bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterologous Expression of theTreponema pallidumLaminin-Binding Adhesin Tp0751 in the Culturable SpirocheteTreponema phagedenis
Authors: Cameron Caroline E. , Kuroiwa Janelle M. Y. , Yamada Mitsunori , Francescutti Teresa , Chi Bo , Kuramitsu Howard K.
Published: February 2008

Treponema pallidumsubsp.pallidum, the causative agent of syphilis, is an unculturable, genetically intractable bacterium. Here we report the use of the shuttle vector pKMR4PEMCS for the expression of a previously identifiedT.pallidumlaminin-binding adhesin, Tp0751, in the nonadherent, culturable spirocheteTreponema phagedenis. Heterologous expression of Tp0751 inT.phagedeniswas confirmed via reverse transcriptase PCR analysis with tp0751 gene-specific primers and immunofluorescence analysis with Tp0751-specific antibodies; the latter assay verified the expression of the laminin-binding adhesin on the treponemal surface. Expression of Tp0751 withinT.phagedeniswas functionally confirmed via laminin attachment assays, in which heterologous Tp0751 expression conferred uponT.phagedenisthe capacity to attach to laminin. Further, specific inhibition of the attachment ofT.phagedenisheterologously expressing Tp0751 to laminin was achieved by using purified antibodies raised against recombinantT.pallidumTp0751. This is the first report of heterologous expression of a gene from an unculturable treponeme inT.phagedenis. This novel methodology will significantly advance the field of syphilis research by allowing targeted investigations ofT.pallidumproteins purported to play a role in pathogenesis, and specifically host cell attachment, in the nonadherent spirocheteT.phagedenis.
Naturally Occurring Nonpathogenic Isolates of the Plant Pathogen
Pseudomonas syringae
Lack a Type III Secretion System and Effector Gene Orthologues
Authors: Mohr Toni J. , Liu Haijie , Yan Shuangchun , Morris Cindy E. , Castillo José A. , Jelenska Joanna , Vinatzer Boris A.
Published: February 2008

Pseudomonas syringae
causes plant diseases, and the main virulence mechanism is a type III secretion system (T3SS) that translocates dozens of effector proteins into plant cells. Here we report the existence of a subgroup of
P. syringae
isolates that do not cause disease on any plant species tested. This group is monophyletic and most likely evolved from a pathogenic
P. syringae
ancestor through loss of the T3SS. In the nonpathogenic isolate
P. syringae
508 the genomic region that in pathogenic
P. syringae
strains contains the
hrp-hrc
cluster coding for the T3SS and flanking effector genes is absent.
P. syringae
508 was also surveyed for the presence of effector orthologues from the closely related pathogenic strain
P. syringae
pv. syringae B728a, but none were detected. The absence of the
hrp-hrc
cluster and effector orthologues was confirmed for other nonpathogenic isolates. Using the AvrRpt2 effector as reporter revealed the inability of
P. syringae
508 to translocate effectors into plant cells. Adding a plasmid-encoded T3SS and the
P. syringae
pv. syringae 61 effector gene
hopA1
increased in planta growth almost 10-fold. This suggests that
P. syringae
508 supplemented with a T3SS could be used to determine functions of individual effectors in the context of a plant infection, avoiding the confounding effect of other effectors with similar functions present in effector mutants of pathogenic isolates.
A Novel Chromate Reductase from
Thermus scotoductus
SA-01 Related to Old Yellow Enzyme
Authors: Opperman Diederik Johannes , Piater Lizelle Ann , van Heerden Esta
Published: February 2008

Bacteria can reduce toxic and carcinogenic Cr(VI) to insoluble and less toxic Cr(III).
Thermus scotoductus
SA-01, a South African gold mine isolate, has been shown to be able to reduce a variety of metals, including Cr(VI). Here we report the purification to homogeneity and characterization of a novel chromate reductase. The oxidoreductase is a homodimeric protein, with a monomer molecular mass of approximately 36 kDa, containing a noncovalently bound flavin mononucleotide cofactor. The chromate reductase is optimally active at a pH of 6.3 and at 65°C and requires Ca
2+
or Mg
2+
for activity. Enzyme activity was also dependent on NADH or NADPH, with a preference for NADPH, coupling the oxidation of approximately 2 and 1.5 mol NAD(P)H to the reduction of 1 mol Cr(VI) under aerobic and anaerobic conditions, respectively. The

