C-terminal region regulates the functional expression of human noradrenaline transporter splice variants

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl--dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.     

Purification,Crystallization and Properties of Tyrosine Phenol Lyase from Erwinia herbicola

Crystalline tyrosine phenol lyase was prepared from the cell extract of Erwinia herbicola grown in a medium supplemented with l-tyrosine. The crystalline enzyme was homogeneous by the criteria of ultracentrifugation and acrylamide gel electrophoresis. The molecular weight was determined to be approximately 259,000. The crystalline enzyme catalyzed the conversion of l-tyrosine into phenol, pyruvate and ammonia, in the presence of added pyridoxal phosphate. The enzyme also catalyzed pyruvate formation from d-tyrosine, S-methyl-l-cysteine, 3, 4-dihydroxyphenyl-l-alanine, l- and d-serine, and l- and d-cysteine, but at lower rates than from l-tyrosine. l-Phenyl-alanine, l-alanine, phenol and pyrocatechol inhibited pyruvate formation from l-tyrosine.

Crystalline tyrosine phenol lyase from Erwinia herbicola is inactive in the absence of added pyridoxal phosphate. Binding of pyridoxal phosphate to the apoenzyme is accompanied by pronounced increase in absorbance at 340 and 425 mμ. The amount of pyridoxal phosphate bound to the apoenzyme was determined by equilibrium dialysis to be 2 moles per mole of enzyme. Addition of the substrate, l-tyrosine, or the competitive inhibitors, l-alanine and l-phenyl-alanine, to the holoenzyme causes appearance of a new absorption peak near 500 mμ which disappears as the substrate is decomposed but remains unchanged in the presence of the inhibitor.     

Interleukin-1β Induces Substance P Release from Primary Afferent Neurons Through the Cyclooxygenase-2 System

Substance P (SP) is synthesized in the dorsal root ganglion (DRG) and released from primary afferent neurons to convey information regarding noxious stimuli. The effects of the proinflammatory cytokine interleukin-1 (IL-1) beta on the release of SP were investigated using primary cultured rat DRG cells. Recombinant mouse IL-1beta added to the cells at 0.1 ng/ml increased the SP-like immunoreactivity (SPLI) in the culture medium after incubation for 6 h by approximately 50% as compared with that of nontreated DRG cells. The effect of IL-1beta was Ca(2+)-dependent and significantly inhibited by 100 ng/ml IL-1 receptor-specific antagonist (IL-1r antagonist), cyclooxygenase (COX) inhibitors such as 0.1 mM aspirin, 1 microg/ml indomethacin, and 1 microM NS-398 (specific for COX-2), and 1 microM dexamethasone. Furthermore, a 1-h incubation with IL-1beta markedly increased the inducible COX-2 mRNA level, which was inhibited by an IL-1r antagonist and dexamethasone, whereas IL-1beta showed no effect on the level of constitutive COX-1 mRNA. These observations indicated that IL-1beta induced the release of SP from the DRG cells via specific IL-1 receptors, the mechanism of which might involve prostanoid systems produced by COX-2. This could be responsible for the hyperalgesic action with reference to inflammatory pain in the primary afferent neuron to spinal cord pathway.     

Cigarette smoke extract induces endothelial cell injury via JNK pathway

Cigarette smoking is the most crucial factor responsible for chronic obstructive pulmonary disease (COPD). The precise mechanisms of the development of the disease have, however, not been fully understood. Recently, impairment of pulmonary endothelial cells has been increasingly recognized as a critical pathophysiological process in COPD. To verify this hypothesis, we examined how cigarette smoke extract (CSE) damages human umbilical vein endothelial cells (HUVECs). CSE activated c-Jun N-terminal kinase (JNK), and treatment of HUVECs with SP600125, a specific inhibitor of the JNK pathway, significantly suppressed endothelial cell damage by CSE. In contrast, inhibition of the extracellular-regulated kinase or the p38 pathway did not affect the cytotoxicity of CSE. Furthermore, anti-oxidants superoxide dismutase and catalase reduced CSE-induced JNK phosphorylation and endothelial cell injury. These results indicate that CSE damages vascular endothelial cells through the JNK pathway activated, at least partially, by oxidative stress.     

Selective Enrichment with a Resuscitation Step for Isolation of Freeze-Injured Escherichia coli O157:H7 from Foods

We studied injury of Escherichia coli O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured E. coli O157:H7 cells from foods. Food samples inoculated with E. coli O157:H7 were stored for 16 weeks at −20°C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of E. coli O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detect E. coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified E. coli broth without bile salts at 25°C for 2 h and then selectively enriched at 42°C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating E. coli O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25°C in nonselective broth improved recovery of E. coli O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating E. coli O157:H7 from frozen foods contaminated with injured E. coli O157:H7 cells.     

