bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of an Archaeal Type II Isopentenyl Diphosphate Isomerase in
Methanothermobacter thermautotrophicus
Authors: Barkley Sam J. , Cornish Rita M. , Poulter C. Dale 
Published: March 2004

ABSTRACT

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in
Streptomyces
sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon
Methanothermobacter thermautotrophicus
encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in
Salmonella enterica
serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (
idi
) and 1-deoxy-
d
-xylulose 5-phosphate (
dxs
). The
dxs
gene was interrupted with a synthetic operon containing the
Saccharomyces cerevisiae
genes
erg8
,
erg12
, and
erg19
allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His
6
-tagged
M. thermautotrophicus
type II IPP isomerase was produced in
Escherichia coli
and purified by Ni
2+
chromatography. The purified protein was characterized by matrix-assisted laser desorption ionization mass spectrometry. The enzyme has optimal activity at 70°C and pH 6.5. NADPH, flavin mononucleotide, and Mg
2+
are required cofactors. The steady-state kinetic constants for the archaeal type II IPP isomerase from
M. thermautotrophicus
are as follows:

K
m

, 64 μM; specific activity, 0.476 μmol mg
−1
min
−1
; and
k
cat
, 1.6 s
−1
.
The pKO2 Linear Plasmid Prophage ofKlebsiella oxytoca
Authors: Casjens Sherwood R. , Gilcrease Eddie B. , Huang Wai Mun , Bunny Kim L. , Pedulla Marisa L. , Ford Michael E. , Houtz Jennifer M. , Hatfull Graham F. , Hendrix Roger W. 
Published: March 2004

ABSTRACTTemperate bacteriophages with plasmid prophages are uncommon in nature, and of these only phages N15 and PY54 are known to have a linear plasmid prophage with closed hairpin telomeres. We report here the complete nucleotide sequence of the 51,601-bpKlebsiella oxytocalinear plasmid pKO2, and we demonstrate experimentally that it is also a prophage. We call this bacteriophage φKO2. An analysis of the 64 predicted φKO2 genes indicate that it is a fairly close relative of phage N15; they share a mosaic relationship that is typical of different members of double-stranded DNA tailed-phage groups. Although the head, tail shaft, and lysis genes are not recognizably homologous between these phages, other genes such as the plasmid partitioning, replicase, prophage repressor, and protelomerase genes (and their putative targets) are so similar that we predict that they must have nearly identical DNA binding specificities. The φKO2 virion is unusual in that its phage λ-like tails have an exceptionally long (3,433 amino acids) central tip tail fiber protein. The φKO2 genome also carries putative homologues of bacterialdinIandumuDgenes, both of which are involved in the host SOS response. We show that these divergently transcribed genes are regulated by LexA protein binding to a single target site that overlaps both promoters.
Antibiotic-Sensitive TolC Mutants and Their Suppressors
Authors: Augustus Anne Marie , Celaya Teresa , Husain Fasahath , Humbard Matthew , Misra Rajeev 
Published: March 2004

ABSTRACT

The TolC protein of
Escherichia coli
, through its interaction with AcrA and AcrB, is thought to form a continuous protein channel that expels inhibitors from the cell. Consequently,
tolC
null mutations display a hypersensitive phenotype. Here we report the isolation and characterization of
tolC
missense mutations that direct the synthesis of mutant TolC proteins partially disabled in their efflux role. All alterations, consisting of single amino acid substitutions, were localized within the periplasmic α-helical domain. In two mutants carrying an I106N or S350F substitution, the hypersensitivity phenotype may be in part due to aberrant TolC assembly. However, two other alterations, R367H and R390C, disrupted efflux function by affecting interactions among the helices surrounding TolC's periplasmic tunnel. Curiously, these two TolC mutants were sensitive to a large antibiotic, vancomycin, and exhibited a Dex
+
phenotype. These novel phenotypes of TolC
R367H
and TolC
R390C
were likely the result of a general influx of molecules through a constitutively open tunnel aperture, which normally widens only when TolC interacts with other proteins during substrate translocation. An intragenic suppressor alteration (T140A) was isolated from antibiotic-resistant revertants of the hypersensitive TolC
R367H
mutant. T140A also reversed, either fully (R390C) or partially (I106N and S350F), the hypersensitivity phenotype of other TolC mutants. Our data suggest that this global suppressor phenotype of T140A is the result of impeded antibiotic influx caused by tapering of the tunnel passage rather than by correcting individual mutational defects. Two extragenic suppressors of TolC
R367H
, mapping in the regulatory region of
acrAB
, uncoupled the AcrR-mediated repression of the
acrAB
genes. The resulting overexpression of AcrAB reduced the hypersensitivity phenotype of all the TolC mutants. Similar results were obtained when the chromosomal
acrR
gene was deleted or the
acrAB
genes were expressed from a plasmid. Unlike the case for the intragenic suppressor T140A, the overexpression of AcrAB diminished hypersensitivity towards only erythromycin and novobiocin, which are substrates of the TolC-AcrAB efflux pump, but not towards vancomycin, which is not a substrate of this pump. This showed that the two types of suppressors produced their effects by fundamentally different means, as the intragenic suppressor decreased the general influx while extragenic suppressors increased the efflux of TolC-AcrAB pump-specific antibiotics.
Engineered Single-Chain, Antiparallel, Coiled CoilMimics the MerR Metal BindingSite
Authors: Song Lingyun , Caguiat Jonathan , Li Zhongrui , Shokes Jacob , Scott Robert A. , Olliff Lynda , Summers Anne O. 
Published: March 2004

