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1.
Neuroscience ; 324: 407-19, 2016 Jun 02.
Article in English | MEDLINE | ID: mdl-26979053

ABSTRACT

Maternal consumption of a high-fat diet (HFD) during pregnancy is found to stimulate the genesis of hypothalamic orexigenic peptide neurons in the offspring, while HFD intake in adult animals produces a systemic low-grade inflammation which increases neuroimmune factors that may affect neurogenesis and neuronal migration. Building on this evidence and our recent study showing that the inflammatory chemokine, CCL2, stimulates the migration of hypothalamic neurons and expression of orexigenic neuropeptides, we tested here the possibility that prenatal exposure to a HFD in rats affects this chemokine system, both CCL2 and its receptors, CCR2 and CCR4, and alters its actions on hypothalamic neurons, specifically those expressing the neuropeptides, enkephalin (ENK) and galanin (GAL). Using primary dissociated hypothalamic neurons extracted from embryos on embryonic day 19, we found that prenatal HFD exposure compared to chow control actually reduces the expression of CCL2 in these hypothalamic neurons, while increasing CCR2 and CCR4 expression, and also reduces the sensitivity of hypothalamic neurons to CCL2. The HFD abolished the dose-dependent, stimulatory effect of CCL2 on the number of migrated neurons and even shifted its normal stimulatory effect on migrational velocity and distance traveled by control neurons to an inhibition of migration. Further, it abolished the dose-dependent, stimulatory effect of CCL2 on neuronal expression of ENK and GAL. These results demonstrate that prenatal HFD exposure greatly disturbs the functioning of the CCL2 chemokine system in embryonic hypothalamic neurons, reducing its endogenous levels and ability to promote the migration of neurons and their expression of orexigenic peptides.


Subject(s)
Chemokine CCL2/metabolism , Dietary Fats/adverse effects , Hypothalamus/embryology , Hypothalamus/metabolism , Neurons/metabolism , Pregnancy Complications/metabolism , Animals , Cell Movement/physiology , Cells, Cultured , Enkephalins/metabolism , Female , Galanin/metabolism , Pregnancy , Protein Precursors/metabolism , RNA, Messenger/metabolism , Rats, Sprague-Dawley , Receptors, CCR2/metabolism , Receptors, CCR4/metabolism
2.
Clin Microbiol Infect ; 14(3): 221-7, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18070129

ABSTRACT

While increasing numbers of cytomegalovirus (CMV)-associated diseases are occurring in patients undergoing conventional chemotherapy, information regarding CMV reactivation is limited. This pilot study was conducted to investigate CMV reactivation induced by chemotherapy. Seven blood samples were collected from each of 15 patients with newly diagnosed malignant disease, at baseline before chemotherapy, and once every month after chemotherapy was commenced. CMV viral loads in leukocytes were determined by real-time PCR. Host responses to changes in viral loads were assessed by assaying CMV-specific IgG titres and tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma levels in each of the blood samples, and by scoring the number of CMV-associated clinical symptoms that developed. All except one patient experienced CMV reactivation during the course of chemotherapy, with the average viral load peaking after the third course of treatment. Titres of CMV-specific IgG increased in line with the increase in viral load. Plasma levels of TNF-alpha and IFN-gamma initially decreased from baseline, and then rose to peak levels at the same time as, or shortly after, the highest viral loads were recorded. Clinical symptoms potentially attributable to CMV infection appeared as the viral load increased. It was concluded that the incidence of CMV reactivation in patients receiving conventional chemotherapy is high. Reactivation is not asymptomatic, but was self-limiting in most of these cases. Increases in plasma TNF-alpha and IFN-gamma occur after reactivation, but not before.


