Cytosolic phospholipase A2: biochemical properties and physiological roles

Phosphatidylcholine (PC) is a major constituent of biological membranes and a component of serum lipoproteins and pulmonary surfactants. The PC and other glycerophospholipid compositions of membranes change dynamically through stimulus-dependent and independent pathways, principally by the action of two different types of enzymes; phospholipase A2 [EC 3.1.1.4] and acyl-CoA:lysophospholipid acyltransferase [EC 2.3.1.23]. Phospholipase A2 is a key enzyme that catalyzes deacylation of the sn-2 position of glycerophospholipids. This enzyme is critical in the remodeling of membrane lipids and formation of two subclasses of lipid mediators, fatty acid derivatives and lysophospholipids. Among many different subtypes of phospholipase A2 enzymes, we found that cytosolic phospholipase A2alpha (cPLA2alpha) is important in various pathological and physiological responses. Here, we summarize the phenotypes resulting from genetic ablation of cPLA2alpha, and the properties of newly discovered enzymes in the cPLA2 family. Comprehensive analysis of lipid mediators using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is useful for understanding the roles of individual mediators in physiological and pathological processes.     

Human autologous serum obtained using a completely closed bag system as a substitute for foetal calf serum in human mesenchymal stem cell cultures

The major problem in cell therapy is the possibility of viral or bacterial infection and immune reactions. Therefore, it is expected of culture cells which are intended to be re-implanted with autologous serum rather than conventional bovine serum. Cell therapy with human mesenchymal stem cells (hMSC), differentiating to various cells, is thought to be curative. To culture hMSC with human autologous serum (HAS) and re-implant them for cell therapy, we developed a completely closed bag system separating serum, comparing proliferation and multipotency of hMSC cultured in HAS with those in foetal calf serum (FCS). HAS was simply, safely and efficiently obtained with the developed closed bag system. Cell proliferation of hMSC cultured in HAS was greater than that in FCS. hMSC, exposed to the defined induction medium containing HAS as well as FCS, differentiated into osteoblasts and adipocytes. These findings suggest that HAS obtained with the developed closed bag system is advantageous in a point of decrease in risk of virus or bacterial infection and foreign protein contamination and enhancement of proliferation of hMSC.     

cDNA-derived amino acid sequences of myoglobins from nine species of whales and dolphins

We determined the myoglobin (Mb) cDNA sequences of nine cetaceans, of which six are the first reports of Mb sequences: sei whale (Balaenoptera borealis), Bryde's whale (Balaenoptera edeni), pygmy sperm whale (Kogia breviceps), Stejneger's beaked whale (Mesoplodon stejnegeri), Longman's beaked whale (Indopacetus pacificus), and melon-headed whale (Peponocephala electra), and three confirm the previously determined chemical amino acid sequences: sperm whale (Physeter macrocephalus), common minke whale (Balaenoptera acutorostrata) and pantropical spotted dolphin (Stenella attenuata). We found two types of Mb in the skeletal muscle of pantropical spotted dolphin: Mb I with the same amino acid sequence as that deposited in the protein database, and Mb II, which differs at two amino acid residues compared with Mb I. Using an alignment of the amino acid or cDNA sequences of cetacean Mb, we constructed a phylogenetic tree by the NJ method. Clustering of cetacean Mb amino acid and cDNA sequences essentially follows the classical taxonomy of cetaceans, suggesting that Mb sequence data is valid for classification of cetaceans at least to the family level.     

cDNA cloning and characterization of Type I procollagen alpha1 chain in the skate Raja kenojei

A full-length cDNA of the Type I procollagen alpha1 [pro-alpha1(I)] chain (4388 bp), coding for 1463 amino acid residues in the total length, was determined by RACE PCR using a cDNA library constructed from 4-week embryo of the skate Raja kenojei. The helical region of the skate pro-alpha1(I) chain consisted of 1014 amino acid residues - the same as other fibrillar collagen alpha chains from higher vertebrates. Comparison on denaturation temperatures of Type I collagens from the skate, rainbow trout (Oncorhynchus mykiss) and rat (Rattus norvegicus) revealed that the number of Gly-Pro-Pro and Gly-Gly in the alpha1(I) chains could be directly related to the thermal stability of the helix. The expression property of the skate pro-alpha1(I) chain mRNA and phylogenetic analysis with other vertebrate pro-alpha1(I) chains suggested that skate pro-alpha1(I) chain could be a precursor form of the skate Type I collagen alpha1 chain. The present study is the first evidence for the primary structure of full-length pro-alpha1(I) chain in an elasmobranch.     