ABSTRACT

The
repressor-activator MerR that controls transcription of the mercury
resistance (
mer
) operon is unusual for its high sensitivity
and specificity for Hg(II) in in vivo and in vitro transcriptional
assays. The metal-recognition domain of MerR resides at the homodimer
interface in a novel antiparallel arrangement of α-helix 5 that
forms a coiled-coil motif. To facilitate the study of this novel metal
binding motif, we assembled this antiparallel coiled coil into a single
chain by directly fusing two copies of the 48-residue α-helix 5
of MerR. The resulting 107-residue polypeptide, called the metal
binding domain (MBD), and wild-type MerR were overproduced and
purified, and their metal-binding properties were determined in vivo
and in vitro. In vitro MBD bound ca. 1.0 equivalent of Hg(II) per pair
of binding sites, just as MerR does, and it showed only a slightly
lower affinity for Hg(II) than did MerR. Extended X-ray absorption fine
structure data showed that MBD has essentially the same Hg(II)
coordination environment as MerR. In vivo, cells overexpressing MBD
accumulated 70 to 100% more
203
Hg(II) than cells
bearing the vector alone, without deleterious effects on cell growth.
Both MerR and MBD variously bound other thiophilic metal ions,
including Cd(II), Zn(II), Pb(II), and As(III), in vitro and in vivo. We
conclude that (i) it is possible to simulate in a single polypeptide
chain the in vitro and in vivo metal-binding ability of dimeric,
full-length MerR and (ii) MerR's specificity in transcriptional
activation does not reside solely in the metal-binding
step.
DNA Interaction and Phosphotransfer of the C
4
-Dicarboxylate- Responsive DcuS-DcuR Two-Component Regulatory System from
Escherichia coli
Authors: Abo-Amer Aly E. , Munn Jonathan , Jackson Kerry , Aktas Murat , Golby Paul , Kelly David J. , Andrews Simon C. 
Published: March 2004

ABSTRACT

The DcuS-DcuR system of
Escherichia coli
is a two-component sensor-regulator that controls gene expression in response to external C
4
-dicarboxylates and citrate. The DcuS protein is particularly interesting since it contains two PAS domains, namely a periplasmic C
4
-dicarboxylate-sensing PAS domain (PASp) and a cytosolic PAS domain (PASc) of uncertain function. For a study of the role of the PASc domain, three different fragments of DcuS were overproduced and examined: they were PASc-kinase, PASc, and kinase. The two kinase-domain-containing fragments were autophosphorylated by [γ-
32
P]ATP. The rate was not affected by fumarate or succinate, supporting the role of the PASp domain in C
4
-dicarboxylate sensing. Both of the phosphorylated DcuS constructs were able to rapidly pass their phosphoryl groups to DcuR, and after phosphorylation, DcuR dephosphorylated rapidly. No prosthetic group or significant quantity of metal was found associated with either of the PASc-containing proteins. The DNA-binding specificity of DcuR was studied by use of the pure protein. It was found to be converted from a monomer to a dimer upon acetylphosphate treatment, and native polyacrylamide gel electrophoresis suggested that it can oligomerize. DcuR specifically bound to the promoters of the three known DcuSR-regulated genes (
dctA
,
dcuB
, and
frdA
), with apparent