Subject(s)
Antineoplastic Agents/adverse effects , Antineoplastic Agents/therapeutic use , Cytomegalovirus Infections/diagnosis , Cytomegalovirus/isolation & purification , Neoplasms/drug therapy , Virus Activation , Adult , Aged , Antibodies, Viral/blood , Cytomegalovirus Infections/immunology , DNA, Viral/blood , Female , Humans , Interferon-gamma/blood , Leukocytes/virology , Male , Middle Aged , Neoplasms/complications , Polymerase Chain Reaction/methods , Tumor Necrosis Factor-alpha/blood , Viral Load
3.
Kidney Int ; 72(1): 63-71, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17410095

ABSTRACT

The sodium-dependent dicarboxylate cotransporter (NaDC1) has a proposed function of reabsorbing various Krebs cycle intermediates in the kidney and the small intestine. Since Krebs cycle intermediates have been suggested to be important for renal cell survival and recovery after hypoxia and reoxygenation, the transporter may play a role in the recovery of the kidney. Additionally, mutations in the transporter homolog in Drosophila led to fly longevity which was thought to be similar to that induced by caloric restriction (CR). To clarify the role of the sodium dicarboxylate cotransporter in vivo we generated cotransporter-deficient mice. These knockout mice excreted significantly higher amounts of various Krebs cycle intermediates in their urine; thus confirming the proposed function to reabsorb these metabolic intermediates in the kidney. No other phenotypic change was identified in these mice, however. Transporter deficiency did not affect renal function under normal physiological conditions, nor did it have an effect on renal damage and recovery from ischemic injury. Additionally, the absence of the transporter did not lead to metabolic or physiological changes associated with CR. Our results suggest that although the sodium dicarboxylate cotransporter is involved in regulating levels of various Krebs cycle intermediates in the kidney, impaired uptake of these intermediates does not significantly affect renal function under normal or ischemic stress.


Subject(s)
Citric Acid Cycle/physiology , Dicarboxylic Acid Transporters/genetics , Dicarboxylic Acid Transporters/physiology , Kidney/physiology , Organic Anion Transporters, Sodium-Dependent/genetics , Organic Anion Transporters, Sodium-Dependent/physiology , Symporters/genetics , Symporters/physiology , Animals , Apoptosis/physiology , Caloric Restriction , Cell Differentiation/physiology , Cell Proliferation , Citrates/blood , Creatinine/blood , Kidney/pathology , Kidney/physiopathology , Kidney Tubules, Proximal/pathology , Kidney Tubules, Proximal/physiology , Kidney Tubules, Proximal/physiopathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Reperfusion Injury/pathology , Reperfusion Injury/physiopathology
4.
Eur Respir J ; 28(6): 1178-85, 2006 Dec.
Article in English | MEDLINE | ID: mdl-16899477

ABSTRACT

A high protein concentration in a pleural effusion makes it more likely to be a malignant than a transudative effusion. However, the variability in protein composition between these two forms of pleural effusion is not well understood. In order to compare their protein compositions, the proteomic profiles of 14 malignant and 13 transudative pleural effusions were studied using two-dimensional gel electrophoresis. Protein spots with differential expression were identified by matrix-assisted laser desorption/ionisation quadrupole time-of-flight mass spectrometry and liquid chromatography/tandem mass spectrometry. Targeted proteins were further examined by ELISA and Western immunoassay in all samples. Two-dimensional gel electrophoresis revealed seven spots whose expression was reduced in malignant pleural effusions. Four of the abnormal spots were identified as fibrinogen gamma-chain precursor, two as fibrinogen beta-chain precursor and one as pigment epithelium-derived factor. ELISA and Western immunoassay showed that pigment epithelium-derived factor levels were significantly lower in malignant than in transudative pleural effusions. It has been demonstrated that proteomic technologies may help in the elucidation of variable expression of proteins with particular functions. By applying these technologies, the level of pigment epithelium-derived factor, a potent anti-angiogenic factor, was found to be significantly lower in malignant than in transudative pleural effusions. This finding allows for further exploration regarding how underexpression of pigment epithelium-derived factor may relate to the pathogenesis of malignant pleural effusions.


