Localization and regulation of mouse pantothenate kinase 2

Coenzyme A (CoA) biosynthesis is initiated by pantothenate kinase (PanK) and CoA levels are controlled through differential expression and feedback regulation of PanK isoforms. PanK2 is a mitochondrial protein in humans, but comparative genomics revealed that acquisition of a mitochondrial targeting signal was limited to primates. Human and mouse PanK2 possessed similar biochemical properties, with inhibition by acetyl-CoA and activation by palmitoylcarnitine. Mouse PanK2 localized in the cytosol, and the expression of PanK2 was higher in human brain compared to mouse brain. Differences in expression and subcellular localization should be considered in developing a mouse model for human PanK2 deficiency.     

Characterization of the bile acid profile in developing male and female hamsters in response to dietary cholesterol challenge

The Syrian golden hamster is a frequently used model to study cholesterol and bile acid metabolism as well as cholesterol-induced cholelithiasis. However, diet-induced gallstones seem limited to young male hamsters of certain strains that develop depressed cholate/chenodeoxycholate bile acid ratios. To further elucidate gender and age specific aspects of cholesterol and bile acid metabolism, i.e. a possible age-related bile acid/gallstone relationship, plasma and biliary lipids and bile acid composition were analyzed in male and female hamsters under various physiological conditions of age and diet, the latter formulated with and without dietary cholesterol. During normal development (no cholesterol challenge) the percentage of cholic acid decreased while chenodeoxycholate increased, the shift being more pronounced in males. Furthermore, female hamsters had higher total plasma cholesterol than in males, while hepatic and biliary lipids did not differ. When challenged with excessive dietary cholesterol, female hamsters again developed significantly higher total plasma and hepatic cholesterol concentrations. Biliary lipids and cholesterol gallstone incidence revealed a significant gender effect with male hamsters developing a higher lithogenic index and more gallstones (cholesterol and pigment stones) than females. Female hamsters revealed a lower percentage of chenodeoxycholate and a higher percentage of cholate resulting in a more protective, higher cholate/cheno ratio (1.5 +/- 1.0) than in males (1.0 +/- 0.2). In summary, the bile acid pattern in developing and cholesterol-fed hamsters renders females less susceptible to gallstones, in part because they maintain more favorable biliary lipid and bile acid profiles, characterized by lower molar percentages of biliary cholesterol and chenodeoxycholate.     

BAREing it all: the adoption of LXR and FXR and their roles in lipid homeostasis.

During the last three years there have been a plethora of publications on the liver X-activated receptors (LXRalpha, NR1H3, and LXRbeta, NR1H2), the farnesoid X-activated receptor (FXR, NR1H4), and the pregnane X receptor (PXR, NR1I2) and the role these nuclear receptors play in controlling cholesterol, bile acid, lipoprotein and drug metabolism. The current interest in these nuclear receptors is high, in part, because they appear to be promising therapeutic targets for new drugs that have the potential to control lipid homeostasis.In this review we emphasize i) the role of LXR in controlling many aspects of cholesterol and fatty acid metabolism, ii) the expanded role of FXR in regulating genes that control not only bile acid metabolism but also lipoprotein metabolism, and iii) the regulation of bile acid transport/metabolism in response to bile acid-activated PXR.     

Feedback inhibition of pantothenate kinase regulates pantothenol uptake by the malaria parasite

To survive, the human malaria parasite Plasmodium falciparum must acquire pantothenate (vitamin B5) from the external medium. Pantothenol (provitamin B5) inhibits parasite growth by competing with pantothenate for pantothenate kinase, the first enzyme in the coenzyme A biosynthesis pathway. In this study we investigated pantothenol uptake by P. falciparum and in doing so gained insights into the regulation of the parasite's coenzyme A biosynthesis pathway. Pantothenol was shown to enter P. falciparum-infected erythrocytes via two routes, the furosemide-inhibited "new permeation pathways" induced by the parasite in the infected erythrocyte membrane (the sole access route for pantothenate) and a second, furosemide-insensitive pathway. Having entered the erythrocyte, pantothenol is taken up by the intracellular parasite via a mechanism showing functional characteristics distinct from those of the parasite's pantothenate uptake mechanism. On reaching the parasite cytosol, pantothenol is phosphorylated and thereby trapped by pantothenate kinase, shown here to be under feedback inhibition control by coenzyme A. Furosemide reduced this inherent feedback inhibition by competing with coenzyme A for binding to pantothenate kinase, thereby increasing pantothenol uptake.     

