Purification and partial characterization of candidate antidiuretic hormone water channel proteins of M(r) 55,000 and 53,000 from toad urinary bladder

Antidiuretic hormone (ADH) increases toad bladder granular cell apical membrane osmotic water permeability (Pf) by insertion of cytoplasmic vesicles containing water channels into the apical membrane. Termination of ADH stimulation results in endocytosis of water channel-containing membrane. In previous work, we have purified water channel-containing vesicles and demonstrated that they contain 12 major protein bands when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of vectorial labeling studies of granular cells and purified vesicles, we have proposed previously that vesicle proteins of 55, 53, and 17 kDa are ADH water channel components. In this report, we have purified and analyzed these three proteins using a combination of SDS-PAGE, peptide mapping, amino acid composition, and amino-terminal analyses. The 55- and 53-kDa proteins are distinct protein species possessing a high degree of structural similarity. Both possess a large content of cysteine. The 17-kDa protein appears to be a proteolytic fragment of the 53-kDa protein. None of these three proteins is phosphorylated or contains large amounts of covalently linked carbohydrate. ADH-elicited Pf is inhibited by the organic mercurial reagent fluorescein mercuric acetate (FMA). Exposure of water channel-containing vesicles to FMA labels selectively four vesicle proteins of 92, 55, 53, and 29 kDa while reducing vesicle Pf by 82%. The combination of FMA and 2-mercaptoethanol or exposure to another mercurial reagent, n-ethylmaleimide, does not inhibit vesicle Pf. Together, these data provide additional evidence for the role of the 55- and 53-kDa proteins as components of the ADH water channel. These candidate ADH water channel proteins are distinct from a 28-kDa candidate water channel protein (CHIP 28) isolated recently from human erythrocyte membranes and kidney proximal tubule by Agre and co-workers (Preston, G. M., Carroll, T. P., Guggino, W. B., and Agre, P. (1992) Science 256, 385-387).     

Reduction of recurrent ischemia with abciximab during continuous ECG-ischemia monitoring in patients with unstable angina refractory to standard treatment (CAPTURE)

BACKGROUND: In the CAPTURE (c7E3 Fab Anti Platelet Therapy in Unstable REfractory angina) trial, 1265 patients with refractory unstable angina were treated with abciximab or placebo, in addition to standard treatment from 16 to 24 hours preceding coronary intervention through 1 hour after intervention. To investigate the incidence of recurrent ischemia and the ischemic burden, a subset of 332 patients (26%) underwent continuous vector-derived 12-lead ECG-ischemia monitoring. METHODS and RESULTS: Patients were monitored from start of treatment through 6 hours after coronary intervention. Ischemic episodes were detected in 31 (18%) of the 169 abciximab and in 37 (23%) of the 163 placebo patients (NS). Only 9 (5%) of abciximab versus 22 (14%) of placebo patients had >/=2 ST episodes (P<0.01). In patients with ischemia, abciximab significantly reduced total ischemic burden (P<0.02), which was calculated alternatively as the total duration of ST episodes per patient, the area under the curve of the ST vector magnitude during episodes, or the sum of the areas under the curves of 12 leads during episodes. Twenty-one patients (6%) suffered a myocardial infarction (MI) (18) or died (3) within 5 days of treatment. The presence of asymptomatic and symptomatic ST episodes during the monitoring period preceding coronary intervention was associated with an increased relative risk of these events of 3.2 (95% CI 1.4, 7.4) and 4.1 (95% CI 1.4, 12.2), respectively. CONCLUSIONS: Recurrent ischemia predicts MI or death within 5 days of follow-up. Treatment with abciximab is associated with a reduction of frequent ischemia and a reduction of total ischemic burden in patients with refractory unstable angina. As such, patients with ischemia derive particularly high benefit from abciximab.     

An audit of magnetic resonance imaging in the paediatric orthopaedic setting

AIMS: To determine when magnetic resonance imaging (MRI) is helpful in the management of a selected group of paediatric orthopaedic patients. METHODS: A retrospective analysis of 131 MRI scans was undertaken with allocation into seven categories based on clinical presentation. RESULTS: MRIs performed for spinal, congenital and intra-articular pathology, as well as for growth plate assessment correlated well with subsequent clinical and/or surgical findings. Three of ten MRIs (30%) incorrectly assessed whether a foreign body was present with a sensitivity of 0.60 and specificity of 0.80. Three of 20 MRIs (15%) could not accurately distinguish between oedema/effusion and frank infection. Here sensitivity was 1.00 and specificity was 0.73. MRIs performed for assessment of tumours were accurate with respect to margins and extent. Assessment of pathology with MRI was never the indication for MRI, thus it was not surprising that in four of 27 soft tissue tumours (sensitivity of 0.88 and specificity of 0.63) MRI did not correlate with subsequent histological findings. CONCLUSIONS: MRI plays an important role in the assessment of a wide range of musculo-skeletal pathology. MRI does not, and could not be expected to, replace the need for incisional biopsy for tumour diagnosis. It must be used with caution where tissues have been previously explored for foreign bodies. Interpretation of MRI in musculoskeletal infection must consider its timing in the evolution of the patient's infection.     