Subject(s)
Neoplasm Proteins/metabolism , Pleural Effusion/metabolism , Proteomics/methods , Adenocarcinoma/metabolism , Databases, Protein , Electrophoresis, Gel, Two-Dimensional , Enzyme-Linked Immunosorbent Assay , Humans , Image Processing, Computer-Assisted , Lung Neoplasms/metabolism , Peptide Fragments/chemistry , Silver Staining , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trypsin/pharmacology
5.
Arch Dis Child Fetal Neonatal Ed ; 91(6): F434-8, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16849367

ABSTRACT

OBJECTIVE: To evaluate the use of a new transcutaneous bilirubinometer (JM-103 Minolta Airshields) for detection of hyperbilirubinaemia in term or near-term healthy Chinese newborns. METHODS: Transcutaneous bilirubin (TcB) was used to screen for severe hyperbilirubinaemia in newborn infants. Blood was taken for total serum bilirubin (TSB) measurement if the initial TcB level was higher than the 40th centile in Bhutani's nomogram. Paired TcB and TSB results were then reviewed over 6 months. The correlation as well as the mean difference between the two methods were calculated. The clinical application of TcB with Bhutani's nomogram in the prediction of severe hyperbilirubinaemia in low-risk, medium-risk and high-risk thresholds for phototherapy was also analysed. RESULTS: 997 paired TcB and TSB measurements were evaluated in term or near-term newborns. TcB was significantly correlated with TSB, with a correlation coefficient of 0.83 (p<0.001). Their mean difference was 21.7 micromol/l (SD 21.2, p<0.001), with the 95% limits of agreement between -19.9 and 63.3 micromol/l. In both low-risk and medium-risk thresholds for phototherapy, using the 75th centile of Bhutani's nomogram as threshold, TcB could identify all cases and had a sensitivity and negative predictive value of 100% each, a specificity of 56% and positive predictive value of 23%. For high-risk cases, using the 75th centile as cut-off, the sensitivity and negative predictive value were reduced to 86.7% and 97.0%, respectively. CONCLUSION: An accurate point-of-care bilirubin analyser facilitates bilirubin screening and avoids unnecessary blood tests. Although using the transcutaneous bilirubinometer JM-103 might result in a significant difference between TcB and TSB measured in Chinese newborns, combining the use of TcB and the 75th centile in Bhutani's nomogram as the cut-off level can identify all cases of significant hyperbilirubinaemia.


Subject(s)
Bilirubin/blood , Hyperbilirubinemia, Neonatal/diagnosis , Neonatal Screening/instrumentation , Humans , Hyperbilirubinemia, Neonatal/therapy , Infant, Newborn , Neonatal Screening/standards , Phototherapy/methods , Predictive Value of Tests , Retrospective Studies , Sensitivity and Specificity
6.
Arch Dis Child Fetal Neonatal Ed ; 89(4): F356-9, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15210675

ABSTRACT

BACKGROUND: Low blood glucose in newborns is difficult to detect clinically. Hence a reliable "point of care" device (glucometer) for early detection and treatment of low glucose is needed. OBJECTIVE: To evaluate the performance of five readily available glucometers for the detection of low blood glucose in newborn infants. METHOD: Glucostix measurements were taken for newborns with risk factors using a Reflolux S (Boehringer) glucometer. If the initial reading was low (< 2.6 mmol/l), further measurements were taken with two other glucometers (phase I, Advantage and Glucotrend (Roche); phase II, Elite XL (Bayer) and Precision (Abbott)), and plasma glucose was measured in the laboratory (Aeroset; Abbott). RESULTS: Over 10 months, 101 specimens were collected from 71 newborns (57 in phase I; 44 in phase II). The Advantage glucometer usually overestimated blood glucose with a mean difference of 1.07 mmol/l (p < 0.01) at all low glucose ranges. The Glucotrend, Precision, and Elite XL glucometers performed better; the mean differences were not significantly different from the laboratory measured value (0.17 mmol/l (p = 0.37); -0.12 mmol/l (p = 0.13), and 0.24 mmol/l (p = 0.13) respectively). For detection of glucose concentrations < 2.6 mmol/l, the Precision glucometer had the highest sensitivity (96.4%) and negative predictive value (90%). For lower glucose concentrations (< 2.0 mmol/l), the Glucotrend glucometer performed even better (sensitivity 92.3%, negative predictive value 96.3%). CONCLUSION: Point of care devices should have good precision in the low glucose concentration range, sensitivity, and accuracy for early detection of neonatal hypoglycaemia. None of the five glucometers was satisfactory as the sole measuring device. The Glucotrend and Precision glucometers have the greatest sensitivity and negative predictive value. However, confirmation with laboratory measurements of plasma glucose and clinical assessment are still of the utmost importance.


