l-Glutamic Acid Fermentation with Molasses

The relation between oleate and biotin to the extracellular accumulation of l-glutamate in Microbacterium ammoniaphilum was studied. And it was suggested that oleate was the essential constituent for the bacterial cell structure, and, at the same time, it participated in the cellular permeability of l-glutamate. On the other hand, biotin was recognized to play a role on the synthesis of cellular fatty acid, mainly oleate and palmitate. Through the discussion above mentioned, the reason was made clear that biotin was not necessary for the bacterial growth or the extracellular accumulation of l-glutamate, if oleate had been added.     

Epidermal growth factor receptor is modulated by redox through multiple mechanisms. Effects of reductants and H2O2.

The cellular redox state has been shown to play an essential role in cellular signaling systems. Here we investigate the effects of reductants and H2O2 on the signaling of epidermal growth factor (EGF) in cells. H2O2 induced the phosphorylation of the EGF receptor and the formation of a receptor complex comprising Shc, Grb2, Sos, and the EGF receptor. Dimerization or oligomerization of the EGF receptor was not induced by H2O2. Protein tyrosine phosphatase (PTP) assay showed that H2O2 suppressed dephosphorylation of the EGF receptor in cell lysates, suggesting that inactivation of PTP was involved in H2O2-induced activation of the EGF receptor. In contrast, the reductants N-acetyl-L-cysteine [Cys(Ac)] and dithiothreitol markedly suppressed EGF-induced dimerization and activation of the EGF receptor in cells. In accordance with suppression of the EGF receptor, Cys(Ac) suppressed EGF-induced activation of Ras, phosphatidylinositol 3-kinase and mitogen-activated protein kinase. Dithiothreitol completely inhibited EGF binding and kinase activation of the EGF receptor both in vitro and in vivo. In contrast, Cys(Ac) suppressed high-affinity EGF-binding sites on the cells, but had no effect on low-affinity binding sites. Furthermore, Cys(Ac) did not suppress EGF-induced kinase activation or dimerization of the EGF receptor in vitro, indicating that it suppressed the EGF receptor through a redox-sensitive cellular process or processes. Thus, the EGF receptor is regulated by redox through multiple steps including dephosphorylation by PTP, ligand binding, and a Cys(Ac)-sensitive cellular process or processes.     

Siphonogobius nue, a new genus and species of gobiid fish from japan

A new genus and species of gobiid fish,Siphonogobius nue, based on 15 specimens, is described from temperate Pacific coasts of Japan. It is unique amongst gobioids in having a simple tube-like infraorbital canal extending below the eye and the oculoscapular canal between pores A' and L' without openings, except for the terminal pores and pore D. Although well-developed infraorbital canals occur in two other gobioid genera, the rhyacichthyidRhyacichthys and the gobiidLophiogobius, such are distinct from that ofSiphonogobius in having several branches and openings. Furthermore, the new taxon is distinguished from all other gobioids by the following combination of characters: first dorsal-fin spines 7–9 (usually 8); pectoral-fin rays 24–26, the upper 7–9 rays free from the fin membrane; pelvic frenum thick, robust and slightly emarginate posteriorly; scales small and cycloid, 87–96 in longitudinal series; vertebrae 14–15+19–20=33–34 (usually 14+20=34); P-V usually 3/I II II I I I 0/11; anal-fin pterygiophores anterior to the first haemal spine 3–4 (usually 3); a row of short fleshy flaps on the ventral surface of the head, extending from the lower edge of the preopercle to the chin; anterior nostril with a minute skin flap. Axial skeletal features indicated thatSiphonogobius is closely related to a group which includesAcanthogobius and its relatives.     

