Characterization of Rat8 localization and mRNA export in Saccharomyces cerevisiae during the brewing of Japanese sake

Ethanol affects the nuclear export of mRNA in a similar way to heat shock in Saccharomyces cerevisiae. We recently reported that the nuclear accumulation of Rat8 caused by ethanol stress correlates well with blocking of the export of bulk poly(A)⁺ mRNA. Here, we characterize the localization of Rat8 and bulk poly(A)⁺ mRNA in sake (Japanese rice wine) yeast during the brewing of sake. In wine must and synthetic dextrose medium, sake yeast showed the same responses to ethanol regarding changes in the localization of Rat8 as wine yeast and a laboratory strain: i.e., cells began the nuclear accumulation of Rat8 at an ethanol concentration of 6% and completed it at 9%. In contrast, during the sake-brewing process, sake yeast showed unique phenomena: i.e., cells did not start the nuclear accumulation of Rat8 until the ethanol concentration of the sake mash reached around 12% and they showed a normal localization of Rat8 around the nuclear envelope at the late stage of fermentation. These results provide new information about the transport of mRNA in yeast cells during actual alcoholic fermentation.     

Branching patterns of trunk lateral line nerves in Pleuronectiformes: uniformity and diversity

Branching patterns of the trunk lateral line nerves were studied in 46 pleuronectiform species, representing nine families in two suborders. The dorsal fin longitudinal ramus (DFLR), derived from the main nerve (horizontal septum lateral line nerve), passed closely along the course of the middle trunk lateral line in all specimens examined, the dorsal longitudinal collector nerve (DLCN) partly coalescing with the DFLR along the arched part of the lateral line in Psettodes erumei (Psettodoidei), compared with the entire length of the latter in all other species (Pleuronectoidei). Citharidae, Paralichthyidae, and Pleuronectidae were characterized by having a simple, elongated dorsal ramule; Bothidae was unique in having more than one dorsal ramule, forming a ladder-like network and peripherally giving off numerous minute branches; Poecilopsettidae and Samaridae possessed a few, short dorsal ramules; Soleidae and Cynoglossidae were characteristic in having a dendritic dorsal ramule. Secondary modifications of the course of the middle trunk lateral line were detected by nerve information, the arched part of the lateral line having been secondarily highly elevated in some genera of Pleuronectidae, but secondarily straightened in Samaridae.     

Distinct Rab27A binding affinities of Slp2-a and Slac2-a/melanophilin: Hierarchy of Rab27A effectors

The small GTPase Rab27A has recently been shown to regulate melanosome transport in mammalian skin melanocytes through sequentially interacting with two Rab27A effectors, Slac2-a/melanophilin and Slp2-a. Although Slac2-a and Slp2-a contain a similar N-terminal Rab27A-binding domain (named SHD, Slp homology domain), nothing is known about the functional differences between the Slac2-a SHD and Slp2-a SHD. In this study, the Rab27A-binding affinity of ten putative Rab27A effector proteins has been investigated. It has been found that they could be classified into a low-affinity group (e.g., Slac2-a) and a high-affinity group (e.g., Slp2-a and Slp4-a) based on their Rab27A-binding affinity. Kinetic analysis of the GTP-Rab27A-binding to the SHD of Slp2-a, Slp4-a, and Slac2-a by surface plasmon resonance further indicated that the kinetic parameters of Rab27A for the Slp2-a SHD, Slp4-a SHD, and Slac2-a SHD consisted of a fast association rate constant (3.35 x 10(4), 2.06 x 10(4), and 2.11 x 10(4) M(-1) s(-1), respectively) and a slow dissociation rate constant (4.48 x 10(-4), 3.96 x 10(-4), and 2.37 x 10(-3) s(-1) respectively), and their equilibrium dissociation constants were determined to be 13.4, 19.2, and 112 nM, respectively. Our data suggest that distinct Rab27A binding activities of Slac2-a and Slp2-a ensure the order (or hierarchy) of Rab27A effectors that sequentially function in melanosome transport in melanocytes.     

The Slp4-a linker domain controls exocytosis through interaction with Munc18-1.syntaxin-1a complex

Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a is specifically localized on dense-core vesicles in certain neuroendocrine cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27A. However, the precise molecular mechanism of its inhibitory effect on exocytosis has never been elucidated and is still a matter of controversy. Here we show by deletion and chimeric analyses that the linker domain of Slp4-a interacts with the Munc18-1.syntaxin-1a complex by directly binding to Munc18-1 and that this interaction promotes docking of dense-core vesicles to the plasma membrane in PC12 cells. Despite increasing the number of plasma membrane docked vesicles, expression of Slp4-a strongly inhibited high-KCl-induced dense-core vesicle exocytosis. The inhibitory effect by Slp4-a is absolutely dependent on the linker domain of Slp4-a, because substitution of the linker domain of Slp4-a by that of Slp5 (the closest isoform of Slp4-a that cannot bind the Munc18-1.syntaxin-1a complex) completely abrogated the inhibitory effect. Our findings reveal a novel docking machinery for dense-core vesicle exocytosis: Slp4-a simultaneously interacts with Rab27A and Munc18-1 on the dense-core vesicle and with syntaxin-1a in the plasma membrane.     