K
m

values for Cr(VI) reduction were 3.5 and 8.4 μM for utilizing NADH and NADPH as electron donors, respectively, with corresponding
V
max
values of 6.2 and 16.0 μmol min
−1
mg
−1
. The catalytic efficiency (
k
cat
/

K
m

) of chromate reduction was 1.14 × 10
6
M
−1
s
−1
, which was >50-fold more efficient than that of the quinone reductases and >180-fold more efficient than that of the nitroreductases able to reduce Cr(VI). The chromate reductase was identified to be encoded by an open reading frame of 1,050 bp, encoding a single protein of 38 kDa under the regulation of an
Escherichia coli
σ
70
-like promoter. Sequence analysis shows the chromate reductase to be related to the old yellow enzyme family, in particular the xenobiotic reductases involved in the oxidative stress response.
Secretome Analysis Uncovers an Hcp-Family Protein Secreted via a Type VI Secretion System inAgrobacterium tumefaciens
Authors: Wu Hung-Yi , Chung Pei-Che , Shih Hsiao-Wei , Wen Sy-Ray , Lai Erh-Min
Published: February 2008

Agrobacterium tumefaciensis a plant-pathogenic bacterium capable of secreting several virulence factors into extracellular space or the host cell. In this study, we used shotgun proteomics analysis to investigate the secretome ofA. tumefaciens, which resulted in identification of 12 proteins, including 1 known secretory protein (VirB1*) and 11 potential secretory proteins. Interestingly, one unknown protein, which we designated hemolysin-coregulated protein (Hcp), is a predicted soluble protein without a recognizable N-terminal signal peptide. Western blot analysis revealed thatA. tumefaciensHcp is expressed and secreted when cells are grown in both minimal and rich media. Further biochemical and immunoelectron microscopy analysis demonstrated that intracellular Hcp is localized mainly in the cytosol, with a small portion in the membrane system. To investigate the mechanism of secretion of Hcp inA. tumefaciens, we generated mutants with deletions of a conserved gene,icmF, or the entire putative operon encoding a recently identified type VI secretion system (T6SS). Western blot analysis indicated that Hcp was expressed but not secreted into the culture medium in mutants with deletions oficmFor the t6ss operon. The secretion deficiency of Hcp in theicmFmutant was complemented by heterologoustransexpression oficmF, suggesting thaticmFis required for Hcp secretion. In tumor assays with potato tuber disks, deletion ofhcpresulted in approximately 20 to 30% reductions in tumorigenesis efficiency, while no consistent difference was observed whenicmFor the t6ss operon was deleted. These results increase our understanding of the conserved T6SS used by both plant- and animal-pathogenic bacteria.
Basis of Arginine Sensitivity of Microbial
N
-Acetyl-
l
-Glutamate Kinases: Mutagenesis and Protein Engineering Study with the
Pseudomonas aeruginosa
and
Escherichia coli
Enzymes
Authors: Fernández-Murga M. Leonor , Rubio Vicente
Published: February 2008

