Continuous production of L-tryptophan from indole and L-serine by immobilized Escherichia coli cells

Escherichia coli B 10, which has high activity of tryptophan synthetase, was grown in a 50-L batch culture in order to determine in which growth phase the cells have the highest specific tryptophan productivity. Accordingly, whole cells of the stationary phase were used for immobilization in polyacrylamide beads. After immobilization, these immobilized cells had 56% activity of tryptophan synthetase compared with that of free cells. First, the properties of immobilized cells were investigated. Next, discontinuous productions of L-tryptophan were carried out by using immobilized cells. In discontinuous production of L-tryptophan by the batch, the activity remaining of immobilized cells was 76-79% after 30 times batchwise use. In continuous production of L-tryptophan with a continuous stirred tank reactor (CSTR), the activity remaining of the immobilized cells was 80% after continuous use for 50 days. The maximum productivity of L-tryptophan in this CSTR system was 0.12 g tryptophan L(-1) h(-1).     

Phosphorylation states greatly regulate the activity and gating properties of Cav3.1 T-type Ca2+ channels

Ca_v3.1 T-type Ca²⁺ channels play pivotal roles in neuronal low-threshold spikes, visceral pain, and pacemaker activity. Phosphorylation has been reported to potently regulate the activity and gating properties of Ca_v3.1 channels. However, systematic identification of phosphorylation sites (phosphosites) in Ca_v3.1 channel has been poorly investigated. In this work, we analyzed rat Ca_v3.1 protein expressed in HEK-293 cells by mass spectrometry, identified 30 phosphosites located at the cytoplasmic regions, and illustrated them as a Ca_v3.1 phosphorylation map which includes the reported mouse Ca_v3.1 phosphosites. Site-directed mutagenesis of the phosphosites to Ala residues and functional analysis of the phospho-silent Ca_v3.1 mutants expressed in Xenopus oocytes showed that the phospho-silent mutation of the N-terminal Ser18 reduced its current amplitude with accelerated current kinetics and negatively shifted channel availability. Remarkably, the phospho-silent mutations of the C-terminal Ser residues (Ser1924, Ser2001, Ser2163, Ser2166, or Ser2189) greatly reduced their current amplitude without altering the voltage-dependent gating properties. In contrast, the phosphomimetic Asp mutations of Ca_v3.1 on the N- and C-terminal Ser residues reversed the effects of the phospho-silent mutations. Collectively, these findings demonstrate that the multiple phosphosites of Ca_v3.1 at the N- and C-terminal regions play crucial roles in the regulation of the channel activity and voltage-dependent gating properties.     

Internal pH crisis, lysine decarboxylase and the acid tolerance response of Salmonella typhimurium

Salmonella typhimurium possesses an adaptive response to acid that increases survival during exposure to extremely low pH values. The acid tolerance response (ATR) includes both log-phase and stationary-phase systems. The log-phase ATR appears to require two components for maximum acid tolerance, namely an inducible pH homeostasis system, and a series of acid-shock proteins. We have discovered one of what appears to be a series of inducible exigency pH homeostasis systems that contribute to acid tolerance in extreme acid environments. The low pH-inducible lysine decarboxylase was shown to contribute significantly to pH homeostasis in environments as low as pH 3.0. Under the conditions tested, both lysine decarboxylase and σ^s-dependent acid-shock proteins were required for acid tolerance but only lysine decarboxylase contributed to pH homeostasis. The cadBA operon encoding lysine decarboxylase and a lysine/cadaverine antiporter were cloned from S. typhimurium and were found to be 79% homologous to the cadBA operon from Escherichia coli . The results suggest that S. typhimurium has a variety of means of fulfilling the pH homeostasis requirement of the ATR in the form of inducible amino acid decarboxylases.     

Lipase-catalyzed synthesis of lysophosphatidic acid in a solvent free system

Summary The direct, lipase-catalyzed esterifications of glycerol-3-phosphate in an organic solvent system and in a solvent free system were carried out. In a solvent free system only, LPA synthesis could be achieved within the acceptable reaction time. Open reaction system was preferable to closed reaction system for LPA synthesis. Yield of LPA isolated by silica gel column chromatography was 32.3%.     

Host-cell cyclophilin is important for the intracellular replication of Leishmania major

The antiparasitic effects of cyclosporin A were examined in leishmanial infection by analysing the role of CsA-binding proteins (cyclophilins) in the host–parasite interaction. We hypothesized that the leishmanicidal effects of CsA on Leishmania major infected macrophages might be mediated through a cyclophilin of either the parasite or the host cell. Two cyclophilins (20 and 22 kDa) were purified from L. major parasites and N-terminally sequenced. Although enzyme activity of these cyclophilins was inhibited by CsA, pretreatment of L. major parasites with CsA did not result in reduction of a subsequent macrophage infection, arguing against a role of L. major cyclophilins as infectivity potentiators. However, host-cell cyclophilin A (CypA) was found to be critically involved in the intracellular replication of L. major parasites in murine macrophages. An antisense oligonucleotide to murine CypA was constructed and added to cultures of peritoneal macrophages prior to infection with L. major parasites. This treatment strongly reduced the expression of CypA in macrophages and resulted in the inhibition of the intracellular replication of L. major amastigotes. These data indicate that interaction of amastigotes with host-cell cyclophilin is an important part of the intracellular replication machinery of L. major and define, for the first time, a direct involvement of a cyclophilin in the survival strategies of an intracellular parasite.     

Properties of fructose-1,6-diphosphate phosphatase and fructose-1,6-diphosphate aldolase fromPseudomonas putida

The fructose-1,6-P₂ (FDP) phosphatase, (FDPase) and FDP aldolase fromPseudomonas putida were partially purified by a combination of (NH₄)₂SO₄ fractionation and DEAE-Sephadex column chromatography. Michaelis-Menten kinetics were observed with, respect to FDP in both FDPase and FDP aldolase. TheK _m for FDP at pH 8.0 was 1.2×10⁻⁵M for FDPase and 3.0×10⁻⁵M for FDP aldolase. The specific activities of these two enzymes (assayed under optimal conditions in cell-free extracts ofP. putida grown ond-fructose), as well as their kinetic properties, are consistent with the suggestion that during growth ond-fructose most, of the FDP generated is converted to fructose-6-P (F-6-P), which is subsequently utilized via the Entner-Doudoroff pathway (EDP).     

Perfusion rate dependent prostaglandin release from isolated rabbit kidneys

Isolated rabbit kidneys were perfused with 37°C Krebs-Henseleit solution aerated with 95% O₂ + 5% CO₂. Perfusion rate was varied from 1 to 10 ml/min. This was accompanied by parallel changes of perfusion pressure, prostaglandin excretion and release of radioactivity from kidneys with ¹⁴C-arachidonic acid incorporated into the tissue lipid pool. It is suggested that enhancement of perfusion rate raises the intrarenal pressure which increases renal prostaglandin release due to increased substrate availability.