Antithrombins Wibble and Wobble (T85M/K): archetypal conformational diseases with in vivo latent-transition, thrombosis, and heparin activation

The inherent variability of conformational diseases is demonstrated by two families with different mutations of the same conserved aminoacid in antithrombin. Threonine 85 underlies the opening of the main beta-sheet of the molecule and its replacement, by the polar lysine, in antithrombin Wobble, resulted in a plasma deficiency of antithrombin with an uncharacteristically severe onset of thrombosis at 10 years of age, whereas the replacement of the same residue by a nonpolar methionine, antithrombin Wibble, gave near-normal levels of plasma antithrombin and more typical adult thromboembolic disease. Isolated antithrombin Wibble had a decreased thermal stability (Tm 56.2, normal 57.6 degreesC) but was fully stabilized by the heparin pentasaccharide (Tm 71.8, normal 71.0 degreesC), indicating that the prime abnormality is a laxity in the transition of the main sheet of the molecule from the 5- to 6-stranded form, as was confirmed by the ready conversion of antithrombin Wibble to the 6-stranded latent form on incubation. That this transition can occur in vivo was shown by the finding of nearly 10% of the proband's plasma antithrombin in the latent form and also, surprisingly, of small but definitive amounts of latent antithrombin in normal plasma. The latent transition will be predictably accelerated not only by gross mutations, as with antithrombin Wobble, to give severe episodic thrombosis, but also by milder mutations, as with antithrombin Wibble, to trigger thrombosis in the presence of other predisposing factors, including the conformational stress imposed by the raised body temperatures of fevers. Both antithrombin variants had an exceptional (25-fold) increase in heparin affinity and this, together with an increased inhibitory activity against factor Xa, provides evidence of the direct linkage of A-sheet opening to the conformational basis of heparin binding and activation.     

Treatment with progesterone and 17 beta-oestradiol to induce emergence of a newly-recruited dominant ovulatory follicle during oestrus synchronisation with long-term use of norgestomet in Brahman heifers

The aim of this study was to determine the effect on ovarian follicular growth and atresia, of acute treatment with either 100 mg of progesterone (n = 10), 200 mg of progesterone (n = 10), 10 mg of oestradiol + 100 mg of progesterone (n = 10), 10 mg of oestradiol (n = 10) or no treatment (n = 10), given on Day 10 of a 17-day treatment with a norgestomet implant in randomly cycling Bos indicus heifers. The fate of the dominant follicle on Day 10, emergence of the new cohort of follicles and the intervals from implant removal to ovulation were recorded by ultrasonography. Plasma concentrations of Luteinizing hormone (LH), progesterone and oestradiol were determined during the time when the norgestomet implant was in place. All treatments resulted in the emergence of a new cohort of follicles within 5 days of administration. The day of emergence of the ovulatory follicle tended to be delayed after treatment with 100 mg of progesterone (2.7 +/- 0.3 days after treatment), 200 mg of progesterone (3.7 +/- 0.5 days after treatment), 10 mg of oestradiol + 100 mg of progesterone (4.4 +/- 0.2 days after treatment) and 10 mg of oestradiol (4.6 +/- 0.4 days after treatment) compared to control heifers (1.4 +/- 1.4 days after time of treatment). The mean interval from implant removal to onset of oestrus was significantly shorter after treatment with 100 mg of progesterone (38.4 +/- 2.6 h) than after treatment with 200 mg of progesterone (61.5 +/- 3.9 h) but otherwise, the mean interval from implant removal to onset of oestrus did not differ. Oestrus synchrony, measured by the sample standard deviation of oestrus onset, was tighter in all treatment groups compared to untreated control heifers. The mean interval from implant removal to ovulation did not differ significantly between groups. The synchrony of ovulation, measured by the sample standard deviation of the interval from implant removal to ovulation, was significantly tighter after treatment with 100 mg of progesterone, 200 mg of progesterone and 10 mg of oestradiol compared to control heifers. Treatment with 10 mg of oestradiol resulted in ovulation in seven of 10 heifers before implant removal, three of which failed to ovulate after implant removal. Progesterone administered on Day 10 lowered plasma LH concentrations (P < 0.05), whereas treatment with oestradiol caused a surge of LH and ovulation. Progesterone administered with oestradiol prevented the LH surge. A combination treatment of oestradiol and progesterone given on Day 10 of a 17-day norgestomet treatment in a range of follicular states resulted in the consistent emergence of a new cohort of follicles which included the eventual ovulatory follicle.     

