Insulin suppresses PDK-4 expression in skeletal muscle independently of plasma FFA

Starvation and experimental diabetes induce a stable increase in pyruvate dehydrogenase kinase (PDK) activity in skeletal muscle, which is largely due to a selective upregulation of PDK-4 expression. Increased free fatty acid (FFA) level has been suggested to be responsible for the upregulation. Because these metabolic states are also characterized by insulin deficiency, the present study was designed to examine whether insulin has a significant effect to regulate PDK mRNA expression in rat skeletal muscle. In study 1, overnight-fasted rats received an infusion of saline or insulin for 5 h (n = 6 each). During the insulin infusion, plasma glucose was clamped at basal levels (euglycemic hyperinsulinemic clamp). A third group (n = 6) received Intralipid infusion during the clamp to prevent a fall in plasma FFA. PDK-2 mRNA level in gastrocnemius muscle was not altered by insulin or FFA (i.e., Intralipid infusion). In contrast, PDK-4 mRNA level was decreased 72% by insulin (P < 0.05), and Intralipid infusion prevented only 20% of the decrease. PDK-4 protein level was decreased approximately 20% by insulin (P < 0.05), but this effect was not altered by Intralipid infusion. In study 2, overnight-fasted rats were refed or received an infusion of saline or nicotinic acid (NA, 30 micromol/h) for 5 h (n = 5 each). During the refeeding and NA infusion, plasma FFA levels were similarly (i.e., 60-70% vs. saline control) lowered. Muscle PDK-4 mRNA level decreased 77% after the refeeding (P < 0.05) but not after the NA infusion. In conclusion, the present data indicate that insulin had a profound effect to suppress PDK-4 expression in skeletal muscle and that, contrary to previous suggestions, circulating FFA had little impact on PDK-4 mRNA expression, at least within 5 h.     

Synthesis and antibacterial activity of arylpiperazinyl oxazolidinones with diversification of the N-substituents

A series of 4-arylpiperazin-1-yl-3-phenyloxazolidin-2-one derivatives with diversification of the N-substituents such as methylene O-linked heterocycles, thioamide, dithiocarbamate, thiourea, and thiocarbamate were synthesized and evaluated as antibacterial agents. Their in vitro activities (MIC) were evaluated against MRSA and VRE resistant Gram-positive strains such as Staphylococcus and Enterococcus. Most of the compounds were more potent in vitro but less active in vivo than linezolid.     

Mutational analysis of OGG1, MYH, MTH1 in FAP, HNPCC and sporadic colorectal cancer patients: R154H OGG1 polymorphism is associated with sporadic colorectal cancer patients

MYH, OGG1 and MTH1 are members of base excision repair (BER) families, and MYH germline mutations were recently identified in patients with multiple adenomas or familial adenomatous polyposis (FAP). A total of 20 APC-negative Korean FAP patients were analyzed for OGG1, MYH and MTH1 germline mutations. A total of 19 hereditary nonpolyposis colorectal cancer (HNPCC), 86 suspected HNPCC, and 246 sporadic colorectal cancer cases were investigated for OGG1 and MYH mutations. A total of 14 R154H OGG1 polymorphisms were identified in hereditary, sporadic colorectal cancers, and normal controls. For the case-control analysis of OGG1 R154H, a total of 625 hereditary or sporadic colorectal cancer patients and 527 normal controls were screened. R154H was a rare polymorphism associated with sporadic colorectal cancer patents (OR: 3.586, P= 0.053). R154H does not segregate with cancer phenotypes. Upon examining the possibility of recessive inheritance of R154H, we could not identify any complementary mutations in OGG1, MYH or MTH1. Samples with R154H were further screened for mutations of K-ras, -catenin, APC, p53, BRAF and the microsatellite instability (MSI) status. Eight somatic mutations were identified in these genes and G:C to T:A transversion mutations were not dominant in samples harboring R154H. This result raises the possibility that OGG1 R154H may function as a low/moderate-penetrance modifier for colorectal cancer development.I.-J. Kim and J.-L. Ku contributed equally to this work     

Functional characterization and molecular modeling of methylcatechol 2,3-dioxygenase from o-xylene-degrading Rhodococcus sp. strain DK17

Rhodococcus sp. strain DK17 is known to metabolize o-xylene and toluene through the intermediates 3,4-dimethylcatechol and 3- and 4-methylcatechol, respectively, which are further cleaved by a common catechol 2,3-dioxygenase. A putative gene encoding this enzyme (akbC) was amplified by PCR, cloned, and expressed in Escherichia coli. Assessment of the enzyme activity expressed in E. coli combined with sequence analysis of a mutant gene demonstrated that the akbC gene encodes the bona fide catechol 2,3-dioxygenase (AkbC) for metabolism of o-xylene and alkylbenzenes such as toluene and ethylbenzene. Analysis of the deduced amino acid sequence indicates that AkbC consists of a new catechol 2,3-dioxygenase class specific for methyl-substituted catechols. A computer-aided molecular modeling studies suggest that amino acid residues (particularly Phe177) in the beta10-beta11 loop play an essential role in characterizing the substrate specificity of AkbC.     

