Sequence analysis of the coding region of human methionine synthase: relevance to hyperhomocysteinaemia in neural-tube defects and vascular disease

Elevated homocysteine (Hcy) levels are observed in two apparently unrelated diseases: neural-tube defects (NTD) and premature vascular disease. Defective human methionine synthase (MS) could result in elevated Hcy levels. We sequenced the coding region of MS in 8 hyperhomocysteinaemic patients (4 NTD patients and 4 patients with pregnancies complicated by spiral arterial disease, SAD). We identified only one mutation resulting in an amino acid substitution: an A-->G transition at bp 2756, converting an aspartic acid (D919) into a glycine (G). We screened genomic DNA for the presence of this mutation in 56 NTD patients, 69 mothers of children with NTD, 108 SAD patients and 364 controls. There was no increased prevalence of the GG and AG genotypes in NTD patients, their mothers or SAD patients. The D919G mutation does not seem to be a risk factor for NTD or vascular disease. We then examined the mean Hcy levels for each MS genotype. There was no correlation between GG- or AG-genotype and Hcy levels. The D919G mutation is thus a fairly prevalent, and probably benign polymorphism. This study, though limited, provides no evidence for a major involvement of MS in the aetiology of homocysteine-related diseases such as NTD or vascular disease.      

Nitric oxide synthase inhibition increases venular leukocyte rolling and adhesion in septic rats

OBJECTIVE: Excess production of nitric oxide (NO) has been implicated in hypotension and blood flow abnormalities in sepsis, but NO is also an important inhibitor of leukocyte rolling and adhesion. Leukocyte adhesion is increased in sepsis despite elevated NO production. We hypothesized that inhibition of NO synthase (NOS) could increase leukocyte adhesion in sepsis. DESIGN: Prospective animal study. SETTING: Experimental animal laboratory. SUBJECTS: Twenty-five male rats, anesthetized with ketamine and acepromazine. INTERVENTIONS: Topical superfusion of the nonselective NOS inhibitor N(G)-monomethyl-L-arginine (NMA) on skeletal muscle postcapillary venules. MEASUREMENTS AND MAIN RESULTS: Rats made septic by cecal ligation and puncture were compared with controls that underwent sham ligation. Leukocyte rolling and adhesion were measured in cremasteric postcapillary venules of septic and control rats using in vivo videomicroscopy. The effects of NOS inhibition on leukocyte rolling and adhesion were also measured. After a stable baseline was reached, 1 microM of the nonselective NOS inhibitor NMA was suffused topically followed by physiologic buffer. The effects of L-arginine on leukocyte rolling and adhesion were also measured, both before and after suffusion of NMA. Leukocyte rolling and adhesion was increased in septic rats as compared with controls (control 5.5+/-0.9 rolling cells/min, 1.0+/-0.3 adherent cells/min; septic 13.7+/-2.0 rolling cells/min, 3.1+/-0.6 adherent cells/min; p < .001), and NOS inhibition further increased leukocyte rolling and adhesion in both septic and control rats (control 14.0+/-1.7 rolling cells/min, 2.8+/-0.5 adherent cells/min; septic 25+/-2.1 rolling cells/min, 5.4+/-0.5 adherent cells/min; both p < .001 vs. baseline). Prior suffusion of excess L-arginine prevented the increase in leukocyte adhesion with NMA in septic rats (2.6+/-0.4 adherent cells/min vs. 3.0+/-0.6 adherent cells/min; n = 3; p > .05). When administered after NMA, excess L-arginine partially reversed leukocyte adhesion in septic rats (5.4+/-0.7 adherent cells/min, with NMA vs. 4.3+/-0.7 adherent cells/min, after L-arginine; n = 5; p < .05). Venular shear did not differ between septic and control rats (600+/-109 (sec(-1)) vs. 620+/-37 (sec(-1)); p > .05). CONCLUSIONS: Although NOS inhibition may ameliorate hypotension in sepsis, such therapy may be deleterious by increasing leukocyte adhesion.     

Separate photosensitizers mediate degradation of the 32-kDa photosystem II reaction center protein in the visible and UV spectral regions.

A component of the photosystem II reaction center, the 32-kDa protein, is rapidly turned over in the light. The mechanism of its light-dependent metabolism is largely unknown. We quantified the rate of 32-kDa protein degradation over a broad spectral range (UV, visible, and far red). The quantum yield for degradation was highest in the UVB (280-320 nm) region. Spectral evidence demonstrates two distinctly different photosensitizers for 32-kDa protein degradation. The data implicate the bulk photosynthetic pigments (primarily chlorophyll) in the visible and far red regions, and plastoquinone (in one or more of its redox states) in the UV region. A significant portion of 32-kDa protein degradation in sunlight is attributed to UVB irradiance.     

Identification of a unique isoform of 1-aminocyclopropane-1-carboxylic acid synthase by monoclonal antibody

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14) is a key enzyme regulating ethylene biosynthesis in higher plants. A monoclonal antibody (mAb T20C) that immunoprecipitates the ACC synthase activity from tomato pericarp tissue extracts revealed that mAb T20C immunodecorates an ≈67-kDa polypeptide. On isoelectric focusing gels, ACC synthase activity in cell-free preparations was resolved into three distinct activity peaks with pI values 5.3, 7, and 9. mAb T20C specifically recognized the pI 7 form of the enzyme on electrophoretic transfer (Western) blots. When analyzed by sodium dodecyl sulfate gel electrophoresis under reducing conditions, the eluted pI 7 form was confirmed to migrate as a polypeptide of 67 kDa. The 67-kDa pI 7 isoform is a previously undescribed form of ACC synthase.     

