Endurance training does not alter the level of neuronal nitric oxide synthase in human skeletal muscle.

The effect of endurance training on neuronal nitric oxide synthase (nNOS) content and distribution in muscle was investigated. Seven male subjects performed 6 wk of one-legged knee-extensor endurance training (protocol A). Muscle biopsies, obtained from vastus lateralis muscle in the untrained and the trained leg, were analyzed for nNOS protein and activity as well as immunohistochemical distribution of nNOS and endothelial nitric oxide synthase (eNOS). Muscle biopsies were also obtained from another seven male subjects before and after 6 wk of training by endurance running (protocol B) and analyzed for nNOS protein. No difference was found in the amount of nNOS protein in the untrained and the trained muscle either with protocol A or protocol B (P > 0.05). In protocol A, the activity of nNOS was 4.76 +/- 0.56 pmol. mg protein(-1). min(-1) in the control leg, and the level was not different in the trained leg (P > 0.05). nNOS was present in the sarcolemma and cytosol of type I and type II muscle fibers, and the qualitative distribution was similar in untrained and trained muscle. The number of eNOS immunoreactive structures and the number of capillaries per muscle fiber were higher (P < 0.05) after training than before. The present findings demonstrate that, in contrast to findings on animals, nNOS levels remain unaltered with endurance training in humans. Evidence is also provided that endurance training may increase the amount of eNOS, in parallel with an increase in capillaries in human muscle.     

Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

Background  

The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene.     

Caleosins: Ca2+-binding proteins associated with lipid bodies

We have previously identified a rice gene encoding a 27 kDa protein with a single Ca²⁺-binding EF-hand and a putative membrane anchor. We report here similar genes termed caleosins, CLO, in other plants and fungi; they comprise a multigene family of at least five members in Arabidopsis (AtClo1–5). Northern hybridization demonstrated that AtClo2–4 mRNAs levels were low in various tissues, while AtClo1 mRNA levels were high in developing embryos and mature seeds. Analysis of transgenic Arabidopsis plants expressing the GUS reporter under control of the AtClo1 promoter showed strong levels of expression in developing embryos and also in root tip cells. Antibodies raised against AtCLO1 were used to detect caleosin in cellular fractions of Arabidopsis and rapeseed. This indicated that caleosins are a novel class of lipid body proteins, which may also be associated with an ER subdomain.     

AMPA Receptor–Mediated Neurotoxicity: Role of Ca2+ and Desensitization

Glutamate-induced neurodegeneration is the result of excessive stimulation of the different subtypes of glutamate receptors. With regard to the AMPA ((RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate) receptors it has been clear from numerous studies that in addition to the Ca²⁺ permeability of the receptor complexes, their desensitization properties may play a determining role in the neurodegeneration mediated by this subtype of the glutamate receptors. Recent studies have revealed important amino acid residues in the AMPA receptor subunits that control the desensitization kinetics and that may constitute important targets for drugs that may alter the desensitization of the AMPA receptor complexes.     

Cloning,heterologous expression,and enzymatic characterization of a thermostable glucoamylase from Talaromyces emersonii

The gene encoding a thermostable glucoamylase from Talaromyces emersonii was cloned and, subsequently, heterologously expressed in Aspergillus niger. This glucoamylase gene encodes a 618 amino acid long protein with a calculated molecular weight of 62,827Da. T. emersonii glucoamylase fall into glucoside hydrolase family 15, showing approximately 60% sequence similarity to glucoamylase from A. niger. The expressed enzyme shows high specific activity towards maltose, isomaltose, and maltoheptaose, having 3-6-fold elevated k(cat) compared to A. niger glucoamylase. T. emersonii glucoamylase showed significantly improved thermostability with a half life of 48h at 65 degrees C in 30% (w/v) glucose, compared to 10h for glucoamylase from A. niger. The ability of the glucoamylase to hydrolyse amylopectin at 65 degrees C is improved compared to A. niger glucoamylase, giving a significant higher final glucose yield at elevated temperatures. The increased thermal stability is thus reflected in the industrial performance, allowing T. emersonii glucoamylase to operate at a temperature higher than the A. niger enzyme.     