N
-Acetylglutamate kinase (NAGK) catalyzes the second step of arginine biosynthesis. In
Pseudomonas aeruginosa
, but not in
Escherichia coli
, this step is rate limiting and feedback and sigmoidally inhibited by arginine. Crystal structures revealed that arginine-insensitive
E. coli
NAGK (EcNAGK) is homodimeric, whereas arginine-inhibitable NAGKs, including
P. aeruginosa
NAGK (PaNAGK), are hexamers in which an extra N-terminal kinked helix (N-helix) interlinks three dimers. By introducing single amino acid replacements in PaNAGK, we prove the functionality of the structurally identified arginine site, as arginine site mutations selectively decreased the apparent affinity for arginine. N-helix mutations affecting R24 and E17 increased and decreased, respectively, the apparent affinity of PaNAGK for arginine, as predicted from enzyme structures that revealed the respective formation by these residues of bonds favoring inaccessible and accessible arginine site conformations. N-helix N-terminal deletions spanning ≥16 residues dissociated PaNAGK to active dimers, those of ≤20 residues decreased the apparent affinity for arginine, and complete N-helix deletion (26 residues) abolished arginine inhibition. Upon attachment of the PaNAGK N-terminal extension to the EcNAGK N terminus, EcNAGK remained dimeric and arginine insensitive. We concluded that the N-helix and its C-terminal portion after the kink are essential but not sufficient for hexamer formation and arginine inhibition, respectively; that the N-helix modulates NAGK affinity for arginine and mediates signal transmission between arginine sites, thus establishing sigmoidal arginine inhibition kinetics; that the mobile αH-β16 loop of the arginine site is the modulatory signal receiver; and that the hexameric architecture is not essential for arginine inhibition but is functionally essential for physiologically relevant arginine control of NAGK.
The Global Responses of
Mycobacterium tuberculosis
to Physiological Levels of Copper
Authors: Ward Sarah K. , Hoye Elizabeth A. , Talaat Adel M.
Published: February 2008

Copper (Cu) is a required micronutrient, but it is highly toxic at high concentrations. Therefore, the levels of Cu must be tightly regulated in all living cells. The phagosome of
Mycobacterium tuberculosis
has been shown to have variable levels of Cu. Previously, we showed that
M. tuberculosis
contains a copper-sensitive operon,
cso
, that is induced during early infection in mice. In this study, we showed that
ctpV
, a gene in the
cso
operon, is a copper-responsive gene and most likely encodes an efflux pump for Cu. Furthermore, the transcription of key genes in the
cso
operon is induced by Cu ions and not by other ions, such as Ni and Zn ions. To elucidate copper-responsive genes other than those in the
cso
operon, we utilized DNA microarrays to profile mycobacterial responses to physiological levels of Cu. A transcriptome analysis identified a novel set of 30 copper-responsive genes in
M. tuberculosis
, one-half of which were induced only when toxic levels of Cu were added. Interestingly, several transcriptional regulators, including the
furA
gene, were induced during toxic Cu exposure, indicating that there was a generalized response to oxidative stressors rather than a Cu-specific response. In general, the Cu-induced transcriptome generated should help elucidate the role of the Cu response in maintaining
M. tuberculosis
survival during infection and could provide novel targets for controlling this virulent pathogen.
TrpY Regulation of
trpB2
Transcription in
Methanothermobacter thermautotrophicus
Authors: Karr Elizabeth A. , Sandman Kathleen , Lurz Rudi , Reeve John N.
Published: February 2008

TrpY binds specifically to TRP box sequences upstream of
trpB2
, but the repression of
trpB2
transcription requires additional TrpY assembly that is stimulated by but not dependent on the presence of tryptophan. Inhibitory complex formation is prevented by insertions within the regulatory region and by a G149R substitution in TrpY, even though TrpY(G149R) retains both TRP box DNA- and tryptophan-binding abilities.
Acetamido Sugar Biosynthesis in the Euryarchaea
Authors: Namboori Seema C. , Graham David E.
Published: February 2008

