Specific substitutions at amino acid 256 of the sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPase mediate resistance to thapsigargin in thapsigargin-resistant hamster cells

High levels of resistance to thapsigargin (TG), a specific inhibitor of intracellular Ca2+ transport ATPases (SERCAs), can be developed in culture by stepwise exposure of mammalian cells to increasing concentrations of TG. We have identified, in two independently selected TG-resistant hamster cell lines of different lineages, mutant forms of SERCA. In the TG-resistant Chinese hamster lung fibroblast cell line DC-3F/TG, a T --> C change at nucleotide 766 introduces a Phe256 --> Leu alteration within the first cytosolic loop of the SERCA. In contrast, in the TG-resistant Syrian hamster smooth muscle cell line DDT/TG 4 microM, a T --> C change at nucleotide 767 introduces a Phe256 --> Ser mutation at that position. When these specific mutations are introduced into a wild-type full-length avian SERCA1 cDNA, transfection experiments reveal that Ca2+ transport function and ATP hydrolytic activity are not altered by such mutations. However, a 4-5-fold resistance to TG inhibition of Ca2+ transport function occurs upon the introduction of either the Phe256 --> Leu or the Phe256 --> Ser mutation into wild-type SERCA1. These specific mutations also render the hydrolytic activity of the ATPase resistant to inhibition by TG. Our results not only implicate amino acid 256 in TG-SERCA interactions, but also demonstrate that specific mutations within SERCA can mediate resistance to TG.     

A study of the diagnostic accuracy and reliability of the Scoliometer and Adam's forward bend test

STUDY DESIGN: Study of the diagnostic accuracy and interexaminer reliability of scoliosis diagnostic tests. OBJECTIVES: To estimate the sensitivity, specificity, and predictive value of the Scoliometer (National Scoliosis Foundation, Watertown, MA) and Adam's forward bend test in diagnosing scoliosis, and to determine the interexaminer reliability of the Scoliometer and Adam's forward bend test. SUMMARY OF BACKGROUND DATA: Exposure to diagnostic radiation in patients with adolescent idiopathic scoliosis may result in a small but significant increase in cancer rates. The full-spine radiographic examination remains the standard procedure for the assessment of scoliosis. There is a need for a valid and reliable noinvasive test to assess scoliosis. METHODS: Two examiners independently assessed 105 patients presenting to a scoliosis clinic for trunk asymmetry with Adam's forward bend test and axial trunk rotation with the Scoliometer. The Cobb method served as the gold standard. RESULTS: The interexaminer agreement for the Scoliometer is excellent in the thoracic spine and substantial in the lumbar spine. The interexaminer measurement error shows poor precision for thoracic and lumbar Scoliometer measurements. The interexaminer agreement for Adam's forward bend test is substantial in the thoracic spine and poor in the lumbar spine. Adam's forward bend test is more sensitive than the Scoliometer in detecting thoracic curves measuring 20 degrees or more by the Cobb method. Receiver operating characteristic curve analysis suggests that the use of the Scoliometer marginally improves the ability of diagnosing a scoliosis in the thoracic spine. CONCLUSIONS: The Scoliometer and Adam's forward bend tests have adequate interexaminer reliability for the assessment of thoracic curves. The Scoliometer has better interexaminer agreement in the lumbar spine. However, the Scoliometer has a high level of interexaminer measurement error that limits its use as an outcome instrument. Because Adam's forward bend test is more sensitive than the Scoliometer, the authors believe that it remains the best noninvasive clinical test to evaluate scoliosis.     

Influence of fibrinolytic factors on scar formation after lumbar discectomy. A magnetic resonance imaging follow-up study with clinical correlation performed 7 years after surgery

STUDY DESIGN: Prospective cohort study. OBJECTIVES: To assess the amount of scar tissue by viewing magnetic resonance images, and to evaluate the correlation between the amount of scar tissue and clinical outcome, surgical technique, and fibrinolytic factors. SUMMARY OF BACKGROUND DATA: The influence of fibrinolytic factors on magnetic resonance images has not been investigated previously. The relation between clinical outcome and findings on magnetic resonance imaging remains uncertain. METHODS: Magnetic resonance imaging at 0.5 Tesla was performed to produce sagittal and axial spin-echo T1-weighted images before and after contrast enhancement on 78 patients 7 years after traditional lumbar discectomy with partial or full laminectomy. Before surgery all patients had been tested for fibrinolytic factors. RESULTS: The overall clinical success rate of the surgery was 73%. No evidence of scar formation was seen in 19 patients, a small amount was seen in 36 patients, a moderate amount in 17 patients, and a large amount was observed in 6 patients. Ten patients who had undergone surgery at two disc levels and 18 who had been treated with full laminectomy exhibited more scar tissue than those patients who had undergone surgery on a single level (P = 0.033) and those who had undergone a partial laminectomy, respectively (P = 0.017). The amount of scar formation also was associated with a poor outcome (P = 0.017) and with low preoperative values of tissue plasminogen activator antigen (P = 0.003) and tissue plasminogen activity (P = 0.048) in samples collected after venous occlusion. The intensity of contrast enhancement, however, was not influenced by these or any other parameters. CONCLUSION: The amount of scar formation after lumbar discectomy seems to be related to the clinical outcome, the size of the surgical exposure, and some fibrinolytic factors.     

Evaluation of biologic gastrin activity of compound CI-988 in the isolated, vascularly perfused rat stomach

BACKGROUND: The peptoid CI-988 has previously been shown to have high affinity for the cholecystokinin (CCK)-B/gastrin receptor and has been reported to be a powerful CCK antagonist in many systems, although it has agonist activity on histidine decarboxylase in the rat. METHODS: In the present study the effect of CI-988 on acid secretion and histamine release in the totally isolated, vascularly perfused rat stomach was assessed. RESULTS: CI-988 was found to be a gastrin agonist with regard to the stimulation of both histamine release and acid secretion. CONCLUSION: Thus, in this stomach model CI-988 behaved as a CCKB/gastrin agonist. The present study underlines the importance of testing the biologic activity of ligands in models with sufficient sensitivity.     

