bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phospho-
N
-Acetyl-Muramyl-Pentapeptide Translocase from
Escherichia coli
: Catalytic Role of Conserved Aspartic Acid Residues
Authors: Lloyd Adrian J. , Brandish Philip E. , Gilbey Andrea M. , Bugg Timothy D. H. 
Published: March 2004

ABSTRACT

Phospho-
N
-acetyl-muramyl-pentapeptide translocase (translocase 1) catalyzes the first of a sequence of lipid-linked steps that ultimately assemble the peptidoglycan layer of the bacterial cell wall. This essential enzyme is the target of several natural product antibiotics and has recently been the focus of antimicrobial drug discovery programs. The catalytic mechanism of translocase 1 is believed to proceed via a covalent intermediate formed between phospho-
N
-acetyl-muramyl-pentapeptide and a nucleophilic amino acid residue. Amino acid sequence alignments of the translocase 1 family and members of the related transmembrane phosphosugar transferase superfamily revealed only three conserved residues that possess nucleophilic side chains: the aspartic acid residues D115, D116, and D267. Here we report the expression and partial purification of
Escherichia coli
translocase 1 as a C-terminal hexahistidine (C-His
6
) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His
6
fusions. Enzymatic analysis established that all three mutations eliminated translocase 1 activity, and this finding verified the essential role of these residues. By analogy with the structural environment of the double aspartate motif found in prenyl transferases, we propose a model whereby D115 and D116 chelate a magnesium ion that coordinates with the pyrophosphate bridge of the UDP-
N
-acetyl-muramyl-pentapeptide substrate and in which D267 therefore fulfills the role of the translocase 1 active-site nucleophile.
In-Depth Profiling of Lysine-Producing
Corynebacterium glutamicum
by Combined Analysis of the Transcriptome, Metabolome, and Fluxome
Authors: Krömer Jens Olaf , Sorgenfrei Oliver , Klopprogge Kai , Heinzle Elmar , Wittmann Christoph 
Published: March 2004

ABSTRACT

An in-depth analysis of the intracellular metabolite concentrations, metabolic fluxes, and gene expression (metabolome, fluxome, and transcriptome, respectively) of lysine-producing
Corynebacterium glutamicum
ATCC 13287 was performed at different stages of batch culture and revealed distinct phases of growth and lysine production. For this purpose,
13
C flux analysis with gas chromatography-mass spectrometry-labeling measurement of free intracellular amino acids, metabolite balancing, and isotopomer modeling were combined with expression profiling via DNA microarrays and with intracellular metabolite quantification. The phase shift from growth to lysine production was accompanied by a decrease in glucose uptake flux, the redirection of flux from the tricarboxylic acid (TCA) cycle towards anaplerotic carboxylation and lysine biosynthesis, transient dynamics of intracellular metabolite pools, such as an increase of lysine up to 40 mM prior to its excretion, and complex changes in the expression of genes for central metabolism. The integrated approach was valuable for the identification of correlations between gene expression and in vivo activity for numerous enzymes. The glucose uptake flux closely corresponded to the expression of glucose phosphotransferase genes. A correlation between flux and expression was also observed for glucose-6-phosphate dehydrogenase, transaldolase, and transketolase and for most TCA cycle genes. In contrast, cytoplasmic malate dehydrogenase expression increased despite a reduction of the TCA cycle flux, probably related to its contribution to NADH regeneration under conditions of reduced growth. Most genes for lysine biosynthesis showed a constant expression level, despite a marked change of the metabolic flux, indicating that they are strongly regulated at the metabolic level. Glyoxylate cycle genes were continuously expressed, but the pathway exhibited in vivo activity only in the later stage. The most pronounced changes in gene expression during cultivation were found for enzymes at entry points into glycolysis, the pentose phosphate pathway, the TCA cycle, and lysine biosynthesis, indicating that these might be of special importance for transcriptional control in
C. glutamicum
.
Roles of Genes 44, 50, and 51 in Regulating Gene Expression and Host Takeover during Infection of
Bacillus subtilis
by Bacteriophage SPO1
Authors: Sampath Aruna , Stewart Charles R. 
Published: March 2004