Archaea and eukaryotes share a dolichol phosphate-dependent system for protein N-glycosylation. In both domains, the acetamido sugar
N
-acetylglucosamine (GlcNAc) forms part of the core oligosaccharide. However, the archaeal
Methanococcales
produce GlcNAc using the bacterial biosynthetic pathway. Key enzymes in this pathway belong to large families of proteins with diverse functions; therefore, the archaeal enzymes could not be identified solely using comparative sequence analysis. Genes encoding acetamido sugar-biosynthetic proteins were identified in
Methanococcus maripaludis
using phylogenetic and gene cluster analyses. Proteins expressed in
Escherichia coli
were purified and assayed for the predicted activities. The MMP1680 protein encodes a universally conserved glucosamine-6-phosphate synthase. The MMP1077 phosphomutase converted α-
d
-glucosamine-6-phosphate to α-
d
-glucosamine-1-phosphate, although this protein is more closely related to archaeal pentose and glucose phosphomutases than to bacterial glucosamine phosphomutases. The thermostable MJ1101 protein catalyzed both the acetylation of glucosamine-1-phosphate and the uridylyltransferase reaction with UTP to produce UDP-GlcNAc. The MMP0705 protein catalyzed the C-2 epimerization of UDP-GlcNAc, and the MMP0706 protein used NAD
+
to oxidize UDP-
N
-acetylmannosamine, forming UDP-
N
-acetylmannosaminuronate (ManNAcA). These two proteins are similar to enzymes used for proteobacterial lipopolysaccharide biosynthesis and gram-positive bacterial capsule production, suggesting a common evolutionary origin and a widespread distribution of ManNAcA. UDP-GlcNAc and UDP-ManNAcA biosynthesis evolved early in the euryarchaeal lineage, because most of their genomes contain orthologs of the five genes characterized here. These UDP-acetamido sugars are predicted to be precursors for flagellin and S-layer protein modifications and for the biosynthesis of methanogenic coenzyme B.
Dual Role of LldR in Regulation of the
lldPRD
Operon, Involved in
l
-Lactate Metabolism in
Escherichia coli
Authors: Aguilera Laura , Campos Evangelina , Giménez Rosa , Badía Josefa , Aguilar Juan , Baldoma Laura
Published: February 2008

The
lldPRD
operon of
Escherichia coli
, involved in
l
-lactate metabolism, is induced by growth in this compound. We experimentally identified that this system is transcribed from a single promoter with an initiation site located 110 nucleotides upstream of the ATG start codon. On the basis of computational data, it had been proposed that LldR and its homologue PdhR act as regulators of the
lldPRD
operon. Nevertheless, no experimental data on the function of these regulators have been reported so far. Here we show that induction of an
lldP-lacZ
fusion by
l
-lactate is lost in an Δ
lldR
mutant, indicating the role of LldR in this induction. Expression analysis of this construct in a
pdhR
mutant ruled out the participation of PdhR in the control of
lldPRD
. Gel shift experiments showed that LldR binds to two operator sites, O1 (positions −105 to −89) and O2 (positions +22 to +38), with O1 being filled at a lower concentration of LldR.
l
-Lactate induced a conformational change in LldR that did not modify its DNA binding activity. Mutations in O1 and O2 enhanced the basal transcriptional level. However, only mutations in O1 abolished induction by
l
-lactate. Mutants with a change in helical phasing between O1 and O2 behaved like O2 mutants. These results were consistent with the hypothesis that LldR has a dual role, acting as a repressor or an activator of
lldPRD
. We propose that in the absence of
l
-lactate, LldR binds to both O1 and O2, probably leading to DNA looping and the repression of transcription. Binding of
l
-lactate to LldR promotes a conformational change that may disrupt the DNA loop, allowing the formation of the transcription open complex.
The intestine as source of cytotoxic mediators in shock: free fatty acids and degradation of lipid-binding proteins
Authors: Penn Alexander H. , Schmid-Schönbein Geert W.
Published: February 2008