A pantothenate kinase from Staphylococcus aureus refractory to feedback regulation by coenzyme A

The key regulatory step in CoA biosynthesis in bacteria and mammals is pantothenate kinase (CoaA), which governs the intracellular concentration of CoA through feedback regulation by CoA and its thioesters. CoaA from Staphylococcus aureus (SaCoaA) has a distinct primary sequence that is more similar to the mammalian pantothenate kinases than the prototypical bacterial CoaA of Escherichia coli. In contrast to all known pantothenate kinases, SaCoaA activity is not feedback-regulated by CoA or CoA thioesters. Metabolic labeling of S. aureus confirms that CoA levels are not controlled by CoaA or at steps downstream from CoaA. The pantothenic acid antimetabolite N-heptylpantothenamide (N7-Pan) possesses potent antimicrobial activity against S. aureus and has multiple cellular targets. N7-Pan is a substrate for SaCoaA and is converted to the inactive butyldethia-CoA analog by the downstream pathway enzymes. The analog is also incorporated into acyl carrier protein and D-alanyl carrier protein, the prosthetic groups of which are derived from CoA. The inactivation of acyl carrier protein and the cessation of fatty acid synthesis are the most critical causes of growth inhibition by N7-Pan because the toxicity of the drug is ameliorated by supplementing the growth medium with fatty acids. The absence of feedback regulation at the pantothenate kinase step allows the accumulation of high concentrations of intracellular CoA, consistent with the physiology of S. aureus, which lacks glutathione and relies on the CoA/CoA disulfide reductase redox system for protection from oxidative damage.     

Armenian hamster female protein (serum amyloid P component). Comparison with the sex-regulated homolog in Syrian hamster

Complementary DNA clones for Armenian hamster female protein (FP) were isolated and the complete nucleotide sequence and derived amino acid sequence were determined and compared with relevant data for the closely related Syrian hamster. Although biosynthesis of preSAP is directed by a 1.0-kb mRNA in both genera and the molecular mass of the primary translation product of FP is identical, the FP gene structure and regulation of expression of FP are different in Syrian and Armenian hamsters. Whereas the direction of alteration in FP mRNA levels is divergent in Syrian hamsters during an acute phase reaction, hepatic FP mRNA levels increase in both male and female Armenian hamsters during inflammation. Regulation of expression of Armenian and Syrian hamster FP genes occurs at a pretranslational level.     

3-Hydroxy-3-methylglutaric acid (HMGA) reduces dietary cholesterol induction of saturated bile in hamster

3-Hydroxy-3-methylglutaric acid (HMGA) is a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR) and strongly reduces cholesterol biosynthesis both in vitro and in vivo. Since the effects of this compound on biliary lipid composition are hitherto unknown, we have investigated whether it prevents dietary cholesterol induction of saturated bile in the hamster. Female Golden Syrian hamsters have been divided into four groups and treated for 10 weeks as follows: I) Standard diet, containing 0.8 mg cholesterol/g food; II) Standard diet plus HMGA (100 mg/kg b.w./day per os); III) Lithogenic diet containing 2.4 mg cholesterol/g food; IV) Lithogenic diet plus HMGA as above. The results indicate that HMGA is effective in reducing both bile cholesterol supersaturation and hypercholesterolemia. Inhibition of hepatic cholesterogenesis at the level of mevalonate synthesis by HMGCoAR and reduction of intestinal cholesterol absorption may be responsible for these effects.     

