bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Essential Residues in Apolipoprotein
N
-Acyl Transferase, a Member of the CN Hydrolase Family
Authors: Vidal-Ingigliardi Dominique , Lewenza Shawn , Buddelmeijer Nienke
Published: April 2007

Apolipoprotein
N
-acyl transferase (Lnt) is an essential membrane-bound protein involved in lipid modification of all lipoproteins in gram-negative bacteria. Essential residues in Lnt of
Escherichia coli
were identified by using site-directed mutagenesis and an in vivo complementation assay. Based on sequence conservation and known protein structures, we predict a model for Lnt, which is a member of the CN hydrolase family. Besides the potential catalytic triad E267-K335-C387, four residues that directly affect the modification of Braun's lipoprotein Lpp are absolutely required for Lnt function. Residues Y388 and E389 are part of the hydrophobic pocket that constitutes the active site. Residues W237 and E343 are located on two flexible arms that face away from the active site and are expected to open and close upon the binding and release of phospholipid and/or apolipoprotein. Substitutions causing temperature-dependent effects were located at different positions in the structural model. These mutants were not affected in protein stability. Lnt proteins from other proteobacteria, but not from actinomycetes, were functional in vivo, and the essential residues identified in Lnt of
E. coli
are conserved in these proteins.
Characterization of a Small Mobilizable Transposon, MTnSag1, inStreptococcus agalactiae
Authors: Achard Adeline , Leclercq Roland
Published: April 2007

The transposon MTnSag1 fromStreptococcus agalactiaecarried an IS1-like transposase gene and thelnu(C) gene, which encoded a lincosamide nucleotidyltransferase. MTnSag1 could be mobilized by the conjugative transposon Tn916. An intermediate circular form of MTnSag1 and a putative origin of transfer at the 3′ end of thelnu(C) gene were characterized.
Distribution of Protein A on the Surface ofStaphylococcus aureus
Authors: DeDent Andrea C. , McAdow Molly , Schneewind Olaf
Published: April 2007

Surface proteins ofStaphylococcus aureusfulfill many important roles during the pathogenesis of human infections and are anchored to the cell wall envelope by sortases. Although the chemical linkage of proteins to cell wall cross bridges is known, the mechanisms whereby polypeptides are distributed on the staphylococcal surface have not been revealed. We show here that protein A, the ligand of immunoglobulin, is unevenly distributed over the staphylococcal surface. Upon removal with trypsin, newly synthesized polypeptide is deposited at two to four discrete foci. During subsequent growth, protein A appears to be slowly distributed from these sites. When viewed through multiple focal planes by laser scanning microscopy, protein A foci are arranged in a circle surrounding the bacterial cell. This pattern of distribution requires the LPXTG sorting signal of protein A as well as sortase A, the transpeptidase that anchors polypeptides to cell wall cross bridges. A model is presented whereby protein A deposition at discrete sites coupled with cell wall synthesis enables distribution of protein A on the staphylococcal surface.
Functional Genomics Enables Identification of Genes of the Arginine Transaminase Pathway in
Pseudomonas aeruginosa
Authors: Yang Zhe , Lu Chung-Dar
Published: April 2007

Arginine utilization in
Pseudomonas aeruginosa
with multiple catabolic pathways represents one of the best examples of the metabolic versatility of this organism. To identify genes involved in arginine catabolism, we have employed DNA microarrays to analyze the transcriptional profiles of this organism in response to
l
-arginine. While most of the genes involved in arginine uptake, regulation, and metabolism have been identified as members of the ArgR (arginine-responsive regulatory protein) regulon in our previous study, they did not include any genes of the arginine dehydrogenase (ADH) pathway. In this study, 18 putative transcriptional units of 38 genes, including the two known genes of the ADH pathway,
kauB
and
gbuA
, were found to be inducible by exogenous
l
-arginine in the absence of ArgR. To identify the missing genes that encode enzymes for the initial steps of the ADH pathway, the potential physiological functions of those candidate genes in arginine utilization were studied by growth phenotype analysis of knockout mutants. Expression of these genes was induced by
l
-arginine in an
aruF
mutant strain devoid of a functional arginine succinyltransferase pathway, the major route of arginine utilization. Disruption of
dadA
, a putative catabolic alanine dehydrogenase-encoding gene, in the
aruF
mutant produced no growth on
l
-arginine, suggesting the involvement of
l
-alanine in arginine catabolism. This hypothesis was further supported by the detection of an
l
-arginine-inducible arginine:pyruvate transaminase activity in the
aruF
mutant. Knockout of
aruH
and
aruI
, which encode an arginine:pyruvate transaminase and a 2-ketoarginine decarboxylase in an operon, also abolished the ability of the
aruF
mutant to grow on
l
-arginine. The results of high-performance liquid chromatography analysis demonstrated consumption of 2-ketoarginine and suggested that generation of 4-guanidinobutyraldehyde occurred in the
aruF
mutant but not in the
aruF aruI
mutant. These results led us to propose the arginine transaminase pathway that removes the α-amino group of
l
-arginine via transamination instead of oxidative deamination by dehydrogenase or oxidase as originally proposed. In the same genetic locus, we also identified a two-component system, AruRS, for the regulation of arginine-responsive induction of the arginine transaminase pathway. This work depicted a wider network of arginine metabolism than we previously recognized.
Characterization of an Arginine:Pyruvate Transaminase in Arginine Catabolism of
Pseudomonas aeruginosa
PAO1
Authors: Yang Zhe , Lu Chung-Dar
Published: April 2007