Shock and multiple organ failure remain primary causes of late-stage morbidity and mortality in victims of trauma. During shock, the intestine is subject to extensive cell death and is the source of inflammatory factors that cause multiorgan failure. We ( 34 ) showed previously that ischemic, but not nonischemic, small intestines and pancreatic protease digested homogenates of normal small intestine can generate cytotoxic factors capable of killing naive cells within minutes. Using chloroform/methanol separation of rat small intestine homogenates into lipid fractions and aqueous and sedimented protein fractions and measuring cell death caused by those fractions, we found that the cytotoxic factors are lipid in nature. Recombining the lipid fraction with protein fractions prevented cell death, except when homogenates were protease digested. Using a fluorescent substrate, we found high levels of lipase activity in intestinal homogenates and cytotoxic levels of free fatty acids. Addition of albumin, a lipid binding protein, prevented cell death, unless the albumin was previously digested with protease. Homogenization of intestinal wall in the presence of the lipase inhibitor orlistat prevented cell death after protease digestion. In vivo, orlistat plus the protease inhibitor aprotinin, administered to the intestinal lumen, significantly improved survival time compared with saline in a splanchnic arterial occlusion model of shock. These results indicate that major cytotoxic mediators derived from an intestine under in vitro conditions are free fatty acids. Breakdown of free fatty acid binding proteins by proteases causes release of free fatty acids to act as powerful cytotoxic mediators.
A positive inotropic effect of ATP in the human cardiac atrium
Authors: Gergs Ulrich , Boknik Peter , Schmitz Wilhelm , Simm Andreas , Silber Rolf-Edgar , Neumann Joachim
Published: February 2008

We studied contractile effects in isolated electrically driven (1 Hz) atrial preparations from patients undergoing cardiac bypass surgery. ATP concentration dependently (10, 30, and 100 μM) and rapidly decreased force of contraction (negative inotropic effect, NIE) and thereafter more slowly increased force of contraction. The maximum positive inotropic effect (PIE) at 100 μM ATP amounted to 152% of the predrug value ( n = 9) and was stable and could be washed out fast and completely. The PIE did not affect time parameters of contraction (time to peak tension and time of relaxation). Moreover, a similar NIE and PIE were noted with adenosine 5′- O-(2-thiotriphosphate) (100 μM). In contrast 2-methyl-thio-ATP did not exert a NIE but only a PIE. In a second set of experiments, preparations were first incubated for 30 min with purinoreceptor antagonists and, in their continuous presence, 100 μM ATP was applied. However, the PIE and NIE of ATP could neither be blocked with suramin (100 and 500 μM), pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (50 μM), nor reactive blue 2 (30, 100, and 500 μM), which are known blockers for subtypes of P2receptors, or 1,3-dipropyl-cyclopentvl-xanthine (1 and 10 μM), a subtype (A1adenosine) P1receptor blocker. Likewise, the inhibitor of phospholipase C (PLC) activity (U-73122) and the inhibitor of adenylate cyclase activity (SQ-022563) (10 μM each) failed to affect the NIE and the PIE of ATP. We tentatively suggest that the PIE of ATP might be mediated via P2X4-like receptors. In summary, we describe a novel biphasic effect of ATP on force contraction in the isolated human atrium. It is conceivable that ATP plays a physiological role in the human heart, for instance, after cardiac injury to sustain contractility.
Sex differences to myocardial ischemia and β-adrenergic receptor blockade in conscious rats
Authors: Lujan Heidi L. , DiCarlo Stephen E.
Published: February 2008

We recently documented sex differences in the susceptibility to reperfusion-induced sustained ventricular tachycardia and β-adrenergic receptor blockade in conscious rats. However, the effect of sex on ischemia-induced ventricular arrhythmias and β-adrenergic receptor blockade is underinvestigated. Therefore, we tested the hypothesis that gonadal hormones influence the ventricular arrhythmia threshold (VAT) induced by coronary artery occlusion as well as the response to β-adrenergic receptor blockade. The VAT was defined as the time from coronary occlusion to sustained ventricular tachycardia resulting in a reduction in arterial pressure. Male and female intact and gonadectomized (GnX) rats were instrumented with a radiotelemetry device for recording arterial pressure, temperature, and ECG, as well as a Doppler ultrasonic flow probe to measure cardiac output and a snare around the left main coronary artery. The VAT was determined in conscious rats by pulling on the snare. The VAT was significantly longer in intact females (5.56 ± 0.19) vs. intact males (4.31 ± 0.14 min). This sex difference was abolished by GnX. Specifically, GnX decreased the VAT in females (4.55 ± 0.22) and increased the VAT in males (5.14 ± 0.30 min). Thus male sex hormones increase and female sex hormones decrease the susceptibility to ischemia-induced sustained ventricular tachycardia. β-Adrenergic receptor blockade increased the VAT in intact males and GnX females only. Thus gonadal hormones influence the response to β-adrenergic receptor blockade. Uncovering major differences between males and females in the pathophysiology of the cardiovascular system may result in sex-specific optimization of patient treatments.
HSP27 regulates p53 transcriptional activity in doxorubicin-treated fibroblasts and cardiac H9c2 cells: p21 upregulation and G2/M phase cell cycle arrest
Authors: Venkatakrishnan C. D. , Dunsmore Kathy , Wong Hector , Roy Sashwathi , Sen Chandan K. , Wani Altaf , Zweier Jay L. , Ilangovan Govindasamy
Published: February 2008

