Structural and electrophysiological analysis of annexin V mutants. Mutagenesis of human annexin V, an in vitro voltage-gated calcium channel, provides information about the structural features of the ion pathway, the voltage sensor and the ion selectivity filter

Annexin V binds to phospholipids in a calcium-dependent manner and exhibits calcium channel activity in vitro. We prepared a variety of mutants yielding information about the structure-function relationship of the ion channel activity. All mutants were characterized by X-ray crystallography, electron microscopy and electrophysiological measurements. Their structures are insignificantly changed whereas their electrophysiological properties are drastically different. Glu95, located in the central hydrophilic pore of the molecule, is crucial for the ion selectivity filter as its exchange leads to reduced calcium and increased sodium conductance. The removal of Glu17, located on the protein surface and far from the ion conduction pathway, leads to the appearance of a second conductance level of 9 pS in addition to the conductance level of about 30 pS in the wild-type molecule. This was also the case for Glu78, which is part of a weak calcium binding site. The exchange of Glu17 and Glu78 produced a mutant retaining only the smaller conductance level. We conclude that these two residues influence the angle between the two halves of the molecule, which determines the diameter of the ion conduction pathway, thereby leading to the occurrence of a second conductance level.     

Subtractive isolation of phage-displayed single-chain antibodies to thymic stromal cells by using intact thymic fragments

In the murine thymus, the stroma forms microenvironments that control different steps in T cell development. To study the architecture of such microenvironments and more particularly the nature of communicative signals in lympho-stromal interaction during T cell development, we have employed the phage antibody display technology, with the specific aim of isolating thymic stromal cell-specific single-chain antibodies from a semisynthetic phage library. A subtractive approach using intact, mildly fixed thymic fragments as target tissue and lymphocytes as absorber cells generated monoclonal phages (MoPhabs) detecting subsets of murine thymic stromal cells. In the present paper we report on the reactivity of single-chain antibodies derived from three MoPhabs, TB4-4, TB4-20, and TB4-28. While TB4-4 and TB4-20 are both epithelium specific, TB4-28 detects an epitope expressed on both epithelial- and mesenchymal-derived stromal cells. TB4-4 reacts with all cortical epithelial cells and with other endoderm-derived epithelia, but this reagent leaves the majority of medullary epithelial cells unstained. In contrast, MoPhab TB4-20 detects both cortical and medullary thymic epithelial cells, as well as other endoderm- and ectoderm-derived epithelial cells. Cross-reaction of single-chain antibodies to human thymic stromal cells shows that our semisynthetic phage antibody display library, in combination with the present subtractive approach, permits detection of evolutionary conserved epitopes expressed on subsets of thymic stromal cells.     

Cumulus cloud base height estimation from high spatial resolutionLandsat data: a Hough transform approach

This study develops a semiautomated methodology for estimating cumulus cloud base heights using high-spatial-resolution Landsat multispectral scanner data. The approach employs a variety of image processing techniques to match cloud edges with their corresponding shadow edges. Cloud base height is then estimated by computing the separation between the corresponding generalized Hough transform reference points. Sixteen subregions, each 30 km×30 km in size, are selected for four Landsat scenes. Standard deviations of cloud base height within each of the subregion range from about 100 m to 150 m. Differences between cloud base heights computed using the Hough transform and a manual verification technique are small. It is estimated that cloud base height accuracies of 50-70 m may be possible using HIRIS and ASTER instruments in the Earth Observation Satellite (EOS) Global Climate Change program     

Increased plasma concentrations of serum amyloid A: an indicator of the acute-phase response after cardiopulmonary bypass

