The tryptophanase from Proteus rettgeri, improved purification and properties of crystalline holotryptophanase.

The inducible tryptophanase (L-tryptophan indole-lyase (deaminating) EC 4.1.99.1) was crystallized in holoenzyme from the cell extract of Proteus rettgeri. The purification procedure included ammonium sulfate fractionation, heat treatment at 60 degrees C, DEAE-Sephadex and hydroxylapatite column chromatographies. Crystallization was performed by the addition of ammonium sulfate to the purified enzyme solution containing 20% (v/v) glycerol, 0.1 mM pyridoxal phosphate and 10 mM mercaptoethanol. The crystallized enzyme was yellow and showed absorption maxima at 340 and 420 nm. The crystalline holotryptophanase preparation was homogeneous by the criteria of ultracentrifugation and disc gel electrophoresis. The molecular weight of the enzyme was calculated as approx. 222 000. The amount of pyridoxal phosphate bound to the enzyme was determined to be 4 mol per mol of the enzyme. The enzyme is composed of four subunits of identical molecular size (mol. wt 55 000) and irreversibly dissociates into these subunits in the presence of a high concentration of sodium dodecylsulfate or guanidine hydrochloride. The NH2-terminal amino acid of the enzyme was identified as alanine.     

Purification and characterization of thiol aminopeptidase from the cytosolic fraction of human placenta

A thiol-dependent aminopeptidase was purified from the cytosolic fraction of human placenta. The purified enzyme consisted of a single polypeptide chain with a mol wt of 95,000. The enzyme was most active in the neutral region with Ala-pNA as substrate, and the activity was increased about 20-fold in the presence of some -SH compounds. The results of substrate specificity studies indicated that the enzyme hydrolyzes bonds involving the amino groups of neutral and basic amino acid residues. However, higher thiol-dependent activity was only detected with neutral ones. The enzyme was strongly inhibited by microbial aminopeptidase inhibitors, puromycin, o-phenanthroline, and sulfhydryl reactive-reagents. As to several naturally occurring peptides tested, the enzyme showed N-terminal Tyr-releasing activity toward enkephalins and kinin-converting activity.     

Thiazolidinediones increase arachidonic acid release and subsequent prostanoid production in a peroxisome proliferator-activated receptor gamma-independent manner

Thiazolidinedione, peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, has been used as an anti-diabetic drug and as an useful tool to elucidate multiple PPARgamma functions by in vitro and in vivo studies. We investigated the effects of thiazolidinediones on prostanoid production in lipopolysaccharide-stimulated cells. The high concentrations (>10 microM) of rosiglitazone and pioglitazone significantly increased lipopolysaccharide-stimulated prostanoid production such as thromboxane A2 and prostaglandin E2. However, PPARgamma antagonist could not inhibit them. In PPARgamma-deficient cells, thiazolidinediones increased prostaglandin E2 production. Thiazolidinediones increased arachidonic acid (AA) release from the cell membrane by not stimulating AA releasing process involving several phospholipase A2s but inhibiting AA reuptaking process. The expression of cyclooxygenase-1 and cyclooxygenase-2 were not affected by thiazolidinediones. In this study, we demonstrated that high concentrations of TZDs increased AA release by the inhibition of AA reuptaking process, leading to subsequent increase in the prostanoid production in a PPARgamma-independent manner. This mechanism provides useful information for the elucidation of multiple PPARgamma functions and diabetic drug therapy.     

Plasma brain natriuretic peptide as a surrogate marker for cardioembolic stroke

Background

Cardioembolic stroke generally results in more severe disability, since it typically has a larger ischemic area than the other types of ischemic stroke. However, it is difficult to differentiate cardioembolic stroke from non-cardioembolic stroke (atherothrombotic stroke and lacunar stroke). In this study, we evaluated the levels of plasma brain natriuretic peptide in acute ischemic stroke patients with cardioembolic stroke or non-cardioembolic stroke, and assessed the prediction factors of plasma brain natriuretic peptide and whether we could differentiate between stroke subtypes on the basis of plasma brain natriuretic peptide concentrations in addition to patient's clinical variables.

Methods

Our patient cohort consisted of 131 consecutive patients with acute cerebral infarction who were admitted to Kagawa University School of Medicine Hospital from January 1, 2005 to December 31, 2007. The mean age of patients (43 females, 88 males) was 69.6 ± 10.1 years. Sixty-two patients had cardioembolic stroke; the remaining 69 patients had non-cardioembolic stroke (including atherothrombotic stroke, lacunar stroke, or the other). Clinical variables and the plasma brain natriuretic peptide were evaluated in all patients.