Different molecular events result in low protein levels of mannan-binding lectin in populations from southeast Africa and South America

Previous studies have shown that three point mutations in exon 1 and a particular promoter haplotype of the mannan-binding lectin (MBL) gene lead to a dramatic decrease in the serum concentration of MBL. In this study, MBL genotypes and serum concentrations were determined in unrelated individuals in a population from Mozambique (n = 154) and in two native Indian tribes from Argentina (i.e., the Chiriguanos (n = 43) and the Mapuches (n = 25)). In both populations, the MBL concentrations were low compared with those found in Eskimo, Asian, and European populations. In Africans, the low serum concentrations were due to a high allele frequency (0.24) of the codon 57 (C) variant, which resulted in a high frequency of individuals with MBL deficiency (0.06), and were also due to the effect of a relatively high frequency (0.13) of low-producing promoter haplotypes. The low concentrations in the South American populations were primarily due to an extremely high allele frequency of the codon 54 (B) variant in both the Chiriguanos (0.42) and the Mapuches (0.46), resulting in high frequencies of individuals with MBL deficiency (0.14 and 0.16, respectively). In the search for additional genetic variants, we found five new promoter mutations that might help to elucidate the evolution of the MBL gene. Taken together, the results of this study show that different molecular mechanisms are the basis for low MBL levels on the two continents.     

Feeding colostrum increases circulating insulin-like growth factor I in newborn pigs independent of endogenous growth hormone secretion

Our objective was to examine the influence of feeding and endogenous GH secretion on circulating IGF-I in colostrum-deprived newborn pigs fed colostrum (n = 4), formula (control, n = 4), or water (n = 4). In another four formula-fed pigs, GH was ablated (GRF-A) with two intravenous injections of a GH releasing-factor antagonist (N-Ac-Tyr1,D-Arg2)-GRF(1-29)-NH2. Blood was serially sampled in all pigs to measure plasma IGF-I and GH profiles. Feeding increased plasma IGF-I concentration two- to fourfold and decreased GH secretion. Despite a more than 80% decrease in the plasma GH in GRF-A pigs, the circulating IGF-I concentration was similar to that in control pigs. In colostrum-fed pigs, plasma IGF-I was higher than that in control pigs, despite equal nutrient intake and lower circulating GH. There were no differences in plasma IGF binding protein (IGFBP)-3 levels among the treatment groups. However, the relative abundance of plasma IGFBP-4 was lower, and that of IGFBP-1 higher, in unfed pigs than in any of the three fed groups. The plasma insulin concentration was not different among fed pigs, but it was lower in unfed pigs. Our results indicate that the circulating IGF-I concentration is more dependent on nutrient intake than on GH in newborn pigs, despite relatively high GH concentrations. However, because the nutrient content in the formula was designed to match that of colostrum, a factor other than nutrient intake and GH was responsible for the maximal increase in circulating IGF-I concentration observed in colostrum-fed pigs.     

Operative videothoracoscopy in the surgical treatment of penetrating firearms wounds of the chest

