bluuebunny/crossref_metadata_2025 huggingface dataset:
HUMAN EMBRYONIC STEM CELL RESEARCH: WHY THE DISCARDED‐CREATED‐DISTINCTION CANNOT BE BASED ON THE POTENTIALITY ARGUMENT
Authors: DEVOLDER KATRIEN
Published: April 2005

Discussions about the use and derivation of pluripotent human embryonic stem cells are a stumbling block in developing public policy on stem cell research. On the one hand there is a broad consensus on the benefits of these cells for science and biomedicine; on the other hand there is the controversial issue of killing human embryos. I will focus on the compromise position that accepts research on spare embryos, but not on research embryos (‘discarded‐created‐distinction’, from now on d‐c‐d). I will point out that this viewpoint is hard to maintain. The main reason is that the ‘revealed beliefs’ of its defenders are inconsistent with their ‘professed beliefs’, more specifically with their main argument, i.e. the potentiality argument. I will point out that (1) the defenders of d‐c‐d actually grant a relative moral status to the human embryo, (2) this moral status is dependent on internal and external criteria of potentiality, (3) potentiality seen as a variable value that also depends on external criteria cannot justify d‐c‐d, and (4) an approach to human embryonic stem cell‐research that would also allow the use of research embryos is more compatible with the feelings, attitudes and values of those who currently defend d‐c‐d and, therefore, could lead to a broader consensus and to actions that alleviate individual human suffering.
THE DETERMINATION OF ‘BEST INTERESTS’ IN RELATION TO CHILDHOOD VACCINATIONS  (published in Bioethics 19(1))
Authors: DAWSON ANGUS
Published: April 2005

ERRATUM

We regret that, due to a technical error, the uncorrected version of Angus Dawson's article was printed in 19:1. We apologise to the author and reprint in full the corrected version of the paper on the following pages.

A. Dawson et al. .
Bioethics
2005; 19: 72–89.

There are many different ethical arguments that might be advanced for and against childhood vaccinations. In this paper I will explore one particular argument that focuses on the idea that childhood vaccinations are justifiable because they are held to be in the best interests of a particular child. Two issues arise from this idea. The first issue is how best interests are to be determined in the case of childhood vaccinations. The second issue is what follows from this to justify potential interventions within the family in relation to such vaccinations. I argue that best interests must be characterised objectively in such situations and that this means that, in at least some cases, parental decision‐making about vaccinating their children may be overridden.
CEEC growth projections: Certainly necessary and necessarily uncertain*
Authors: Wagner Martin , Hlouskova Jaroslava
Published: April 2005

In this paper we discuss the necessity for an indirect approach to assess the growth and convergence prospects of ten Central and Eastern European countries (CEEC10). Ongoing structural changes in these countries and the recent European Union membership of eight countries in the sample have to be taken into account in growth projections.

Our indirect approach consists of basing growth projections for the CEEC10 on growth equations estimated for the incumbent EU member states. The study improves upon current practice in two ways. First, growth equations are estimated for the EU14 and not on a large heterogeneous panel that includes many countries unrelated to the CEEC10. Second, by means of a variety of equations and scenarios we assess the uncertainty inherent in such projections.

We present growth‐rate and convergence time distributions. The mean convergence times are in line with previous findings. The growth‐rate and convergence time distributions are bi‐modal, reflecting the possibility of two distinct growth paths, depending upon economic policy choices.
Ionic dialysance: Principle and review of its clinical relevance for quantification of hemodialysis efficiency
Authors: Mercadal Lucile , Ridel Christophe , Petitclerc Thierry
Published: April 2005

Ionic dialysance (D) is an online measured variable now available on several dialysis monitors to evaluate small‐solute clearance. Based on conductivity measurements in the inlet and outlet dialysate, the principle of the measurement and the different measurement methods are described. Studies that have evaluated the reliability of ionic dialysance to assess dialysis efficiency are discussed. These studies are divided into two groups: the first comparing ionic dialysance to urea clearance and the second comparing Dt/V to Kt/Vurea, in which the uncertainties of the measurement of Vurea could have misrepresented the relationship between Dt/V and Kt/Vurea. When Kt/Vurea via the Daugirdas second‐generation equation taking the rebound into account is considered, slight—even nonsignificant—differences are evidenced between Kt/Vurea and Dt/V. Therefore, ionic dialysance should be considered as a valid measure in future guidelines for dialysis efficiency.
Does provision of a higher Kt/Vurea make a difference? A hemodialysis controversial issue
Authors: Abbas Tarek M. , Sheashaa Hussein A. , Saad Mohamed A. , Sobh Mohamed A.
Published: April 2005

Background:  Adequate dialysis cannot be ascertained on the sole base of a normal or even a high Kt/Vurea so the impetus of this study was to use the neurophysiologic studies as a marker of the biologic status of the hemodialysis patients to assess the optimum level of Kt/Vurea.

Methods:  This study was carried out on 20 patients (15 men and 5 women) on maintenance hemodialysis; their ages ranged from 18 to 66 years. Initially, the patients were subjected to thorough clinical and laboratory investigations, and their dialysis adequacy was assessed by studying their urea kinetic modeling and neurophysiologic studies (Phase I). Dialysis was optimized to achieve a target Kt/Vurea of 1.3 in Phase II and 1.5 in Phase III. The duration of each phase was six months at the end of which all patients were thoroughly reevaluated. Nutrition was not manipulated during the study.

Results:  A neurophysiologic study showed a significant improvement of polyphasicity pattern of both proximal and distal muscles of the upper and lower limbs concomitant with improvement of quality of life on achieving a Kt/Vurea of 1.5 (p < 0.001). There was no significant change of the duration and amplitude of all studied muscles, however.

