Serum but not myocardial TNF-alpha concentration is increased in pacing-induced heart failure in rabbits

In animals and patients with severe heart failure (HF), the serum tumor necrosis factor-alpha (TNF-alpha) concentration is increased. It is, however, still controversial whether or not such increased serum TNF-alpha originates from the heart itself or is of peripheral origin secondary to gastrointestinal congestion and increased endotoxin concentration. We therefore now examined TNF-alpha in serum, myocardium, and liver of sham-operated and HF rabbits. In nine rabbits in which HF was induced by left ventricular (LV) pacing at 400 beats/min for 3 wk, LV end-diastolic diameter was increased and systolic shortening fraction (9.4 +/- 1.0 vs. 28.5 +/- 1.3%, echocardiography, P < 0.05) was reduced. Serum TNF-alpha was higher in HF than in sham-operated rabbits (240 +/- 24 vs. 150 +/- 22 U/ml, WEHI-cell assay, P < 0.05). In the heart, TNF-alpha was located mainly in the vascular endothelium (immunohistochemistry), and TNF-alpha protein (920 +/- 160 vs. 900 +/- 95 U/g) did not differ between groups. In the liver of HF rabbits, hepatocytes expressed TNF-alpha, and TNF-alpha protein was increased compared with sham-operated rabbits (2,390 +/- 310 vs. 1,220 +/- 135 U/g, P < 0.05) and correlated to the number of hepatic leukocytes (r = 0.85) and serum TNF-alpha (r = 0.69). The intestinal endotoxin concentration was 24.5 +/- 1.2 vs. 17.0 +/- 3.1 endotoxin units/g wet wt (P < 0.05) in HF compared with sham-operated rabbits. In this HF model, serum but not myocardial TNF-alpha is increased. The increased serum TNF-alpha originates from peripheral sources.     

An experimental study on the formation of anterior-posterior polarity in the early development of the marine hydroid Dynamena pumila

Elements of evolutionarily initial morphogenesis providing for the formation of main body axes could have been preserved in embryogenesis of lower Metazoa animals, Cnidaria. However, the information on the morphological bases of axes formation in their normal development is not yet complete. When studying the normal development of Dynamena pumila (Hydrozoa, Thecaphora, Sertulariidae), it has been proposed that the region, where the embryonic ectoblast remained unclosed for the longest time, determines the position of the posterior pole of the larval anteroposterior axis. In the experiments, the formation of closed ectoblast in an arbitrarily chosen region of the embryonic surface was delayed artificially, for example, by incisions. The fate of this region was traced with the help of a mark consisting of carmine particles. It was shown that the posterior pole did differentiate near the region of surface, which was the last to epithelize and redetermination of the anteroposterior axis orientation was only possible before the formation of closed ectoblast in the normal development. The morphogeneses involved in the formation of anteroposterior axis and its poles in Dynamena embryos were reconstructed by means of observations over the displacement of mark particles. It was shown that the establishment of this axis and appearance of morphological differences between the anterior and posterior planula poles are morphogenetic consequences of the closed ectoblast formation. The region, in which the closure of ectoblast is delayed, is a functional analog of the blastopore of higher Metazoa.     

Increase of functional diversity by alternative splicing

A large-scale analysis of protein isoforms arising from alternative splicing shows that alternative splicing tends to insert or delete complete protein domains more frequently than expected by chance, whereas disruption of domains and other structural modules is less frequent. If domain regions are disrupted, the functional effect, as predicted from 3D structure, is frequently equivalent to removal of the entire domain. Also, short alternative splicing events within domains, which might preserve folded structure, target functional residues more frequently than expected. Thus, it seems that positive selection has had a major role in the evolution of alternative splicing.     

Early changes in pancreatic acinar cell calcium signaling after pancreatic duct obstruction

Intracellular Ca(2+)-changes not only participate in important signaling pathways but have also been implicated in a number of disease states including acute pancreatitis. To investigate the underlying mechanisms in an experimental model mimicking human gallstone-induced pancreatitis, we ligated the pancreatic duct of Sprague-Dawley rats and NMRI mice for up to 6 h and studied intrapancreatic changes including the dynamics of [Ca(2+)](i) in isolated acini. In contrast to bile duct ligation, pancreatic duct obstruction induced intra-pancreatic trypsinogen activation, leukocytosis, hyperamylasemia, and pancreatic edema and increased lung myeloperoxidase activity. Although resting [Ca(2+)](i) in isolated acini rose by 45% to 205 +/- 7 nmol, the acetylcholine- and cholecystokinin (CCK)-stimulated calcium peaks as well as the amylase secretion declined, but neither the [Ca(2+)](i)-signaling pattern nor the amylase output in response to the Ca(2+)-ATPase inhibitor thapsigargin nor the secretin-stimulated amylase release were impaired by pancreatic duct ligation. On the single cell level pancreatic duct ligation reduced the percentage of cells in which submaximal secretagogue stimulation was followed by a physiological response (i.e. Ca(2+) oscillations) and increased the percentage of cells with a pathological response (i.e. peak plateau or absent Ca(2+) signal). Moreover, it reduced the frequency and amplitude of Ca(2+) oscillation as well as the capacitative Ca(2+) influx in response to secretagogue stimulation. Serum pancreatic enzyme elevation as well as trypsinogen activation was significantly reduced by pretreatment of animals with the calcium chelator BAPTA-AM. These experiments suggest that pancreatic duct obstruction rapidly changes the physiological response of the exocrine pancreas to a Ca(2+)-signaling pattern that has been associated with premature digestive enzyme activation and the onset of pancreatitis, both of which can be prevented by administration of an intracellular calcium chelator.     