The arginine transaminase (ATA) pathway represents one of the multiple pathways for
l
-arginine catabolism in
Pseudomonas aeruginosa
. The AruH protein was proposed to catalyze the first step in the ATA pathway, converting the substrates
l
-arginine and pyruvate into 2-ketoarginine and
l
-alanine. Here we report the initial biochemical characterization of this enzyme. The
aruH
gene was overexpressed in
Escherichia coli
, and its product was purified to homogeneity. High-performance liquid chromatography and mass spectrometry (MS) analyses were employed to detect the presence of the transamination products 2-ketoarginine and
l
-alanine, thus demonstrating the proposed biochemical reaction catalyzed by AruH. The enzymatic properties and kinetic parameters of dimeric recombinant AruH were determined by a coupled reaction with NAD
+
and
l
-alanine dehydrogenase. The optimal activity of AruH was found at pH 9.0, and it has a novel substrate specificity with an order of preference of Arg > Lys > Met > Leu > Orn > Gln. With
l
-arginine and pyruvate as the substrates, Lineweaver-Burk plots of the data revealed a series of parallel lines characteristic of a ping-pong kinetic mechanism with calculated
V
max
and
k
cat
values of 54.6 ± 2.5 μmol/min/mg and 38.6 ± 1.8 s
−1
. The apparent

K
m

and catalytic efficiency (
k
cat
/

K
m

) were 1.6 ± 0.1 mM and 24.1 mM
−1
s
−1
for pyruvate and 13.9 ± 0.8 mM and 2.8 mM
−1
s
−1
for
l
-arginine. When
l
-lysine was used as the substrate, MS analysis suggested Δ
1
-piperideine-2-carboxylate as its transamination product. These results implied that AruH may have a broader physiological function in amino acid catabolism.
Eukaryotic-Type Serine/Threonine Protein Kinase StkP Is a Global Regulator of Gene Expression inStreptococcus pneumoniae
Authors: Sasková Lenka , Nováková Linda , Basler Marek , Branny Pavel
Published: April 2007

Signal transduction pathways in both prokaryotes and eukaryotes utilize protein phosphorylation as a key regulatory mechanism. Recent studies have proven that eukaryotic-type serine/threonine protein kinases (Hank's type) are widespread in many bacteria, although little is known regarding the cellular processes they control. In this study, we have attempted to establish the role of a single eukaryotic-type protein kinase, StkP ofStreptococcus pneumoniae, in bacterial survival. Our results indicate that the expression of StkP is important for the resistance ofS. pneumoniaeto various stress conditions. To investigate the impact of StkP on this phenotype, we compared the whole-genome expression profiles of the wild-type and ΔstkPmutant strains by microarray technology. This analysis revealed that StkP positively controls the transcription of a set of genes encoding functions involved in cell wall metabolism, pyrimidine biosynthesis, DNA repair, iron uptake, and oxidative stress response. Despite the reduced transformability of thestkPmutant, we found that the competence regulon was derepressed in thestkPmutant under conditions that normally repress natural competence development. Furthermore, the competence regulon was expressed independently of exogenous competence-stimulating peptide. In summary, our studies show that a eukaryotic-type serine/threonine protein kinase functions as a global regulator of gene expression inS. pneumoniae.
Interspecies DNA Microarray Analysis Identifies WblA as a Pleiotropic Down-Regulator of Antibiotic Biosynthesis in
Streptomyces
Authors: Kang Seung-Hoon , Huang Jianqiang , Lee Han-Na , Hur Yoon-Ah , Cohen Stanley N. , Kim Eung-Soo
Published: April 2007

Using
Streptomyces coelicolor
microarrays to discover regulators of gene expression in other
Streptomyces
species, we identified
wblA
, a
whiB
-like gene encoding a putative transcription factor, as a down-regulator of doxorubicin biosynthesis in
Streptomyces peucetius
. Further analysis revealed that
wblA
functions pleiotropically to control antibiotic production and morphological differentiation in streptomycetes. Our results reveal a novel biological role for
wblA
and show the utility of interspecies microarray analysis for the investigation of streptomycete gene expression.
The Zinc-Responsive Regulator Zur Controls a Zinc Uptake System and Some Ribosomal Proteins inStreptomyces coelicolorA3(2)
Authors: Shin Jung-Ho , Oh So-Young , Kim Soon-Jong , Roe Jung-Hye
Published: April 2007

In various bacteria, Zur, a zinc-specific regulator of the Fur family, regulates genes for zinc transport systems to maintain zinc homeostasis. It has also been suggested that Zur controls zinc mobilization by regulating some ribosomal proteins. The antibiotic-producing soil bacteriumStreptomyces coelicolorcontains four genes for Fur family regulators, and one (namedzur) is located downstream of theznuACBoperon encoding a putative zinc uptake transporter. We found that zinc specifically repressed the level ofznuAtranscripts and that this level was derepressed in a Δzurmutant. Purified Zur existing as homodimers bound to theznuApromoter region in the presence of zinc, confirming the role of Zur as a zinc-responsive repressor. We analyzed transcripts for paralogous forms of ribosomal proteins L31 (RpmE1 and RpmE2) and L33 (RpmG2 and RpmG3) for their dependence on Zur and found that RpmE2 and RpmG2 with no zinc-binding motif of conserved cysteines (C's) were negatively regulated by Zur. C-negative RpmG3 and C-positive RpmE1 were not regulated by Zur. Instead, they were regulated by the sigma factor σRas predicted from their promoter sequences. TherpmE1andrpmG3genes were partially induced by EDTA in a manner dependent on σR, suggesting that zinc depletion may stimulate the σRregulatory system. This finding reflects a link between thiol-oxidizing stress and zinc depletion. We determined the Zur-binding sites withinznuAandrpmG2promoter regions by footprinting analyses and identified a consensus inverted repeat sequence (TGaaAatgatTttCA, where uppercase letters represent the nucleotides common to all sites analyzed). This sequence closely matches that for mycobacterial Zur and allows the prediction of more genes in the Zur regulon.
Characterization of a Novel Bifunctional Dihydropteroate Synthase/Dihydropteroate Reductase Enzyme from
Helicobacter pylori
Authors: Levin Itay , Mevarech Moshe , Palfey Bruce A.
Published: April 2007

