Specific expression of annexin III in rat-small-hepatocytes

Small hepatocytes are cells that express characteristic phenotypes such as a high growth potential and differentiation capacity. In order to identify rat-small-hepatocyte specific proteins, we separated the cellular proteins of isolated small and parenchymal hepatocytes by 2D polyacrylamide gel electrophoresis. Comparison of their profiles revealed a protein with a molecular mass of 37 kDa in the small hepatocytes that was not present in the parenchymal hepatocytes. Proteolytic peptide mass fingerprinting was used to identify the protein and it was found to be annexin III. The validity of the identification was confirmed by Western blot analysis with anti-annexin III antibody.     

Proposed mechanism and functional amino acid residues of malonyl-CoA:anthocyanin 5-O-glucoside-6'''-O-malonyltransferase from flowers of Salvia splendens,a member of the versatile plant acyltransferase family

The versatile plant acyltransferase (VPAT) family is a recently identified protein family consisting of acyltransferases involved in secondary metabolism in plants along with numerous homologues with as yet unidentified biochemical functions. Malonyl-CoA:anthocyanin 5-O-glucoside-6' "-O-malonyltransferase of Salvia splendens flowers (Ss5MaT1) is a member of this family that catalyzes the regiospecific transfer of the malonyl group from malonyl-CoA to the 6' "-hydroxyl group of the 5-glycosyl moiety of anthocyanins. To elucidate the mechanism and functional amino acid residues of VPAT family enzymes, steady-state kinetic analyses and site-directed mutagenesis of Ss5MaT1 guided by sequence comparison studies were carried out. On the basis of the results of product and dead-end inhibition studies as well as sequence comparison studies, the kinetic mechanism of Ss5MaT1 could be most consistently described in terms of a ternary complex mechanism in which both substrates and the enzyme form a complex before catalysis can occur, as in the case of chloramphenicol O-acetyltransferase (CAT) and histone acetyltransferase (HAT). Eight polar or ionizable amino acid residues that are invariant among 12 VPAT family enzymes were replaced by alanine, and the mutant enzymes were kinetically characterized. A significant diminution of the k(cat) value was observed with the substitution of His167 (relative k(cat), 0.02%) and Asp390 (<0.01%), strongly suggesting that His167 and Asp390 are very important for catalytic activity. The log k(cat) versus pH plots of the Ss5MaT1-catalyzed malonyl transfer suggested that a deprotonated active site group of pK(a) = 7.0 +/- 0.1 may be involved in the catalytic steps of the "substrate to product" conversion in the ternary enzyme-substrate complex. Taking these lines of evidence together with the suggested similarity of the kinetic and catalytic mechanisms of Ss5MaT1 to those of CAT and HAT, the following Ss5MaT1 mechanism based on general acid/base catalysis was proposed: in the ternary complex, a general base deprotonates the 6' "-hydroxyl group of the anthocyanin substrate, thereby promoting a nucleophilic attack on the carbonyl of the thioester of malonyl-CoA; His167 and Asp390 appear to be involved in the general acid/base mechanism of Ss5MaT1.     

Human umbilical vein endothelium-derived cells retain potential to differentiate into smooth muscle-like cells

Mouse embryonic stem-derived cells were recently shown to differentiate into endothelial and smooth muscle cells. In the present study, we investigated whether human umbilical vein endothelium-derived cells retain the potential to differentiate into smooth muscle cells. Examination of biochemical markers, including basic calponin, SM22alpha, prostaglandin E synthase, von Willebrand factor, and PECAM-1, as well as cell contractility, showed that whereas endothelium-derived cells cultured with fibroblast growth factor can be characterized as endothelial cells, when deprived of fibroblast growth factor, a significant fraction differentiates into smooth muscle-like cells. Reapplication of fibroblast growth factor reversed this differentiation. Activin A was up-regulated in fibroblast growth factor-deprived, endothelium-derived cells; moreover, the inhibitory effects of exogenous follistatin and overexpressed Smad7 on smooth muscle-like differentiation confirmed that the differentiation was driven by activin A signaling. These findings indicate that when deprived of fibroblast growth factor, human umbilical vein endothelium-derived cells are capable of differentiating into smooth muscle-like cells through activin A-induced, Smad-dependent signaling, and that maintenance of the endothelial cell phenotype and differentiation into smooth muscle-like cells are reciprocally controlled by fibroblast growth factor-1 and activin A.     

