DHEA administration increases brown fat uncoupling protein 1 levels in obese OLETF rats

We found that UCP-1 and UCP-3 mRNA expression levels and the UCP-1 protein content in brown adipose tissue (BAT) were reduced in prediabetic OLETF rats than the lean LETO rats. Administration of dehydroepiandrosterone (DHEA) for 17 days induced remarkable weight loss, which was in part attributed to an enhanced utilization of ingested energy. DHEA administration significantly increased the levels of BAT UCP-1 and UCP-3 mRNA expression. Among the upstream signals for UCP-1 regulation, expression levels of the beta 3 adrenergic receptor (beta(3)AR) and PPAR gamma coactivator-1 (PGC-1) were significantly decreased in the OLETF rats and increased by DHEA administration. The decreased expression levels of UCP-1 and its upstream regulators, beta(3)AR and PGC-1, in BAT may contribute to inefficient energy utilization and obesity in OLETF rats, which was corrected by DHEA treatment.     

Maintenance of mouse male germ line stem cells in vitro

The proliferation and differentiation of a stem cell are regulated intrinsically by the stem cell and extrinsically by the stem cell niche. Elucidation of regulatory mechanisms of spermatogonial stem cells (SSCs), the stem cell of the postnatal male germ line, would be facilitated by in vitro studies that provide a defined microenvironment reconstituted ex vivo. We analyzed the effect of in vitro environment on the maintenance of adult and immature SSCs in a 7-day culture system. Although the number of adult and immature SSCs decreased in a time-dependent manner, nearly one in four stem cells (24%) could be maintained in vitro for 7 days. Stem cell maintenance was enhanced by coculture with OP9 bone marrow stroma or L fibroblast cell lines, addition of glial cell line-derived neurotrophic factor, or utilization of specific culture medium. In contrast, coculture with TM4 or SF7 Sertoli cell lines and addition of activin A or bone morphogenetic protein 4 (BMP4) reduced stem cell maintenance in vitro. Only 4% of the stem cells remained when cultured with TM4 cells or activin A, and 6% remained when cultured with SF7 cells or BMP4. These results lead to the hypothesis that suppression of germ cell differentiation improves in vitro maintenance of SSCs by interrupting the unidirectional cascade of spermatogenesis and blocking stem cell differentiation.     

gly gene cloning and expression and purification of glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines 8ra

Glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines, inhibits the growth of X. campestris pv. vesicatoria. We have cloned and expressed the genes encoding glycinecin A in Escherichia coli. Recombinant glycinecin A was purified from cell extracts by ammonium sulfate precipitation followed by chromatography on Q-Sepharose, Mono Q (ion exchange), and size exclusion columns. Purified glycinecin A is composed of two polypeptides, is active over a wide pH range (6 to 9), and is stable at temperatures up to 60 degrees C. Glycinecin A is a heterodimer consisting of 39- and 14-kDa subunits, as revealed through size exclusion chromatography and cross-linking analysis. Two genes, glyA and glyB, encoding the 39- and 14-kDa subunits, respectively, were identified based on the N-terminal sequences of the subunits. From the nucleotide sequences of glyA and glyB, we conclude that both genes are translated as bacteriocin precursors that include N-terminal leader sequences. When expressed in E. coli, recombinant glycinecin A was found primarily in cell extracts. In contrast, most glycinecin A from Xanthomonas was found in the culture media. E. coli transformed with either glyA or glyB separately did not show the bacteriocin activity.     

Hwangryun-Hae-Dok-tang (Huanglian-Jie-Du-Tang) extract and its constituents reduce ischemia-reperfusion brain injury and neutrophil infiltration in rats

The preventive effect of Hwangryun-Hae-Dok-tang (HHDT, Huanglian-Jie-Du-Tang), a Chinese herbal medicine, and its ingredients on ischemia/reperfusion-induced brain injury was evaluated in the rat brain. HHDT consists of four herbs, namely, Coptidis rhizoma, Scutellariae radix, Phellodendri cortex, and Gardeniae fructus. Ischemia was induced by intraluminal occlusion of the right middle cerebral artery for 120 min and reperfusion was continued for 22 h. HHDT (200 mg/kg), Coptidis rhizoma (100 mg/kg), Scutellariae radix (100 mg/kg), Phellodendri cortex (100 mg/kg), and Gardeniae fructus (100 mg/kg) were orally administered, promptly prior to reperfusion and 2 h after reperfusion. Baicalein, a component of Scutellariae radix, was also examined at a dosage of 50 mg/kg given 2 h apart, promptly prior to and 2 h after reperfusion. Total infarction volume in the ipsilateral hemisphere of ischemia/reperfusion rats was significantly lowered by treatment with HHDT, Scutellariae radix, and balicalein. However, the other ingredient of HHDT did not show any ameliorating effects on total infarction volume. The inhibiting effect of Scutellariae radix on total infarction volume was much higher than that of the others. In addition, HHDT, Scutellariae radix, and baicalein significantly inhibited myeloperoxidase (MPO) activity, an index of neutrophil infiltration in ischemic brain tissue at about the same rate (30%). There was marked mismatch between total infarction volume and MPO activity in the Scutellariae radix-treated rats but not in the HHDT- and baicalein-treated groups. Our findings suggest that Scutellariae radix as an ingredient of HHDT plays a crucial protective role in ischemia-induced brain injury. In addition, it is apparent that the effect of Scutellariae radix is the result, in part, of baicalein, a compound contained in Scutellariae radix.     