Subject(s)
Blood Glucose/analysis , Hypoglycemia/diagnosis , Monitoring, Physiologic/instrumentation , Point-of-Care Systems/standards , Humans , Infant, Newborn , Monitoring, Physiologic/standards , Neonatal Screening/instrumentation , Sensitivity and Specificity
7.
Dig Dis Sci ; 46(11): 2374-7, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11713938

ABSTRACT

Altered gallbladder motility by progesterone has been recognized as an important factor in the development of gallstones. There are two types of hepatolithiasis, that occurs de novo in the intrahepatic ducts with an intact gallbladder (primary hepatolithiasis) and that which originates in the gallbladder and the stones migrate into the intrahepatic duct (secondary hepatolithiasis). This study was designed to evaluate the possible role of the progesterone receptor of gallbladder in the pathogenesis of hepatolithiasis. Eighty-four patients with hepatolithiasis (34 patients had primary hepatolithiasis and the other 50 patients secondary hepatolithiasis) were included. Paraffin-embedded specimens of gallbladder were processed to have an immunohistochemical staining for progesterone receptor. Positivity for progesterone receptor of gallbladder specimens was noted in eight patients (23.5%) with primary hepatolithiasis and in 23 patients (46%) with secondary hepatolithiasis. There is significant difference (P = 0.031) in positive rate for progesterone receptor between the primary hepatolithiasis and secondary hepatolithiasis groups. In conclusion, many more patients (46%) with secondary hepatolithiasis show positivity for progesterone receptor of gallbladder specimens than patients with primary hepatolithiasis (23.5%) (P = 0.031). This phenomenon is intriguing and should be of further evaluation and elucidated.


Subject(s)
Bile Ducts, Intrahepatic , Cholelithiasis/metabolism , Gallbladder/metabolism , Liver Diseases/metabolism , Receptors, Progesterone/metabolism , Cholelithiasis/etiology , Humans , Liver Diseases/etiology
9.
Mol Cell Biol ; 20(16): 5840-6, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10913167

ABSTRACT

Aldose reductase (ALR2) is thought to be involved in the pathogenesis of various diseases associated with diabetes mellitus, such as cataract, retinopathy, neuropathy, and nephropathy. However, its physiological functions are not well understood. We developed mice deficient in this enzyme and found that they had no apparent developmental or reproductive abnormality except that they drank and urinated significantly more than their wild-type littermates. These ALR2-deficient mice exhibited a partially defective urine-concentrating ability, having a phenotype resembling that of nephrogenic diabetes insipidus.


Subject(s)
Aldehyde Reductase/deficiency , Aldehyde Reductase/genetics , Diabetes Insipidus, Nephrogenic/genetics , Mice, Knockout , Animals , Diabetes Insipidus, Nephrogenic/etiology , Diabetes Insipidus, Nephrogenic/metabolism , Disease Models, Animal , Mice
10.
Stat Med ; 19(8): 1029-37, 2000 Apr 30.
Article in English | MEDLINE | ID: mdl-10790678

ABSTRACT

In clinical trials, appropriate designs are often chosen to address scientific/medical questions of particular interest to the investigator. For a chosen statistical design, however, standard statistical procedures may not be applicable owing to the nature of the design. In this paper we examine statistical methods for analysis of data collected from a placebo-challenging design which is often considered for assessment of the efficacy of drug products for indication of erectile dysfunction. An example concerning a clinical trial conducted with 120 male patients with erectile dysfunction is used to illustrate the derived statistical methods. Some recommendations to the randomization procedure for the study design of this kind are also made.