Treatment of cancer patients with anthracyclin-based chemotherapeutic drugs induces congestive heart failure by a mechanism involving p53. However, it is not known how p53 aggravates doxorubicin (Dox)-induced toxicity in the heart. On the basis of in vitro acute toxicity assay using heat shock factor-1 (HSF-1) wild-type (HSF-1+/+) and HSF-1-knockout (HSF-1−/−) mouse embryonic fibroblasts and neonatal rat cardiomyocyte-derived H9c2 cells, we demonstrate a novel mechanism whereby heat shock protein 27 (HSP27) regulates transcriptional activity of p53 in Dox-treated cells. Inhibition of p53 by pifithrin-α (PFT-α) provided different levels of protection from Dox that correlate with HSP27 levels in these cells. In HSF-1+/+cells, PFT-α attenuated Dox-induced toxicity. However, in HSF-1−/−cells (which express a very low level of HSP27 compared with HSF-1+/+cells), there was no such attenuation, indicating an important role of HSP27 in p53-dependent cell death. On the other hand, immunoprecipitation of p53 was found to coimmunoprecipitate HSP27 and vice versa (confirmed by Western blotting and matrix-assisted laser desorption/ionization time of flight), demonstrating HSP27 binding to p53 in Dox-treated cells. Moreover, upregulation of p21 was observed in HSF-1+/+and H9c2 cells, indicating that HSP27 binding transactivates p53 and enhances transcription of p21 in response to Dox treatment. Further analysis with flow cytometry showed that increased expression of p21 results in G2/M phase cell cycle arrest in Dox-treated cells. Overall, HSP27 binding to p53 attenuated the cellular toxicity by upregulating p21 and prevented cell death.
Gene expression in the developing diaphragm: significance for congenital diaphragmatic hernia
Authors: Clugston Robin D. , Zhang Wei , Greer John J.
Published: February 2008

Congenital diaphragmatic hernia (CDH) is a frequently occurring birth defect and a source of potentially fatal neonatal respiratory distress. Recently, through the application of detailed karyotyping methods, several CDH-critical regions within the human genome have been identified. These regions typically contain several genes. Here we focused on genes from 15q26, the best-characterized CDH-critical region, as well as FOG2 and GATA4, genes singled out from CDH-critical regions at 8q22–8q23 and 8p23.1, respectively. We tested the hypothesis that these putative CDH-related genes are expressed within the developing diaphragm at the time of the hypothesized initial defect. Our results show that 15q26 contains a cluster of genes that are expressed in the developing rodent diaphragm, consistent with an association between deletions in this region and CDH. We then examined the protein expression pattern of positively identified genes within the developing diaphragm. Two major themes emerged. First, those factors strongly associated with CDH are expressed only in the nonmuscular, mesenchymal component of the diaphragm, supporting the hypothesis that CDH has its origins in a mesenchymal defect. Second, these factors are all coexpressed in the same cells. This suggests that cases of CDH with unique genetic etiology may lead to a common defect in these cells and supports the hypothesis that these factors may be members of a common pathway. This study is the first to provide a detailed examination of how genes associated with CDH are expressed in the developing diaphragm and provides an important foundation for understanding how the deletion of specific genes may contribute to abnormal diaphragm formation.
Chronic hypoxia induces Rho kinase-dependent myogenic tone in small pulmonary arteries
Authors: Broughton Brad R. S. , Walker Benjimen R. , Resta Thomas C.
Published: February 2008