Differential CCR1-mediated chemotaxis signaling induced by human CC chemokine HCC-4/CCL16 in HOS cells

Human CC chemokine-4 (HCC-4)/CCL16 is a chemoattractant for monocytes and lymphocytes. Although HCC-4 binds to multiple CC chemokine receptors, the receptor-mediated signal transduction pathway induced by HCC-4 has not been characterized. Human osteogenic sarcoma cells stably expressing CCR1 were used to investigate HCC-4-mediated chemotaxis signaling events via CCR1. The chemotactic activity of HCC-4 as well as those of other CCR1-dependent chemokines including MIP-1alpha/CCL3, RANTES/CCL5, and Lkn-1/CCL15 was inhibited by the treatment of pertussis toxin, an inhibitor of Gi/Go protein, U73122, an inhibitor of phospholipase C (PLC), and rottlerin, a specific inhibitor of protein kinase Cdelta (PKCdelta). These results indicate that HCC-4-induced chemotaxis signaling is mediated through Gi/Go protein, PLC, and PKCdelta. SB202190, an inhibitor of p38 mitogen activated protein kinase, only blocked the chemotactic activity of HCC-4, but not those of other CCR1-dependent chemokines. SB202190 inhibited HCC-4-induced chemotaxis in a dose-dependent manner (P < 0.01). HCC-4 induces p38 activation in both a time and dose-dependent manner. However, such p38 activation was not induced by other CCR1-dependent chemokines. To further investigate the differential effect of HCC-4, the Ca2+ mobilization was examined. HCC-4 induced no intracellular Ca2+ flux in contrast to other CCR1-dependent chemokines. These results indicate that HCC-4 transduces signals differently from other CCR1-dependent chemokines and may play different roles in the immune response.     

Identification of parathyroid hormone-regulated proteins in mouse bone marrow cells by proteomics

The ability of parathyroid hormone (PTH) to enhance bone formation has recently been exploited in the treatment of osteoporosis. Several studies have suggested that the activation of bone marrow stromal cells could be preceded to show the anabolic effect of PTH on bone formation, but little is known of PTH-regulated proteins in bone marrow cells. Therefore, protein profiling in the intermittent PTH-treated bone marrow cells was evaluated using proteomics. Daily treatment for 5 days consisting of subcutaneous injection of either 150 microg/kg per day of mouse PTH (1-84) or vehicle (0.9% normal saline) was performed on the ICR mouse. At the end of the treatment period, bone marrow cells were separated and used in proteomics. The expression levels of seven proteins including vimentin were decreased, but those of four proteins including calreticulin and thioredoxin domain containing 7 protein (Txnde7) were increased. Among these, the decrease of vimentin and the increase of both calreticulin Txnde7 in mRNA levels were confirmed by semi-quantitative RT-PCR. In PTH-treated mouse MC3T3-E1 osteoblast cells, mRNA expression levels were not totally consistent with the results observed in proteomics. In conclusion, the differentially expressed proteins in bone marrow cells depending on PTH could be highly linked to the differentiation of osteoprogenitor cells in the bone marrow into preosteoblast cells.     

Effect of jasmonic acid on endogenous gibberellins and abscisic acid in rice under NaCl stress

Content of endogenous abscisic acid (ABA) increased in rice plants under salt stress. Pre- or post-treatment by jasmonic acid (JA) mostly further increased ABA content. In the presence of salt stress also content of gibberellins (GAs) mostly increased more after treatment by JA. Endogenous content of bioactive GA₁ was higher in post-treatment by JA than in pre-treatment by JA.This study was supported by the Korea Science and Engineering Foundation (2000-2-20100-001-3)     

Equilibrium and kinetic analysis of folding of ketosteroid isomerase from Comamonas testosteroni

Equilibrium and kinetic analyses have been carried out to elucidate the folding mechanism of homodimeric ketosteroid isomerase (KSI) from Comamonas testosteroni. The folding of KSI was reversible since the activity as well as the fluorescence and CD spectra was almost completely recovered after refolding. The equilibrium unfolding transitions monitored by fluorescence and CD measurements were almost coincident with each other, and the transition midpoint increased with increasing protein concentration. This suggests that the KSI folding follows a simple two-state mechanism consisting of native dimer and unfolded monomer without any thermodynamically stable intermediates. Sedimentation equilibrium analysis and size-exclusion chromatography of KSI at different urea concentrations supported the two-state model without any evidence of folded monomeric intermediates. Consistent with the two-state model, (1)H-(15)N HSQC spectra obtained for KSI in the unfolding transition region could be reproduced by a simple addition of the spectra of the native and the unfolded KSI. The KSI refolding kinetics as monitored by fluorescence intensity could be described as a fast first-order process followed by a second-order and a subsequent slow first-order processes with rate constants of 60 s(-)(1), 5.4 x 10(4) M(-)(1).s(-)(1), and 0.017 s(-)(1), respectively, at 0.62 M urea, suggesting that there may be a monomeric folding intermediate. After a burst phase that accounts for >83% of the total amplitude, the negative molar ellipticity at 225 nm increased slowly in a single phase at a rate comparable to that of the bimolecular intermediate step. The kinetics of activity recovery from the denatured state were markedly dependent upon the protein concentration, implying that the monomers are not fully active. Taken together, our results demonstrate that the dimerization induces KSI to fold into the complete structure and is crucial for maintaining the tertiary structure to perform efficient catalysis.