Subject(s)
Models, Statistical , Randomized Controlled Trials as Topic , Cross-Over Studies , Double-Blind Method , Erectile Dysfunction/drug therapy , Humans , Male , Penile Erection/drug effects , Placebos
11.
Bioorg Med Chem Lett ; 10(8): 715-7, 2000 Apr 17.
Article in English | MEDLINE | ID: mdl-10782671

ABSTRACT

4-Thiazolidinones were synthesized and evaluated for their ability to inhibit the bacterial enzyme MurB. Selected 4-thiazolidinones displayed activity against the enzyme in vitro. This activity, coupled with the design principles of the thiazolidinones, supports the postulate that 4-thiazolidinones may be recognized as diphosphate mimics by a biological selector.


Subject(s)
Bacteria/enzymology , Carbohydrate Dehydrogenases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Thiazoles/pharmacology
12.
J Clin Microbiol ; 38(3): 1127-30, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10699007

ABSTRACT

The autoSCAN-W/A (W/A; Dade Behring Microscan Inc., West Sacramento, Calif.) and Vitek AutoMicrobic System (Vitek AMS; bioMérieux Vitek Systems, Inc., Hazelwood, Mo.) are both fully automated microbiology systems. We evaluated the accuracy of these two systems in identifying nonglucose-fermenting gram-negative bacilli. We used the W/A with conventional-panel Neg Combo type 12 and Vitek GNI+ identification systems. A total of 301 isolates from 25 different species were tested. Of these, 299 isolates were identified in the databases of both systems. The conventional biochemical methods were used for reference. The W/A correctly identified 215 isolates (71. 4%) to the species level at initial testing with a high probability of >/=85%. The Vitek GNI+ correctly identified 216 isolates (71.8%) to the species level at initial testing with a high probability of >/=90%. After additional testing that was recommended by the manufacturer's protocol, the correct identifications of the W/A and Vitek GNI+ improved to 96.0 and 92.3%, respectively. The major misidentified species were Sphingomonas paucimobilis and Agrobacterium radiobacter in the W/A system and Acinetobacter lwoffii, Chryseobacterium indologenes, and Comamonas acidovorans in the Vitek GNI+ system. The error rates were 4.0 and 7.6%, respectively. The overall accuracy for both systems was above 90% if the supplemental tests were applied. There was no significant difference in accuracy (P > 0.05) between the two systems.


Subject(s)
Bacterial Typing Techniques , Gram-Negative Bacteria/classification , Gram-Negative Bacteria/isolation & purification , Gram-Negative Bacterial Infections/diagnosis , Automation/methods , False Negative Reactions , False Positive Reactions , Fermentation , Glucose/metabolism , Gram-Negative Bacteria/growth & development , Humans , Probability , Reproducibility of Results
14.
J Bacteriol ; 181(17): 5395-401, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10464212

ABSTRACT

The biochemical properties of the D-glutamate-adding enzymes (MurD) from Escherichia coli, Haemophilus influenzae, Enterococcus faecalis, and Staphylococcus aureus were investigated to detect any differences in the activity of this enzyme between gram-positive and gram-negative bacteria. The genes (murD) that encode these enzymes were cloned into pMAL-c2 fusion vector and overexpressed as maltose-binding protein-MurD fusion proteins. Each fusion protein was purified to homogeneity by affinity to amylose resin. Proteolytic treatments of the fusion proteins with factor Xa regenerated the individual MurD proteins. It was found that these fusion proteins retain D-glutamate-adding activity and have Km and Vmax values similar to those of the regenerated MurDs, except for the H. influenzae enzyme. Substrate inhibition by UDP-N-acetylmuramyl-L-alanine, the acceptor substrate, was observed at concentrations greater than 15 and 30 microM for E. coli and H. influenzae MurD, respectively. Such substrate inhibition was not observed with the E. faecalis and S. aureus enzymes, up to a substrate concentration of 1 to 2 mM. In addition, the two MurDs of gram-negative origin were shown to require monocations such as NH4+ and/or K+, but not Na+, for optimal activity, while anions such as Cl- and SO4(2-) had no effect on the enzyme activities. The activities of the two MurDs of gram-positive origin, on the other hand, were not affected by any of the ions tested. All four enzymes required Mg2+ for the ligase activity and exhibited optimal activities around pH 8. These differences observed between the gram-positive and gram-negative MurDs indicated that the two gram-negative bacteria may apply a more stringent regulation of cell wall biosynthesis at the early stage of peptidoglycan biosynthesis pathway than do the two gram-positive bacteria. Therefore, the MurD-catalyzed reaction may constitute a fine-tuning step necessary for the gram-negative bacteria to optimally maintain its relatively thin yet essential cell wall structure during all stages of growth.