Construction of mini-Tn4001tet and its use in Mycoplasma gallisepticum

The Mollicutes are a group of cell-wall-less bacteria and are important plant and animal pathogens. Progress toward analyzing their pathogenic mechanisms has been hampered by the few available genetic tools. Of the two transposons shown to function in mycoplasmas, only Tn4001 is readily amenable to modification and development. One disadvantage of using Tn4001 in mycoplasmas has been independent insertion of the insertion sequence, IS256, probably as a result of inadequate control of the transposase expression in mycoplasmas. In this study, we describe the construction of a mini-Tn4001 containing the tetM antibiotic resistance gene from Tn916. The transposase gene was placed outside the inverted repeats to lower the frequency of independent transposition events. Transposition of mini-Tn4001tet in Mycoplasma gallisepticum occurred at a frequency of 1-8 x 10(-6), a frequency similar to that of the parent transposon. Insertions of mini-Tn4001tet were random and only single insertions were observed. Several unique restriction sites between the inverted repeat sequences provide for further development of mini-Tn4001.     

Dizocilpine Inhibits Amphetamine-Induced Formation of Nitric Oxide and Amphetamine-Induced Release of Amino Acids and Acetylcholine in the Rat Brain

Glutamate receptor activation participates in mediation of neurotoxic effects in the striatum induced by the psychomotor stimulant amphetamine. The effects of the non-competitive NMDA receptor antagonist dizocilpine (MK-801) on amphetamine-induced toxicity and formation of nitric oxide (NO) in both striatum and cortex and on induced transmitter release in the nucleus accumbens were investigated. Repeated, systemic application of amphetamine elevated striatal and cortical lipid peroxidation and NO production. Moreover, amphetamine caused an immediate release of acetylcholine and aspartate and a delayed release of GABA in the nucleus accumbens. Surprisingly, glutamate release was not affected. Dizocilpine abolished the amphetamine-induced lipid peroxidation and NO production in striatum and cortex and diminished the elevation of neurotransmitter release. These findings suggest that amphetamine evokes neurotoxic effects in both striatal and cortical brain areas that are prevented by inhibiting NMDA receptor activation. The amphetamine-induced acetylcholine, aspartate and GABA release in the nucleus accumbens is also mediated through NMDA receptor-dependent mechanisms. Interestingly, the enhanced aspartate release might contribute to NMDA receptor activation in the nucleus accumbens, while glutamate does not seem to mediate amphetamine-evoked transmitter release in this striatal brain area.     

Theoretical analysis of alternative splice forms using computational methods

Nowadays understanding alternative splicing is one of the greatest challenges in biology, because it is a genetic process much more important than thought at the time of its discovery. In this paper, we explain the approach of using the different available databases and software tools to start a large scale investigation of alternative splice forms. To collect information about alternative splicing we investigated known data in the databases using different computational methods. The investigations proceeded from the genomic sequence data to structural protein data. Then, we interpreted those data to find the relationship between alternative splice forms and protein function and structure. We found some interesting features of alternative splicing which are presented here. We discuss the results of one chosen example. They concern the coverage quality of the protein sequence of a known structure, an EST analysis, the validation of splice variants, the determination of the alternative splice type, and finally the link between alternative splicing and disease.     

Cystathionine beta-synthase is coordinately regulated with proliferation through a redox-sensitive mechanism in cultured human cells and Saccharomyces cerevisiae

Cystathionine beta-synthase (CBS) catalyzes the condensation of serine with homocysteine to form cystathionine and occupies a crucial regulatory position between the methionine cycle and the biosynthesis of cysteine by transsulfuration. Analysis of CBS activity under a variety of growth conditions indicated that CBS is coordinately regulated with proliferation in both yeast and human cells. In batch cultures of Saccharomyces cerevisiae, maximal CBS activities were observed in the exponential phase of cells grown on glucose, while growth-arrested cultures or those growing non-fermentatively on ethanol or glycerol had approximately 3-fold less activity. CBS activity assays and Western blotting indicated that growth-specific regulation of CBS is evolutionarily conserved in a range of human cell lines. CBS activity was found to be maximal during proliferation and was reduced two- to five-fold when cells became quiescent at confluence. In cultured HepG2 cells, the human CBS gene is induced by serum and basic fibroblast growth factor and is downregulated, but not abolished, by contact inhibition, serum-starvation, nutrient depletion, or the induction of differentiation. Consequently, for certain cell types, CBS may represent a novel marker of both differentiation and proliferation. The intracellular level of the CBS regulator compound, S-adenosylmethionine, was found to reflect the proliferation status of both yeast and human cells, and as such, constitutes an additional mechanism for proliferation-specific regulation of human CBS. Our data indicates that screening compounds for the ability to affect transsulfuration in cultured cell models must take proliferation status into account to avoid masking regulatory interactions that may be of significance in vivo.