K
D

s of 6 to 32 μM for untreated DcuR and ≤1 to 2 μM for the acetylphosphate-treated form. The binding sites were located by DNase I footprinting, allowing a putative DcuR-binding motif [tandemly repeated (T/A)(A/T)(T/C)(A/T)AA sequences] to be identified. The DcuR-binding sites of the
dcuB
,
dctA
, and
frdA
genes were located 27, 94, and 86 bp, respectively, upstream of the corresponding +1 sites, and a new promoter was identified for
dcuB
that responds to DcuR.
Deletion of a Cyclic AMP Receptor Protein Homologue Diminishes Germination and Affects Morphological Development of
Streptomyces coelicolor
Authors: Derouaux A. , Halici S. , Nothaft H. , Neutelings T. , Moutzourelis G. , Dusart J. , Titgemeyer F. , Rigali S. 
Published: March 2004

ABSTRACT

Open reading frame SCO3571 of
Streptomyces coelicolor
encodes a protein of the cyclic AMP (cAMP) receptor protein (CRP) superfamily of regulatory proteins. A mutant revealed a dramatic defect in germination, followed by growth delay and earlier sporulation. This phenotype correlates with those of an adenylate cyclase (
cya
) mutant that cannot synthesize cAMP. This finding suggests that
S. coelicolor
may use a Cya-cAMP-CRP system to trigger complex physiological processes such as morphogenesis.
Cellular XylS Levels Are a Function of Transcription of
xylS
from Two Independent Promoters and the Differential Efficiency of Translation of the Two mRNAs
Authors: González-Pérez M.-Mar , Ramos Juan L. , Marqués Silvia 
Published: March 2004

ABSTRACT

XylS controls the expression of the
meta
-cleavage pathway for the metabolism of benzoates in
Pseudomonas putida
KT2440. The
xylS
gene is expressed from two promoters, Ps1 and Ps2. Transcription from Ps2 is low and constitutive, whereas transcription from Ps1 is induced in the presence of toluene. In this study, we also show that translation of mRNA generated from Ps1 is 10 times more efficient than that generated from Ps2. This pattern of transcription and translation of
xylS
gives rise to two modes of activation of the promoter of the
meta
pathway operon (Pm) according to the concentration of XylS in the cell. In cells growing with benzoate, with small amounts of XylS, the activated XylS regulator binds the effector and stimulates transcription from Pm, whereas in cells growing with toluene, the high levels of XylS suffice to stimulate transcription from Pm even in the absence of XylS effectors.
Seventeen Cases of Central Serous Chorioretinopathy Associated with Systemic Corticosteroid Therapy
Authors: Koyama Makoto , Mizota Atsushi , Igarashi Yoshinori , Adachi-Usami Emiko 
Published: March 2004

<i>Purpose:</i> To determine the relationship between the clinical characteristics of patients with central serous chorioretinopathy (CSC) and systemic corticosteroid therapy. <i>Methods:</i> The medical records of 17 cases of CSC that developed during systemic corticosteroid treatment from 1987 to 1999 at Chiba University Hospital were reviewed. The relationship of CSC to the age, gender, laterality, and disease requiring the corticosteroid treatment, and the dose and duration of corticosteroid therapy were examined. <i>Results:</i> There were 6 men and 11 women, and 2 of these developed bilateral and 15 developed unilateral CSC. The duration from the beginning of corticosteroid treatment to the onset of CSC ranged from 3 days to 23 years; 9 patients developed CSC within 1 year after the beginning of the corticosteroid medication and 6 patients after more than 8 years. The amount of corticosteroid medication at the onset of CSC ranged from 5 to 1,000 mg/day equivalent prednisolone units. There was a significant correlation between age at the onset of CSC and the daily dosage of corticosteroid. <i>Conclusion:</i> Even small amounts of daily corticosteroids (5–10 mg/day) can cause CSC, especially in elderly patients. These findings indicate that we need to monitor patients undergoing corticosteroid treatment carefully.
The Efficacy and Safety of Reteplase for Thrombolysis of Hemodialysis Catheters at a Community and Academic Regional Medical Center
Authors: Hyman Gary , England Michael , Kibede Sosena , Lee Patti , Willets George 
Published: March 2004