Recombinant Rhodococcus sp. strain T09 can desulfurize DBT in the presence of inorganic sulfate

The putative Rhodococcus rrn promoter region was cloned from the benzothiophene desulfurizing Rhodococcus sp. strain T09, and the dibenzothiophene desulfurizing gene, dsz, was expressed under the control of the putative rrn promoter in the strain T09 using a Rhodococcus–E.coli shuttle vector. Strain T09 harboring the expression vector, pNT, could desulfurize dibenzothiophene in the presence of inorganic sulfate, methionine, or cysteine, while the Dsz phenotype was completely repressed in recombinant cells carrying the gene under the control of the native dsz promoter under the same conditions. Among the sulfur sources examined, no intermediates were detected and only the final desulfurized product, 2-hydroxy-biphenyl, was produced using ammonium sulfate as the sulfur source. Received: 4 December 2001 / Accepted: 7 January 2002     

High-cut characteristics of the baroreflex neural arc preserve baroreflex gain against pulsatile pressure

A transfer function from baroreceptor pressure input to sympathetic nerve activity (SNA) shows derivative characteristics in the frequency range below 0.8 Hz in rabbits. These derivative characteristics contribute to a quick and stable arterial pressure (AP) regulation. However, if the derivative characteristics hold up to heart rate frequency, the pulsatile pressure input will yield a markedly augmented SNA signal. Such a signal would saturate the baroreflex signal transduction, thereby disabling the baroreflex regulation of AP. We hypothesized that the transfer gain at heart rate frequency would be much smaller than that predicted from extrapolating the derivative characteristics. In anesthetized rabbits (n = 6), we estimated the neural arc transfer function in the frequency range up to 10 Hz. The transfer gain was lost at a rate of -20 dB/decade when the input frequency exceeded 0.8 Hz. A numerical simulation indicated that the high-cut characteristics above 0.8 Hz were effective to attenuate the pulsatile signal and preserve the open-loop gain when the baroreflex dynamic range was finite.     

Strain-specific patterns of autonomic nervous system activity and heart failure susceptibility in mice

Transgenic mice are widely used to study cardiac function, but strain-dependent differences in autonomic nervous system activity (ANSA) have not been explored. We compared 1) short-term pharmacological responses of cardiac rhythm in FVB vs. C57Black6/SV129 wild-type mice and 2) long-term physiological dynamics of cardiac rhythm and survival in tumor necrosis factor (TNF)-alpha transgenic mice with heart failure (TNF-alpha mice) on defined backgrounds. Ambulatory telemetry electrocardiographic recordings and response to saline, adrenergic, and cholinergic agents were examined in FVB and C57Black6/SV129 mice. In FVB mice, baseline heart rate (HR) was higher and did not change after injection of isoproterenol or atropine but decreased with propranolol. In C57Black6/SV129 mice, HR did not change with propranolol but increased with isoproterenol or atropine. Mean HR, but not indexes of HR variability, was an excellent predictor of response to autonomic agents. The proportion of surviving animals was higher in TNF-alpha mice on an FVB background than on a mixed FVB/C57Black6 background. The homeostatic states of ANSA are strain specific, which can explain the interstrain differences in mean HR, pharmacological responses, and survival of animals with congestive heart failure. Strain-specific differences should be considered in selecting the strains of mice used for transgenic and gene targeting experiments.     

Morphology and physiology of the serotonin-immunoreactive putative antennal lobe feedback neuron in the male silkmoth Bombyx mori

In the male silkmoth Bombyx mori, olfactory information is relayed from olfactory receptor neurons in the antennae to the antennal lobe, and then to a variety of protocerebral neuropils. Currently, very little is known about neuromodulators that may affect the dynamics of this olfactory neural network. Immunocytochemical studies have revealed the presence of a serotonin-immunoreactive (SI) neuron that, in several insect species, is thought to provide feedback to the antennal lobe. To date, no studies have revealed details of this neuron's physiology. Using intracellular recording and staining, the silkmoth SI neuron (in two individuals) was first characterized physiologically and then stained with Lucifer Yellow to reveal morphological details. Immunocytochemical methods were also used to confirm the presence of serotonin. The silkmoth SI neuron branched in many important brain neuropils such as the mushroom body, central body, lateral accessory lobe and antennal lobe. The SI neuron in both individuals fired spontaneous, long duration action potentials, and responded to mechanosensory stimuli to the antennae.     