Conclusion:  Achieving a Kt/Vurea of 1.5 is a more suitable target for hemodialysis patients because it may be an avenue for improving the neuromuscular functions of these patients.
Uremic pruritus: A review
Authors: Lugon Jocemir R.
Published: April 2005

Pruritus is a major disorder among the skin derangements in advanced renal failure. Its prevalence seems to be diminishing perhaps because of improvements in dialysis treatment. Recent information suggests that interactions between dermal mast cells and distal ends of nonmyelinated C fibers may be important in the precipitation and regulation of the sensory stimuli. The knowledge as to the control of pruritus transmission to cortex areas is still incomplete but endogenous opioid and opioid receptors may have a role in this regard. A recent classification was proposed for pruritus based on the level of its origin. Uremic pruritus, however, seems to be too complex to fit perfectly in any of the suggested modalities. Inflammation and malnutrition are recognized risk factors for cardiovascular death in end‐stage renal disease patients, which may be related to the genesis of pruritus. Consistent with this concept, lower serum levels of albumin and higher serum levels of ferritin were found in pruritic patients when compared to nonpruritic ones. Newer treatments for this difficult clinical problem are being developed and tested.
EFFECT OF CALCIUM CHLORIDE MARINATION AND COLLAGEN CONTENT ON BEEF, HORSE, RABBIT AND HEN MEAT HARDNESS
Authors: PEREZ‐CHABELA M.L. , GUERRERO I. , GUTIERREZ‐RUIZ M.C. , BETANCOURT‐RULE J.M.
Published: April 2005

Overall, meat texture is composed of two types of toughness: primary toughness, because of mechanical resistance of the myofibrillar structure, and secondary toughness, by reason of connective tissue content. Primary toughness can be reduced during aging by intrinsic and extrinsic protease activity. Secondary toughness is determined by a given amount of epimysial and perimysial connective tissue, which cannot be reduced as no collagenases are naturally present in the muscle or produced by native meat microbial populations. The objective of this work was to study primary toughness reduction in meat from four animal species, i.e., beef, horse, rabbit and hen, employing M. Biceps femoris, by calpain activation with calcium chloride. Secondary toughness, expressed as hydroxyproline content, and contribution of both parameters to overall hardness were also studied. Marination with 150 mM CaCl

2

increased enzymatic activity in pre‐rigor meat (horse and beef), but higher concentrations (250 mM) reduced enzymatic activity increments. Although beef marination considerably increased enzymatic activity, it did not reduce hardness, probably because of high collagen content. The high hydroxyproline concentration in beef and horse caused an overall hardness. Marinated horse meat had lower collagen content and a higher enzyme activity, resulting in less hard samples. As expected, pre‐rigor meats, hen and rabbit, had low hardness values for both control and treated samples. Because CaCl

2

in high concentrations inhibits calpain activity, it was assumed that rabbit meat calpains were inhibited at concentrations as low as 150 mM. Therefore, calpains were active in control pre‐rigor rabbit meat but were inactivated by CaCl

2

addition.
IMMUNOHISTOCHEMICAL LOCALIZATION OF PREADIPOCYTE FACTOR‐1: POTENTIAL MARKER OF PREADIPOCYTES IN BOVINE MUSCLE TISSUE
Authors: HUFF PHILLIP W. , LOZEMAN FRED J. , WESELAKE RANDALL J. , WEGNER JOCHEN
Published: April 2005

The objective of the present study was to develop an immunohistochemical technique to locate preadipocytes in bovine muscle tissue. Preadipocyte factor‐1 (pref‐1) is a transmembrane protein that is part of the family of epidermal growth factor‐like repeat‐containing proteins that are involved in cell fate determination. Pref‐1 is highly expressed in preadipocytes, but expression is completely abolished during differentiation into an adipocyte. In the present study, reverse transcription polymerase chain reaction demonstrated that bovine intramuscular adipose tissue contains the three splice forms of pref‐1 (A, C2 and E). Western blots were used to confirm that the protein for pref‐1 was expressed in intramuscular adipose tissue. Polyclonal antibodies against pref‐1 were tested against a cell culture of bovine preadipocytes from an embryo source to confirm that the antibody would immunolocate bovine preadipocytes. The antibody was applied to sections of longissimus dorsi muscle from Charolais and Holstein cattle. Immunohistochemical results showed that pref‐1 is expressed in the perimycium near mature adipocytes and blood vessels. The pool size of preadipocytes appeared to be low. Previous reports, however, have demonstrated that preadipocytes are known to divide and/or migrate, providing a potentially endless source of adipogenic precursor cells.
Non‐invasive pulsatile arterial pressure and stroke volume changes from the human finger
Authors: Bogert Lysander W. J. , van Lieshout Johannes J.
Published: April 2005

In this paper we review recent developments in the methodology of non‐invasive finger arterial pressure measurement and the information about arterial flow that can be obtained from it. Continuous measurement of finger pressure based on the volume‐clamp method was introduced in the early 1980s both for research purposes and for clinical medicine. Finger pressure tracks intra‐arterial pressure but the pressure waves may differ systematically both in shape and magnitude. Such bias can, at least partly, be circumvented by reconstruction of brachial pressure from finger pressure by using a general inverse anti‐resonance model correcting for the difference in pressure waveforms and an individual forearm cuff calibration. The Modelflow method as implemented in the Finometer computes an aortic flow waveform from peripheral arterial pressure by simulating a non‐linear three‐element model of the aortic input impedance. The methodology tracks fast changes in stroke volume (SV) during various experimental protocols including postural stress and exercise. If absolute values are required, calibration against a gold standard is needed. Otherwise, Modelflow‐measured SV is expressed as change from control with the same precision in tracking. Beat‐to‐beat information on arterial flow offers important and clinically relevant information on the circulation beyond what can be detected by arterial pressure.
Post‐stimulus potentiation of transmission in pelvic ganglia enhances sympathetic dilatation of guinea‐pig uterine artery in vitro
Authors: Morris Judy L. , Gibbins Ian L. , Jobling Phillip
Published: April 2005

