Cloning and expression of a chicken alpha-amylase gene

The effects of cabbage leaf protein concentrate (CLPC) on serum and liver lipid concentrations were determined in rats fed cholesterol-enriched and cholesterol-free diets. In rats fed the cholesterol-enriched diet with CLPC, total cholesterol, triacylglycerol and phospholipid concentrations in both the serum and liver, as well as the atherogenic index diet were significantly lower than those of the rats fed a casein diet. A supplement of methionine to the CLPC diet raised serum HDL-cholesterol and body weight gain, indicating that the addition of methionine to the CLPC diet is not only available to improve the nutritive value of CLPC but also to lower the atherogenic index. In rats fed the cholesterol-free diet, the liver total cholesterol and triacylglycerol concentrations of the CLPC-fed rats also showed lower values than those of the casein-fed rats, however, the serum total cholesterol concentration of the CLPC-fed rats did not differ from that of the casein-fed rats.     

An in vitro model of human red blood cell production from hematopoietic progenitor cells

Hemoglobinopathies, such as beta-thalassemias and sickle cell anemia (SCA), are among the most common inherited gene defects. Novel models of human erythropoiesis that result in terminally differentiated red blood cells (RBCs) would be able to address the pathophysiological abnormalities in erythrocytes in congenital RBC disorders and to test the potential of reversing these problems by gene therapy. We have developed an in vitro model of production of human RBCs from normal CD34(+) hematopoietic progenitor cells, using recombinant growth factors to promote terminal RBC differentiation. Enucleated RBCs were then isolated to a pure population by flow cytometry in sufficient numbers for physiological studies. Morphologically, the RBCs derived in vitro ranged from early polylobulated forms, resembling normal reticulocytes to smooth biconcave discocytes. The hemoglobin pattern in the in vitro-derived RBCs mimicked the in vivo adult or postnatal pattern of beta-globin production, with negligible gamma-globin synthesis. To test the gene therapy potential using this model, CD34(+) cells were genetically marked with a retroviral vector carrying a cell-surface reporter. Gene transfer into CD34(+) cells followed by erythroid differentiation resulted in expression of the marker gene on the surface of the enucleated RBC progeny. This model of human erythropoiesis will allow studies on pathophysiology of congenital RBC disorders and test effective therapeutic strategies.     

Differences in muscle endurance and recovery between fallers and nonfallers, and between young and older women

BACKGROUND: Significant morbidity and mortality are associated with falls in older adults. We tested the hypothesis that older women with a history of falls demonstrate decreased muscle endurance and longer recovery times following fatiguing exercise. METHODS: We evaluated dynamic endurance and recoverability of the quadriceps femoris of 29 young women (YW) (M age = 21.7), 26 older women with a history of falls (FA) (M age = 73.3), and 27 older women with no history of falls (NF) (M age = 71.2) using an isokinetic dynamometer. Subjects performed repeated maximal concentric knee extensions until the force output of two consecutive repetitions fell below 50% of their maximal voluntary contraction (MVC). Recovery was defined as the time required for the return of force output > or = 80% MVC for 2 consecutive repetitions, within a set consisting of 3 maximal contractions. One minute rest was allowed between sets. We collected electromyographic (EMG) data from the quadriceps during all testing to evaluate spectral shifts. RESULTS: ANOVA with a post-hoc Bonferroni-Dunn test revealed time to fatigue was significantly faster in FA than YW (p < .02) and in FA than NF (p < .05), but not different between YW and NF. Time to recovery was significantly slower in FA than YW (p = .01), but not different between YW and NF, or between FA and NF, EMG median frequency power shift (from the beginning to the end of the test) was significantly less in FA (p < .001) than either YW (p < .002) or NF (p < .05). CONCLUSIONS: Older women with a history of falls demonstrate decreased muscular endurance compared to YW and NF, and increased time to recover from fatiguing exercise when compared to young women.     