In occluded hemodialysis catheters, thrombolytic agents are used to dissolve fibrin clots, reestablish blood flow and allow the patient to continue with hemodialysis treatment. Prior to 2001, urokinase was the indicated fibrinolytic for hemodialysis catheter thrombolysis. However, when urokinase became unavailable in the United States, New Hanover Regional Medical Center developed and implemented a protocol for the use of another fibrinolytic, reteplase, to lyse catheter occlusions. The purpose of this retrospective analysis was to assess the safety and efficacy of reteplase in opening occluded catheters in a series of patients receiving hemodialysis. Between January 1 and June 30, 2002, 59 patients could not complete dialysis, because of either poor arterial blood flow or elevated venous resistance. Reteplase, 0.4 U, was administered to the lumen of occluded catheters. After 30 min dwell times, the lumens were aspirated. If flow could not be sufficiently reestablished, a second reteplase dose was administered. Efficacy endpoints were defined as the ability to complete hemodialysis and achieve flow rates of ≧250 ml/min. Safety endpoints were defined as the occurrence of allergic reactions or bleeding. Eighty-five percent (50/59) of the patients were able to complete their hemodialysis session following reteplase administration, with 70% (41/59) able to sustain blood flow rates of ≧250 ml/min. Of the 50 patients who successfully completed dialysis, 66% (33/50) required only one 0.4-unit dose of reteplase per lumen while 34% (17/50) required a second dose. No instances of bleeding or allergic reactions were noted.
Intracellular Cyclic Nucleotide Analogues Inhibit in vitro Mitogenesis and Activation of Fibroblasts Derived from Obstructed Rat Kidneys
Authors: Hewitson T.D. , Martic M. , Darby I.A. , Kelynack K.J. , Bisucci T. , Tait M.G. , Becker G.J. 
Published: March 2004

As several studies indirectly suggest that inhibiting the intracellular breakdown of cyclic nucleotides may inhibit fibrogenesis, this study used membrane permeable cyclic nucleotide analogues to examine the role of cAMP and cGMP signaling pathways in the regulation of renal fibroblast function. Fibroblasts were isolated by explant outgrowth culture of rat kidneys post unilateral ureteric obstruction. Subcultured cells were exposed to 10– 1,000 µ<i>M</i> of the cyclic nucleotide analogues 8-bromo-cAMP (8br-cAMP) and 8-bromo-cGMP (8br-cGMP). Functional parameters examined included mitogenesis (thymidine incorporation), collagen synthesis (proline incorporation), myofibroblast differentiation (Western blotting for α-smooth muscle actin; α-SMA) and expression of CTGF (Northern blotting), a TGF-β<sub>1</sub>-driven immediate early response gene. Serum-stimulated mitogenesis was decreased 27 ± 4% by 100 µ<i>M</i> 8br-cAMP (p < 0.01), 49 ± 6% by 1,000 µ<i>M</i> 8br-cAMP (p < 0.001) and 43 ± 7% by 1,000 µ<i>M</i> 8br-cGMP (p < 0.01). 1,000 µ<i>M</i> 8br-cAMP and 8br-cGMP reduced basal collagen synthesis by 80 ± 5 and 60 ± 21% respectively (both p < 0.05). Maximum dose of 8br-cAMP but not 8br-cGMP inhibited basal expression of the differentiation marker α-SMA by 43 ± 33 (p < 0.05), resulted in a more rounded cell morphology and reduced expression of CTGF by 39 ± 24% (p < 0.05). Measurement of mitochondrial activity confirmed that effects were independent of cell toxicity. In conclusion, cyclic nucleotides inhibit fibrogenesis in vitro. Strategies which elevate intracellular cyclic nucleotide concentrations may therefore be therapeutically valuable in preventing the proliferation and activation of fibroblasts in progressive renal disease.
The Biagram Vector: A Graphical Relation between Reactance and Phase Angle Measured by Bioelectrical Analysis in Infants
Authors: Savino F. , Cresi F. , Grasso G. , Oggero R. , Silvestro L. 
Published: March 2004