Biochemical site of regulation of bile acid biosynthesis in the rat

The production of bile salts by rat liver is regulated by a feedback mechanism, but it is not known which enzyme controls endogenous bile acid synthesis. In order to demonstrate the biochemical site of this control mechanism, bile fistula rats were infused intravenously with (14)C-labeled bile acid precursors, and bile acid biosynthesis was inhibited as required by intraduodenal infusion of sodium taurocholate. The infusion of taurocholate (11-14 mg/100 g of rat per hr) inhibited the incorporation of acetate-1-(14)C, mevalonolactone-2-(14)C, and cholesterol-4-(14)C into bile acids by approximately 90%. In contrast, the incorporation of 7alpha-hydroxycholesterol-4-(14)C into bile acids was reduced by less than 10% during taurocholate infusion. These results indicate that the regulation of bile acid biosynthesis is exerted via cholesterol 7alpha-hydroxylase provided that hepatic cholesterol synthesis is adequate.     

Cloning and activation of the Syrian hamster neu proto-oncogene

Point mutations of the Syrian hamster neu proto-oncogene have been observed in the transmembrane domain of N-nitroso-N-ethylurea(ENU)-induced neurofibromas, Genomic DNA derived from tumor tissue showed transforming activity in an NIH 3T3 assay and a cDNA clone of the hamster neu gene, containing the entire protein-coding region, was isolated from a transformant cDNA library. The encoded product is 92 and 88% homologous to the rat neu and the human c-erbB-2, respectively. The product of the mutated hamster neu gene showed increased autophosphorylation of Tyr residues.     

Casein kinase I: another cog in the circadian clockworks

Multiple components of the circadian central clock are phosphoproteins, and it has become increasingly clear that posttranslational modification is an important regulator of circadian rhythm in diverse organisms, from dinoflagellates to humans. Genetic studies in Drosophila have identified double-time (dbt), a serine/threonine protein kinase that is highly homologous to human casein kinase I epsilon (CKIε), as the first kinase linked to behavioral rhythms. Identification of a missense mutation in CKIε as the tau mutation in the Syrian hamster places CKIε within the core clock machinery in mammals. Most recently, identification of a phosphorylation site mutant of hPER2 in a family with an inherited circadian rhythm abnormality strongly suggests that PER2 is a physiologically relevant substrate of CKI. Phosphorylation may regulate multiple properties of clock proteins, including stability and intracellular localization. (Chronobiology International, 18(3), 389-398, 2001)     

Qualitative and quantitative aspects of the biosynthesis of ribonucleic acid and of protein in the liver and the lung of the Syrian golden hamster

1. The incorporation of orotic acid and of uridine into total RNA was measured in vivo in liver and lung of the Syrian golden hamster. Specific activities of total acid-soluble UMP were measured in both organs. An estimation of the rate of RNA biosynthesis showed that hamster lung synthesizes RNA at about one-half of the rate of that of hamster liver. 2. The apparent K(m) and V(max.) values of a few enzymes involved in pyrimidine biosynthesis were measured in the 100000g supernatants of liver and lung. The apparent K(m) values were very similar in both organs. From the estimated V(max.,) it was concluded that hamster lung cells have less capacity to metabolize orotic acid than have liver cells. 3. A time-response and a dose-response study showed that actinomycin D inhibits pulmonary RNA synthesis as efficiently as hepatic RNA synthesis. 4. Protein synthesis, measured as the incorporation of leucine, was inhibited in both organs 30min after a dose of 2mg of cycloheximide/kg. The dose-response patterns were similar in both liver and lung 3h after cycloheximide. 5. It is concluded that RNA and protein synthesis in vivo in hamster lung are very similar to the corresponding reactions in liver. Alterations of RNA and protein synthesis by toxic agents can therefore be evaluated in lung with a similar approach to that used to study the pathological biochemistry of liver.     