Dependence of peroxisomal beta-oxidation on cytosolic sources of NADPH

Growth of Saccharomyces cerevisiae with a fatty acid as carbon source was shown previously to require function of either glucose-6-phosphate dehydrogenase (ZWF1) or cytosolic NADP+-specific isocitrate dehydrogenase (IDP2), suggesting dependence of beta-oxidation on a cytosolic source of NADPH. In this study, we find that DeltaIDP2DeltaZWF1 strains containing disruptions in genes encoding both enzymes exhibit a rapid loss of viability when transferred to medium containing oleate as the carbon source. This loss of viability is not observed following transfer of a DeltaIDP3 strain lacking peroxisomal isocitrate dehydrogenase to medium with docosahexaenoate, a nonpermissive carbon source that requires function of IDP3 for beta-oxidation. This suggests that the fatty acid- phenotype of DeltaIDP2DeltaZWF1 strains is not a simple defect in utilization. Instead, we propose that the common function shared by IDP2 and ZWF1 is maintenance of significant levels of NADPH for enzymatic removal of the hydrogen peroxide generated in the first step of peroxisomal beta-oxidation in yeast and that inadequate levels of the reduced form of the cofactor can produce lethality. This proposal is supported by the finding that the sensitivity to exogenous hydrogen peroxide previously reported for DeltaZWF1 mutant strains is less pronounced when analyses are conducted with a nonfermentable carbon source, a condition associated with elevated expression of IDP2. Under those conditions, similar slow growth phenotypes are observed for DeltaZWF1 and DeltaIDP2 strains, and co-disruption of both genes dramatically exacerbates the H2O2s phenotype. Collectively, these results suggest that IDP2, when expressed, and ZWF1 have critical overlapping functions in provision of reducing equivalents for defense against endogenous or exogenous sources of H2O2.     

Properties of calcium spikes revealed during GABAA receptor antagonism in hippocampal CA1 neurons from guinea pigs

A potential energy function for unsaturated hydrocarbons is proposed and is shown to agree well with experiment, using molecular dynamics simulations of a water/octene interface and a dioleoyl phosphatidylcholine (DOPC) bilayer. The simulation results verify most of the assumptions used in interpreting the DOPC experiments, but suggest a few that should be reconsidered. Comparisons with recent results of a simulation of a dipalmitoyl phosphatidylcholine (DPPC) lipid bilayer show that disorder is comparable, even though the temperature, hydration level, and surface area/lipid for DOPC are lower. These observations highlight the dramatic effects of unsaturation on bilayer structure.     

Interactions of nonsteroidal anti-inflammatory drugs with rat renal organic anion transporter, OAT-K1

We recently cloned and characterized the rat kidney-specific organic anion transporter, OAT-K1, which was suggested to mediate renal tubular transport of methotrexate. In this study, we investigated the interactions of nonsteroidal anti-inflammatory drugs (NSAIDs) with OAT-K1 by evaluating the effects of these drugs on renal distribution of methotrexate in vivo, and on methotrexate accumulation in the stably transfected LLC-PK1 cells expressing OAT-K1 (LLC-OAT-K1). NSAIDs such as indomethacin and ketoprofen had significant inhibitory effects on renal accumulation of methotrexate in rats after coadministration. Indomethacin and ketoprofen inhibited methotrexate accumulation by LLC-OAT-K1 cells in a competitive manner with the apparent inhibition constant values of 1. 0 mM and 1.9 mM, respectively. Other NSAIDs including ibuprofen, flufenamate and phenylbutazone also showed potent inhibitory effects on methotrexate accumulation. However, indomethacin was not transported via OAT-K1. These results indicate that NSAIDs have potent inhibitory effects against the OAT-K1-mediated methotrexate transport, which suggests that the OAT-K1 may be one of interaction sites for methotrexate and NSAIDs in the kidney.     