Vasodilatation produced by stimulation of preganglionic neurones in lumbar and sacral pathways to pelvic ganglia was studied using an in vitro preparation of guinea‐pig uterine artery and associated nerves in a partitioned bath allowing selective drug application to the ganglia or artery. Arterial diameter was monitored using real time video imaging. Vasodilatations produced by hypogastric nerve stimulation (HN; 300 pulses, 10 Hz) were significantly larger and longer in duration than with pelvic nerve stimulation (N= 18). Stimulation of ipsilateral lumbar splanchnic nerves or ipsilateral third lumbar ventral roots also produced prolonged vasodilatations. Blockade of ganglionic nicotinic receptors (0.1–1 mm hexamethonium) delayed the onset and sometimes reduced the peak amplitude of dilatations, but slow dilatations persisted in 16 of 18 preparations. These dilatations were not reduced further by 3 μm capsaicin applied to the artery and ganglia, or ganglionic application of 1 μm hyoscine, 30–100 μm suramin or 10 μm CNQX. Dilatations were reduced slightly by ganglionic application of NK1 and NK3 receptor antagonists (SR140333, SR142801; 1 μm), but were reduced significantly by bathing the ganglia in 0.5 mm Ca2+ and 10 mm Mg2+. Intracellular recordings of paracervical ganglion neurones revealed fast excitatory postsynaptic potentials (EPSPs) in all neurones on HN stimulation (300 pulses, 10 Hz), and slow EPSPs (3–12 mV amplitude) in 25 of 37 neurones. Post‐stimulus action potential discharge associated with slow EPSPs occurred in 16 of 37 neurones (firing rate 9.4 ± 1.5 Hz). Hexamethonium (0.1–1 mm) abolished fast EPSPs. Hexamethonium and hyoscine (1 μm) did not reduce slow EPSPs and associated post‐stimulus firing in identified vasodilator neurones (with VIP immunoreactivity) or non‐vasodilator paracervical neurones. These results demonstrate a predominantly sympathetic origin of autonomic pathways producing pelvic vasodilatation in females. Non‐cholinergic mediators of slow transmission in pelvic ganglia produce prolonged firing of postganglionic neurones and long‐lasting dilatations of the uterine artery. This mechanism would facilitate maintenance of pelvic vasodilatation on stimulation of preganglionic neurones during sexual activity.
Sensory transduction of pulmonary reactive oxygen species by capsaicin‐sensitive vagal lung afferent fibres in rats
Authors: Ruan Ting , Shuei Lin You , Lin Kae‐Shin , Ru Kou Yu
Published: April 2005

The mechanisms of sensory transduction of pulmonary reactive oxygen species (ROS) by capsaicin‐sensitive vagal lung afferent fibres are unclear. To investigate the role of transient receptor potential vanilloid 1 (TRPV1) receptors and P2X purinoceptors in this sensory transduction, we recorded fibre activity (FA) from 132 fibres of this type in 132 anaesthetized and ventilated rats. Airway challenge of aerosolized H2O2 (0, 0.2 and 0.4%) produced a concentration‐dependant fibre stimulation. The fibre responses to 0.4% H2O2 were attenuated by dimethylthiourea (a hydroxyl radical (·OH) scavenger; change in fibre activity (ΔFA), −55 ± 9%) or deferoxamine (an iron‐chelator that prevents formation of ·OH; ΔFA, −59 ± 9%), were prevented by catalase (an enzyme catalysing H2O2; ΔFA, −96 ± 3%) and were unaffected by the vehicle for dimethylthiourea, iron‐saturated deferoxamine or heat‐inactivated catalase. The fibre responses to 0.4% H2O2 were attenuated by capsazepine (a TRPV1 receptor antagonist; ΔFA, −39 ± 9%) or iso‐pyridoxalphosphate‐6‐azophenyl‐2′,5′‐disulphonate (iso‐PPADS, a P2X receptor antagonist; ΔFA, −51 ± 9%), were further reduced by capsazepine and iso‐PPADS in combination (ΔFA, −70 ± 13%), and were unaltered by their vehicles. The fibre responses to cigarette smoke (20 ml), an irritant that generates ROS, were attenuated by dimethylthiourea (ΔFA, −61 ± 9%) or capsazepine and iso‐PPADS in combination (ΔFA, −67 ± 9%). These results suggest that both the TRPV1 and P2X receptors mediate the sensory transduction of ROS, especially H2O2 and ·OH, by capsaicin‐sensitive vagal lung afferent fibres.
Human Mpp11 J Protein: Ribosome-Tethered Molecular Chaperones Are Ubiquitous
Authors: Hundley Heather A. , Walter William , Bairstow Shawn , Craig Elizabeth A.
Published: April 2005