Effect of some heavy metal ions on copper-induced metallothionein synthesis in the yeast Saccharomyces cerevisiae

Copper-induced metallothionein (MT) synthesis in Saccharomyces cerevisiae was investigated in order to associate this exclusively with Cu²⁺ in vivo, when cultured in nutrient medium containing other heavy metal ions. Expression of the CUP1 promoter/lacZ fusion gene was inhibited by all heavy metal ions tested, especially Cd²⁺ and Mn²⁺. By adding Cd²⁺ and Mn²⁺ at 10 M concentration, the -galactosidase activity decreased by about 80% and 50% of the maximum induction observed with 1 mM CuSO₄, respectively. Furthermore, cell growth was markedly inhibited by combinations of 1 mM-Cu²⁺ and 1 M-Cd²⁺. Therefore, the yeast S. cerevisiae could not rely on MT synthesis as one of the copper-resistance mechanisms, when grown in a Cd²⁺ environment. In contrast, the presence of Mn²⁺ in the nutrient medium showed alleviation rather than growth inhibition by high concentrations of Cu²⁺. The recovery from growth inhibition by Mn²⁺ was due to decreased Cu²⁺ accumulation. Inhibitory concentrations of Co²⁺, Ni²⁺ and Zn²⁺ on expression of the CUP1p/lacZ fusion gene were at least one order of magnitude higher than that of Cd²⁺ and Mn²⁺. These results are discussed in relation to Cu²⁺ transport and Cu-induced MT synthesis in the copper-resistance mechanism of the yeast S. cerevisiae.     

Splenic contraction-induced reversible increase in hemoglobin concentration in intermittent hypoxia

The effect of intermittent hypoxia (IHx) on blood hemoglobinconcentration ([Hb]) and the underlying mechanisms werestudied in rats exposed to 10%O₂, 1 h/day, for up to 5 wk. IHxprotocols with longer daily hypoxic exposure show persistentpolycythemia; however, it is unknown whether [Hb] increasestransiently during hypoxia in protocols without polycythemia. Hypoxiaproduced a reversible [Hb] increase after 4 days of IHx butnot in normoxic controls (NxC) or after shorter period of IHx.Splenectomy abolished the phenomenon. Plasma epinephrine andnorepinephrine levels during hypoxia were comparable in IHx and NxCgroups, but the epinephrine-induced [Hb] increase waslarger in IHx. The ₁- and₂-adrenoreceptor blockade(phentolamine) and ₂-blockade(yohimbine) abolished the [Hb] increase of IHx rats.Conversely, ₂-receptorstimulation (oxymetazoline) increased [Hb] during normoxiain IHx but not in NxC. In conclusion, this IHx protocol results inreversible [Hb] increases during hypoxia via spleniccontraction mediated by increased₂-adrenoreceptor response. Thismay protect O₂ supply duringhypoxia without the cardiovascular burden of polycythemia during normoxia.

     

Acute and Chronic D-Fenfluramine Treatments Have Different Effects on Serotonin Synthesis Rates in the Rat Brain: An Autoradiographic Study

The effects of acute and chronic treatments with D-fenfluramine on the regional rates of serotonin (5-hydroxy-tryptamine; 5-HT) synthesis were investigated using the -[¹⁴C]methyl-L-tryptophan (-[¹⁴C]MTrp) autoradiographic method. In the first series of experiments, acute D-fenfluramine treatment (5 mg/kg; i.p.) given 20 min before the tracer injection significantly (p < 0.05) decreased 5-HT synthesis in the dorsal raphe, and significantly (p < 0.05) increased the rates in the cerebral cortices and caudate nucleus, when compared to the rates in the control rats (saline treated). In a second series of experiments, following a 7-day treatment with D-fenfluramine (5 mg/kg/day; i.p.), a significant (p < 0.05) decrease of 5-HT synthesis, in the dorsal raphe was observed, and significant (p < 0.05) increases were observed in the hypothalamus, the dorsal thalamus, the medial and lateral geniculate body and some brain stem regions (locus ceruleus, inferior and superior colliculus). No significant changes were observed in the cerebral cortices.     

First determination of the inhibitor complex structure of human hematopoietic prostaglandin D synthase

Hematopoietic prostaglandin (PG) D synthase (H-PGDS) is responsible for the production of PGD(2) as an allergy or inflammation mediator in mast and Th2 cells. We determined the X-ray structure of human H-PGDS complexed with an inhibitor, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT) at 1.9 A resolution in the presence of Mg(2+). The styryl group of the inhibitor penetrated to the bottom of the active site cleft, and the tetrazole ring was stabilized by the stacking interaction with Trp104, inducing large movement around the alpha5-helix, which caused the space group of the complex crystal to change from P2(1) to P1 upon binding of BSPT. The phthalhydrazidyl group of BSPT exhibited steric hindrance due to the cofactor, glutathione (GSH), increasing the IC(50) value of BSPT for human H-PGDS from 36.2 micro M to 98.1 micro M upon binding of Mg(2+), because the K(m) value of GSH for human H-PGDS was decreased from 0.60 micro M in the presence of EDTA to 0.14 micro M in the presence of Mg(2+). We have to avoid steric hindrance of the GSH molecule that was stabilized by intracellular Mg(2+) in the mM range in the cytosol for further development of structure-based anti-allergic drugs.