ABSTRACT

We show that the products of SPO1 genes 44, 50, and 51 are required for the normal transition from early to middle gene expression during infection of
Bacillus subtilis
by bacteriophage SPO1; that they are also required for control of the shutoff of host DNA, RNA, and protein synthesis; and that their effects on host shutoff could be accounted for by their effects on the regulation of gene expression. These three gene products had four distinguishable effects in regulating SPO1 gene expression: (i) gp44-50-51 acted to restrain expression of all SPO1 genes tested, (ii) gp44 and/or gp50-51 caused additional specific repression of immediate-early genes, (iii) gp44 and/or gp50-51 stimulated expression of middle genes, and (iv) gp44 and/or gp50-51 stimulated expression of some delayed-early genes. Shutoff of immediate-early gene expression also required the activity of gp28, the middle-gene-specific sigma factor. Shutoff of host RNA and protein synthesis was accelerated by either the 44
−
single mutant or the 50
−
51
−
double mutant and more so by the 44
−
50
−
51
−
triple mutant. Shutoff of host DNA synthesis was accelerated by the mutants early in infection but delayed by the 44
−
50
−
51
−
triple mutant at later times. Although gp50 is a very small protein, consisting almost entirely of an apparent membrane-spanning domain, it contributed significantly to each activity tested. We identify SPO1 genes 41 to 51 and 53 to 60 as immediate-early genes; genes 27, 28, and 37 to 40 as delayed-early genes; and gene 52 as a middle gene.
Cloning and Sequence Analysis of Genes Encoding
Staphylococcus hyicu
s Exfoliative Toxin Types A, B, C, and D
Authors: Ahrens Peter , Andresen Lars Ole 
Published: March 2004

ABSTRACT

Exfoliative toxins produced by certain strains of
Staphylococcus hyicus
mediate exudative epidermitis in pigs. In this study the genes coding for four different exfoliative toxin from
S. hyicus
(ExhA, ExhB, ExhC, and ExhD) were cloned and sequenced. The coding sequence of the four toxin genes ranged from 816 to 834 bp. The amino acid sequences of these four toxins were homologous to the earlier described exfoliative toxins SHETB from
S. hyicus
and ETA, ETB, and ETD from
Staphylococcus aureus
. The homology between the
S. hyicus
toxins was at the same level as the homology to the exfoliative toxins from
S. aureus
. The toxins showed similarity to serine proteases, including preservation of the catalytic tract in ExhA, ExhB, and ExhC. However, in ExhD, Asp in the putative catalytic tract was replaced with Glu. The recombinant toxins could be expressed in
Escherichia coli
, and three of the four toxins were recognized by monoclonal antibodies raised against native exfoliative toxins.
Quorum Sensing in
Staphylococcus aureus
Biofilms
Authors: Yarwood Jeremy M. , Bartels Douglas J. , Volper Esther M. , Greenberg E. Peter 
Published: March 2004

ABSTRACT

Several serious diseases are caused by biofilm-associated
Staphylococcus aureus
, infections in which the accessory gene regulator (
agr
) quorum-sensing system is thought to play an important role. We studied the contribution of
agr
to biofilm development, and we examined
agr
-dependent transcription in biofilms. Under some conditions, disruption of
agr
expression had no discernible influence on biofilm formation, while under others it either inhibited or enhanced biofilm formation. Under those conditions where
agr
expression enhanced biofilm formation, biofilms of an
agr
signaling mutant were particularly sensitive to rifampin but not to oxacillin. Time lapse confocal scanning laser microscopy showed that, similar to the expression of an
agr
-independent fluorescent reporter, biofilm expression of an
agr
-dependent reporter was in patches within cell clusters and oscillated with time. In some cases, loss of fluorescence appeared to coincide with detachment of cells from the biofilm. Our studies indicate that the role of
agr
expression in biofilm development and behavior depends on environmental conditions. We also suggest that detachment of cells expressing
agr
from biofilms may have important clinical implications.
Isolation and Characterization of β-Ketoacyl-Acyl Carrier Protein Reductase (
fabG
) Mutants of
Escherichia coli
and
Salmonella enterica
Serovar Typhimurium
Authors: Lai Chiou-Yan , Cronan John E. 
Published: March 2004