Omeprazole improves peak expiratory flow rate and quality of life in asthmatics with gastroesophageal reflux

OBJECTIVE: The aim of this study was to determine if omeprazole improves pulmonary function and quality of life in asthmatics with gastroesophageal reflux. METHODS: This was a double blind, randomized, placebo-controlled cross-over trial. After a 4-wk lead-in period, nine patients with documented asthma and gastroesophageal reflux, were prescribed either omeprazole 20 mg, daily or placebo for 8 wk and then crossed over to the alternate treatment. Outcome measurements included: forced expiratory volume at 1 s (FEV1), peak expiratory flow rate (PEFR), and responses on the Asthma Quality of Life Questionnaire, a validated disease specific measure of functional status. RESULTS: After omeprazole treatment, compared with placebo, patients had higher mean morning and evening PEFR, mean absolute difference (95% CI): morning: 37.8 L/min. (10.9-64.6), evening: 31.2 (3.2-59.2). Omeprazole treatment led to higher mean overall scores on the Asthma Quality of Life Questionnaire, and on the subdomains of activity limitation, symptoms, and emotions (p = 0.039, 0.049, 0.024, 0.040). A trend toward higher FEV1 (mean: 15.6% difference) with omeprazole failed to reach statistical significance (p > 0.2). CONCLUSIONS: After taking omeprazole for 8 wk, asthmatics with GER have better PEFR and quality of life than after placebo.     

Quantification of phosphorus metabolites from chemical shift imaging spectra with corrections for point spread effects and B1 inhomogeneity

Neospora caninum is an apicomplexan parasite which is morphologically and ultrastructurally very similar to Toxoplasma gondii. In order to identify molecules involved in host cell entry and subsequent modification of the parasitophorous vacuole, a polyclonal antiserum directed against N. caninum tachyzoites was raised in a rabbit. Subcellular fractionation of tachyzoites was performed using the non-ionic detergent Triton-X-114. Membrane fractions were analysed by immunoblotting using the polyclonal antiserum. One of the immunoreactive protein bands had a mol. wt of 33,000 and was subsequently named Nc-p33. Affinity-purified anti-Nc-p33 antibodies were used to characterise this polypeptide using SDS-PAGE, isoelectric focusing, Western blot analysis and immuno-EM. Nc-p33 was found in two isolates of N. caninum (NC-1 and Liverpool), but could not be detected in T. gondii tachyzoites. Immunogold EM revealed that Nc-p33 constituted a dense granule-associated protein, and Western blotting demonstrated that Nc-p33 was most likely identical to the recently described antigen NCDG1. Shortly after invasion, this dense granule protein was targeted to the parasitophorous vacuole membrane, and, at later timepoints after infection, was also found on the parasitophorous vacuolar network. This suggested that Nc-p33 could play a functional role in the modification of the parasitophorous vacuole and its membrane.     

Serum HIV-1 RNA levels and time to development of AIDS in the Multicenter Hemophilia Cohort Study

Normal women produce small amounts of active androgens. When androgen levels are elevated, such as for example in the polycystic ovary syndrome, this is followed by the development of male physical characteristics and muscle mass, structure and function as well as android adipose tissue distribution and function. Psychological features and stress reactions also seem similar to those of men. Such women have an increased risk of developing hypertension, non-insulin-dependent diabetes mellitus and cardiovascular disease. Recent data have shown that these physical, and psychological characteristics, as well as risk of ill health, are also found in the population of women selected at random. Women in the lowest quintiles of levels of sex-hormone-binding globulin--an indicator inversely related to active androgens--are at risk of developing hypertension, non-insulin-dependent diabetes mellitus and cardiovascular mortality. The mechanism probably includes muscular insulin resistance, following a relative androgen excess. It is thus apparent that androgens, even within the highest levels of the nonselected population of women, are powerful predictors of serious disease development. The population at risk might be as large as about 20% of middle-aged women. This is an area of female disease risk which requires more attention in screening and intervention procedures.     

Intracellular accumulation, subcellular distribution, and efflux of tilmicosin in chicken phagocytes

Tilmicosin is a semi-synthetic macrolide antibiotic, currently approved for veterinary use in cattle and swine respiratory disease, and is in development for use in poultry mycoplasma air sacculitis. In order to provide an understanding of clinical efficacy, the in vitro interaction of tilmicosin with three types of chicken phagocytes (MQ-NCSU macrophages, monocyte-macrophages, and heterophils) was evaluated. After incubation with radiolabeled tilmicosin, uptake was determined and expressed as the ratio of the cellular (Cc) to the extracellular (Ce) drug concentration (Cc:Ce). Tilmicosin was avidly accumulated by heterophils (Cc: Ce 138 at 4 h incubation vs 32 and 66, respectively, in MQ-NCSU and monocyte-macrophages) with 61 to 88% localized in the lysosomes. Uptake was dependent on cell viability, temperature, and pH, but was not influenced by metabolic inhibitors. However, phagocytosis of Pasteurella multocida and lipopolysaccharide exposure increased tilmicosin uptake by the chicken phagocytes. Upon removal of extracellular tilmicosin, 50% of the intracellular tilmicosin was effluxed within the first 30 min, but after 4 h of incubation in antibiotic-free medium, 30% remained cell-associated. Opsonized P. multocida significantly enhanced the release of tilmicosin from all three types of chicken phagocytes. Tilmicosin uptake was observed to increase lysosomal enzyme (acid phosphatase, lysozyme, avidin, and beta-glucuronidase) production. Finally, neutrophils were shown to transport and efflux bioactive tilmicosin in a test system measuring both neutrophil chemotaxis under agarose and a bioassay measuring inhibition of bacterial growth in the presence of antibiotic in agar. These in vitro observations of cellular pharmacology suggest a complex interaction between phagocytes and tilmicosin that contribute to clinical efficacy.