A longitudinal study of male buprenorphine addicts attending an addiction clinic in India

Aim. There is a lack of longitudinal studies of buprenorphine dependence, an important opioid dependence in several countries. We investigated the course and outcome of buprenorphine dependence in an Indian clinic-attending cohort. Design. Retrospective longitudinal study. Setting. An addiction clinic in northern India. Participants. Ninety-four male patients with buprenorphine dependence, registered for treatment between 1987 and 1993. Follow-up analyses were conducted for the 52 patients (55% of the index cohort) who completed more than a year of follow-up. In 48% of these 52 patients data were obtained from their clinical records of follow-up, while 52% were contacted specifically to obtain the required data on follow up. Measurement. Baseline demographic and clinical variables; time spent in various phases of use or abstinence; outcome at the latest follow up; transition to other drugs during follow-up period. Findings. Over an average follow-up duration of 3 years, 56% of the time was spent in dependent use, 12% in non-dependent use and 32% in abstinence. By the end of follow-up, 6% of patients were dead (annual death rate 1.9%), 33% were unchanged and 61% were classified as 'improved'. The proportion of patients with 'improved' outcome increased over the years. Patients with poor outcome had shorter follow-up and hospital stay, and had used pentazocine and/or antihistaminic injections in the buprenorphine 'cocktail' more often than those with better outcome. Thirty-two patients shifted to other drugs over the years, notably heroin or polydrug use. These 'transition' patients had a family history of drug use more often, started their drug career earlier, had marital and legal complications more often, spent more time in dependent phase of drug use, underwent multiple hospital admissions but stayed for a shorter period and faced more deaths, when compared to those who did not shift. Conclusion. In clinic-attending male patients with buprenorphine dependence who were followed-up although dependent pattern of use of the drug continued for a long time in their career, there was a slow but progressive improvement. Transition to other drugs was associated with a worse course and outcome as compared to being stable on buprenorphine.     

Changes in neutrophil surface protein composition accompany phagocytosis

Phagocytosis is a critical host defense mechanism of neutrophils. In this study, membrane protein changes occurring during phagocytosis were studied in human neutrophils using surface radiolabeling before or after phagocytosis of various target particles. Cells were labeled at the cell surface using lactoperoxidase-catalyzed iodination or neuraminidase-galactose oxidase-NaB3H4, galactose oxidase-NaB3H4, or periodate-NaB3H4 techniques. Such studies are complicated by the fact that these techniques identify many surface proteins on the phagocyte, and labeling after phagocytosis occurs often results in radiolabeling proteins of the target particle, thus making changes in cell-surface proteins more difficult to detect. Immunoprecipitation with monoclonal antibody AHN-1, which reacts with a carbohydrate present on several human neutrophil surface proteins and inhibits phagocytosis, eliminated interference caused by radiolabeled proteins of the target particle and simplified analysis by restricting the study to a limited number of proteins. AHN-1 immunoprecipitated less radiolabeled protein from neutrophils labeled after phagocytosis of particles opsonized with IgG or complement than from cells labeled before phagocytosis. Isolation of phagocytic vesicles containing opsonized emulsified paraffin oil demonstrated that three proteins of mol wt 105,000, 140,000, and 170,000 recognized by AHN-1 were internalized in the phagocytic vesicle during phagocytosis.     

Procoagulant activity of lymphocyte-macrophage populations in rabbits: selective increases in marrow, blood, and spleen cells during Shwartzman reactions

The present experiments examine leukocyte procoagulant activity using mononuclear cell populations purified or enriched from rabbit bone marrow, blood, spleen, lymph node, thymus, and pulmonary alveoli. Cells from these six sites, obtained from control and endotoxemic animals and assayed without an intermediate culture step, were found to have procoagulant activity identified as tissue factor. Under control conditions, tissue factor activity was found to be at low levels in marrow and blood populations compared to median activities 3- and 11- fold higher in populations from spleen and lymph node, and 33- and 45- fold higher in thymus and alveolar populations. By contrast to respective controls, significantly increased amounts of tissue factor (35-, 15-, and 12-fold at median levels) were found in marrow, blood, and spleen populations from endotoxemic animals. The types of leukocytes in these latter three populations were morphologically and histochemically indistinguishable from respective controls, indicating that endotoxin induced increases of activity in cells with relatively low amounts under control conditions. Activity did not change significantly in lymph node, thymus, or alveolar populations after endotoxemia. These studies show that tissue factor is present in a range of leukocyte populations not previously reported to have procoagulant activity. In addition, the finding of widespread gains of tissue factor in the marrow-blood-spleen pool due to endotoxemia provides new evidence supporting the importance of leukocyte procoagulants in Shwartzman-like reactions.     

Oral delivery of Hyperimmune bovine serum antibodies against CS6-expressing enterotoxigenic Escherichia coli as a prophylactic against diarrhea

ABSTRACT

Background

. Oral administration of bovine antibodies active against enterotoxigenic Escherichia coli (ETEC) have demonstrated safety and efficacy against diarrhea in human challenge trials. The efficacy of bovine serum immunoglobulins (BSIgG) against recombinant colonization factor CS6 or whole cell ETEC strain B7A was assessed against challenge with the CS6-expressing B7A.