The existence of specialized molecular chaperones that interact directly with ribosomes is well established in microorganisms. Such proteins bind polypeptides exiting the ribosomal tunnel and provide a physical link between translation and protein folding. We report that ribosome-associated molecular chaperones have been maintained throughout eukaryotic evolution, as illustrated by Mpp11, the human ortholog of the yeast ribosome-associated J protein Zuo. When expressed in yeast, Mpp11 partially substituted for Zuo by partnering with the multipurpose Hsp70 Ssa, the homolog of mammalian Hsc70. We propose that in metazoans, ribosome-associated Mpp11 recruits the multifunctional soluble Hsc70 to nascent polypeptide chains as they exit the ribosome.
Structure of the Rotor of the V-Type Na
+
-ATPase from
Enterococcus hirae
Authors: Murata Takeshi , Yamato Ichiro , Kakinuma Yoshimi , Leslie Andrew G. W. , Walker John E.
Published: April 2005

The membrane rotor ring from the vacuolar-type (V-type) sodium ion–pumping adenosine triphosphatase (Na
+
-ATPase) from
Enterococcus hirae
consists of 10 NtpK subunits, which are homologs of the 16-kilodalton and 8-kilodalton proteolipids found in other V-ATPases and in F
1
F
o
- or F-ATPases, respectively. Each NtpK subunit has four transmembrane α helices, with a sodium ion bound between helices 2 and 4 at a site buried deeply in the membrane that includes the essential residue glutamate-139. This site is probably connected to the membrane surface by two half-channels in subunit NtpI, against which the ring rotates. Symmetry mismatch between the rotor and catalytic domains appears to be an intrinsic feature of both V- and F-ATPases.
A Novel Mutation in
kaiC
Affects Resetting of the Cyanobacterial Circadian Clock
Authors: Kiyohara Yota B. , Katayama Mitsunori , Kondo Takao
Published: April 2005

Light is the most important factor controlling circadian systems in response to day-night cycles. In order to better understand the regulation of circadian rhythms by light in
Synechococcus elongatus
PCC 7942, we screened for mutants with defective phase shifting in response to dark pulses. Using a 5-h dark-pulse protocol, we identified a mutation in
kaiC
that we termed
pr1
, for phase response 1. In the
pr1
mutant, a 5-h dark pulse failed to shift the phase of the circadian rhythm, while the same pulse caused a 10-h phase shift in wild-type cells. The rhythm in accumulation of KaiC was abolished in the
pr1
mutant, and the rhythmicity of KaiC phosphorylation was reduced. Additionally, the
pr1
mutant was defective in mediating the feedback inhibition of
kaiBC
. Finally, overexpression of mutant KaiC led to a reduced phase shift compared to that for wild-type KaiC. Thus, KaiC appears to play a role in resetting the cellular clock in addition to its documented role in the feedback regulation of circadian rhythms.
Characterization of LtsA from
Rhodococcus erythropolis
, an Enzyme with Glutamine Amidotransferase Activity
Authors: Mitani Yasuo , Meng XianYing , Kamagata Yoichi , Tamura Tomohiro
Published: April 2005

The nocardioform actinomycete
Rhodococcus erythropolis
has a characteristic cell wall structure. The cell wall is composed of arabinogalactan and mycolic acid and is highly resistant to the cell wall-lytic activity of lysozyme (muramidase). In order to improve the isolation of recombinant proteins from
R. erythropolis
host cells (N. Nakashima and T. Tamura, Biotechnol. Bioeng.
86:
136-148, 2004), we isolated two mutants, L-65 and L-88, which are susceptible to lysozyme treatment. The lysozyme sensitivity of the mutants was complemented by expression of
Corynebacterium glutamicum ltsA
, which codes for an enzyme with glutamine amidotransferase activity that results from coupling of two reactions (a glutaminase activity and a synthetase activity). The lysozyme sensitivity of the mutants was also complemented by
ltsA
homologues from
Bacillus subtilis
and
Mycobacterium tuberculosis
, but the homologues from
Streptomyces coelicolor
and
Escherichia coli
did not complement the sensitivity. This result suggests that only certain LtsA homologues can confer lysozyme resistance. Wild-type recombinant LtsA from
R. erythropolis
showed glutaminase activity, but the LtsA enzymes from the L-88 and L-65 mutants displayed drastically reduced activity. Interestingly, an
ltsA
disruptant mutant, which expressed the mutated LtsA, changed from lysozyme sensitive to lysozyme resistant when NH
4
Cl was added into the culture media. The glutaminase activity of the LtsA mutants inactivated by site-directed mutagenesis was also restored by addition of NH
4
Cl, indicating that NH
3
can be used as an amide donor molecule. Taken together, these results suggest that LtsA is critically involved in mediating lysozyme resistance in
R. erythropolis
cells.
Expression of
fnr
Is Constrained by an Upstream IS
5
Insertion in Certain
Escherichia coli
K-12 Strains
Authors: Sawers R. Gary
Published: April 2005

FNR is a global transcriptional regulator that controls anaerobic gene expression in
Escherichia coli
. Through the use of a number of approaches it was shown that
fnr
gene expression is reduced approximately three- to fourfold in
E. coli
strain MC4100 compared with the results seen with strain MG1655. This reduction in
fnr
expression is due to the insertion of IS
5
(
is5F
) in the regulatory region of the gene at position −41 relative to the transcription initiation site. Transcription of the
fnr
gene nevertheless occurs from its own promoter in strain MC4100, but transcript levels are reduced approximately fourfold compared with those seen with strain MG1655. Remarkably, in strains bearing
is5F
the presence of Hfq prevents IS
5
-dependent transcriptional silencing of
fnr
expression. Thus, an
hfq
mutant of MC4100 is devoid of FNR protein and has the phenotype of an
fnr
mutant. In strain MG1655, or a derivative of MC4100 lacking
is5F
, mutation of
hfq
had no effect on
fnr
transcript levels. This finding indicates that IS
5
mediates the effect of Hfq on
fnr
expression in MC4100. Western blot analysis revealed that cellular levels of FNR were reduced threefold in strain MC4100 compared with strain MG1655 results. A selection of FNR-dependent genes fused to
lacZ
were analyzed for the effects of reduced FNR levels on anaerobic gene expression. Expression of some operons, e.g.,
focA
-
pfl
and
fdnGHJI
, was unaffected by reduction in the level of FNR, while the expression of other genes such as
ndh
and
nikA
was clearly affected.
An FNR-Type Regulator Controls the Anaerobic Expression of Hyn Hydrogenase in
Thiocapsa roseopersicina
Authors: Kovács Ákos T. , Rákhely Gábor , Browning Douglas F. , Fülöp András , Maróti Gergely , Busby Stephen J. W. , Kovács Kornél L.
Published: April 2005