Activation of mitogen-activated protein kinase and its activator by ras in intact cells and in a cell-free system.

Mitogen-activated protein (MAP) kinase is a serine/threonine kinase whose function is thought to be essential for the transduction of mitogenic signals. MAP kinase is activated by phosphorylation induced by a variety of extracellular stimuli, and its direct upstream activator has been identified. Using amphibian and mammalian systems, we show here that ras can activate MAP kinase and its activator. Injection of v-Ha-ras p21 into Xenopus immature oocytes activated both MAP kinase and maturation-promoting factor (MPF) activities. The activation of MAP kinase preceded that of MPF, demonstrating that ras activates MAP kinase in an MPF-independent pathway. Moreover, we found that the MAP kinase activator is also activated in ras-injected oocytes. Activation of MAP kinase and its activator occurred also when the v-Ki-ras gene was conditionally induced in rat fibroblastic 3Y1 cells. Furthermore, we observed that ras activated MAP kinase and its activator in a cell-free system prepared from Xenopus oocytes. Using an antibody against the Xenopus 45-kDa MAP kinase activator, we demonstrated that the 45-kDa activator molecule was activated by ras. These findings suggest that the MAP kinase activator/MAP kinase system may be the downstream components of ras signal transduction pathways.     

6-formylpterin, a xanthine oxidase inhibitor, intracellularly generates reactive oxygen species involved in apoptosis and cell proliferation

The chemical property of 6-formylpterin and its biological functions were examined. Polarographic studies revealed that 6-formylpterin reacted with NAD(P)H and consumed oxygen. In contrast, other conjugated pterins, such as biopterin and neopterin, showed no consumption of oxygen. The production analysis using high-performance liquid chromatography documented that 6-formylpterin catalyzes the conversion from NADH to NAD. Electroparamagnetic resonance spin trapping experiments demonstrated that this reaction is accompanied with the generation of reactive oxygen species (ROS), superoxide anion and hydrogen peroxide. When 6-formylpterin was administered to HL-60 cells, intracellular ROS generation was observed and apoptosis was induced. In contrast, other conjugated pterins induced neither intracellular ROS generation nor apoptosis in HL-60 cells. The intracellular ROS generation by 6-formylpterin was observed in other cells, such as PanC-1 cells and Jurkat cells. 6-formylpterin suppressed cell proliferation in PanC-1 cells and inhibited Fas-mediated apoptosis in Jurkat cells. These findings indicate that, among conjugated pterins, 6-formylpterin has the unique property to transfer electron from NAD(P)H to oxygen and that the property brings about intracellular ROS generation, which exerts various biological functions such as induction of apoptosis, suppression of cell proliferation, and inhibition of Fas-mediated apoptosis.     

Two kinds of chlorocatechol 1,2-dioxygenase from 2,4-dichlorophenoxyacetate-degrading Sphingomonas sp. strain TFD44

Two kinds of chlorocatechol 1,2-dioxygenase (CCD), TfdC and TfdC2 were detected in Sphingomonas sp. strain TFD44. These two CCDs could be simultaneously synthesized in TFD44 during its growth with 2,4-D as the sole carbon and energy sources. The apparent subunit molecular masses of TfdC and TfdC2 estimated by SDS-PAGE analysis were 33.8 and 33.1 kDa, respectively. The genes encoding the two CCDs were cloned and expressed in Escherichia coli. The two purified CCDs showed broad substrate specificities but had different specificity patterns. TfdC showed the highest specificity constant for 3-chlorocatechol and TfdC2 showed the highest specificity constant for 3,5-dichlorocatechol. The substrate specificity difference seemed to correlate with the alternation of amino acid supposed to be involved in the interaction with substrates. Whereas phylogenetic analysis indicated that the CCDs of Sphingomonas constitute a distinctive group among Gram-negative bacteria, TfdC and TfdC2 of TFD44 have divergently evolved in terms of their substrate specificity.     

Changes in the antibiotic production by co-culture of Rhizopus peka P8 and Bacillus subtilis IFO3335

The co-culture of Bacillus subtilis IFO 3335 with Rhizopus peka P8 or Rhizopus oligosporus P12 in liquid medium was found to increase production of antibiotic activity and to alter the spectrum of activity relative to the pure cultures. However, a mixed culture of Rhizopus arrhizus P7 and Rhizopus oryzae P17 did not produce antibiotic activity. The concentration, ratio, and time of addition of B. subtilis to the R. peka culture was found to influence antibiotic yields. Solid-state fermentations using mixed cultures of R. peka and B. subtilis were investigated. The growth of Escherichia coli IFO 3792 as a target bacterium was inhibited by the mixed culture. These results suggest the possibility of biopreservation of fermented foods by novel co-culture systems.