Subject(s)
Enterococcus faecalis/metabolism , Escherichia coli/metabolism , Glutamates/metabolism , Haemophilus influenzae/metabolism , Peptide Synthases/metabolism , Staphylococcus aureus/metabolism , Cations, Divalent , Cations, Monovalent , Gene Expression , Peptide Synthases/antagonists & inhibitors , Peptide Synthases/genetics , Peptide Synthases/isolation & purification , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism
15.
Eur J Biochem ; 259(3): 726-30, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10092857

ABSTRACT

Aldose reductase (AR), best known as the first enzyme in the polyol pathway of sugar metabolism, has been implicated in a wide variety of physiological functions and in the etiology of diabetic complications. We have determined the structures and chromosomal locations of the mouse AR gene (Aldor1) and of two genes highly homologous to Aldor1: the fibroblast growth factor regulated protein gene (Fgfrp) and the androgen regulated vas deferens protein gene (Avdp). The number of introns and their locations in the mouse Aldor1 gene are identical to those of rat and human AR genes and also to those of Fgfrp and Avdp. Mouse Aldor1 gene was found to be located near the Cald1 (Caldesmon) and Ptn (Pleiotropin) loci at the proximal end of chromosome 6. The closely related genes Fgfrp and Avdp were also mapped in this region of the chromosome, suggesting that these three genes may have arisen by a gene duplication event.


Subject(s)
Aldehyde Reductase/genetics , Epidermal Growth Factor , Animals , Base Sequence , Chromosome Mapping , Chromosome Segregation , Diabetes Mellitus/etiology , Fibroblast Growth Factors/genetics , Gene Duplication , Mice , Proteins/genetics , Pseudogenes/genetics , Sequence Homology, Nucleic Acid
16.
Br J Radiol ; 72(859): 712-3, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10624332

ABSTRACT

An unusual case of systemic vein to pulmonary vein communication in superior vena cava obstruction is reported. This was a right-to-left shunt, demonstrated by spiral CT and aided by three-dimensional reconstruction. The pulmonary venous shunts were mainly seen in fibro-atelectatic lung where prominent bridging veins were concentrated.


Subject(s)
Image Processing, Computer-Assisted , Pulmonary Veins/diagnostic imaging , Superior Vena Cava Syndrome/diagnostic imaging , Tomography, X-Ray Computed/methods , Adult , Collateral Circulation , Female , Humans , Pulmonary Veins/abnormalities
17.
Biochemistry ; 35(5): 1417-22, 1996 Feb 06.
Article in English | MEDLINE | ID: mdl-8634271

ABSTRACT

In the peptidoglycan biosynthesis pathway in Escherichia coli, UDP-N-acetylmuramate:L-alanine ligase (MurC) catalyzes the formation of UDP-N-acetylmuramyl-L-alanine. A peptide bond is formed in this reaction and an ATP molecule is hydrolyzed concomitantly to produce ADP and orthophosphate. A biochemical approach was devised to elucidate the role of ATP in this reaction. A fusion construct pMAL::murC was prepared and the maltose binding protein--UDP-N-acetylmuramyl:L-alanine ligase fusion protein was overproduced in E. coli/pMal::murC upon isopropyl beta-thiogalactoside induction. The fusion protein was purified to > or = 90% homogeneity by a single-step affinity chromatography. Subsequently, the ligase was released from the maltose binding protein by proteolytic cleavage and was purified to > or = 95% homogeneity by an ion-exchange chromatographic step. The kinetic parameters of the regenerated ligase are comparable to those of the purified native enzyme. This ligase was used to investigate the role that ATP plays in the formation of UDP-N-acetylmuramyl-L-alanine. UDP-N-acetyl[18O]muramate (with 18O located at the carboxylate function only) was prepared by a combination of chemical and enzymatic processes and was used as the substrate of the ligase to probe the reaction mechanism. All reaction products were purified and subjected to liquid chromatographic-mass spectrometric analysis. A single [18O]oxygen was transferred from UDP-N-acetyl[18O]muramate to the orthophosphate produced in the reaction. No [18O]oxygen was detected in the adenosine nucleotides recovered from the reaction. These results strongly suggest that this ligase-catalyzed peptide formation proceeds through an activated acyl-phosphate linkage during the reaction process. ATP therefore assists in the process of the peptide bond formation by donating its gamma-phosphoryl group to activate the carboxyl group of UDP-N-acetylmuramic acid.