Overexpression of active Syrian golden hamster prion protein PrPc as a glutathione S-transferase fusion in heterologous systems.

This article describes a procedure which permits for the first time the isolation of the prion protein PrPc from the Syrian golden hamster in heterologous systems. Using a glutathione S-transferase (GST) fusion approach, milligram amounts of stable, soluble, and homogeneous GST::PrPc protein were obtained in Escherichia coli and with baculovirus-infected insect cells. Authentic PrPc was released from the immobilized fusion protein by direct cleavage with thrombin. GST::PrPc expressed in these two expression systems and also authentic PrPc released by thrombin cleavage were recognized by a polyclonal antibody directed against amino acid 95 to 110 of the golden hamster PrPc protein. GST::PrPc was not detected by a monoclonal antibody recognizing the region encompassing amino acids 138 to 152 of the human prion protein. The fusion protein was sensitive to proteinase K digestion, demonstrating that the cellular rather than the proteinase K-resistant scrapie isoform was produced.     

Carnosic acid: A potent chemopreventive agent against oral carcinogenesis

The present study was aimed to investigate the chemopreventive potential of carnosic acid in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. The chemopreventive potential was assessed by analyzing the tumor incidence, tumor volume and burden as well as by measuring the status of lipid peroxidation, non-enzymatic and enzymatic antioxidants and phase I and phase II detoxification enzymes. Oral squamous cell carcinoma was developed in the buccal pouch of golden Syrian hamsters by painting with 0.5% DMBA in liquid paraffin three times a week for 14 weeks. In the present study, 100% tumor formation was observed in hamsters treated with DMBA alone. Also, the status of lipid peroxidation, antioxidants and phase I and phase II detoxification enzymes were significantly altered during DMBA-induced oral carcinogenesis. Oral administration of carnosic acid at a dose of 10 mg/kg body weight/day to DMBA-treated animals completely prevented the tumor formation in the hamsters’ buccal pouches. Also, carnosic acid exerted potent anti-lipid peroxidative function and stimulated the detoxification cascade during DMBA-induced hamster buccal pouch carcinogenesis. The results of the present study suggest that the chemopreventive potential of carnosic acid is probably due to its anti-lipid peroxidative potential and modulating effect on carcinogen detoxification enzymes during DMBA-induced oral carcinogenesis.     

Enhanced sialylation of recombinant human erythropoietin in Chinese hamster ovary cells by combinatorial engineering of selected genes

Therapeutic glycoproteins with exposed galactose (Gal) residues are cleared rapidly from the bloodstream by asialoglycoprotein receptors in hepatocytes. Various approaches have been used to increase the content of sialic acid, which occupies terminal sites of N- or O-linked glycans and thereby increases the half-life of therapeutic glycoproteins. We enhanced sialylation of human erythropoietin (EPO) by genetic engineering of the sialylation pathway in Chinese hamster ovary (CHO) cells. The enzyme GNE (uridine diphosphate-N-acetyl glucosamine 2-epimerase)/MNK (N-acetyl mannosamine kinase), which plays a key role in the initial two steps of sialic acid biosynthesis, is regulated by cytidine monophosphate (CMP)-sialic acid through a feedback mechanism. Since sialuria patient cells fail in regulating sialic acid biosynthesis by feedback mechanism, various sialuria-like mutated rat GNEs were established and subjected to in vitro activity assay. GNE/MNK-R263L-R266Q mutant showed 93.6% relative activity compared with wild type and did not display feedback inhibition. Genes for sialuria-mutated rat GNE/MNK, Chinese hamster CMP-sialic acid transporter and human α2,3-sialyltransferase (α2,3-ST) were transfected simultaneously into recombinant human (rh) EPO-producing CHO cells. CMP-sialic acid concentration of engineered cells was significantly (>10-fold) increased by sialuria-mutated GNE/MNK (R263L-R266Q) expression. The sialic acid content of rhEPO produced from engineered cells was 43% higher than that of control cells. Ratio of tetra-sialylated glycan of rhEPO produced from engineered cells was increased ～32%, but ratios of asialo- and mono-sialylated glycans were decreased ～50%, compared with control. These findings indicate that sialuria-mutated rat GNE/MNK effectively increases the intracellular CMP-sialic acid level. The newly constructed host CHO cell lines produced more highly sialylated therapeutic glycoproteins through overexpression of sialuria-mutated GNE/MNK, CMP-SAT and α2,3-ST.     