Tetrahydrofolate is a ubiquitous C
1
carrier in many biosynthetic pathways in bacteria, importantly, in the biosynthesis of formylmethionyl tRNA
fMet
, which is essential for the initiation of translation. The final step in the biosynthesis of tetrahydrofolate is carried out by the enzyme dihydrofolate reductase (DHFR). A search of the complete genome sequence of
Helicobacter pylori
failed to reveal any sequence that encodes DHFR. Previous studies demonstrated that the
H. pylori
dihydropteroate synthase gene
folP
can complement an
Escherichia coli
strain in which
folA
and
folM
, encoding two distinct DHFRs, are deleted. It was also shown that
H. pylori
FolP possesses an additional N-terminal domain that binds flavin mononucleotide (FMN). Homologous domains are found in FolP proteins of other microorganisms that do not possess DHFR. In this study, we demonstrated that
H. pylori
FolP is also a dihydropteroate reductase that derives its reducing power from soluble flavins, reduced FMN and reduced flavin adenine dinucleotide. We also determined the stoichiometry of the enzyme-bound flavin and showed that half of the bound flavin is exchangeable with the soluble flavins. Finally, site-directed mutagenesis of the most conserved amino acid residues in the N-terminal domain indicated the importance of these residues for the activity of the enzyme as a dihydropteroate reductase.
Rho kinase is an effector underlying Ca2+-desensitizing hypoxic relaxation in porcine coronary artery
Authors: Wardle Robert L. , Gu Min , Ishida Yukisato , Paul Richard J.
Published: April 2007

Acute hypoxia dilates most systemic arteries leading to increased tissue perfusion. We have previously shown that at high-stimulus conditions, porcine coronary artery was relaxed by hypoxia without a change in intracellular [Ca2+] ( 27 ). This Ca2+-desensitizing hypoxic relaxation (CDHR) was validated in permeabilized porcine coronary artery smooth muscle (PCASM) in which hypoxia decreased force and myosin regulatory light chain phosphorylation (p-MRLC) despite fixed [Ca2+] ( 10 ). Rho kinase-dependent phosphorylation of myosin phosphatase-targeting subunit 1 (p-MYPT1) is associated with decreased MRLC phosphatase activity and increased Ca2+sensitivity of both p-MRLC and force. We recently reported that p-MYPT1 dephosphorylation was a key effector in CDHR ( 33 ). In the current study, we tested the hypothesis that Rho kinase and not p-MYPT1 phosphatase is the regulated enzyme involved in CDHR. We used α-toxin to permeabilize deendothelialized PCASM. CDHR was attenuated in contractions attributable to myosin light chain kinase (MLCK, in the presence of the Rho kinase inhibitor Y-27632). In contrast, hypoxia relaxed contractions attributable to Rho kinase phosphorylation of MYPT1 and MRLC or MRLC alone (in the presence of the MLCK inhibitor ML7). Using an in situ assay, we showed that Rho kinase activity, measured as thiophosphorylation of MYPT1 and MRLC, was nearly abolished by hypoxia. The in vitro activity of the catalytically active fragment of Rho kinase was not affected by hypoxia. Our evidence strongly implicates that hypoxia directly inhibits Rho kinase-dependent phosphorylation of MYPT1. This underlies the decreases in both p-MYPT1 and p-MRLC and thereby leads to the Ca2+-desensitizing hypoxic relaxation.
Rescue of tropomyosin-induced familial hypertrophic cardiomyopathy mice by transgenesis
Authors: Jagatheesan Ganapathy , Rajan Sudarsan , Petrashevskaya Natalia , Schwartz Arnold , Boivin Greg , Arteaga Grace M. , John Solaro R. , Liggett Stephen B. , Wieczorek David F.
Published: April 2007

Familial hypertrophic cardiomyopathy (FHC) is a disease caused by mutations in contractile proteins of the sarcomere. Our laboratory developed a mouse model of FHC with a mutation in the thin filament protein α-tropomyosin (TM) at amino acid 180 (Glu180Gly). The hearts of these mice exhibit dramatic systolic and diastolic dysfunction, and their myofilaments demonstrate increased calcium sensitivity. The mice also develop severe cardiac hypertrophy, with death ensuing by 6 mo. In an attempt to normalize calcium sensitivity in the cardiomyofilaments of the hypertrophic mice, we generated a chimeric α-/β-TM protein that decreases calcium sensitivity in transgenic mouse cardiac myofilaments. By mating mice from these two models together, we tested the hypothesis that an attenuation of myofilament calcium sensitivity would modulate the severe physiological and pathological consequences of the FHC mutation. These double-transgenic mice “rescue” the hypertrophic phenotype by exhibiting a normal morphology with no pathological abnormalities. Physiological analyses of these rescued mice show improved cardiac function and normal myofilament calcium sensitivity. These results demonstrate that alterations in calcium response by modification of contractile proteins can prevent the pathological and physiological effects of this disease.
Pulmonary arterial hypertension: a disease of tethers, SNAREs and SNAPs?
Authors: Sehgal Pravin B. , Mukhopadhyay Somshuvra
Published: April 2007