Purification and crystallization of complexes modeling the active state of the fragile histidine triad protein

Fragile histidine triad protein (Fhit) is a diadenosine triphosphate (ApppA) hydrolase encoded at the human chromosome 3 fragile site which is frequently disrupted in tumors. Reintroduction of FHIT coding sequences to cancer cell lines with FHIT deletions suppressed the ability of these cell lines to form tumors in nude mice even when the reintroduced FHIT gene had been mutated to allow ApppA binding but not hydrolysis. Because this suggested that the tumor suppressor activity of Fhit protein depends on substrate-dependent signaling rather than ApppA catabolism, we prepared two crystalline forms of Fhit protein that are expected to model its biologically active, substrate-bound state. Wild-type and the His96Asn forms of Fhit were overexpressed in Escherichia coli, purified to homogeneity and crystallized in the presence and absence of ApppA and an ApppA analog. Single crystals obtained by vapor diffusion against ammonium sulfate diffracted X-rays to beyond 2.75 A resolution. High quality native synchrotron X-ray data were collected for an orthorhombic and a hexagonal crystal form.      

Degradation in bulk heterojunction organic solar cells: changes in electrode interface and reduction in the occupation probability of the interface states

We have investigated the degradation of P3HT:PCBM (poly(3-hexylthiophene):6,6-phenylC61 butyric acid methyl ester) solar cell beyond 150?h of fabrication in continuation to our earlier reported work up to 150?h of fabrication. The current- voltage characteristics of degraded Indium tin oxide/poly(3,4-ethylenedioxythiopene):poly(styrenesulfonate)/poly(3-hexylthiophene):6,6-phenylC61 butyric acid methyl ester/Aluminum (ITO/PEDOT:PSS/P3HT:PCBM/Al) solar cell can be explained by considering the tunneling current through electrode interfaces, increase in both the interface states density and the thickness of interface with time for150-200?h. Beyond 200?h of fabrication, a significant reduction in the occupation probability at the electrode interfaces explains the experimental results up to 300?h fairly well. Calculations based on realistic parameters and activity at both the electrode interfaces (ITO/PEDOT:PSS and P3HT:PCBM/Al) confirm that degradation at P3HT:PCBM/Al interface is more prominent than that at ITO/PEDOT:PSS interface.     

Fully dense hot pressed calcium cobalt oxide ceramics

The processing parameters have been optimized to achieve highly pure and fully dense pellets of calcium cobalt oxide (Ca₃Co₄O₉) from solid-state ball milled calcium carbonate and cobalt oxide mixtures, calcined at optimized temperature and time, and consolidated by hot-pressing. The microscopic, spectroscopic, and thermal analysis suggest samples with longer ball-milling time require less calcination time for synthesizing highly pure crystalline phases of Ca₃Co₄O₉, and provide 99.2 ± 0.5% relative density.     

EFFECT OF LIPASE ENZYME ON THE RIPENING OF WHITE PICKLED CHEESE

The effect of addition of pregastric lipase enzyme on the accelerated ripening of white pickled cheese was investigated. Commercial pregastric lipase was added to milk before rennet addition at a level of 0,5, 8, 11 g per 100 L of milk and cheeses were made from this milk. Total solids, fat, total nitrogen, salt, titratable acidity, pH and free fatty acids (C₂-C_18:1) were analysed in the samples during 1–90 days of ripening period at 15 days intervals. Total solids, fat, total nitrogen, salt, titratable acidity, and pH of cheeses slightly increased during the ripening period. Free fatty acids and volatile free fatty acid contents in cheeses made from pregastric lipase added milk were affected by pregastric lipase and their contents were increased significantly (P<0.01) during the ripening period. Particularly, when cheese had a high level (11 g per 100 L milk) pregastric lipase, the amounts of butyric, caproic and caprylic acids in white pickled cheese were quite high. The relative amounts of volatile free fatty acids varied with storage time and pregastric lipase levels.     

Geomorphic diversity as a river management tool and its application to the Ganga River,India

Understanding of geomorphic processes and the determination of geomorphic diversity in catchments are prerequisites for the sustainable rehabilitation of river systems and for reach‐scale assessment of river health. The Ganga River system in India is a large, complex system consisting of several long tributaries, some >1,000 km, originating from 2 distinct hinterlands—the Himalaya to the north and the cratons to the south. Traversing through a diverse climatic regime across the Plain and through precipitation zones ranging from 600 mm/year near Delhi to 1,200 mm/year in the eastern plains, the Ganga River system has formed very diverse landform assemblages in 3 major geomorphic domains. We have recognized 10 different river classes for the trunk river from Gangotri (source) to Farakka (upstream of its confluence with the Brahmaputra) based on (a) landscape setting, (b) channel and active floodplain properties, and (c) channel planform parameters. The mountainous stretch is characterized by steep valleys and bedrock channels and is dominated by large‐scale sediment production and transport through hill slope processes. The alluvial part of the river is characterized by 8 different river classes of varying reach lengths (60–300 km) many of which show sharp transitions in landscape setting. We have highlighted the application of this approach for the assessment of habitat suitability, environmental flows, and flood risk all of which have been significantly modified during the last few decades due to large‐scale anthropogenic disturbances. We suggest that the diversity embedded in this geomorphic framework can be useful for developing a sustainable river management programme to “work with” the contemporary character and behaviour of rivers.