Results

Plasma brain natriuretic peptide was linearly associated with atrial fibrillation, heart failure, chronic renal failure, and left atrial diameter, independently (F_4,126 = 27.6, p < 0.0001; adjusted R² = 0.45). Furthermore, atrial fibrillation, mitral regurgitation, plasma brain natriuretic peptide (> 77 pg/ml), and left atrial diameter (> 36 mm) were statistically significant independent predictors of cardioembolic stroke in the multivariable setting (Χ² = 127.5, p < 0.001).

Conclusion

It was suggested that cardioembolic stroke was strongly predicted with atrial fibrillation and plasma brain natriuretic peptide. Plasma brain natriuretic peptide can be a surrogate marker for cardioembolic stroke.     

Molecular identification of the causative agent of human strongyloidiasis acquired in Tanzania: Dispersal and diversity of Strongyloides spp. and their hosts

In order to identify the causative agent of imported strongyloidiasis found in a Japanese mammalogist, who participated in a field survey in Tanzania, the hyper-variable region IV (HVR-IV) of 18S ribosomal DNA and partial mitochondrial cytochrome c-oxidase subunit 1 gene (cox1) were analyzed and compared with Strongyloides fuelleborni collected from apes and monkeys of Africa and Japan, and S. stercoralis from humans, apes and dogs. The HVR-IV and cox1 of the patient's worms were identical to or only slightly differed from those of worms parasitic in Tanzanian chimpanzees and yellow baboons, demonstrating that the patient acquired the infection during her field survey in Tanzania. Phylogenetic analysis with the maximum-likelihood method largely divided isolates of S. fuelleborni into three groups, which corresponded to geographical localities but not to host species. Meanwhile, isolates of S. stercoralis were grouped by the phylogenetic analysis into dog-parasitic and primate-parasitic clades, and not to geographical regions. It is surmised that subspeciation has occurred in S. fuelleborni during the dispersal of primates in Africa and Asia, while worldwide dispersal of S. stercoralis seems to have occurred more recently by migration and the activities of modern humans.     

The interactions between calcium-dependent regulator protein of cyclic nucleotide phosphodiesterase and microtubule proteins. II. Association of calcium-dependent regulator protein with tubulin dimer.

The Ca2+-dependent regulator protein (CDR) of cyclic nucleotide phosphodiesterase (PDE) was reported to be a Ca2+-dependent regulator of microtubule (MT) assembly in the preceding paper. In this paper, the binding of Ca2+-CDR complex to tubulin dimer was investigated in order to elucidate the Ca2+-dependent inhibitory action of CDR on MT assembly. Purified microtubular proteins (PMPs) isolated from porcine brain did not affect the ability of CDR to activate Ca2+-activatable PDE, and did not include any inhibitory protein of Ca2+-activatable PDE. The binding of CDR to the tubulin dimer was observed on Sephadex G-200 gel filtration and ammonium sulfate fractionation in a Ca2+-dependent manner. CDR did not bind to microtubule associated proteins. We now assume that Ca2+-dependent inhibition of MT assembly by CDR is due to the binding of CDR to tubulin dimer in a Ca2+-dependent manner.     

Characteristics of halorhodopsin-bacterioruberin complex from Natronomonas pharaonis membrane in the solubilized system

Halorhodopsin is a retinal protein with a seven-transmembrane helix and acts as an inward light-driven Cl(-) pump. In this study, structural state of the solubilized halorhodopsin (NpHR) from the biomembrane of mutant strain KM-1 of Natronomonas pharaonis in nonionic detergent was investigated. A gel filtration chromatography monitored absorbances at 280 and 504 nm corresponding to the protein and a lipid soluble pigment of bacterioruberin (BR), respectively, has clearly detected an oligomer formation of the NpHRs and a complex formation between the NpHR and BR in the solubilized system. A molar ratio of NpHR:BR in the solubilized complex was close to 1:1. Further SDS-PAGE analysis of the solubilized NpHR cross-linked by 1% glutaraldehyde has revealed that the NpHR forms homotrimer in detergent system. Although this trimeric structure was stable in the presence of NaCl, it was dissociated to the monomer by the heat treatment at 45 °C in the desalted condition. The same tendency has been reported in the case of trimeric NpHR expressed heterologously on the E. coli membrane, leading to a conclusion that the change of strength of the trimeric association dependent on the ion binding is a universal feature of the NpHR. Interestingly, the trimer dissociation on the NpHR was accompanied by the complete dissociation of the BR molecule from the protein, indicated that the cavity formed by the NpHR protomers in the trimeric conformation is important for tight binding of the BR. Because the binding affinity for Cl(-) and the resistance to hydroxylamine under light illumination showed only minor differences between the NpHR in the solubilized state and that on the biomembrane, the influences of solubilization to the tertiary structure and function of the protein are thought to be minor. This NpHR-BR complex in the solubilized system has a potential to be a good model system to investigate the intermolecular interaction between the membrane protein and lipid.