Histological and electron microscopic studies over the past four decades have highlighted “plump,” “enlarged” endothelial, smooth muscle, and fibroblastic cellular elements with increased endoplasmic reticulum, Golgi stacks, and vacuolation in pulmonary arterial lesions in human and in experimental (hypoxia and monocrotaline) pulmonary arterial hypertension. However, the contribution of disrupted intracellular membrane trafficking in the pathobiology of this disease has received insufficient attention. Recent studies suggest a pathogenetic role of the disruption of intracellular trafficking of vasorelevant proteins and cell-surface receptors in the development of this disease. The purpose of this essay is to highlight the molecular regulation of vesicular trafficking by membrane tethers, SNAREs and SNAPs, and to suggest how their dysfunction, directly and/or indirectly, might contribute to development of pulmonary arterial hypertension in experimental models and in humans, including that due to mutations in bone morphogenetic receptor type 2.
Similar but not the same: normobaric and hyperbaric pulmonary oxygen toxicity, the role of nitric oxide
Authors: Demchenko Ivan T. , Welty-Wolf Karen E. , Allen Barry W. , Piantadosi Claude A.
Published: April 2007

Pulmonary manifestations of oxygen toxicity were studied and quantified in rats breathing >98% O2at 1, 1.5, 2, 2.5, and 3 ATA to test our hypothesis that different patterns of pulmonary injury would emerge, reflecting a role for central nervous system (CNS) excitation by hyperbaric oxygen. At 1.5 atmosphere absolute (ATA) and below, the well-recognized pattern of diffuse pulmonary damage developed slowly with an extensive inflammatory response and destruction of the alveolar-capillary barrier leading to edema, impaired gas exchange, respiratory failure, and death; the severity of these effects increased with time over the 56-h period of observation. At higher inspired O2pressures, 2–3 ATA, pulmonary injury was greatly accelerated but less inflammatory in character, and events in the brain were a prelude to a distinct lung pathology. The CNS-mediated component of this lung injury could be attenuated by selective inhibition of neuronal nitric oxide synthase (nNOS) or by unilateral transection of the vagus nerve. We propose that extrapulmonary, neurogenic events predominate in the pathogenesis of acute pulmonary oxygen toxicity in hyperbaric oxygenation, as nNOS activity drives lung injury by modulating the output of central autonomic pathways.
Power, Knowledge and Authority in Public Theology
Authors: Graham Elaine
Published: April 2007

One of the most distinctive movements within Christian theology to have emerged over the past generation has been the various theologies of liberation which originated in Latin America but which now span a diversity of styles, including feminist and womanist, Black, Asian and lesbian/gay/bisexual and transgendered (LGBT) theologies. All theologies of liberation purport to give a voice to the experiences of those formerly silenced or marginalized by society and/or the Church. This is essentially an issue of power, since one of the authenticating marks of such theologies is the extent to which they enable such groups to move from powerlessness to empowerment. Yet theologies of liberation also represent, potentially, another redistribution of power, by enabling previously excluded groups to bring their interpretations and testimonies into theological discourse. This article examines the background to this intersection of power and knowledge in theology, and asks how public theology might assist such a process of theological empowerment.
God and Our Public Life: A Scriptural Wisdom
Authors: Ford David
Published: April 2007

This article recognises both the need for wisdom for the flourishing of public life and the value of the contribution that Christian wisdom, founded on Scripture, has to offer. However, this article also notes that the contemporary world is a complexly religious and secular environment, and hence if Christian wisdom is to realise its potential, there is a need for the creation and nurture of attitudes, groups and institutions within which fruitful dialogue between faiths and ideologies in public life can occur. The article observes that Britain currently has a particular opportunity to work towards this kind of wisdom-embracing religious and secular society, and the practice of scriptural reasoning is explored as an exemplary practice that promotes the kind of inter-faith collegiality, collaboration and friendships that enhance public life. Finally, the article offers some brief reflections on Job and the role of wisdom in an authentic and biblical Christian faith.
Russia, The Turks and Europe: Legitimations of War and the Formation of European Identity in the Early Modern Period
Authors: Piirimäe Pärtel
Published: April 2007

An essential criterion of belonging to a community is the expressed willingness to play by its rules. "Europe" in the Early Modern period can be seen as a moral community of "Christian" and "civilized" states which abided by the principles of ius gentium. The core of this code was the limitation and regulation of warfare. Although moral and legal principles of bellum iustum were often overruled by considerations of interest, there was at least one thing common to all European wars: the states always took pains to prove publicly that they were waging a just war. This essay examines the significance of printed legitimations of war for the formation of European identity. It focuses on the case of Muscovy, which before the end of the seventeenth century had not been concerned with its image in Europe, and was thus left at the mercy of the propaganda of its western neighbors who were instrumental in constructing the image of Muscovites as Asiatic barbarians, more similar to the Turks than to Christians. Tsar Peter the Great, however, took a novel decision to launch a campaign of public legitimation of Muscovy's attack on Sweden in 1700. The legitimations of war published during Peter's reign can be seen as essential components of his quest for the acknowledgement of Russia as a fully-fledged member of the European moral, legal, and political community.
Max Weber und die ,,deutsche Gemütlichkeit": Amerika, Du hast es besser … (Goethe, 1827)
Authors: Arndt Martin
Published: April 2007