Neuronal nitric oxide synthase (NNOS) catalyzes one-electron reduction of 2,4,6-trinitrotoluene, resulting in decreased nitric oxide production and increased nNOS gene expression: implication for oxidative stress

To determine the mechanism of 2,4,6-trinitrotoluene (TNT)-induced oxidative stress involving neuronal nitric oxide synthase (nNOS), we examined alterations in enzyme activity and gene expression of nNOS by TNT, with an enzyme preparation and rat cerebellum primary neuronal cells. TNT inhibited nitric oxide formation (IC(50) = 12.4 microM) as evaluated by citrulline formation in a 20,000 g cerebellar supernatant preparation. A kinetic study revealed that TNT was a competitive inhibitor with respect to NADPH and a noncompetitive inhibitor with respect to L-arginine. It was found that purified nNOS was capable of reducing TNT, with a specific activity of 3900 nmol of NADPH oxidized/mg/min, but this reaction required CaCl(2)/calmodulin (CaM). An electron spin resonance (ESR) study indicated that superoxide (O(2)(.-)) was generated during reduction of TNT by nNOS. Exposure of rat cerebellum primary neuronal cells to TNT (25 microM) caused an intracellular generation of H(2)O(2), accompanied by a significant increase in nNOS mRNA levels. These results indicate that CaM-dependent one-electron reduction of TNT is catalyzed by nNOS, leading to a reduction in NO formation and generation of H(2)O(2) derived from O(2)(.-). Thus, it is suggested that upregulation of nNOS may represent an acute adaptation to an increase in oxidative stress during exposure to TNT.     

Molecular cloning of Mucor hiemalis endo-beta-N-acetylglucosaminidase and some properties of the recombinant enzyme

Endo-M, endo-beta-N-acetylglucosaminidase from Mucor hiemalis, is known as a useful enzyme for the synthesis of neoglycopeptides due to its transglycosylation activity. We cloned the Endo-M gene encoding a putative 744 amino acids, which shows high identity to glycoside hydrolase family 85 endo-beta-N-acetylglucosaminidases. The gene encoding Endo-M was expressed in protease-deficient Candida boidinii with a molecular mass of 85 kDa as a monomeric form. Recombinant Endo-M could liberate both high-mannose type and biantennary complex type oligosaccharides from glycopeptides, which was same as the native enzyme. The Km and Kcat values for DNS-Man6GlcNAc2Asn were 0.51 mM and 8.25 s(-1), respectively. Recombinant Endo-M also exhibited transglycosylation activity toward high-mannose type and biantennary complex type oligosaccharides, which were transferred to alcohols, monosaccharides, oligosaccharides, and glycosides. To investigate about the catalytically essential amino acids of Endo-M, site-directed mutagenesis was performed, and it was found that mutants E177G and E177Q completely abolished the hydrolytic activity and W228R partially abolished the transglycosylation activity.     

Sex-biased dispersal and growth in sablefish (Anoplopoma fimbria) in the northeastern Pacific Ocean

To examine the potential linkage between sex-biased growth and dispersal in demersal fish, we studied the movement distance and growth of tag-recaptured sablefish (Anoplopoma fimbria). Tagging was conducted by teams from Japan and the United States. From 1980 to 1984, 58,918 sablefish were captured by Japanese vessels and tagged with anchor tags, and between 1980 and 2007, 3319 sablefish were recaptured in the Bering Sea and northeastern Pacific. Of these 3319 fish, we analyzed data on 659 individuals for which we have reliable data on recapture location, sex and fork length at the time of release and recapture, and elapsed time longer than 10?days. Annual growth increments (mean±S.D.) were 2.5?±?6.5?cm (males) and 3.5?±?8.6?cm (females), and the movement distances (assumed to be straight) were 712?±?800?km (males) and 877?±?920?km (females). Females grew larger and moved longer distances than males. We calculated the growth increment deviation that accounted for the effects of elapsed time, curvilinear growth, and initial fork length at release using a von Bertalanffy growth model. The growth increment deviation increased significantly with the distance moved over elapsed time. In addition, the relationship between distance moved over elapsed time and the growth deviation differed significantly between sexes; females grew faster than males at a given movement distance. We suggest that female-biased dispersal is a factor generating female-biased growth in sablefish.     

VopB1 and VopD1 are essential for translocation of type III secretion system 1 effectors of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a pathogenic Vibrio species that causes food-borne acute gastroenteritis, often related to the consumption of raw or undercooked seafood. Vibrio parahaemolyticus has 2 type III secretion systems (T3SS1 and T3SS2). Here, we demonstrate that VP1657 (VopB1) and VP1656 (VopD1), which share sequence similarity with Pseudomonas genes popB (38%) and popD (36%), respectively, are essential for translocation of T3SS1 effectors into host cells. A VP1680CyaA fusion reporter system was constructed to observe effector translocation. Using this reporter assay we showed that the VopB1 and VopD1 deletion strains were unable to translocate VP1680 to host cell but that the secretion of VP1680 into the culture medium was not affected. VopB1 or VopD1 deletion strains did not enhance cytotoxicity and failed to activate mitogen-activated protein kinases and secretion of interleukin-8, which depend on VP1680. Thus, we conclude that VopB1 and VopD1 are essential components of the translocon. To target VopB1 and VopD1 may have therapeutic potential for the treatment or prevention in V.?parahaemolyticus infection.     

Effect of hypophysectomy and growth hormone administration on somatostatin and gastrin content in the stomach of rats

The effect of hypophysectomy and bovine growth hormone (GH) administration on somatostatin (SRIF) content as well as gastrin content in the rat stomach was investigated. SRIF content was determined by a specific radioimmunoassay. The total SRIF content in the stomach had decreased 4 weeks after hypophysectomy but was restored significantly in those rats which were subjected to bovine GH administration for 7 days after hypophysectomy. Furthermore, in control rats, an increase in SRIF content in the stomach was observed after 7 days of GH administration. Similar changes in total content of gastrin were observed after hypophysectomy and bovine GH administration, although these changes were not significant. These results indicate that GH may influence gastric function through changes in SRIF and gastrin content in the stomach.