The purple sulfur photosynthetic bacterium
Thiocapsa roseopersicina
BBS contains a heat-stable membrane-associated hydrogenase encoded by the
hyn
operon. Expression from the
hyn
operon regulatory region is up-regulated under anaerobic conditions.
cis
elements were mapped between positions −602 and −514 upstream from the
hynS
gene. Within this region two sequences that resemble DNA sites for FNR were recognized. The gene of an FNR homologue, FnrT, was identified in the genome of
T. roseopersicina
, and an
fnrT
knockout mutant was constructed. Anaerobic induction of
hynS
expression was abolished in the
fnrT
mutant, suggesting that FnrT is an activator of the
hynS
promoter. The
T. roseopersicina hynS
promoter could be activated in
Escherichia coli
, and this regulation was dependent on
E. coli
FNR. In vitro experiments with purified
E. coli
Ala154 FNR protein and purified
E. coli
RNA polymerase showed that FNR bound to two sites in the
hyn
regulatory region, that FNR could activate transcription initiation at the
hynS
promoter, and that FNR bound at the two target sites activated to different extents.
Flavobacterium johnsoniae
GldJ Is a Lipoprotein That Is Required for Gliding Motility
Authors: Braun Timothy F. , McBride Mark J.
Published: April 2005

Cells of
Flavobacterium johnsoniae
glide rapidly over surfaces by an unknown mechanism. Eight genes required for gliding motility have been described. Complementation of the nonmotile mutant UW102-48 identified another gene,
gldJ
, that is required for gliding.
gldJ
mutants formed nonspreading colonies, and individual cells were completely nonmotile. Like previously described nonmotile mutants,
gldJ
mutants were deficient in chitin utilization and were resistant to bacteriophages that infect wild-type cells. Cell fractionation and labeling studies with [
3
H]palmitate indicated that GldJ is a lipoprotein. Mutations in
gldA
,
gldB
,
gldD
,
gldF
,
gldG
,
gldH
, or
gldI
resulted in normal levels of
gldJ
transcript but decreased levels of GldJ protein. Expression of truncated GldJ protein in wild-type cells resulted in a severe motility defect. GldJ was found in regular bands that suggest the presence of a helical structure within the cell envelope.
Translational Control of Tetracycline Resistance and Conjugation in the
Bacteroides
Conjugative Transposon CTnDOT
Authors: Wang Yanping , Rotman Ella R. , Shoemaker Nadja B. , Salyers Abigail A.
Published: April 2005

The
tetQ-rteA-rteB
operon of the
Bacteroides
conjugative transposon CTnDOT is responsible for tetracycline control of the excision and transfer of CTnDOT. Previous studies revealed that tetracycline control of this operon occurred at the translational level and involved a hairpin structure located within the 130-base leader sequence that lies between the promoter of
tetQ
and the start codon of the gene. This hairpin structure is formed by two sequences, designated Hp1 and Hp8. Hp8 contains the ribosome binding site for
tetQ
. Examination of the leader region sequence revealed three sequences that might encode a leader peptide. One was only 3 amino acids long. The other two were 16 amino acids long. By introducing stop codons into the peptide coding regions, we have now shown that the 3-amino-acid peptide is the one that is essential for tetracycline control. Between Hp1 and Hp8 lies an 85-bp region that contains other possible RNA hairpin structures. Deletion analysis of this intervening DNA segment has now identified a sequence, designated Hp2, which is essential for tetracycline regulation. This sequence could form a short hairpin structure with Hp1. Mutations that made the Hp1-Hp2 structure more stable caused nearly constitutively high expression of the operon. Thus, stalling of ribosomes on the 3-amino-acid leader peptide could favor formation of the Hp1-Hp2 structure and thus preclude formation of the Hp1-Hp8 structure, releasing the ribosome binding site of
tetQ
. Finally, comparison of the CTnDOT
tetQ
leader regions with upstream regions of five
tetQ
genes found in other elements reveals that the sequences are virtually identical, suggesting that translational attenuation is responsible for control of tetracycline resistance in these other cases as well.
Import of the Transfer RNase Colicin D Requires Site-Specific Interaction with the Energy-Transducing Protein TonB
Authors: Mora Liliana , Diaz Nancy , Buckingham Richard H. , de Zamaroczy Miklos
Published: April 2005

The transfer RNase colicin D and ionophoric colicin B appropriate the outer membrane iron siderophore receptor FepA and share a common translocation requirement for the TonB pathway to cross the outer membrane. Despite the almost identical sequences of the N-terminal domains required for the translocation of colicins D and B, two spontaneous
tonB
mutations (Arg158Ser and Pro161Leu) completely abolished colicin D toxicity but did not affect either the sensitivity to other colicins or the FepA-dependent siderophore uptake capacity. The sensitivity to colicin D of both
tonB
mutants was fully restored by specific suppressor mutations in the TonB box of colicin D, at Ser18(Thr) and Met19(Ile), respectively. This demonstrates that the interaction of colicin D with TonB is critically dependent on certain residues close to position 160 in TonB and on the side chains of certain residues in the TonB box of colicin D. The effect of introducing the TonB boxes from other TonB-dependent receptors and colicins into colicins D and B was studied. The results of these and other changes in the two TonB boxes show that the role of residues at positions 18 and 19 in colicin D is strongly modulated by other nearby and/or distant residues and that the overall function of colicin D is much more dependent on the interaction with TonB involving the TonB box than is the function of colicin B.
Nucleotide Substitution and Recombination at Orthologous Loci in
Staphylococcus aureus
Authors: Hughes Austin L. , Friedman Robert
Published: April 2005

