Cloning and characterization of a novel fold-type I branched-chain amino acid aminotransferase from the hyperthermophilic archaeon Thermococcus sp. CKU-1

We successfully cloned a novel branched-chain amino acid aminotransferase (Ts-BcAT; EC 2.6.1.42) gene from the Thermococcus sp. CKU-1 genome and expressed it in the soluble fraction of Escherichia coli Rosetta (DE3) cells. Ts-BcAT is a homodimer with an apparent molecular mass of approximately 92 kDa. The primary structure of Ts-BcAT showed high homology with the fold-type I, subgroup I aminotransferases, but showed little homology with BcATs known to date, i.e., those of Escherichia coli and Salmonella typhimurium, which belong to the fold-type IV, subgroup III aminotransferases. The maximum enzyme activity of Ts-BcAT was detected at 95 °C, and Ts-BcAT did not lose any enzyme activity, even after incubation at 90 °C for 5 h. Ts-BcAT was active in the pH range from 4.0 to 11.0, the optimum pH was 9.5, and the enzyme was stable between pH 6 and 7. The exceptionally low pK _a of the nitrogen atom in the Lys258 ε-amino group in the internal aldimine bond of Ts-BcAT was determined to be 5.52 ± 0.05. Ts-BcAT used 21 natural and unnatural amino acids as a substrate in the overall transamination reaction. l-Leucine and other aliphatic amino acids are efficient substrates, while polar amino acids except glutamate were weak substrates. Phylogenetic analysis revealed that Ts-BcAT is a novel fold-type I, subgroup I branched-chain aminotransferase.     

Visualization of erythrocytes in the zebrafish brain

We found that erythrocytes of zebrafish have cytoplasmic peroxidase activity. Blood in the zebrafish brain was visualized using a standard peroxidase staining method after formaldehyde fixation. The erythrocytes in the brain were heavily stained, but neurons and glias were not stained at all. This easy method enables the distribution of erythrocytes in the whole brain to be determined, and enables the actual number of erythrocytes in each area in the brain to be calculated. The paths of major, thick blood vessels in zebrafish brain are similar to those in higher vertebrates, however, the distribution of thin blood vessels is different. We also found that the erythrocytes were unevenly distributed in the brain. For example, the density of erythrocytes in the surface layer of the tectum was more than 30-fold higher than in the deeper granular layer. Very few erythrocytes were found in bundles of axons like cranial nerves and the medial longitudinal fascicle. In general, fewer erythrocytes were found in areas near the ventricle, whereas many more were found closer to the surface of the brain. The distribution of erythrocytes in the brains of sleeping, awake and actively moving fish were compared. In the brains of sleeping fish, most of the erythrocytes were present in large vessels. This was not observed in brains of awake or actively moving fish. We found that the blood supply to motor neurons in the ventral horn of the spinal cord increased during active movement compared to that in awake or sleeping fish.     

Taenia solium taeniasis/cysticercosis in Papua,Indonesia in 2001: detection of human worm carriers

A preliminary study to detect human worm carriers of Taenia solium in Papua (Irian Jaya), Indonesia was carried out using stool examinations for the detection of copro-antigens and adult proglottids after chemotherapy, and confirmation by mitochondrial DNA analysis using expelled proglottids and metacestodes developed in NOD/Shi-scid mice from eggs of expelled proglottids. Approximately 8.6% of the local population in Kama (5/58), 1 km from the local capital city centre, Wamena, were confirmed to harbour adult T. solium using these techniques.     

NBT-II carcinoma behaviour is not dependent on cell-cell communication through gap junctions

To study the mechanism(s) underlying the proliferation of heterogeneous cell populations within a solid tumour, the NBT-II rat bladder carcinoma system was used. It has been first investigated whether the different cell populations are coupled through gap junctions (GJIC). Cells overexpressing the Cx43 were generated to test for any tumour suppressive activity in vivo. To determine whether GJIC is essential for tumour proliferation and the establishment of a cooperative community effect, NBT-II cells that are incompetent for cell coupling were generated. The data report that (i) carcinoma cells expressing or not FGF-1 are coupled through GJIC in vitro and in coculture and express the gap junction protein Cx43, (ii) overexpression of Cx43 in these cells does not affect their in vitro coupling capacities and in vivo tumourigenic growth properties, (iii) inhibition of GJIC through antisense strategy has no in vivo obvious consequence on the tumour growth properties of the carcinoma, and (iv) the community effect between two carcinoma cell populations does not critically involve cell coupling through gap junctions.     