Max Weber is presented as a fervent supporter of the US-American life-style, its love of freedom and capitalist competition. After his return from the United States he makes all efforts at Americanizing Germany and bridging the transatlantic divide that Germany has brought about by its cultivation of "Gemütlichkeit" and "Bildung".
Plasma properties of helium gas tungsten arc with metal vapour
Authors: Tashiro S. , Tanaka M. , Nakata K. , Iwao T. , Koshiishi F. , Suzuki K. , Yamazaki K.
Published: April 2007

The energy source characteristics of gas tungsten arc (GTA) strongly depend on the physical property of arc plasma. In welding processes, it has been experimentally confirmed that metal vapour evaporated from a high temperature weld pool drastically changes the property of arc plasma and decreases its temperature. However, the effect of metal vapour on the characteristics of heat flux into a base metal is still not clear owing to the difficulty in experimental studies of arc plasma. In the present paper, the energy source property of helium GTA mixed with metal vapour was numerically analysed. It was found that the intense radiation generated from dense metal vapour decreases heat flux into a base metal and contracts the current density distribution especially near the arc axis.
Numerical studies on effect of axial pressure in friction stir welding
Authors: Zhang Z. , Zhang H. W.
Published: April 2007

A fully coupled thermomechanical model of friction stir welding (FSW) is developed in the current research to investigate the effect of the axial pressure on the material behaviours and temperature distributions in the FSW process. Results indicate that the axial pressure is a key factor for the successful FSW. Insufficient or excessive axial pressure leads to the failure of FSW. The material flows at the advancing side and the retreating side are different, which can be fitted well with the experimental observations. The material flows in the direction of the thickness become more obvious when the axial pressure is increased. The maximum temperature and plastic contribution to the temperature field in FSW can be both increased with the increase in the axial pressure.
An intra-articular method for assessing topical application to a synovial joint
Authors: Holland T. , Jones R. , Basford A. , Child T. , Smith A.
Published: April 2007

Intra-articular injection of drugs is increasingly used in human medicines. We report a method for the direct administration of a test substance to the synovial fluid of the canine stifle joint. This method caused little distress or pathology, making it suitable for pre-clinical assessment of new drugs in dogs and other species.
Telemetric monitoring of blood pressure in freely moving mice: a preliminary study
Authors: Kramer Klaas , Voss Hans-Peter , Grimbergen Joop A. , Mills Perry A. , Huetteman Daniel , Zwiers Lynn , Brockway Brian
Published: April 2007

This paper describes for the first time the possibility for recording the systolic pressure (SPI, diastolic pressure (DP), and the mean arterial pressure (MAP) as well as the heart rate (HR) and locomotor activity (LA) in freely moving mice, using a commercially available telemetry and data acquisition system. The system comprises a new, small radio-telemetry transmitter implanted in the peritoneal cavity, a receiver board placed underneath the home cage, a multiplexer and a computer-based data acquisition system. The signals from the receiver were consolidated by the multiplexer and were stored and analysed by the computer. The telemetered pressure signals (absolute pressure) were corrected automatically for changes in atmospheric pressure measured by an ambient pressure monitor. The effects of implantation on animal behaviour, and, after the animals had recovered, the effects of handling on the SP, DP, MAP and HR were examined. The radio-telemetry system for recording the SP, DP, MAP and HR provides an accurate and reliable method for monitoring the direct effects of handling on SP, DP, MAP and HR. In addition, by using this new blood pressure (BP) transmitter, we maintain that BP measurements in freely moving mice are more efficient, reliable, and less labour-intensive than the measurement techniques described in the literature thus far.
An alternative method to stereotactic inoculation of transplantable brain tumours in large numbers of rats
Authors: La Regina M. C. , Culbreth V. O. , Higashikubo R. , Roti J. L. Roti , Spitz D. R.
Published: April 2007

The rat 9L gliosarcoma brain tumour model has been widely used in brain cancer studies. Intracerebral implantation of the cells in the parietal lobe of the brain has been performed using the stereotactic or freehand inoculation methods. For large numbers of rats, we wished to develop a method more accurate and precise than the freehand method, but less labour intensive than the stereotactic method. A template implantation technique was developed and compared quantitatively with the stereotactic method. Rats were inoculated with either the template or stereotactic method at doses of 1000, 5000, 10 000, 20 000 or 40 000 cells. Results of this comparison showed that the template method is precise and accurate for tumour placement within the brain cortex, and decreases labour requirements. Mean survival rates between groups were not significantly different at doses of 5000, 20 000 or 40 000 cells inoculated. Significance was seen at the low dose of 1000 cells ( P < 0.001). This was attributable to an absence of tumour growth in five of six stereotactic rats in this group. Significance was also seen at the 10 000 dose level ( P < 0.05) with the stereotactic rats again surviving longer than the template rats. However, in this case all the stereotactic rats had tumour growth. Brain weights did not differ significantly between groups, except at the 1000 dose level where no growth of tumour occurred in five of the six stereotactic animals. Body weight gain within one week following surgery did not differ significantly between any of the groups at α = 0.05. Studies on rat cadavers showed no statistical difference in placement measurements between the stereotactic and template methods. These results indicate that the template method for intra cerebrally implanting tumour cells in rats provides a precise, accurate and rapid procedure that maximizes reproducibility with a significant reduction in labour requirements, when compared with the conventional stereotactic methodology.
Comparison of assays for antibodies to Encephalitozoon cuniculi in rabbits
Authors: Boot R. , Hansen A. K. , Hansen C. K. , Nozari N. , Thuis H. C. W.
Published: April 2007