The pattern of nucleotide substitution was examined at 2,129 orthologous loci among five genomes of
Staphylococcus aureus
, which included two sister pairs of closely related genomes (MW2/MSSA476 and Mu50/N315) and the more distantly related MRSA252. A total of 108 loci were unusual in lacking any synonymous differences among the five genomes; most of these were short genes encoding proteins highly conserved at the amino acid sequence level (including many ribosomal proteins) or unknown predicted genes. In contrast, 45 genes were identified that showed anomalously high divergence at synonymous sites. The latter genes were evidently introduced by homologous recombination from distantly related genomes, and in many cases, the pattern of nucleotide substitution made it possible to reconstruct the most probable recombination event involved. These recombination events introduced genes encoding proteins that differed in amino acid sequence and thus potentially in function. Several of the proteins are known or likely to be involved in pathogenesis (e.g., staphylocoagulase, exotoxin, Ser-Asp fibrinogen-binding bone sialoprotein-binding protein, fibrinogen and keratin-10 binding surface-anchored protein, fibrinogen-binding protein ClfA, and enterotoxin P). Therefore, the results support the hypothesis that exchange of homologous genes among
S. aureus
genomes can play a role in the evolution of pathogenesis in this species.
Smx Nuclease Is the Major, Low-pH-Inducible Apurinic/Apyrimidinic Endonuclease in
Streptococcus mutans
Authors: Faustoferri Roberta C. , Hahn Kristina , Weiss Kellie , Quivey Robert G.
Published: April 2005

The causative agent of dental caries in humans,
Streptococcus mutans
, outcompetes other bacterial species in the oral cavity and causes disease by surviving acidic conditions in dental plaque. We have previously reported that the low-pH survival strategy of
S. mutans
includes the ability to induce a DNA repair system that appears to involve an enzyme with exonuclease functions (K. Hahn, R. C. Faustoferri, and R. G. Quivey, Jr., Mol. Microbiol 31:1489-1498, 1999). Here, we report overexpression of the
S. mutans
apurinic/apyrimidinic (AP) endonuclease, Smx, in
Escherichia coli
; initial characterization of its enzymatic activity; and analysis of an
smx
mutant strain of
S. mutans
. Insertional inactivation of the
smx
gene eliminates the low-pH-inducible exonuclease activity previously reported. In addition, loss of Smx activity renders the mutant strain sensitive to hydrogen peroxide treatment but relatively unaffected by acid-mediated damage or near-UV irradiation. The
smx
strain of
S. mutans
was highly sensitive to the combination of iron and hydrogen peroxide, indicating the likely production of hydroxyl radical by Fenton chemistry with concomitant formation of AP sites that are normally processed by the wild-type allele. Smx activity was sufficiently expressed in
E. coli
to protect an
xth
mutant strain from the effects of hydrogen peroxide treatment. The data indicate that
S. mutans
expresses an inducible, class II-like AP endonuclease, encoded by the
smx
gene, that exhibits exonucleolytic activity and is regulated as part of the acid-adaptive response of the organism. Smx is likely the primary, if not the sole, AP endonuclease induced during growth at low pH values.
Mutants of FtsZ Targeting the Protofilament Interface: Effects on Cell Division and GTPase Activity
Authors: Redick Sambra D. , Stricker Jesse , Briscoe Gina , Erickson Harold P.
Published: April 2005

The bacterial cell division protein FtsZ assembles into straight protofilaments, one subunit thick, in which subunits appear to be connected by identical bonds or interfaces. These bonds involve the top surface of one subunit making extensive contact with the bottom surface of the subunit above it. We have investigated this interface by site-directed mutagenesis. We found nine bottom and eight top mutants that were unable to function for cell division. We had expected that some of the mutants might poison cell division substoichiometrically, but this was not found for any mutant. Eight of the bottom mutants exhibited dominant negative effects (reduced colony size) and four completely blocked colony formation, but this required expression of the mutant protein at four to five times the wild-type FtsZ level. Remarkably, the top mutants were even weaker, most showing no effect at the highest expression level. This suggests a directional assembly or treadmilling, where subunit addition is primarily to the bottom end of the protofilament. Selected pairs of top and bottom mutants showed no GTPase activity up to 10 to 20 μM, in contrast to the high GTPase activity of wild-type FtsZ above 1 μM. Overall, these results suggest that in order for a subunit to bind a protofilament at the 1 μM

K
d

for elongation, it must have functional interfaces at both the top and bottom. This is inconsistent with the present model of the protofilament, as a simple stack of subunits one on top of the other, and may require a new structural model.
cse
, a Chimeric and Variable Gene, Encodes an Extracellular Protein Involved in Cellular Segregation in
Streptococcus thermophilus
Authors: Borges Frédéric , Layec Séverine , Thibessard Annabelle , Fernandez Annabelle , Gintz Brigitte , Hols Pascal , Decaris Bernard , Leblond-Bourget Nathalie
Published: April 2005