Asterropteryx atripes, a new gobiid fish from the Western Pacific Ocean (Perciformes: Gobioidei)

 A new gobiid fish, Asterropteryx atripes, is described on the basis of eight specimens from Iriomote-jima Island, Ryukyu Islands, and El Nido, Philippines. It differs from its congeners by having the following combination of characters: 3rd spine of first dorsal fin long, filamentous, distal tip usually over end of 2nd dorsal fin base when appressed in both sexes; pelvic fins almost separated, innermost (=5th) segmented rays connected by rudimentary low membrane between bases, and no frenum; 4–7 short spines on posterior margin of preopercle (the uppermost spine usually just behind the cephalic sensory canal pore N); eye large, 32.3–35.8% of head length; enlarged haemal arches on 1st two caudal vertebrae; a distinct black band from posterior margin of eye to caudal fin base (indistinct in dark-phase individuals); black pelvic fin (vivid in dark-phase individuals); numerous minute bright blue spots on head and body in life; no distinct dark spots on head and body; iris entirely reddish-brown or dusky (bright white ventrally in pale-phase individuals) when alive or fresh, and entirely black in preservation, without white transverse bar on middorsal surface; hovering habit. The new species appears to be most closely related with the other only known hovering species, A. striata; the latter is readily distinguished from the former in having no long, filamentous dorsal spine; semitranslucent pelvic fin; and a series of small black spots along dorsal fin base and dorsal edge of caudal peduncle. Asterropteryx contains two distinct groups, and the monophyly of the genus is open to question. Received: March 19, 2000 / Revised: February 25, 2002 / Accepted: April 25, 2002     

Identification of Helicobacter pylori and the cagA genotype in gastric biopsies using highly sensitive real-time PCR as a new diagnostic tool

The CagA protein is one of the virulence factors of Helicobacter pylori, and two major subtypes of CagA have been observed, the Western and East Asian type. CagA is injected from the bacteria into gastric epithelial cells, undergoes tyrosine phosphorylation, and binds to Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2. The East Asian type CagA binds to SHP-2 more strongly than the Western type CagA. Here, we tried to distinguish the CagA type by highly sensitive real-time PCR with the objective of establishing a system to detect H. pylori and CagA subtypes from gastric biopsies. We designed primers and probe sets for Western or East Asian-cagA at Western-specific or East Asian-specific sequence regions, respectively, and H. pylori 16S rRNA. We could detect the H. pylori 16S rRNA gene, Western and East Asian-cagA gene from DNA of gastric biopsies. The sensitivity and specificity for H. pylori infection was 100% in this system. In Thai patients, 87.8% (36/41) were cagA-positive; 26.8% (11/41) were Western-cagA positive and 53.7% (22/41) were East Asian-cagA positive, while 7.3% (3/41) reacted with both types of cagA. These results suggest that this real-time PCR system provides a highly sensitive assessment of CagA type as a new diagnostic tool for the pathogenicity of H. pylori infection.     

K+ currents regulate the resting membrane potential, proliferation, and contractile responses in ventricular fibroblasts and myofibroblasts

Despite the important roles played by ventricular fibroblasts and myofibroblasts in the formation and maintenance of the extracellular matrix, neither the ionic basis for membrane potential nor the effect of modulating membrane potential on function has been analyzed in detail. In this study, whole cell patch-clamp experiments were done using ventricular fibroblasts and myofibroblasts. Time- and voltage-dependent outward K(+) currents were recorded at depolarized potentials, and an inwardly rectifying K(+) (Kir) current was recorded near the resting membrane potential (RMP) and at more hyperpolarized potentials. The apparent reversal potential of Kir currents shifted to more positive potentials as the external K(+) concentration ([K(+)](o)) was raised, and this Kir current was blocked by 100-300 muM Ba(2+). RT-PCR measurements showed that mRNA for Kir2.1 was expressed. Accordingly, we conclude that Kir current is a primary determinant of RMP in both fibroblasts and myofibroblasts. Changes in [K(+)](o) influenced fibroblast membrane potential as well as proliferation and contractile functions. Recordings made with a voltage-sensitive dye, DiBAC(3)(4), showed that 1.5 mM [K(+)](o) resulted in a hyperpolarization, whereas 20 mM [K(+)](o) produced a depolarization. Low [K(+)](o) (1.5 mM) enhanced myofibroblast number relative to control (5.4 mM [K(+)](o)). In contrast, 20 mM [K(+)](o) resulted in a significant reduction in myofibroblast number. In separate assays, 20 mM [K(+)](o) significantly enhanced contraction of collagen I gels seeded with myofibroblasts compared with control mechanical activity in 5.4 mM [K(+)](o). In combination, these results show that ventricular fibroblasts and myofibroblasts express a variety of K(+) channel alpha-subunits and demonstrate that Kir current can modulate RMP and alter essential physiological functions.     