Two indirect immunofluorescence (IIF) assays, two enzyme-linked immunosorbent assays (ELISAs)and the carbon immunoassay (CIA) for determination of antibodies to Encephalitozoon cuniculi were compared using 210 sera of rabbits, 135 of which originated from seven infected colonies, while 75 originated from four uninfected colonies. There was no evidence of a difference between the different assays with respect to the number of positive sera. There was a clear correlation between the quantitative response measured by IIF and CIA and the other assays, and between both IIF tests, while no such correlation was found in the quantitative response measured by ELISAs, which might be explained by the less quantitative nature of the ELISA. Therefore quantitative determination of antibodies to E. cuniculi should be performed by IIF and not by ELISA. The nosographic sensitivities N1 and specificities N2 of the assays were ≥ 0.94 and ≥ 0.97 respectively. Small differences in N1 and N2 between the assays, although not statistically significant, were responsible for differences in the calculated predictive values of a positive test and of a negative test. As expected, the magnitude of these differences depended on the fraction of positive sera sampled from a given colony. There was strong evidence of such a difference between the fraction of positive sera found in different colonies, but the sample size from some colonies was too small to allow any conclusion, whether this was due to differences in the prevalences of the infection in the colonies or something else. We conclude that any of the assays will be suitable for the routine health monitoring of laboratory rabbit colonies for E. cuniculi infection, as recommended by the Federation of European Laboratory Animal Science Associations.
Feeding sugar overnight maintains metabolic homeostasis in rats and is preferable to overnight starvation
Authors: Levine Seymour , Saltzman Arthur
Published: April 2007

Rats are often starved overnight for many different reasons. Overnight starvation causes loss of body and liver weights, depletion of liver glycogen, decrease of blood glucose and loss of amino acids because of gluconeogenesis. Providing pure sucrose cubes as the sole overnight nutrient is a simple, inexpensive way to empty the gastrointestinal (GI) tract, while minimizing liver changes and preventing decrease of blood glucose and loss of amino acids. Adding sugars to the overnight drinking water as the sole nutrient has the same beneficial effects, provided the type of sugar and its concentration allow for sufficient intake and provided hyponatremia is avoided. Feeding sucrose cubes or sugar solutions will empty the gastrointestinal tract as effectively as starvation. In all instances, simple precautions against coprophagy and pica should be taken in order to secure optimal benefit.
Effects of various levels of dietary Artemisia abyssinica leaves on rats
Authors: Adam S. E. I. , Al-Qarawi A. A. , Elhag E. A.
Published: April 2007

Artemisia abyssinica leaves, a traditional medicine for the treatment of various disorders, were fed to male Wistar rats at 2% and 10% of the standard diet for 6 weeks. A 2% A. abyssinica leaf diet was not toxic to rats. Depression in growth, hepatopathy and nephropathy were observed in rats fed a diet containing 10% of A. abyssinica leaves. These findings were accompanied by leukopenia, anaemia and alterations of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma glutamyl transferase (GGT) activities with changes in concentrations of total protein, albumin, cholesterol and urea.
Sufentanil and medetomidine anaesthesia in the rat and its reversal with atipamezole and butorphanol
Authors: Hedenqvist P. , Roughan J. V. , Flecknell P. A.
Published: April 2007

Injectable anaesthetics are widely used to anaesthetize rats, but recovery times are often prolonged. Reversible anaesthetic regimens have the advantage that animals may be recovered quickly, thus reducing the incidence of postoperative complications such as hypothermia, and also providing a means of treating inadvertent anaesthetic overdose. This study assessed and compared the characteristics of anaesthesia induced with combinations of sufentanil and medetomidine administered as a single subcutaneous or intraperitoneal dose, and reversal with butorphanol and atipamezole. Combinations of sufentanil/medetomidine at 40 μg/150 μg and 50 μg/150 μg/kg administered subcutaneously, and 80 μg/300 μg/kg by intraperitoneal injection were found to produce surgical anaesthesia for 101 ± 49, 124 ± 45 and 76 ± 23 min (means ± SD) respectively. All three combinations produced marked respiratory depression 30min after injection (< 50% of resting respiratory rate). Oxygen saturation, measured by pulse oximetry, was < 50% in all groups 30 min following drug administration. Subcutaneous administration is recommended since it resulted in a more reliable and more rapid induction of anaesthesia than intraperitoneal administration. The administration of butorphanol and atipamezole (0.2/0.5 mg/kg s.c.) resulted in a rapid (< 7 min) reversal of anaesthesia and an associated respiratory depression. The induction of anaesthesia with sufentanil/medetomidine and its reversal with a combination of atipamezole and butorphanol is an effective technique for anaesthetizing rats. However, due to the marked respiratory depression and the resulting hypoxia, we recommend that this regimen should only be used in animals which are free from respiratory disease and that oxygen should be provided during anaesthesia.
The successful use of fentanyl/fluanisone ('Hypnorm') as an anaesthetic for intracranial surgery in neonatal rats
Authors: Clowry Gavin J. , Flecknell Paul A.
Published: April 2007