OBJECTIVES: To assess the expression of mixed and hepatic venous serum amyloid A (SAA) concentrations and its relationship to plasma concentrations of C-reactive protein, interleukin-6 (IL-6), and endotoxin during and after cardiopulmonary bypass (CPB). DESIGN: Prospective, consecutive sample with repeated measurements. SETTING: Surgical intensive care unit (ICU) in a university hospital. PATIENTS: Twenty patients who underwent elective coronary bypass grafting. INTERVENTIONS: A radial artery catheter, pulmonary artery catheter, and right hepatic vein catheter were inserted. Blood samples were collected to determine the different mediators, lactate concentrations, and oxygen saturations. MEASUREMENTS AND MAIN RESULTS: After induction of anesthesia, baseline values were obtained and the following parameters were determined 20 mins after onset of CPB, 20 mins after termination of CPB, at admission to the ICU, and 6, 8, 12, and 24 hrs later: hemodynamics, body core temperature, hepatic venous oxygen saturation, and mixed and hepatic venous lactate, endotoxin, interleukin (IL)-6, C-reactive protein (CRP), and SAA concentrations. Endotoxin and IL-6 plasma concentrations increased during CPB, peaked 6 hrs after admission to the ICU (endotoxin: 23.1 +/- 6.2 pg/mL; IL-6: 646 +/- 104 pg/mL), and decreased thereafter; SAA and CRP concentrations began to increase after 6 and 8 hrs, respectively, with the highest concentrations reached 24 hrs postoperatively (CRP: 14 +/- 3.6 mg/L; SAA: 668 +/- 114 micrograms/mL). Lactate concentrations began to increase 20 mins after CPB, and continued to increase until 12 hrs postoperatively. There were no significant differences between mixed and hepatic venous values of endotoxin, IL-6, CRP, SAA, and lactate (p < .05). Body core temperature, which was < 37.5 degrees C before surgery for all patients, increased 6 hrs after admission to the ICU and peaked 12 hrs postoperatively (38.3 +/- 1.1 degrees C). Hepatic venous oxygen saturation did not change. Correlations were obtained between IL-6 values and heart rate (r2 = .20; p < .005), and endotoxin concentrations and systemic vascular resistance (r2 = .18; p < .001). Body core temperature correlated significantly closer with SAA (r2 = .52; p < .0001) values than with IL-6 (r2 = .27; p < .0001) or CRP (r2 = .16; p < .001) values (p < .05). CONCLUSIONS: SAA is an additional and sensitive marker of the acute-phase response following CPB; the increase in SAA concentrations parallels the temporary increase in body core temperature and is preceded by endotoxemia and IL-6 secretion.     

Influence of Skydrol immersion at elevated temperatures on the thermo-mechanical properties of a high-Tg anhydride epoxy resin toughened with a hydroxy-terminated polyester

Currently, the application of composites in aerospace parts exposed to higher temperatures and in aggressive media is still severely limited. To replace metal alloys, alternative resins systems with suitable long-term heat resistance are needed. In this study, the effect of the aviation hydraulic fluid Skydrol on the thermal and mechanical properties of a high-T_g, anhydride-cured epoxy resin in the unmodified and toughened state at elevated temperature is investigated. An aliphatic polyester diol was selected as an intrinsic toughener and its impact on the thermal, mechanical, and aging properties was determined. Experimental characterization of the aging effects is carried out with dynamic-mechanical characterization, infrared spectroscopy, and electron dispersion x-ray spectroscopy. In addition, the fracture toughness and the fatigue crack propagation behavior are determined. Initially, the toughened system shows an improved fracture toughness. Since oxidation is blocked by the Skydrol fluid only thermal degradation takes place as determined by the decrease in glass transition temperature T_g and network density. The thermal degradation leads to a tougher behavior, which is observed in both systems in static and dynamic mode with toughness decreasing with aging time again.     

A rapid and efficient purification method for recombinant annexin V for biophysical studies

Annexin V binds in a calcium-dependent manner to acidic phospholipids and exhibits ion channel activity in vitro. We are investigating mutants of annexin V by single channel measurements, X-ray crystallography and electron microscopy in order to understand the structure-function relationships of the ion channel activity. We describe here a method to obtain very pure recombinant annexin V required for such studies. The initial step is the mild opening of the bacterial cells by an osmotic shock. In the purification procedure, use is made of the reversible calcium-mediated binding of annexin V to liposomes. In the last purification step the protein is subjected to ion-exchange chromatography and elutes as a single peak free of any detectable contaminants.     

Causes of reproductive age mortality in low socioeconomic settlements of Karachi

The Maternal and Infant Mortality Survey (MIMS) was conducted in eight squatter settlements of Karachi. The female mortality rate was 151.0 per 100,000 women aged 10-49 years and the maternal mortality ratio was 281 per 100,000 livebirths. The leading causes of deaths among women were complications of pregnancy (28.1%), infectious diseases (24.8%), cardiovascular diseases (20.7%), neoplasia (10.7%) and trauma (10.7%). Hemorrhage (47.1% of all maternal deaths), tuberculosis (40.0% of all infectious disease deaths), oropharyngeal cancer (23.1% of all neoplastic deaths), and burns (61.5% of all trauma deaths) were among the major causes identified. Maternal deaths were associated with young age and nulliparity (p-value < 0.01), and a higher proportion occurred in the hospital or on the way to the hospital as compared to non-maternal deaths.