Identification of effector cells for TNFalpha-mediated cytotoxicity against WEHI164S cells

WEHI164S cells were found to be very sensitive targets for in vitro killing in a 6-h culture when liver or splenic lymphocytes were used as effector cells in mice. Of particular interest, a limiting cell-dilution analysis showed that effector cells were present in the liver with a high frequency (1/4,300). In contrast to YAC-1 cells as NK targets, perforin-based cytotoxicity was not highly associated with WEHI164S killing. The major killer mechanism for WEHI164S targets was TNFalpha-mediated cytotoxicity. By cell sorting experiments, both NK cells and intermediate T cells (i.e., TCR(int) cells) were found to contain effector cells against WEHI164S cells. However, the killer mechanisms underlying these effector cells were different. Namely, NK cells killed WEHI164S cells by perforin-based cytotoxicity, TNFalpha-mediated cytotoxicity, Fas ligand cytotoxicity, and other mechanisms, whereas intermediate T cells did so mainly by TNFalpha-mediated cytotoxicity. These results suggest that TNFalpha-mediated cytotoxicity mediated by so-called natural cytotoxic (NC) cells comprised events which were performed by both NK and intermediate T cells using somewhat different killer mechanisms. Intermediate T cells which were present in the liver were able to produce TNFalpha if there was appropriate stimulation.     

Stationary and time-resolved resonance Raman spectra of His77 and Met95 mutants of the isolated heme domain of a direct oxygen sensor from Escherichia coli

The heme environments of Met(95) and His(77) mutants of the isolated heme-bound PAS domain (Escherichia coli DOS PAS) of a direct oxygen sensing protein from E. coli (E. coli DOS) were investigated with resonance Raman (RR) spectroscopy and compared with the wild type (WT) enzyme. The RR spectra of both the reduced and oxidized WT enzyme were characteristic of six-coordinate low spin heme complexes from pH 4 to 10. The time-resolved RR spectra of the photodissociated CO-WT complex had an iron-His stretching band (nu(Fe-His)) at 214 cm(-1), and the nu(Fe-CO) versus nu(CO) plot of CO-WT E. coli DOS PAS fell on the line of His-coordinated heme proteins. The photodissociated CO-H77A mutant complex did not yield the nu(Fe-His) band but gave a nu(Fe-Im) band in the presence of imidazole. The RR spectrum of the oxidized M95A mutant was that of a six-coordinate low spin complex (i.e. the same as that of the WT enzyme), whereas the reduced mutant appeared to contain a five-coordinate heme complex. Taken together, we suggest that the heme of the reduced WT enzyme is coordinated by His(77) and Met(95), and that Met(95) is displaced by CO and O(2). Presumably, the protein conformational change that occurs upon exchange of an unknown ligand for Met(95) following heme reduction may lead to activation of the phosphodiesterase domain of E. coli DOS.     

Overexpression of the wasabi defensin gene confers enhanced resistance to blast fungus ( Magnaporthe grisea) in transgenic rice

Transgenic rice ( Oryza sativa cv. Sasanishiki) overexpressing the wasabi defensin gene, a plant defensin effective against the rice blast fungus, was generated by Agrobacterium tumefaciens-mediated transformation. Twenty-two T2 homozygous lines harboring the wasabi defensin gene were challenged by the blast fungus. Transformants exhibited resistance to rice blast at various levels. The inheritance of the resistance over generations was investigated. T3 plants derived from two highly blast-resistant T2 lines (WT14-5 and WT43-5) were challenged with the blast fungus using the press-injured spots method. The average size of disease lesions of the transgenic line WT43-5 was reduced to about half of that of non-transgenic plants. The 5-kDa peptide, corresponding to the processed form of the wasabi defensin, was detected in the total protein fraction extracted from the T3 progeny. Transgenic rice plants overproducing wasabi defensin are expected to possess a durable and wide-spectrum resistance (i.e. field resistance) against various rice blast races.