ABSTRACT

FabG, β-ketoacyl-acyl carrier protein (ACP) reductase, performs the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products, the first reductive step in the elongation cycle of fatty acid biosynthesis. We report the first documented
fabG
mutants and their characterization. By chemical mutagenesis followed by a tritium suicide procedure, we obtained three conditionally lethal temperature-sensitive
fabG
mutants. The
Escherichia coli
[
fabG
(Ts)] mutant contains two point mutations: A154T and E233K. The β-ketoacyl-ACP reductase activity of this mutant was extremely thermolabile, and the rate of fatty acid synthesis measured in vivo was inhibited upon shift to the nonpermissive temperature. Moreover, synthesis of the acyl-ACP intermediates of the pathway was inhibited upon shift of mutant cultures to the nonpermissive temperature, indicating blockage of the synthetic cycle. Similar results were observed for in vitro fatty acid synthesis. Complementation analysis revealed that only the E233K mutation was required to give the temperature-sensitive growth phenotype. In the two
Salmonella enterica
serovar Typhimurium
fabG
(Ts) mutants one strain had a single point mutation, S224F, whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the twofold axes of symmetry at the dimer interfaces in this homotetrameric protein, suggesting that the quaternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature.
The
eutD
Gene of
Salmonella enterica
Encodes a Protein with Phosphotransacetylase Enzyme Activity
Authors: Brinsmade Shaun R. , Escalante-Semerena Jorge C. 
Published: March 2004

ABSTRACT

The EutD protein of
Salmonella enterica
is homologous to the catalytic domain of the phosphotransacetylase (Pta) enzyme. The Pta-like activity level of the EutD enzyme compared favorably to that of other Pta enzymes. High-pressure liquid chromatography and mass spectrometry verified that acetyl-coenzyme A was the product of the reaction. The EutD protein restored growth of an
S. enterica pta
strain on acetate as the source of carbon and energy.
Analysis of an Agmatine Deiminase Gene Cluster in
Streptococcus mutans
UA159
Authors: Griswold Ann R. , Chen Yi-Ywan M. , Burne Robert A. 
Published: March 2004

ABSTRACT

An operon encoding enzymes of the agmatine deiminase system (AgDS) has been identified in the cariogenic bacterium
Streptococcus mutans
UA159. The AgDS is regulated by agmatine induction and carbohydrate catabolite repression. Ammonia is produced from agmatine at low pH, suggesting that the AgDS could augment acid tolerance.
Transcriptional Phase Variation at the
flhB
Gene of
Pseudomonas putida
DOT-T1E Is Involved in Response to Environmental Changes and Suggests the Participation of the Flagellar Export System in Solvent Tolerance
Authors: Segura Ana , Hurtado Ana , Duque Estrella , Ramos Juan L. 
Published: March 2004

ABSTRACT

Frameshift mutations in a poly(G) track at the
flhB
gene of
Pseudomonas putida
DOT-T1E are responsible for the diminished swimming of this strain on semisolid medium, which contrasts with the high swimming ability of
P. putida
KT2440, which does not exhibit a poly(G) track at the
flhB
gene. We previously showed that a mutant lacking FlhB was more sensitive to solvents than the wild-type strain (Segura et al., J. Bacteriol., 183:4127-4133, 2001). In this study, we show that swimming ability correlates with solvent tolerance in
P. putida
DOT-T1E, so that growth conditions favoring a functional
flhB
gene (growth on semisolid medium) resulted in increased innate tolerance to a sudden toluene shock.
Restoration of Blood Pressure by Centrally Injected U-46619, a Thromboxane A<sub>2</sub> Analog, in Hemorrhaged Hypotensive Rats: Investigation of Different Brain Areas
Authors: Yalcin Murat , Savci Vahide 
Published: March 2004

In the present study, we investigated the cardiovascular effects of centrally injected U-46619, a thromboxane A<sub>2</sub> (TXA<sub>2</sub>) analog, and the central and peripheral mechanisms of these effects in hemorrhagic shock conditions. Hemorrhage was performed by withdrawing a total volume of 2.1 ml of blood/100 g body weight over a period of 10 min. Injections were made into the lateral cerebral ventricle (LCV), nucleus tractus solitarius (NTS), rostral ventrolateral medulla (RVLM) and paraventricular nucleus of hypothalamus (PVN). U-46619 (0.1, 1 and 2 µg) increased blood pressure and reversed hypotension in hemorrhagic shock. The pressor effect was dose- and time-dependent in all investigated brain areas. Heart rate changes were not significantly different in all groups. Pretreatment of rats with an injection of SQ-29548 (4 or 8 µg), a TXA<sub>2</sub> receptor antagonist, into the LCV, NTS, RVLM and PVN completely blocked the pressor effect of U-46619 (1 µg) injected into respective brain areas. Hemorrhage itself increased plasma adrenaline, noradrenaline, vasopressIN levels and renin activity. U-46619 (1 µg) injected into the LCV, PVN, RVLM and NTS produced additional increases in these hormone levels and in renin activity. Intravenous pretreatments of rats with prazosin (0.5 mg/kg), an α<sub>1</sub>-adrenoceptor antagonist, [β-mercapto-β,β-cyclopentamethylenepropionyl<sup>1</sup>, O-Me-Tyr<sup>2</sup>,Arg<sup>8</sup>]- vasopressin (10 µg/kg), a vasopressin V<sub>1</sub>-receptor antagonist, or saralasin (250 µg/kg), an angiotensin II receptor antagonist, in hemorrhaged rats partially blocked the pressor response to U-46619 (1 µg) injected into the LCV, PVN, RVLM and NTS. Results show that centrally administered U-46619, a TXA<sub>2</sub> analog, increases blood pressure and reverses hypotension in hemorrhagic shock. Activation of central TXA<sub>2</sub> receptors mediates the pressor effect of the drug. Furthermore, the increases in plasma adrenaline, noradrenaline, vasopressin levels and renin activity are involved in these effects.
Endocrinological Effects of High-Dose <i>Hypericum perforatum</i> Extract WS 5570 in Healthy Subjects
Authors: Schüle C. , Baghai T. , Sauer N. , Laakmann G. 
Published: March 2004