Angiotensin II attenuates the natriuresis of water immersion in humans

The hypothesis was tested that suppression of generation of ANG II is one of the mechanisms of the water immersion (WI)-induced natriuresis in humans. In one protocol, eight healthy young males were subjected to 3 h of 1) WI (WI + placebo), 2) WI combined with ANG II infusion of 0.5 ng. kg(-1). min(-1) (WI + ANG II-low), and 3) a seated time control (Con). In another almost identical protocol, 7-10 healthy young males were investigated to delineate the tubular site(s) of action of ANG II by the lithium clearance method (C(Li)) and were on an additional fourth study day subjected to infusion of ANG II at a rate of 1.5 ng. kg(-1). min(-1) (WI + ANG II-high). During WI + placebo, plasma concentration of ANG II decreased from 16 +/- 2 to 8 +/- 1 pg/ml (P < 0.05) and renal sodium excretion increased from 104 +/- 15 to 294 +/- 27 micromol/min (P < 0.05). During WI + ANG II-low, plasma ANG II was not suppressed by WI, and the natriuresis was blunted by 52 +/- 13% (P < 0.05). During WI + ANG II-low and WI + ANG II-high, an increase in C(Li) was prevented that was otherwise observed during WI, and fractional distal reabsorption of sodium was facilitated. In conclusion, maintaining plasma concentration of ANG II unchanged at the level of control attenuates the natriuresis of WI considerably in humans. Therefore, suppression of generation of ANG II is an important mechanism of the natriuresis of WI in humans. Furthermore, infusion of ANG II during WI prevents an otherwise induced increase in C(Li) and facilitates the fractional distal reabsorption of sodium, probably via an effect on aldosterone release.     

Characterization of Glutamate-Induced Formation of N-Acylphosphatidylethanolamine and N-Acylethanolamine in Cultured Neocortical Neurons

Abstract: Glutamate-induced formation of N-acylethanolamine (NAE) and N-acylphosphatidylethanolamine (NAPE) was studied in primary cultures of mouse neocortical neurons prelabeled with [¹⁴C]ethanolamine. The formation of these two lipids was dependent on the maturity of the cell culture; i.e., no glutamate-induced formation was seen in 2-day-old cultures, whereas glutamate induced a pronounced formation in 6-day-old cultures. The calcium ionophore A23187 (2 µM) stimulated, within 2 h, formation of NAPE in 2-day-old cultures (fourfold) as well as in 6-day-old cultures (eightfold). Glutamate exerted its effect via NMDA receptors as seen by the inhibitory action of the NMDA-selective receptor antagonists d -(?)-2-amino-5-phosphonovalerate and N-(1-(2-thienyl)-cyclohexyl)piperidine and the lack of effect of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate-receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In 6-day-old cultures, exposure to NMDA (100 µM for 24 h) induced a linear increase in the formation of NAPE and NAE as well as a 40–50% neuronal death, as measured by a decrease in cellular formazan formation [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay]. The increase in NAPE and NAE could be detected earlier than the neuronal death. Neither cyclic AMP, cyclic GMP, nitric oxide, protein kinase C, nor peroxidation appears to be involved in the formation of NAPE and NAE, as assessed by the use of different pharmacological agents. Exposure to 5 mM NaN₃ for 8 h resulted in a >80% decrease in the cellular MTT staining and a pronounced linear increase in the formation of NAE and NAPE (reaching 25–30% of total labeling). [¹⁴C]Anandamide was also formed in [¹⁴C]arachidonic acid-labeled neurons exposed to NaN₃. No NAPE formation was detected in A23187-stimulated mouse astrocytes, rat Leydig cells and cardiomyocytes, and several other cells. These results suggest that the glutamate-induced formation of NAPE and NAE was mediated by the NMDA receptor and the formation of these lipids may be associated with neuronal death.     

Structure-function relationships in glucagon. Re-evaluation of glucagon-(1-21)

Glucagon-(1-21) was prepared fully synthetically as well as by carboxypeptidase A digestion of natural porcine glucagon. Neither of the two preparations had glucagon agonistic effects with regard to receptor binding or adenylate cyclase activation in purified rat liver plasma membranes. Nor did these preparations contain lipolytic activity in isolated free fat cells. A preliminary batch of glucagon-(1-21) prepared by carboxypeptidase A digestion did, however, contain 1-2% glucagon bioactivity. This activity was separated from glucagon-(1-21) by high-performance liquid chromatography and quantitatively recovered in four minor hind peaks which eluted close to but not in a position identical to the elution position of native glucagon.