K+ currents activated by depolarization in cardiac fibroblasts

K(+) currents expressed in freshly dispersed rat ventricular fibroblasts have been studied using whole-cell patch-clamp recordings. Depolarizing voltage steps from a holding potential of -90 mV activated time- and voltage-dependent outward currents at membrane potentials positive to approximately -30 mV. The relatively slow activation kinetics exhibited strong dependence on the membrane potential. Selected changes in extracellular K(+) concentration ([K(+)](o)) revealed that the reversal potentials of the tail currents changed as expected for a K(+) equilibrium potential. The activation and inactivation kinetics of this K(+) current, as well as its recovery from inactivation, were well-fitted by single exponential functions. The steady-state inactivation was well described by a Boltzmann function with a half-maximal inactivation potential (V(0.5)) of -24 mV. Increasing [K(+)](o) (from 5 to 100 mM) shifted this V(0.5) in the hyperpolarizing direction by -11 mV. Inactivation was slowed by increasing [K(+)](o) to 100 mM, and the rate of recovery from inactivation was decreased after increasing [K(+)](o). Block of this K(+) current by extracellular tetraethylammonium also slowed inactivation. These [K(+)](o)-induced changes and tetraethylammonium effects suggest an important role for a C-type inactivation mechanism. This K(+) current was sensitive to dendrotoxin-I (100 nM) and rTityustoxin Kalpha (50 nM).     

Substitution of a single residue, Asp575, renders the NCKX2 K+-dependent Na+/Ca2+ exchanger independent of K+

The Na(+)/Ca(2+)-K(+) exchanger (NCKX) is a polytopic membrane protein that uses both the inward Na(+) gradient and the outward K(+) gradient to drive Ca(2+) extrusion across the plasma membrane. NCKX1 is found in retinal rod photoreceptors, while NCKX2 is found in retinal cone photoreceptors and is also widely expressed in the brain. Here, we have identified a single residue (out of >100 tested) for which substitution removed the K(+) dependence of NCKX-mediated Ca(2+) transport. Charge-removing replacement of Asp(575) by either asparagine or cysteine rendered the mutant NCKX2 proteins independent of K(+), whereas the charge-conservative substitution of Asp(575) to glutamate resulted in a nonfunctional mutant NCKX2 protein, accentuating the critical nature of this residue. Asp(575) is conserved in the NCKX1-5 genes, while an asparagine is found in this position in the three NCX genes, coding for the K(+)-independent Na(+)/Ca(2+) exchanger.     

Development of novel steroid sulfatase inhibitors; II. TZS-8478 potently inhibits the growth of breast tumors in postmenopausal breast cancer model rats

In postmenopausal breast cancer tissue, steroid sulfatase (STS) activity is high and much estrone sulfate also exists; these facts reveal that estrone sulfate may be involved in the growth of breast cancer as an estrogen source. Steroid sulfatase is an enzyme, which catalyzes hydrolysis from estrone sulfate to estrone, and the development of steroid sulfatase inhibitors is expected as novel therapeutic drugs for postmenopausal breast cancer. We have developed a novel compound 2',4'-dicyanobiphenyl-4-O-sulfamate (TZS-8478), which has potent steroid sulfatase-inhibitory activity and exhibits no estrogenicity in vitro and in vivo. To elucidate its usefulness as a therapeutic drug for postmenopausal breast cancer, we examined the breast cancer cell proliferation- and breast tumor growth-inhibitory activity of TZS-8478 in postmenopausal breast cancer model rats. TZS-8478 dose-dependently suppressed the estrone sulfate-stimulated proliferation of MCF-7 cells. Regarding nitrosomethylurea (NMU)-induced postmenopausal breast cancer models, furthermore, TZS-8478 (0.5 mg/kg per day) markedly inhibited the estrone sulfate-stimulated growth of breast tumors similarly to estrone sulfate-depletion. TZS-8478 completely inhibited steroid sulfatase activity in tumor, uterus and liver, and also markedly lowered plasma concentrations of estrone and estradiol. The above mentioned results suggested that TZS-8478 may be useful as a therapeutic drug for estrogen-dependent postmenopausal breast cancer.