The isolation of a
Streptococcus thermophilus
CNRZ368 mutant displaying a long-chain phenotype allowed us to identify the
cse
gene (for cellular segregation). The N terminus of Cse exhibits high similarity to
Streptococcus agalactiae
surface immunogenic protein (SIP), while its C terminus exhibits high similarity to
S. thermophilus
PcsB. In CNRZ368, deletion of the entire
cse
open reading frame leads to drastic lengthening of cell chains and altered colony morphology. Complementation of the Δ
cse
mutation with a wild-type allele restored both wild-type phenotypes. The central part of Cse is a repeat-rich region with low sequence complexity. Comparison of
cse
from CNRZ368 and LMG18311 strains reveals high variability of this repeat-rich region. To assess the impact of this central region variability, the central region of LMG18311
cse
was exchanged with that of CNRZ368
cse
. This replacement did not affect chain length, showing that divergence of the central part does not modify cell segregation activity of Cse. The structure of the
cse
locus suggests that the chimeric organization of
cse
results from insertion of a duplicated sequence deriving from the
pcsB
3′ end into an ancestral
sip
gene. Thus, the
cse
locus illustrates the module-shuffling mechanism of bacterial gene evolution.
Mycobacterial Lipid II Is Composed of a Complex Mixture of Modified Muramyl and Peptide Moieties Linked to Decaprenyl Phosphate
Authors: Mahapatra Sebabrata , Yagi Tetsuya , Belisle John T. , Espinosa Benjamin J. , Hill Preston J. , McNeil Michael R. , Brennan Patrick J. , Crick Dean C.
Published: April 2005

Structural analysis of compounds identified as lipid I and II from
Mycobacterium smegmatis
demonstrated that the lipid moiety is decaprenyl phosphate; thus,
M. smegmatis
is the first bacterium reported to utilize a prenyl phosphate other than undecaprenyl phosphate as the lipid carrier involved in peptidoglycan synthesis. In addition, mass spectrometry showed that the muropeptides from lipid I are predominantly
N-
acetylmuramyl-
l
-alanine-
d
-glutamate-
meso
-diaminopimelic acid-
d
-alanyl-
d
-alanine, whereas those isolated from lipid II form an unexpectedly complex mixture in which the muramyl residue and the pentapeptide are modified singly and in combination. The muramyl residue is present as
N
-acetylmuramic acid,
N-
glycolylmuramic acid, and muramic acid. The carboxylic functions of the peptide side-chains of lipid II showed three types of modification, with the dominant one being amidation. The preferred site for amidation is the free carboxyl group of the
meso
-diaminopimelic acid residue. Diamidated species were also observed. The carboxylic function of the terminal
d
-alanine of some molecules is methylated, as are all three carboxylic acid functions of other molecules. This study represents the first structural analysis of mycobacterial lipid I and II and the first report of extensive modifications of these molecules. The observation that lipid I was unmodified strongly suggests that the lipid II intermediates of
M. smegmatis
are substrates for a variety of enzymes that introduce modifications to the sugar and amino acid residues prior to the synthesis of peptidoglycan.
Participation of 3′-to-5′ Exoribonucleases in the Turnover of
Bacillus subtilis
mRNA
Authors: Oussenko Irina A. , Abe Teppei , Ujiie Hiromi , Muto Akira , Bechhofer David H.
Published: April 2005

Four 3′-to-5′ exoribonucleases have been identified in
Bacillus subtilis
: polynucleotide phosphorylase (PNPase), RNase R, RNase PH, and YhaM. Mutant strains were constructed that were lacking PNPase and one or more of the other three ribonucleases or that had PNPase alone. Analysis of the decay of mRNA encoded by seven small, monocistronic genes showed that PNPase was the major enzyme involved in mRNA turnover. Significant levels of decay intermediates, whose 5′ ends were at the transcriptional start site and whose 3′ ends were at various positions in the coding sequence, were detected only when PNPase was absent. A detailed analysis of
rpsO
mRNA decay showed that decay intermediates accumulated as the result of a block to 3′-to-5′ processivity at the base of stem-loop structures. When RNase R alone was present, it was also capable of degrading mRNA, showing the involvement of this exonuclease in mRNA turnover. The degradative activity of RNase R was impaired when RNase PH or YhaM was also present. Extrapolation from the seven genes examined suggested that a large number of mRNA fragments was present in the PNPase-deficient mutant. Maintenance of the free ribosome pool in this strain would require a high level of activity on the part of the tmRNA
trans
translation system. A threefold increase in the level of peptide tagging was observed in the PNPase-deficient strain, and selective pressure for increased tmRNA activity was indicated by the emergence of mutant strains with elevated tmRNA transcription.
Catabolite Repression of the Propionate Catabolic Genes inEscherichia coliandSalmonella enterica: Evidence for Involvement of the Cyclic AMP Receptor Protein
Authors: Lee Sung Kuk , Newman Jack D. , Keasling Jay D.
Published: April 2005

Previous studies withSalmonella entericaserovar Typhimurium LT2 demonstrated that transcriptional activation of theprpBCDEoperon requires the function of transcription factor PrpR, sigma-54, and IHF. In this study, we found that transcription from theprpBCDEandprpRpromoters was down-regulated by the addition of glucose or glycerol, indicating that these genes may be regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. Targeted mutagenesis of a putative CRP-binding site in the promoter region betweenprpRandprpBCDEsuggested that these genes are under the control of CRP. Furthermore, cells with defects incyaorcrpexhibited reduced transcriptional activation ofprpRandprpBCDEinEscherichia coli. These results demonstrate that propionate metabolism is subject to catabolite repression by the global transcriptional regulator CRP and that this regulation is effected through control of both the regulator geneprpRand theprpBCDEoperon itself. The unique properties of the regulation of these two divergent promoters may have important implications for mechanisms of CRP-dependent catabolite repression acting in conjunction with a member of the sigma-54 family of transcriptional activators.
Antimutator Role of DNA Glycosylase MutY in Pathogenic
Neisseria
Species
Authors: Davidsen T. , Bjørås M. , Seeberg E. C. , Tønjum T.
Published: April 2005