Cloning and analysis of the telomere and terminal inverted repeat of the linear chromosome of Streptomyces griseus

Cloning and sequencing of the telomere of Streptomyces griseus revealed five palindromic sequences in the terminal 116 nucleotides, all of which can make a hairpin loop structure. However, the end sequence cannot form the foldback secondary structure that is common in Streptomyces telomeres and is suggested to be necessary for terminal replication. Both inside ends of the terminal inverted repeat (TIR) were also cloned and sequenced. The results confirmed the size of the TIR to be 24 kb and identified two almost identical open reading frames that might have been involved in the formation of the TIR.     

Nkx2.5 and Nkx2.6, homologs of Drosophila tinman, are required for development of the pharynx

Nkx2.5 and Nkx2.6 are murine homologs of Drosophila tinman. Their genes are expressed in the ventral region of the pharynx at early stages of embryogenesis. However, no abnormalities in the pharynges of embryos with mutations in either Nkx2.5 or Nkx2.6 have been reported. To examine the function of Nkx2.5 and Nkx2.6 in the formation of the pharynx, we generated and analyzed Nkx2.5 and Nkx2.6 double-mutant mice. Interestingly, in the double-mutant embryos, the pharynx did not form properly. Pharyngeal endodermal cells were largely missing, and the mutant pharynx was markedly dilated. Moreover, we observed enhanced apoptosis and reduced proliferation in pharyngeal endodermal cells of the double-mutant embryos. These results demonstrated a critical role of the NK-2 homeobox genes in the differentiation, proliferation, and survival of pharyngeal endodermal cells. Furthermore, the development of the atrium was less advanced in the double-mutant embryos, indicating that these two genes are essential for both pharyngeal and cardiac development.     

Activation of histamine H3 receptors inhibits renal noradrenergic neurotransmission in anesthetized dogs

To investigate the possible involvement of histamine H(3) receptors in renal noradrenergic neurotransmission, effects of (R)alpha-methylhistamine (R-HA), a selective H3-receptor agonist, and thioperamide (Thiop), a selective H3-receptor antagonist, on renal nerve stimulation (RNS)-induced changes in renal function and norepinephrine (NE) overflow in anesthetized dogs were examined. RNS (0.5-2.0 Hz) produced significant decreases in urine flow and urinary sodium excretion and increases in NE overflow rate (NEOR), without affecting renal hemodynamics. When R-HA (1 microg x kg(-1) x min(-1)) was infused intravenously, mean arterial pressure and heart rate were significantly decreased, and there was a tendency to reduce basal values of urine flow and urinary sodium excretion. During R-HA infusion, RNS-induced antidiuretic action and increases in NEOR were markedly attenuated. Thiop infusion (5 microg x kg(-1) x min(-1)) did not affect basal hemodynamic and excretory parameters. Thiop infusion caused RNS-induced antidiuretic action and increases in NEOR similar to the basal condition. When R-HA was administered concomitantly with Thiop infusion, R-HA failed to attenuate the RNS-induced antidiuretic action and increases in NEOR. However, in the presence of pyrilamine (a selective H1-receptor antagonist) or cimetidine (a selective H2-receptor antagonist) infusion, R-HA attenuated the RNS-induced actions, similarly to the case without these antagonists. Thus functional histamine H3 receptors, possibly located on renal noradrenergic nerve endings, may play the role of inhibitory modulators of renal noradrenergic neurotransmission.     

Characteristics of body heat balance of paraplegics during exercise in a hot environment

The purpose of this investigation was to clarify the characteristics of body temperature regulation in paraplegics due to spinal cord injury (SCI) during an arm cranking exercise in a hot environment. Twelve paraplegics with lesions located between Th3 and L1,2 and seven able-bodied subjects (AB) participated in this study. The subjects were exposed to a hot (33 degrees C) or a moderate temperature (25 degrees C) environment for one hour and during the last 10 min of the exposure, the subjects performed arm cranking exercises at an exercise intensity of 40 W. The skin temperatures at the chest, the upper arm, the thigh and the calf, the tympanic membrane temperature (Tty), and the skin blood flow of the thigh (SBFT) were continuously monitored during the experiment. Although no systematical variation was found in the Tty at 25 degrees C, the Tty at 33 degrees C in paraplegics during exercise was significantly greater than that at rest (P < 0.01), which indicated a pronounced heat stress for paraplegics at 33 degrees C. SBFT of paraplegics with high lesions of the SCI remained unchanged during the experiment at 25 degrees C and 33 degrees C, while paraplegics with low lesions in this study showed consecutive increases in SBFT during exercise in both environmental conditions similar to AB. The increased core temperature in paraplegics with high lesions was considered to be due to a lack of sweat response and vasomotor activity in the paralyzed area. On the basis of the findings in this study, it can be suggested that high core temperature without any increment of SBFT may be characterized as body heat balance of paraplegics with high lesions during exercise in a hot environment.