Role of feedback regulation of pantothenate kinase (CoaA) in control of coenzyme A levels in Escherichia coli

Pantothenate kinase (CoaA) is a key regulator of coenzyme A (CoA) biosynthesis in Escherichia coli, and its activity is controlled by feedback inhibition by CoA and its thioesters. The importance of feedback inhibition in the control of the intracellular CoA levels was tested by constructing three site-directed mutants of CoaA that were predicted to be feedback resistant based on the crystal structure of the CoaA-CoA binary complex. CoaA[R106A], CoaA[H177Q], and CoaA[F247V] were purified and shown to retain significant catalytic activity and be refractory to inhibition by CoA. CoaA[R106A] retained 50% of the catalytic activity of CoaA, whereas the CoaA[H177Q] and CoaA[F247V] mutants were less active. The importance of feedback control of CoaA to the intracellular CoA levels was assessed by expressing either CoaA or CoaA[R106A] in strain ANS3 [coaA15(Ts) panD2]. Cells expressing CoaA[R106A] had significantly higher levels of phosphorylated pantothenate-derived metabolites and CoA in vivo and excreted significantly more 4'-phosphopantetheine into the medium compared to cells expressing the wild-type protein. These data illustrate the key role of feedback regulation of pantothenate kinase in the control of intracellular CoA levels.     

Differential Metabolite Profiles during Fruit Development in High-Yielding Oil Palm Mesocarp

To better understand lipid biosynthesis in oil palm mesocarp, in particular the differences in gene regulation leading to and including de novo fatty acid biosynthesis, a multi-platform metabolomics technology was used to profile mesocarp metabolites during six critical stages of fruit development in comparatively high- and low-yielding oil palm populations. Significantly higher amino acid levels preceding lipid biosynthesis and nucleosides during lipid biosynthesis were observed in a higher yielding commercial palm population. Levels of metabolites involved in glycolysis revealed interesting divergence of flux towards glycerol-3-phosphate, while carbon utilization differences in the TCA cycle were proven by an increase in malic acid/citric acid ratio. Apart from insights into the regulation of enhanced lipid production in oil palm, these results provide potentially useful metabolite yield markers and genes of interest for use in breeding programmes.     

Myometrial characteristics of the syrian hamster uterine smooth muscle cell line,SHM

Summary We describe several characteristics of a novel smooth muscle cell line, SHM (Syrian hamster myometrium) derived from a primary uterine leiomyosarcoma which was induced by chronic estrogen plus androgen treatment of a female Syrian (golden) hamster. To determine the usefulness of the SHM cell line as a model for understanding myometrial function and its regulation, we have examined the morphologic and immunocytochemical properties of these cells, and the ability of uterotonic agonists to activate transmembrane signaling via phosphoinositide hydrolysis. The SHM cells exhibited a spindle-shape, smooth musclelike morphology when subconfluent, and a more compact, stellate shape at confluence. Like primary myocytes, SHM cells expressed the intermediate filament desmin and the contractile protein alpha smooth muscle actin, but not the epithelial antigen cytokeratin. Norepinephrine and bradykinin, which stimulate contraction and inositol polyphosphate production in the uterus, also stimulated inositol polyphosphate production in SHM cells. The maximal phosphoinositide signaling responses were lower in SHM cells compared with primary hamster uterine myocytes. We conclude that the SHM cell line exhibits primary uterine myocyte characteristics, and may therefore be a useful system for examining the mechanisms through which myometrial functions are regulated.