Subject(s)
Adenosine Triphosphate/metabolism , Escherichia coli/enzymology , Organophosphates/metabolism , Peptide Synthases/metabolism , Uridine Diphosphate N-Acetylmuramic Acid/metabolism , Base Sequence , Models, Chemical , Molecular Sequence Data , Peptide Synthases/genetics , Peptidoglycan/biosynthesis , Recombinant Fusion Proteins/metabolism
18.
Biochemistry ; 35(5): 1423-31, 1996 Feb 06.
Article in English | MEDLINE | ID: mdl-8634272

ABSTRACT

Uridine diphosphate N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) adds the first amino acid to the sugar moiety of the peptidoglycan precursor, catalyzing one of the essential steps in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Here, we report our studies on the secondary and quaternary structures of UNAM:L-Ala ligase from Escherichia coli. The molecular weight of the purified recombinant enzyme determined by electrospray ionization mass spectrometry agreed well with the molecular weight deduced from the DNA sequence. Through sedimentation equilibrium analysis, we show that the enzyme exists in equilibrium between monomeric and dimeric forms and that the dissociation constant of the dimer, Kd, was determined to be 1.1 +/- 0.4 microM at 37 degrees C and 0.58 +/- 0.30 microM at 4 degrees C. A very similar Kd value was also obtained at 37 degrees C by gel filtration chromatography. The secondary structure of the enzyme was characterized by circular dichroism spectroscopy. No change in the secondary structure was observed between the monomeric and dimeric forms of the enzyme. The activity assays at enzyme concentrations both below and above the determined Kd value lead to the conclusion that the enzyme is active both as dimers and as monomers and that the specific activity is independent of the oligomerization state.


Subject(s)
Escherichia coli/enzymology , Peptide Synthases/chemistry , Protein Structure, Secondary , Centrifugation, Isopycnic , Mass Spectrometry , Molecular Weight , Peptide Synthases/metabolism , Recombinant Proteins/chemistry
19.
Biochemistry ; 34(16): 5390-402, 1995 Apr 25.
Article in English | MEDLINE | ID: mdl-7727397

ABSTRACT

The Escherichia coli MurB gene encoding UDP-N-acetylenolpyruvylglucosamine reductase was expressed to a level of approximately 100 mg/L as a fusion construct with maltose binding protein. Rapid affinity purification, proteolysis, and anion exchange chromatography yielded homogeneous enzyme containing 1 mol/mol bound FAD. Enzyme was maximally activated by K+, NH4+, and Rb+ at cation concentrations between 10 and 50 mM. Steady-state enzyme kinetics at pH 8.0 and 37 degrees C revealed weak and strong substrate inhibition by NADPH and UDP-N-acetylenolpyruvylglucosamine, respectively, where the KiS were 910 microM and 73 microM. Substrate inhibition was pH dependent for both substrates. Initial velocity measurements as a function of both substrates produced patterns consistent with a ping pong bi bi double competitive substrate inhibition mechanism. Data at pH 8.0 yielded kinetic constants corresponding to Km,UNAGEP = 24 +/- 3 microM, Ki,UNAGEP = 73 +/- 19 microM, Km,NADPH = 17 +/- 3 microM, Ki,NADPH = 910 +/- 670 microM, and kcat = 62 +/- 3 s-1. A slow anaerobic exchange reaction with thio-NADP+ provided evidence for release of NADP+ in the absence of UNAGEP. Alternate reduced nicotinamide dinucleotides, including NHXDPH, 3'-NADPH, and alpha-NADPH, were substrates, whereas NADH was not. Several nucleotides, including ADP and UDP, were weak inhibitors of the enzyme with inhibition constants between 5 and 97 mM. Various analogs of NADP+, including 3'-NADP+, thio-NADP+, APADP+, NEthDP+, and NHXDP+, were inhibitors of the enzyme with respect to NADPH and yielded inhibition constants in the range of 110-1100 microM. Analogs without the 2'- or 3'-phosphate of NADPH or NADP+ were not substrates or inhibitors. Double inhibition experiments with varied APADP+ and UNAG produced inhibition patterns consistent with mutually exclusive inhibitor binding. The data suggest that NADPH and UNAGEP share a subsite that prevents both molecules from binding at once.