Genome alterations due to horizontal gene transfer and stress constantly generate strain on the gene pool of
Neisseria meningitidis
, the causative agent of meningococcal (MC) disease. The DNA glycosylase MutY of the base excision repair pathway is involved in the protection against oxidative stress. MC MutY expressed in
Escherichia coli
exhibited base excision activity towards DNA substrates containing A:7,8-dihydro-8-oxo-2′-deoxyguanosine and A:C mismatches. Expression in
E. coli
fully suppressed the elevated spontaneous mutation rate found in the
E. coli mutY
mutant. An assessment of MutY activity in lysates of neisserial wild-type and
mutY
mutant strains showed that both MC and gonococcal (GC) MutY is expressed and active in vivo. Strikingly, MC and GC
mutY
mutants exhibited 60- to 140-fold and 20-fold increases in mutation rates, respectively, compared to the wild-type strains. Moreover, the differences in transitions and transversions in
rpoB
conferring rifampin resistance observed with the wild type and mutants demonstrated that the neisserial MutY enzyme works in preventing GC→AT transversions. These findings are important in the context of models linking mutator phenotypes of disease isolates to microbial fitness.
Cytochrome Oxidase Deficiency Protects
Escherichia coli
from Cell Death but Not from Filamentation Due to Thymine Deficiency or DNA Polymerase Inactivation
Authors: Strauss Bernard , Kelly Kemba , Ekiert Damian
Published: April 2005

Temperature-sensitive DNA polymerase mutants (
dnaE
) are protected from cell death on incubation at nonpermissive temperature by mutation in the
cydA
gene controlling cytochrome
bd
oxidase. Protection is observed in complex (Luria-Bertani [LB]) medium but not on minimal medium. The
cydA
mutation protects a thymine-deficient strain from death in the absence of thymine on LB but not on minimal medium. Both
dnaE
and
Δthy
mutants filament under nonpermissive conditions. Filamentation per se is not the cause of cell death, because the
dnaE cydA
double mutant forms long filaments after 24 h of incubation in LB medium at nonpermissive temperature. These filaments have multiply dispersed nucleoids and produce colonies on return to permissive conditions. The protective effect of a deficiency of
cydA
at high temperature is itself suppressed by overexpression of cytochrome
bo3
, indicating that the phenomenon is related to energy metabolism rather than to a specific effect of the
cydA
protein. We propose that filamentation and cell death resulting from thymine deprivation or slowing of DNA synthesis are not sequential events but occur in response to the same or a similar signal which is modulated in complex medium by cytochrome
bd
oxidase. The events which follow inhibition of replication fork progression due to either polymerase inactivation, thymine deprivation, or hydroxyurea inhibition differ in detail from those following actual DNA damage.
Polarity of Enteropathogenic
Escherichia coli
EspA Filament Assembly and Protein Secretion
Authors: Crepin Valérie F. , Shaw Robert , Abe Cecilia M. , Knutton Stuart , Frankel Gad
Published: April 2005

Type III secretion systems (TTSS) are sophisticated macromolecular structures that play an imperative role in bacterial infections and human disease. The TTSS needle complex is conserved among bacterial pathogens and shows broad similarity to the flagellar basal body. However, the TTSS of enteropathogenic and enterohemorrhagic
Escherichia coli
, two important human enteric pathogens, is unique in that it has a ∼12-nm-diameter filamentous extension to the needle that is composed of the secreted translocator protein EspA. EspA filaments and flagellar structures have very similar helical symmetry parameters. In this study we investigated EspA filament assembly and the delivery of effector proteins across the bacterial cell wall. We show that EspA filaments are elongated by addition of EspA subunits to the tip of the growing filament. Moreover, EspA filament length is modulated by the availability of intracellular EspA subunits. Finally, we provide direct evidence that EspA filaments are hollow conduits through which effector proteins are delivered to the extremity of the bacterial cell (and subsequently into the host cell).
Clonal Distribution and Phase-Variable Expression of a Major Histocompatibility Complex Analogue Protein in
Staphylococcus aureus
Authors: Buckling Angus , Neilson James , Lindsay Jodi , ffrench-Constant Richard , Enright Mark , Day Nicholas , Massey Ruth C.
Published: April 2005

The
mapW
gene of
Staphylococcus aureus
strain N315 contains a poly(A) tract which truncates translation of the protein. This study demonstrates that
mapW
is an allelic variant of the
map/eap
genes found in other strains and that the variation in the length of this poly(A) tract suggests that it is a contingency locus.
Positive Selection for Loss-of-Function
tat
Mutations Identifies Critical Residues Required for TatA Activity
Authors: Hicks Matthew G. , Lee Philip A. , Georgiou George , Berks Ben C. , Palmer Tracy
Published: April 2005

The Tat system, found in the cytoplasmic membrane of many bacteria, is a general export pathway for folded proteins. Here we describe the development of a method, based on the transport of chloramphenicol acetyltransferase, that allows positive selection of mutants defective in Tat function. We have demonstrated the utility of this method by selecting novel loss-of-function alleles of
tatA
from a pool of random
tatA
mutations. Most of the mutations that were isolated fall in the amphipathic region of TatA, emphasizing the pivotal role that this part of the protein plays in TatA function.