<i>Background:</i> Biagram vector is derived by plotting direct measurements of reactance and phase angle from the analyzer, which are not dependent on anthropometric characteristics of the subject and it can be considered an emerging method for evaluating the nutritional status in clinical practice. <i>Aim:</i> To calculate and plot the Biagram vector in a sample of healthy infants in their first year of life. <i>Methods:</i> 174 healthy infants (99 male, 75 female) aged under 1 year, born at full term and adequate for gestational age, were enrolled in a cross-sectional study. The sample was divided into three age groups: 0–3.99 months (group A), 4–7.99 months (group B), and 8–11.99 months (group C). Bioelectric impedance analysis (BIA) was performed by a tetrapolar single frequence phase-sensitive impedance analyzer (STA/BIA; Akern Srl, Firenze, Italy). <i>Results:</i> Biagram vector showed an increasing trend (p < 0.05) in the first year of life (group A: 27.9 ± 12.6; group B: 32.1 ± 9.7; group C: 37.2 ± 9.5). Females showed Biagram vector values significantly higher than males in group A (34.7 ± 15.0 vs. 24.0 ± 9.1; p < 0.05). 95% confidence ellipses of Biagram vector for each age group were calculated and plotted and a comparison of our results with published data calculated in older children was conducted. <i>Conclusion: </i>The bivariate analysis of BIA measurements, plotted as a vector in relation to the age, showed an increasing trend with a higher gain rate under 1 year with respect to the following age periods. These data could reflect the variation of body composition in the first years of life and could be of interest to assess the nutritional status for clinical practice.
Association of Pontine Small Infarction (Lacuna) with Disturbance of Postural Stability
Authors: Nagahori T. , Hirashima Y. , Umemura K. , Nakagawa H. , Kamisaki Y. , Origasa H. , Endo S. 
Published: March 2004

Neuronal degeneration within the brainstem has been reported in patients with impaired postural stability. However, the functional significance of these abnormalities is unknown at present. In the present study, we evaluated the relationship between the presence of pontine lacunae and postural stability measured by stabilometry. A total of 209 consecutive patients without neurological signs were divided into three groups according to the territory of lacunae on magnetic resonance imaging: (1) non-lacunar group, (2) pontine lacunar group, and (3) non-pontine lacunar group. Stabilometry was performed and statokinesigram measures including each Romberg quotient were compared among the three groups. Using multivariate analysis, postural stability was found to be disturbed in the pontine lacunar group compared with the other groups. The data of stabilometry in this group were compatible with disturbance of the central controlling system for keeping postural stability. Pontine lacuna is associated with patients with postural instability. This result may be related to the deterioration of the central coordination system for posture and locomotion.
Vascular Anomalies in Alagille Syndrome
Authors: Kamath Binita M. , Spinner Nancy B. , Emerick Karan M. , Chudley Albert E. , Booth Carol , Piccoli David A. , Krantz Ian D. 
Published: March 2004

Background—Alagille syndrome (AGS) is a dominantly inherited multisystem disorder involving the liver, heart, eyes, face, and skeleton, caused by mutations inJagged1. Intracranial bleeding is a recognized complication and cause of mortality in AGS. There are multiple case reports of intracranial vessel abnormalities and other vascular anomalies in AGS. The objective of this study was to characterize the nature and spectrum of vascular anomalies in AGS.Methods and Results—Retrospective chart review of 268 individuals with AGS was performed. Twenty-five patients (9%) had noncardiac vascular anomalies or events. Sixteen patients had documented structural vascular abnormalities. Two had basilar artery aneurysms, 7 had internal carotid artery anomalies, and another had a middle cerebral artery aneurysm. Moyamoya disease was described in 1 patient. Three of the 16 patients had aortic aneurysms, and 2 had aortic coarctations. One of the patients with a basilar artery aneurysm also had coarctation of the aorta. One of the individuals with an internal carotid artery anomaly also had renal artery stenosis. Nine more patients had intracranial events without documented vessel abnormalities. Vascular accidents accounted for 34% of the mortality in this cohort.Conclusions—The vascular anomalies described in our cohort of AGS individuals identify an underrecognized and potentially devastating complication of this disorder. It is a major cause of morbidity and mortality in this population, accounting for 34% of the mortality. We have also reviewed the body of evidence supporting a role forJagged1and the Notch signaling pathway in vascular development.
The Cornerstones for a Prohibition of Cloning Human Beings laid down in the European Convention on Human Rights and Biomedicine
Authors: Winter  
Published: March 2004

AbstractThe recent debate on the theoretical possibility of cloning human beings is urging society to develope a global legal barrier in order to prohibit the use of this technique on humans. Some national legislation, e.g. Germany, already bans the cloning of human beings. The European Convention on Human Rights and Biomedicine contains three articles which together form the cornerstones for a prohibition of cloning: Article 1 guarantees the identity of human beings, Article 18.2 explicitly prohibits the creation of human embryos for research purposes and Article 13 contains a prohibition on the modification of the genome of any decendants. The prohibition of cloning human beings in the Protocol on Embryo Protection foreseen by the Council of Europe seems a necessary consequence. Furthermore, the forthcoming UNESCO Declaration on the Human Genome and Human Rights should contain such an explicit prohibition.