This study reports on the successful use of fentanyl citrate and fluanisone ('Hypnorm') anaesthesia for intracranial surgery in neonatal (7-day-old) rats. Provided the anaesthetic was administered subcutaneously, the animals showed a very high survival rate in the short term (81/85, 95%) and showed no ill effects in the long term. The depth of anaesthesia was sufficient to allow the operation to be carried out without the animal reacting to any painful stimuli. However, the animals did make random movements during the period of surgical anaesthesia which were not related to any painful stimuli. Although these movements did not interfere with the surgery performed here, such movements would interfere with operations requiring greater precision, such as the localized micro-injection of neural tracers.
Induction of drug resistance and transformation in human cancer cells by the noncoding RNA CUDR
Authors: Tsang Wing Pui , Wong Timothy W.L. , Cheung Albert H.H. , Co Chloe N.N. , Kwok Tim Tak
Published: April 2007

Refractory to apoptosis induced by anticancer drugs is one of the major causes of drug resistance in human cancers. The involvement of noncoding RNA (ncRNA) in cancer cell drug resistance has not yet been reported. By using the technique of RT-PCR-based differential display, a novel gene, cancer up-regulated drug resistant (CUDR) gene, was found to be overexpressed in a doxorubicin-resistant subline of human squamous carcinoma A431 and A10A cells, which were also more resistant to drug-induced apoptosis. The full-length CUDR mRNA transcript is ∼2.2 kb as detected by Northern blot analysis and has no sequence homology with other genes identified so far. Interestingly, no distinct open reading frame was found throughout the CUDR cDNA sequence, and no recombinant protein was detected from in vitro translation or from a protein lysate of human cancer cells after CUDR transfection. Therefore, CUDR is likely to exert its function as a noncoding RNA. Stable transfection with the CUDR gene was found to induce resistance to doxorubicin and etoposide as well as drug-induced apoptosis in A431 cells. By Western blot analysis, down-regulations of caspase 3 were observed in CUDR transfectants. On the other hand, overexpression of CUDR promoted anchorage-independent growth in A431 cells. Results from the present study suggest that CUDR may likely regulate the drug sensitivity and promote cellular transformation at least through caspase 3-dependent apoptosis.
Role of various elliptical shapes for efficient microwave processing of materials
Authors: Basak Tanmay
Published: April 2007

A detailed theoretical analysis has been carried out to assess the role of various elliptical shapes/cross sections on microwave heating of 2‐D cylinders for beef and oil samples. Two types of elliptical cross sections are considered as type A (ellipse with major axis along the horizontal plane), and type B (ellipse with minor axis along the horizontal plane.) A preliminary analysis on microwave heating of samples has been shown via average power within a sample vs. sample radius of circular cross section for beef and oil samples. Several regimes (I–III) based on small and large radius for circular cross sections have been selected. The effect of elliptical shapes for type A and B configurations has been studied initially via average power vs. aspect ratio distributions for various regimes. The grayscale images of power absorption have been analyzed further for elliptical cross section with varying aspect ratios for regimes I–III where aspect ratio and types of configuration are shown to influence spatial power absorptions. The detailed temperature profiles have also been shown to illustrate the role of elliptical shapes on uniform heating and thermal runaway. Depending on the material dielectric properties and sample dimension, either type A or B or both configuration has been recommended. © 2007 American Institute of Chemical Engineers AIChE J, 2007
EEC syndrome, Arg227Gln TP63 mutation and micturition difficulties: Is there a genotype–phenotype correlation?
Authors: Maclean Kenneth , Holme Stephen A. , Gilmour Elizabeth , Taylor Mark , Scheffer Heide , Graf Nicole , Smith Grahame H.H. , Onikul Ella , van Bokhoven Hans , Moss Celia , Adès Lesley C.
Published: April 2007

We report on two unrelated families with EEC syndrome (ectrodactyly, ectodermal dysplasia, cleft lip/palate), each with an Arg227Gln TP63 gene mutation, where the phenotype overlapped extensively with the allelic disorder, limb‐mammary syndrome (LMS). Features common to both families were an ectodermal dysplasia principally affecting tooth, breast and nipple development, dacryostenosis and severe micturition difficulties. Additional findings included post‐axial digital hypoplasia, cleft uvula, anal stenosis, hypoplasia of the perineal body and biopsy‐proven interstitial cystitis. No individual had cleft lip. Split hand‐split foot malformation (SHFM) occurred in one child—born after the molecular diagnosis was established. Unlike previous reports, the urinary symptoms were refractory to treatment with oral Fibrase and persisted into adulthood. Of the six cases/families now reported with EEC syndrome and Arg227Gln TP63 mutation, four have manifested this distinct urological abnormality, indicative of a genotype‐phenotype correlation. © 2007 Wiley‐Liss, Inc.
Small supernumerary marker chromosome causing partial trisomy 6p in a child with craniosynostosis
Authors: Villa Olaya , del Campo Miguel , Salido Marta , Gener Blanca , Astier Laura , del Valle Jesús , Gallastegui Fátima , Pérez‐Jurado Luis A. , Solé Francesc
Published: April 2007

We report on a child with a small supernumerary marker chromosome (sSMC) causing partial trisomy 6p. The child showed a phenotype consisting of neonatal craniosynostosis, microcephaly, and borderline developmental delay. By molecular techniques the sSMC has been shown to contain ∼16 Mb of genomic DNA from 6p21.1 to 6cen, being de novo and of maternal origin. © 2007 Wiley‐Liss, Inc.
Tracheal occlusion: A review of obstructing fetal lungs to make them grow and mature
Authors: Khan Paul A. , Cloutier Marc , Piedboeuf Bruno
Published: April 2007

