Conflicting evidence regarding the transport of alpha-glutamyl-dipeptides by human erythrocytes.

A novel ninhydrin-positive compound, N-methyl-D-aspartic acid, was identified in the muscle extracts of the blood shell, Scapharca broughtonii. This compound is already known to have potent neuroexcitatory activity, inducing hypermotility and strong releasing action of serum luteinizing hormone in mammals. This may be, however, the first finding of N-methyl-D-aspartic acid in natural products.     

The Na+-independentd-glucose transporter in the enterocyte basolateral membrane: Orientation and cytochalasin B binding characteristics

Summary Phloridzin-insensitive, Na⁺-independentd-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll^® gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed aK _t of 48mm and aV _max of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lowerK _t of 23mm and aV _max of 0.20 nmol glucose/mg protein/sec.d-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. TheK _t for cytochalasin B as an inhibitor of glucose transport was 0.11 m and theK _D for binding to the carrier was 0.08 m.d-glucose-sensitive binding of cytochalasin B to the membrane preparation was maximized withl- andd-glucose concentrations of 1.25m. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na⁺-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and theK _t for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.     

Vitamin E protects proteins against free radical damage in lipid environments

The fragmentation of the membrane protein monoamine oxidase in submitochondrial particles was induced by defined free radicals during radiolysis and by a system dependent on hydrogen peroxide and a transition metal. By injection of alpha-tocopherol in vivo, the levels of this physiological antioxidant in the mitochondrial preparations could be elevated more than ten-fold. In both radical-generating systems the presence of high levels of alpha-tocopherol in the membrane substantially retarded the protein fragmentation, in parallel with lipid peroxidation. It is suggested that membrane-bound proteins are damaged during lipid peroxidation and that alpha-tocopherol protects cells against both types of damage.     

Influence of the Malate-Aspartate Shuttle on Oxidative Metabolism in Synaptosomes

beta-Methyleneaspartate, a specific inhibitor of aspartate aminotransferase (EC 2.6.1.1.), was used to investigate the role of the malate-aspartate shuttle in rat brain synaptosomes. Incubation of rat brain cytosol, "free" mitochondria, synaptosol, and synaptic mitochondria, with 2 mM beta-methyleneaspartate resulted in inhibition of aspartate aminotransferase by 69%, 67%, 49%, and 76%, respectively. The reconstituted malate-aspartate shuttle of "free" brain mitochondria was inhibited by a similar degree (53%). As a consequence of the inhibition of the aspartate aminotransferase, and hence the malate-aspartate shuttle, the following changes were observed in synaptosomes: decreased glucose oxidation via the pyruvate dehydrogenase reaction and the tricarboxylic acid cycle; decreased acetylcholine synthesis; and an increase in the cytosolic redox state, as measured by the lactate/pyruvate ratio. The main reason for these changes can be attributed to decreased carbon flow through the tricarboxylic acid cycle (i.e., decreased formation of oxaloacetate), rather than as a direct consequence of changes in the NAD+/NADH ratio. Malate/glutamate oxidation in "free" mitochondria was also decreased in the presence of 2 mM beta-methyleneaspartate. This is probably a result of decreased glutamate transport into mitochondria as a result of low levels of aspartate, which are needed for the exchange with glutamate by the energy-dependent glutamate-aspartate translocator.     

To breed or not to breed: an analysis of the social and density-dependent constraints on the fecundity of female badgers (Meles meles).

Data from post-mortem examinations, population density estimates and long term capture-mark-recapture studies have been combined to look at the pattern of reproductive behaviour and the social factors leading to reproductive failure in badgers in Britain. The results are used to evaluate whether the hypothesis that the defence of oestrous females (as opposed to defence of food resources) best explains territorial behaviour and the social organization of badgers. Badgers in Britain have two peaks of reproductive activity, one immediately post partum and one in the summer/autumn. These coincide with two peaks of ovulation, and in the late winter/spring there is a steep rise in the number of sows carrying blastocysts, to reach an asymptote in June for yearling sows and April in older sows. Measured by their contribution to overall productivity, winter/spring matings were much more important than summer/autumn matings, contributing 65% of total autumn blastocysts in yearling sows and 71% of autumn blastocysts in older sows. The relative importance of the two mating periods is reflected in the seasonal pattern of bite wounding in adult male badgers; minor bite wounding in January-March was 2.3 times as frequent as in August-October, and moderate-extensive bite wounding was 3.1 times more frequent. In the populations studied, pre- and post-natal losses were high, with reproductive failure occurring at all stages of the breeding cycle, so that less than 30% of potential productivity was achieved. Indeed 22% of sows failed to develop blastocysts; these had a lower body mass, less body fat, larger adrenal glands, poorer health and higher bite wound scores than sows with blastocysts. Only 44% of adult sows implanted their blastocysts and proceeded to the end of pregnancy. However, it was less easy to identify features characteristic of sows that did or did not go on to implant their blastocysts. Finally, 35% of sows that produced cubs ceased lactation early, and this loss of entire litters was thought to be due to infanticide by dominant sows. The presence of annexe setts correlates with increased productivity in younger sows, and this is thought to be because annexe setts enable younger sows and their cubs to avoid the aggression of older, more dominant sows. Living in large social groups has no net reproductive gain for adult males or females, and there was a decline in productivity (per adult) with increasing group size.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolism of Abscisic Acid in Guard Cells of Vicia faba L. and Commelina communis L

