2) at their first visit (1984-1985) were examined retrospectively for the presence of HIV-1. MAIN OUTCOME MEASURES: PBMC samples from groups 1 and 2 were examined for HIV-1 by polymerase chain reaction (PCR) using gag, env and strong-stop (long terminal repeat) specific primers. In addition, fresh PBMC samples from group 1 were examined for HIV-1 by virus culture. RESULTS: None of the 10 PBMC samples from group 1 were positive for virus culture and PCR. Only one of the 26 men from group 2 was positive for gag and strong-stop DNA sequences. This PCR-positive, seronegative subject was found to be negative for HIV-1 by PCR at follow-up visits up to 48 months later. None of 15 seronegative, low-risk homosexual men and 12 seronegative heterosexual men were found to be PCR-positive for HIV-1. However, six HIV-1-seropositive men were positive by PCR for gag, env, and strong-stop HIV-1 DNA sequences. CONCLUSIONS: These results suggest a low prevalence of covert HIV-1 infection in high-risk seronegative homosexual men in our geographic area.