Fetal lung growth and functional differentiation are affected strongly by the extent that pulmonary tissue is distended (expanded) by liquid that naturally fills developing future airspaces. Methods that prevent normal egress of this lung fluid through the trachea magnify mechanical stretching of lung parenchymal cells, thereby promoting lung development. Indeed, experimental observations demonstrate that in utero tracheal occlusion (TO) performed on fetuses during the late canalicular‐early saccular stage potently stimulates pulmonary growth and maturation. In this review, we present the four principle non‐human animal models of TO/obstruction and discuss them in relation to their utility in elucidating lung development, in remedying congenital diaphragmatic hernia (CDH) as well as in investigating the stretching effects on growth and remodeling of the fine vasculature. © 2007 Wiley‐Liss, Inc.
Development of the diaphragm and genetic mouse models of diaphragmatic defects
Authors: Ackerman Kate G. , Greer John J.
Published: April 2007

Improving our understanding of diaphragmatic development is essential to making progress in defining the pathogenesis and genetic etiologies of congenital diaphragmatic defects in humans. As mouse genetic technology has given us new tools to manipulate and observe development, a number of mouse models have recently emerged that provide valuable insight to this field. In this article, we review our current understanding of diaphragmatic embryogenesis including the origin of diaphragmatic tissue. We use rodent models to review the muscularization of the diaphragm and review selected genetic models of abnormal muscularization. We also review models of posterior diaphragmatic defects and discuss evidence for the pleuroperitoneal fold (PPF) tissue contributing to the diaphragm. Finally, we discuss models of anterior and central hernias. It may be simplistic to subdivide this review based on anatomic regions of the diaphragm, as evidence is emerging that defects in different regions of the diaphragm in humans and in mice may be etiologically related. However, at this time we do not have enough knowledge to make more mechanistic or genetic classifications though with time, genetic progress in the field of diaphragm development will allow us to do this. © 2007 Wiley‐Liss, Inc.
Congenital diaphragmatic hernia (CDH) etiology as revealed by pathway genetics
Authors: Kantarci Sibel , Donahoe Patricia K.
Published: April 2007

Congenital diaphragmatic hernia (CDH) is a common birth defect with high mortality and morbidity. Two hundred seventy CDH patients were ascertained, carefully phenotyped, and classified as isolated (diaphragm defects alone) or complex (with additional anomalies) cases. We established different strategies to reveal CDH‐critical chromosome loci and genes in humans. Candidate genes for sequencing analyses were selected from CDH animal models, genetic intervals of recurrent chromosomal aberration in humans, such as 15q26.1–q26.2 or 1q41–q42.12, as well as genes in the retinoic acid and related pathways and those known to be involved in embryonic lung development. For instance, FOG2, GATA4, and COUP‐TFII are all needed for both normal diaphragm and lung development and are likely all in the same genetic and molecular pathway. Linkage analysis was applied first in a large inbred family and then in four multiplex families with Donnai–Barrow syndrome (DBS) associated with CDH. 10K SNP chip and microsatellite markers revealed a DBS locus on chromosome 2q23.3–q31.1. We applied array‐based comparative genomic hybridization (aCGH) techniques to over 30, mostly complex, CDH patients and found a de novo microdeletion in a patient with Fryns syndrome related to CDH. Fluorescence in situ hybridization (FISH) and multiplex ligation‐dependent probe amplification (MLPA) techniques allowed us to further define the deletion interval. Our aim is to identify genetic intervals and, in those, to prioritize genes that might reveal molecular pathways, mutations in any step of which, might contribute to the same phenotype. More important, the elucidation of pathways may ultimately provide clues to treatment strategies. © 2007 Wiley‐Liss, Inc.
Macaque monkey retrosplenial cortex: III. Cortical efferents
Authors: Kobayashi Yasushi , Amaral David G.
Published: April 2007

We have investigated the cortical efferent projections of the macaque monkey retrosplenial and posterior cingulate cortices by using 3H‐amino acids as anterograde tracers. All the injections produced extensive local connections to other portions of this region. There were also a number of extrinsic efferent cortical connections, many of which have not hitherto been reported. Major projections from the retrosplenial cortex were directed to the frontal lobe, with heaviest terminations in areas 46, 9, 10, and 11. There were also very substantial projections to the entorhinal cortex, presubiculum, and parasubiculum of the hippocampal formation, as well as to areas TH and TF of the parahippocampal cortex. Some injections led to labeling of area V4, the dorsal bank of the superior temporal sulcus, and area 7a of the parietal cortex. Projections from the posterior cingulate cortex innervated all these same regions, although the density of termination was different from the retrosplenial projections. The posterior cingulate cortex gave rise to additional projections to parietal area DP and to the cortex along the convexity of the superior temporal gyrus. The ventral portion of the posterior cingulate cortex (area 23v) gave rise to much denser efferent projections to the hippocampal formation than the dorsal portions (areas 23e and i). These connections are discussed in relation to the clinical syndromes of retrosplenial amnesia and topographic disorientation in humans commonly caused by lesions in the caudoventral portions of the retrosplenial and posterior cingulate cortices. J. Comp. Neurol. 502:810–833, 2007. © 2007 Wiley‐Liss, Inc.