Metabolism of abscisic acid (ABA) was investigated in isolated guard cells and in mesophyll tissue of Vicia faba L. and Commelina communis L. After incubation in buffer containing [G-³H]±ABA, the tissue was extracted by grinding and the metabolites separated by thin layer chromatography. Guard cells of Commelina metabolized ABA to phaseic acid (PA), dihydrophaseic acid (DPA), and alkali labile conjugates. Guard cells of Vicia formed only the conjugates. Mesophyll cells of Commelina accumulated DPA while mesophyll cells of Vicia accumulated PA. Controls showed that the observed metabolism was not due to extracellular enzyme contaminants nor to bacterial action.

Metabolism of ABA in guard cells suggests a mechanism for removal of ABA, which causes stomatal closure of both species, from the stomatal complex. Conversion to metabolites which are inactive in stomatal regulation, within the cells controlling stomatal opening, might precede detectable changes in levels of ABA in bulk leaf tissue. The differences observed between Commelina and Vicia in metabolism of ABA in guard cells, and in the accumulation product in the mesophyll, may be related to differences in stomatal sensitivity to PA which have been reported for these species.

     

Effect of external abdominal irradiation on intestinal morphology and brush border membrane enzyme and lipid composition

Previous studies have shown that external abdominal irradiation is associated with alterations in intestinal morphology and function. The activity of the jejunal brush border membrane (BBM) enzyme markers sucrase (S) and alkaline phosphate (AP) were not altered by 600 rad irradiation in the rat. In contrast, ileal BBM, AP, and AP/S were increased 3, 7/8, and 28 days postirradiation. The total lipid composition of the jejunal BBM was lower than in control animals only at 3 days postirradiation; this was due to a decrease in the total free fatty acid content. In addition to a lower total free fatty acid content, the ileal BBM contained an increased amount of total phospholipid (PL) which resulted in an increased phospholipid/cholesterol ratio at 3 days following irradiation. Variations in the BBM phospholipid composition occurred in both jejunum and ileum. In the jejunal BBM, the phospholipid composition changes did not alter the choline or amine phospholipid content; therefore, the choline/amine phospholipid ratio was unaffected by irradiation at 600 rad. In the ileal BBM, the phosphatidyl ethanolamine was increased at 3, 7/8, 14, and 28 days following irradiation. The choline/amine phospholipid ratio was not altered in the ileal BBM due to concomitant increases in lecithin content. Jejunal villus height, villus surface area, and the number of cells per villus were decreased at 3 days postirradiation, but increased by day 7/8 and 14 postirradiation to levels much higher than observed in control jejunal villi. The mucosal surface area was decreased at 3 and 7/8 days following irradiation but returned to control values by Day 14. Jejunal microvillus morphology was unaffected by irradiation. Few significant changes were observed in ileal villus morphology following irradiation at 600 rad. Ileal villus height, villus surface area, and mucosal surface area did not change, but the number of cells per villus initially decreased at 3 days and then increased beyond control values at 7/8 and 14 days postirradiation. Ileal microvillus height was significantly decreased only at 7 days postirradiation, while the number of microvilli per micron was increased only at 3 days postirradiation. This study suggests that changes in intestinal morphology and brush border composition may contribute to the altered passive permeation toward lipids which has been reported following abdominal radiation.     

Application of Deuterated A-Tocopherols to the Biokinetics and Bioavailability of Vitamin E

-Tocopherol, a superior chain-breaking, peroxyl radical-trapping antioxidant and the most active component of vitamin E, is elevated in liver tumor cells, contributing to their greater resistance towards lipid peroxidation compared to cells from normal tissues. Also, in regenerating rat liver the level of vitamin E has been found to fluctuate in phase with the rate of cell division. In order to study the biokinetcis and mechanisms of the distribution of vitamin E in organs and within tissues of animals, deuterated forms of -tocopherol have been synthesized and their uptake into blood and tissues has been measured by gas chromatography-mass spectrometry. Measurement of the competitive uptake from a mixture of the RRR-and SRR--tocopherol stereoisomers labelled with different amounts of deuterium shows that the liver exerts a strong preference for secretion of the natural (RRR) stereoisomer into the plasma. It is suggested that a tocopherol-binding protein plays a key role in this process.     

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.