Effects of resveratrol-related hydroxystilbenes on the nitric oxide production in macrophage cells: structural requirements and mechanism of action

NF-kappaB that plays an important role in iNOS expression is one of the targets of various potential anti-inflammatory agents including resveratrol. Resveratrol contains a structural similarity with estrogen, and there has been speculation about resveratrol as estrogen agonist. In this study, the mechanism and structural requirements of resveratrol and related hydroxystilbenes for the inhibition of LPS-induced nitric oxide production were studied in macrophage cells (RAW 264.7 and J774) by comparing its effect on LPS-induced NF-kappaB translocation and nitric oxide production, and by considering the possibility of involvement of an estrogen receptor. LPS-induced nitric oxide production was inhibited only when cells were treated with resveratrol prior to stimulation with LPS, suggesting that resveratrol does not affect the enzyme itself. A higher concentration of resveratrol than needed for the inhibition of nitric oxide production was required for the inhibition of NF-kappaB mobilization or iNOS expression. Estrogen and diethylstilbesterol, an estrogen agonist, caused only weak inhibition of nitric oxide production, and the effects of resveratrol were not noticeably blocked by ICI-182780, an estrogen antagonist. Structure-activity analysis of resveratrol and nine hydroxystilbenes suggests that the structural balance between oxygen functional groups on the benzene rings is important for their activity. Our results suggest that resveratrol might act on other cellular targets as well as NF-kappaB at the initial stage of gene expression. Unique structural features of hydroxystilbenes are needed for suppression of nitric oxide production and it is unlikely that estrogen receptor is involved in it.     

Purification and characterization of a cytosolic, 42-kDa and Ca2+-dependent phospholipase A2 from bovine red blood cells: its involvement in Ca2+-dependent release of arachidonic acid from mammalian red blood cells

It has become evident that a Ca(2+)-dependent release of arachidonic acid (AA) and subsequent formation of bioactive lipid mediators such as prostaglandins and leukotrienes in red blood cells (RBCs) can modify physiological functions of neighboring RBCs and platelets. Here we identified a novel type of cytosolic PLA(2) in bovine and human RBCs and purified it to apparent homogeneity with a 14,000-fold purification. The purified enzyme, termed rPLA(2), has a molecular mass of 42 kDa and reveals biochemical properties similar to group IV cPLA(2), but shows different profiles from cPLA(2) in several column chromatographies. Moreover, rPLA(2) did not react with any of anti-cPLA(2) and anti-sPLA(2) antibodies and was identified as an unknown protein in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Divalent metal ions tested exhibited similar effects between rPLA(2) and cPLA(2), whereas mercurials inhibited cPLA(2) but had no effect on rPLA(2). Antibody against the 42-kDa protein not only precipitated the rPLA(2) activity, but also reacted with the 42-kDa protein from bovine and human RBCs in immunoblot analysis. The 42-kDa protein band was selectively detected in murine fetal liver cells known as a type of progenitor cells of RBCs. It was found that EA4, a derivative of quinone newly developed as an inhibitor for rPLA(2), inhibited a Ca(2+) ionophore-induced AA release from human and bovine RBCs, indicating that this enzyme is responsible for the Ca(2+)-dependent AA release from mammalian RBCs. Finally, erythroid progenitor cell assay utilizing diaminobenzidine staining of hemoglobinized fetal liver cells showed that rPLA(2) detectable in erythroid cells was down-regulated when differentiated to non-erythroid cells. Together, our results suggest that the 42-kDa rPLA(2) identified as a novel form of Ca(2+)-dependent PLA(2) may play an important role in hemostasis, thrombosis, and/or erythropoiesis through the Ca(2+)-dependent release of AA.     

Novel zinc-binding center and a temperature switch in the Bacillus stearothermophilus L1 lipase

The bacterial thermoalkalophilic lipases optimally hydrolyze saturated fatty acids at elevated temperatures. They also have significant sequence homology with staphylococcal lipases, and both the thermoalkalophilic and staphylococcal lipases are grouped as the lipase family I.5. We report here the first crystal structure of the lipase family I.5, the structure of a thermoalkalophilic lipase from Bacillus stearothermophilus L1 (L1 lipase) determined at 2.0-A resolution. The structure is in a closed conformation, and the active site is buried under a long lid helix. Unexpectedly, the structure exhibits a zinc-binding site in an extra domain that accounts for the larger molecular size of the family I.5 enzymes in comparison to other microbial lipases. The zinc-coordinated extra domain makes tight interactions with the loop extended from the C terminus of the lid helix, suggesting that the activation of the family I.5 lipases may be regulated by the strength of the interactions. The unusually long lid helix makes strong hydrophobic interactions with its neighbors. The structural information together with previous biochemical observations indicate that the temperature-mediated lid opening is triggered by the thermal dissociation of the hydrophobic interactions.     

Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library

Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.