Chest radiographic data acquisition and quality assurance in multicenter studies

Dorsal fusion with the internal fixator has become the standard treatment of instabilities and deformities of the thoracolumbar spine. With our new device, the modular spine fixator (MSF), which has been specially designed for short-distance instrumentations, we have increasingly been treating unstable injuries of the thoracolumbar spine by one-level stabilization. Prerequisite is an accurate evaluation of the indication, including CT and MRI to assess the involvement of the intervertebral disc and the ligamental structures. The operative technique differs in some details from the procedure in more-multi-level instrumentations, especially concerning the application of the pedicle screws. The instrumentation is always combined with posterior allogenic bone grafting. Since the beginning of 1993 we also perform anterior autogenic transpedicular bone grafting. Between January 1991 and July 1995, 57 one-level stabilizations with the MSF were performed. Of the 57 patients operated on 39, 27 men and 12 women, with an average age of 41 years, have had a clinical and radiographic follow-up examination so far, on average, 27 months after the accident. Seventeen patients were completely free of pain and 17 patients (were only) sensitive to weather changes or had minor pain during great physical stress. Five patients had pain even during slight physical stress or at rest. The preoperatively measured Cobb angle was 15.1 degrees on average, after the operation 5.2 degrees, and at the time of the follow-up examination amounted to 8.1 degrees. The patients' range of motion was normal. Only five minor complications have been seen. No implant fatigue failure has been noted in this series. We derive from these results that, for correct indications, one-level stabilization can be performed successfully and should be firmly established in the operative treatment of unstable fractures of the thoracolumbar spine.     

Early ambulation after coronary angioplasty and stenting with six French guiding catheters and low-dose heparin

The safety and feasibility of early ambulation was evaluated prospectively in 907 patients undergoing elective coronary angioplasty and stenting with the use of 6Fr guiding catheters, low-dose heparin (5,000 IU), and immediate postprocedural sheath removal by comparing ambulation after 4 hours with immobilization for at least 12 hours. Because no excess in puncture site complications (2.3% vs 2.2%) could be demonstrated after 4-hour ambulation, it is concluded that early ambulation after 6Fr guiding catheter angioplasty by the femoral route with low-dose heparin is feasible, safe, and may facilitate a shorter hospital stay.     

Red flags and religious coping: identifying some religious warning signs among people in crisis

Particulate and soluble (1-3)-beta-glucans are effective in preventing infections by enhancing macrophage and neutrophil functions. However, the mechanisms triggering these enhanced cellular responses are essentially unknown. We recently demonstrated that zymosan, a particulate (1-3)-beta-glucan receptor agonist, caused an influx of Ca2+ in NR8383 rat alveolar macrophages (AMs) and a resulting increase in intracellular Ca2+ (Zhang et al., J. Leukoc. Biol. 62 (1997) 341-348). Since Ca2+ is important in mediating leukocyte responses, we investigated whether other (1-3)-beta-glucans also alter Ca2+ mobilization in AMs. Particulate and soluble (1-3)-beta-glucans derived from Saccharomyces cerevisiae were used in these studies. Like zymosan, particulate (1-3)-beta-glucan (WGPs) caused a concentration-dependent increase in [Ca2+]i, which was inhibited by removal of extracellular Ca2+ and by SKF96365, an inhibitor of receptor-operated Ca2+ channels. When three different soluble (1-3)-beta-glucans, with molecular weights of approximately 11,000, 150,000, and 1,000,000 Da, were tested alone for effects on Ca2+ responses, the low molecular weight (1-3)-beta-glucan produced no effect and the intermediate and high molecular weight (1-3)-beta-glucans caused only a small increase in [Ca2+]i. Interestingly, however, all three soluble (1-3)-beta-glucans could significantly reduce the Ca2+ responses induced by a subsequent exposure to either WGPs or zymosan. These results demonstrate that: 1) particulate (1-3)-beta-glucan activates Ca2+ influx in NR8383 macrophages through receptor-operated Ca2+ channels; 2) soluble (1-3)-beta-glucans do not strongly activate Ca2+ influx in these cells; and 3) soluble (1-3)-beta-glucans significantly inhibit Ca2+ influx induced by WGPs or zymosan. Soluble (1-3)-beta-glucans are likely to prevent Ca2+ influx by competitively binding to the (1-3)-beta-glucan receptors recognizing zymosan and WGPs. The smaller Ca2+ influx induced by soluble (1-3)-beta-glucans may represent only a partial activation of post-receptor signal transduction pathways necessary for inducing Ca2+ influx.