Subject(s)
Carbohydrate Dehydrogenases/metabolism , Escherichia coli/enzymology , Recombinant Proteins/metabolism , Uridine Diphosphate N-Acetylglucosamine/analogs & derivatives , Anaerobiosis , Carbohydrate Dehydrogenases/biosynthesis , Carbohydrate Dehydrogenases/isolation & purification , Cloning, Molecular , Escherichia coli/genetics , Flavin-Adenine Dinucleotide/metabolism , Genes, Bacterial , Glucosamine/analogs & derivatives , Glucosamine/metabolism , Kinetics , Mathematics , NADP/metabolism , Recombinant Proteins/isolation & purification , Ribonucleotides/pharmacology , Uridine Diphosphate/analogs & derivatives , Uridine Diphosphate/metabolism
20.
Biochemistry ; 34(8): 2464-70, 1995 Feb 28.
Article in English | MEDLINE | ID: mdl-7873525

ABSTRACT

D-Glutamate is an essential component of the bacterial peptidoglycan. In Escherichia coli, the biosynthesis of D-glutamate is catalyzed by a glutamate racemase (encoded by the dga gene) and is regulated by UDP-N-acetylmuramyl-L-alanine [Doublet et al. (1994) Biochemistry 33, 5285], a bacterial peptidoglycan subunit precursor. Investigation was conducted to elucidate the interaction between the enzyme and its regulator. Whole and N-terminal truncated enzymes, encoded by individual constructs containing either a full-length or an N-terminal truncated dga gene, were evaluated. In the absence of the regulator, the purified whole enzyme showed a low-level basal racemase activity for which a Km value of 18.9 mM and a Vmax of 0.4 mumol/(min.mg) were determined, using D-glutamate as the substrate. Using the same substrate, in the presence of 6.5 microM UDP-N-acetylmuramyl-L-alanine, a Km value of 4.2 mM and a Vmax of 34 mumol/(min.mg) were measured. Similar kinetic parameters for the activated enzyme were obtained using L-glutamate as the substrate. The N-terminal truncated E. coli enzyme, with a 21 amino acid region removed, is similar in size to the Pediococcus pentosaceus glutamate racemase. Effects of the regulator on the full-length and the N-terminal truncated enzyme in the dialyzed cell lysate were compared. A host cell line, E. coli WM335 delta recA, containing a nonfunctional chromosomal dga gene was used to minimize the background interference. With 6.5 microM regulator added, the N-terminal truncated enzyme displayed a loss of more than 80% of the activity compared to the full-length enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Amino Acid Isomerases/metabolism , Escherichia coli/enzymology , Uridine Diphosphate N-Acetylmuramic Acid/analogs & derivatives , Amino Acid Isomerases/genetics , Amino Acid Isomerases/isolation & purification , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA Primers/genetics , DNA, Bacterial/genetics , Enzyme Activation/drug effects , Escherichia coli/genetics , Genes, Bacterial , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Pediococcus/enzymology , Pediococcus/genetics , Peptidoglycan/biosynthesis , Peptidoglycan/chemistry , Sequence Homology, Amino Acid , Substrate Specificity , Uridine Diphosphate N-Acetylmuramic Acid/biosynthesis , Uridine Diphosphate N-Acetylmuramic Acid/chemistry , Uridine Diphosphate N-Acetylmuramic Acid/metabolism , Uridine Diphosphate N-Acetylmuramic Acid/pharmacology
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