In this single-blind study, the effects of acute oral administration of high-dose <i>Hypericum perforatum</i> extract WS 5570 on the cortisol (COR), adrenocorticotropic hormone (ACTH), growth hormone (GH), and prolactin (PRL) secretions were examined in 12 healthy male volunteers. In a randomized order, the subjects received placebo or WS 5570 at several dosages (600, 900, and 1,200 mg) at 08.00 h on 4 different days. After insertion of an intravenous catheter, blood samples were drawn 1 h prior to administration of placebo or WS 5570 (600, 900, or 1,200 mg), at the time of administration, and during 5 h thereafter at intervals of 30 min. The serum concentrations of COR, GH, and PRL as well as the plasma levels of ACTH were determined in each blood sample by means of double antibody radioimmunoassay, fluoroimmunoassay, and chemiluminescence immunometric assay methods. The area under the curve value was used as parameter for COR, ACTH, GH, and PRL responses. Repeated-measures Anova revealed a significant stimulatory effect of WS 5570 on the ACTH secretion, whereas COR and PRL secretions were not significantly influenced. Moreover, there was a stimulatory peak of GH release 240 min after challenge with WS 5570 in some but not all volunteers, without reaching statistical significance in comparison with placebo. Mean arterial blood pressure and heart rate remained unchanged after administration of WS 5570. Apparently, WS 5570 at the dosages given in this study inconsistently causes endocrinological effects in healthy subjects by influencing central neurotransmitters.
Management of Atherothrombosis with Clopidogrel in High-Risk Patients with Recent Transient Ischaemic Attack or Ischaemic Stroke (MATCH): Study Design and Baseline Data
Authors: Diener Hans-Christoph , Bogousslavsky Julien , Brass Lawrence M. , Cimminiello Claudio , Csiba Laszlo , Kaste Markku , Leys Didier , Matias-Guiu Jordi , Rupprecht Hans-Jürgen 
Published: March 2004

<i>Background:</i> The CAPRIE study showed the superiority of clopidogrel over acetylsalicylic acid (ASA) for reducing the combined risk of major atherothrombotic events in patients with recent myocardial infarction (MI), recent ischaemic stroke (IS) or established peripheral arterial disease. The benefit of clopidogrel over ASA is amplified in high-risk patients. Proof of concept for the benefit of clopidogrel in addition to ASA in patients with coronary manifestations of atherothrombosis was provided by the CURE trial. <i>Methods:</i> MATCH is a randomized, double-blind, placebo-controlled trial that compares clopidogrel and ASA versus clopidogrel alone in high-risk patients with recently symptomatic cerebrovascular disease. Eligible patients have experienced a transient ischaemic attack (TIA) or IS within the last 3 months and have evidence of at least 1 additional risk factor within the last 3 years (prior IS, MI, stable or unstable angina pectoris, diabetes or symptomatic peripheral arterial disease). Patients were randomized to receive ASA 75 mg once daily or placebo, with both groups receiving clopidogrel 75 mg once daily as part of standard therapy. The primary end point is the composite of IS, MI, vascular death and rehospitalization for an acute ischaemic event. The duration of treatment and follow-up is 18 months for each patient.<i> Results:</i> Enrolment was completed in April 2002, with 7,599 patients randomized to receive the study medication. The mean age at randomization was 66 years, and the qualifying event was IS in 78.9% of patients and TIA in 21.1%. The baseline features of the study cohort indicate a population that is at a high risk for atherothrombotic recurrence. <i>Conclusion:</i> MATCH is a major ongoing trial that will provide important data on the benefit of clopidogrel and ASA compared with clopidogrel alone for reduction of vascular ischaemic events in patients with recent TIA or IS who are at high risk of atherothrombotic event recurrence.
Imatinib Attenuates Diabetes-Associated Atherosclerosis
Authors: Lassila Markus , Allen Terri J. , Cao Zemin , Thallas Vicki , Jandeleit-Dahm Karin A. , Candido Riccardo , Cooper Mark E. 
Published: March 2004