We prospectively analyzed our experience with operative videothoracoscopy (OVT) performed in a field military hospital in cases of penetrating firearms wounds of the thorax (PFAWT) sustained in Chechnya. From February to April 1996, we treated 206 wounded patients, of whom 37 (18.0%) had sustained chest injuries. PFAWT were present in 23 soldiers, accounting for 62.2% of all chest injuries. Twelve injuries were confined to the thorax, eight patients had associated injuries, and three soldiers had thoracoabdominal injuries. Nineteen patients had pleural drainage performed during medical evacuation. The thoracic injuries were right-sided (17), involved bullets or shell splinters (23); were through and through (16), represented solitary wounds (19), and were associated with internal organ injuries (21). Fifteen patients had indications for OVT when they were delivered from the battle-field 1.5 to 22 hours after injury. All patients manifested signs of hemorrhagic shock and hemodynamic instability. Indications for OVT were ongoing intrapleural bleeding (6), clotted hemothorax (6), or marked air leakage (3) preventing lung inflation with the OP-02 apparatus (field modification). OVT revealed 12 lung wounds, nine of which were multiple wounds, pleural bleeding in 6 patients, clotted hemothorax in 11 patients, and foreign bodies in 5 patients. Two patients underwent thoracotomy, one for suspicion of heart injury and the second because we could not adequately visualize and control bleeding revealed at OVT to be from the intercostal artery in the left costovertebral angle. Eight of 23 patients had no indication for operative videothoracoscopy and were managed with continued pleural aspiration and drug therapy. Wedge resection of the lung using an Endo-GIA-30 stapler was necessary in two patients because of parenchymal destruction and bleeding. Evacuation of clotted blood by fragmentation and aspiration was satisfactory in all cases. Satisfactory manual suturing of selected lung injuries was obtained largely with intracorporeal knot tying. The duration of the procedures ranged from 40 to 90 minutes. No morbidity nor mortality was encountered in patients undergoing OVT. Postoperative pain was minimized by using OVT placement of catheters in the chest wall soft tissue with local administration of 2% Trimecain. Patients were able to stand in 10 to 12 hours and to walk by the end of the first postoperative day. All patients who underwent OVT were evacuated without drains by the third or fourth postoperative day, all tolerating sitting and standing positions. We conclude that early OVT in the military field hospital for continued bleeding, clotted hemothorax, and continued major air leakage has several advantages in military patients with PFAWT: early definition and management of organ injury; identification and control of bleeding in most instances; earlier and more accurate assessment for thoracotomy; vigorous lavage and removal of projectiles such as bone fragments and evacuation of clotted hemothorax; early debridement with suture closure of the thoracic wall canal; and minimal postoperative pain with dramatically reduced suppurative sequelae and bronchopleural fistulae.     

Graded balloon dilation atrial septostomy in severe primary pulmonary hypertension. A therapeutic alternative for patients nonresponsive to vasodilator treatment

Endothelial cell cultures were established from several wild African mammalian species. Long-term cultures were established from three ruminants, stable antelope (Hippotragus niger), buffalo (Syncerus caffer), and eland (Tragelaphus oryx), and from an omnivore, the bushpig (Potamochoerus porcus). Cowdria ruminanntium was isolated from plasma of clinically affected animals in these four cell lines and in bovine endothelial cells used routinely for C. ruminantium propagation. Nineteen different strains of C. ruminantium from Africa and the Caribbean region were grown and maintained in these cell lines and their growth was comparable with growth in the bovine endothelial cells. The role of sable antelope, eland, and bushpigs in the epidemiology of heartwater is unknown. However, these results extend the number of cell lines that can be used to isolate and grow C. ruminantium.     

Acceptability of voluntary HIV testing by pregnant women in developing countries: an international survey. Ghent International Working Group on Mother-to-Child Transmission of HIV

OBJECTIVE: To evaluate acceptability of voluntary HIV counselling and testing (VCT) by pregnant women in the context of clinical trials assessing interventions to reduce mother-to-child transmission (MCT) of HIV in developing countries. METHODS: During September-October 1997, 13 studies located in West (Abidjan, Bobo Dioulasso), East (Nairobi, Mombasa, Dar Es Salaam, Blantyre, Lusaka, Harare) and South Africa (Soweto, Durban), and Thailand (Bangkok) were included in a cross-sectional mailing survey about the acceptability of VCT in antenatal clinics. Acceptance rate, return rate, overall acceptability of VCT (acceptance of both pre- and post-VCT sessions) were obtained using a standardized questionnaire. RESULTS: The median overall acceptability of VCT was 69% (range, 33-95%). Overall acceptability of VCT most frequently depended on return rates because acceptance rates were generally high. Where several studies were conducted in parallel in the same city or the same country, overall acceptability rates of HIV testing were generally comparable even if the intervention programmes differed. Overall acceptability rates of VCT were high in antenatal clinics where a particular effort in implementing VCT programmes had been made. CONCLUSIONS: This international survey shows that despite many obstacles, VCT is feasible and acceptable for pregnant women aiming to reduce their risk of transmitting HIV to their children.     

Opioids activate G proteins in REM sleep-related brain stem nuclei of rat

Mu opioid receptors within the pontine reticular formation contribute to opioid-induced rapid eye movement (REM) sleep inhibition. Mu receptors are coupled to guanine nucleotide binding (G) proteins and this study tested the hypothesis that the micro opioid agonist [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) would activate G proteins in rat brain stem nuclei known to regulate REM sleep. In vitro autoradiography of DAMGO-stimulated [35S]GTPgammaS binding showed that, compared with basal [35S]GTPgammaS binding, DAMGO significantly increased G protein activation in the nucleus pontis oralis (56.2%), nucleus pontis caudalis (57.3%), laterodorsal tegmental nucleus (75.8%), pedunculopontine tegmental nucleus (72.4%), nucleus locus coeruleus (77.2%) and dorsal raphe nucleus (73.4%). DAMGO stimulation of [35S]GTPgammaS binding in nuclei regulating REM sleep suggests that opioid-induced REM sleep inhibition involves activation of G proteins.