Objective—Diabetes is associated with accelerated atherosclerosis, the major factor contributing to increased mortality and morbidity in the diabetic population. The molecular mechanisms by which diabetes promotes atherosclerosis are not fully understood. Platelet-derived growth factor has been shown to play a major role in the pathology of vascular diseases, but whether it plays a role in atherosclerosis associated with diabetes remains unknown. The aims of this study were to assess whether platelet-derived growth factor–dependent pathways are involved in the development of diabetes-induced atherosclerosis and to determine the effects of platelet-derived growth factor receptor antagonism on this disorder.Methods and Results—Diabetes was induced by injection of streptozotocin in 6-week-old apolipoprotein E knockout mice. Diabetic animals received treatment with a tyrosine kinase inhibitor that inhibits platelet-derived growth factor action, imatinib (STI-571, 10 mg/kg per day), or no treatment for 20 weeks. Nondiabetic apolipoprotein E knockout mice served as controls. Induction of diabetes was associated with a 5-fold increase in plaque area in association with an increase in aortic platelet-derived growth factor-B expression and platelet-derived growth factor-β receptor phosphorylation as well as other prosclerotic and proinflammatory cytokines. Imatinib treatment prevented the development of atherosclerotic lesions and diabetes-induced inflammatory cytokine overexpression in the aorta.Conclusions—Tyrosine kinase inhibition with imatinib appears to be a novel therapeutic option to retard the development of atherosclerosis, specifically in the context of diabetes.
Depletion of Natural Killer Cell Function Decreases Atherosclerosis in Low-Density Lipoprotein Receptor Null Mice
Authors: Whitman Stewart C. , Rateri Debra L. , Szilvassy Stephen J. , Yokoyama Wayne , Daugherty Alan 
Published: March 2004

Objective—Natural killer (NK) cells are a key component of innate immunity. Despite being identified in human and mouse atherosclerotic lesions, the role of NK cells in the disease process in unknown. To determine this role, we created chimeric atherosclerosis-susceptible low-density lipoprotein (LDL) receptor null (ldl-r−/−) mice that were deficient in functional NK cells through expression of a transgene encoding for Ly49A.Methods and Results—Bone marrow cells from Ly49A transgenic and nontransgenic littermates were used to repopulate the hematopoietic system of lethally-irradiated femaleldl-r−/−mice. After a recovery period to permit sufficient engraftment, mice were placed on a diet enriched in saturated fat and cholesterol. After 8 weeks, there was no difference in either serum total cholesterol concentrations or lipoprotein cholesterol distribution in mice repopulated with nontransgenic versus Ly49A transgenic marrow cells. Using immunohistochemistry, we detected NK cells in atherosclerotic lesions of both groups of mice. However, deficiency of functional NK cells significantly reduced the size of atherosclerosis by 70% (P=0.0002) in cross-sectional analysis of the aortic root and by 38% (P=0.004) inen faceanalysis of the intimal surface of the aortic arch.Conclusion—These studies demonstrate that NK cells infiltrate the vessel wall and promote atherosclerotic lesion development.
Legal safeguards against medical practice by not suitably qualified persons: A comparative study in seven EU-countries
Authors: de Bijl  , Nederveen-van de Kragt  
Published: March 2004

AbstractIn this article the conditions and regulatory systems for access to medical practice in seven countries of the European Union are described with a view to the health protection of patients/consumers. In Belgium and France the legislator has chosen a monopolistic approach to the licensing of medical doctors: only the practice of medicine by legally qualified doctors is lawful. In Germany, Ireland and the United Kingdom a more tolerant system is in force: only medical practitioners with a university degree in medicine are recognized, but also persons who practice 'alternative medicine' without a medical degree are tolerated by law. In Denmark and the Netherlands the system is a mixture of the monopolistic and tolerant system. In principle everyone is allowed to practice medicine, with the exclusion of certain procedures that are reserved to qualified medical doctors. Violation of this limited monopoly can be prosecuted. The restrictions to and preconditions for access to medical practice are compared as well as the maintenance of the respective systems. The role of the legislator is described. From the perspective of the protection of patient's health and safety it seems that a mixed system offers the patient optimal protection.