Myogenic tone in the pulmonary vasculature of normoxic adult animals is minimal or nonexistent. Whereas chronic hypoxia (CH) increases basal tone in pulmonary arteries, it is unclear if a portion of this elevated tone is due to development of myogenicity. Since basal arterial RhoA activity and Rho kinase (ROK) expression are augmented by CH, we hypothesized that CH elicits myogenic reactivity in pulmonary arteries through ROK-dependent vascular smooth muscle (VSM) Ca2+sensitization. To test this hypothesis, we assessed the contribution of ROK to basal tone and pressure-induced vasoconstriction in endothelium-disrupted pulmonary arteries [50–300 μm inner diameter (ID)] from control and CH [4 wk at 0.5 atmosphere (atm)] rats. Arteries were loaded with fura-2 AM to continuously monitor VSM intracellular Ca2+concentration ([Ca2+]i). Basal VSM [Ca2+]iwas not different between groups. The ROK inhibitor, HA-1077 (100 nM to 30 μM), caused a concentration-dependent reduction of basal tone in CH arteries but had no effect in control vessels. In contrast, PKC inhibition with GF109203X (1 μM) did not alter basal tone. Furthermore, significant vasoconstriction in response to stepwise increases in intraluminal pressure (5–45 mmHg) was observed at 12, 15, 25, and 35 mmHg in arteries (50–200 μm ID) from CH rats. This myogenic reactivity was abolished by HA-1077 (10 μM) but not by GF109203X. VSM [Ca2+]iwas unaltered by HA-1077, GF109203X, or increases in pressure in either group. Myogenicity was not observed in larger vessels (200–300 μm ID). We conclude that CH induces myogenic tone in small pulmonary arteries through ROK-dependent myofilament Ca2+sensitization.
FXYD5 modulates Na+absorption and is increased in cystic fibrosis airway epithelia
Authors: Miller Timothy J. , Davis Pamela B.
Published: February 2008

FXYD5, also known as dysadherin, belongs to a family of tissue-specific regulators of the Na+-K+-ATPase. We determined the kinetic effects of FXYD5 on Na+-K+-ATPase pump activity in stably transfected Madin-Darby canine kidney cells. FXYD5 significantly increased the apparent affinity for Na+twofold and decreased the apparent affinity for K+by 60% with a twofold increase in Vmaxof K+, a pattern that would increase activity and Na+removal from the cell. To test the effect of increased Na+uptake on FXYD5 expression, we analyzed Madin-Darby canine kidney cells stably transfected with an inducible vector expressing all three subunits of the epithelial Na+channel (ENaC). Na+-K+-ATPase activity increased sixfold after 48-h ENaC induction, but FXYD5 expression decreased 75%. FXYD5 expression was also decreased in lung epithelia from mice that overexpress ENaC, suggesting that chronic Na+absorption by itself downregulates epithelial FXYD5 expression. Patients with cystic fibrosis (CF) display ENaC-mediated hyperabsorption of Na+in the airways, accompanied by increased Na+-K+-ATPase activity. However, FXYD5 was significantly increased in the lungs and nasal epithelium of CF mice as assessed by RT-PCR, immunohistochemistry, and immunoblot analysis ( P < 0.001). FXYD5 was also upregulated in nasal scrapings from human CF patients compared with controls ( P < 0.02). Treatment of human tracheal epithelial cells with a CFTR inhibitor (I-172) confirmed that loss of CFTR function correlated with increased FXYD5 expression ( P < 0.001), which was abrogated by an inhibitor of NF-κB. Thus FXYD5 is upregulated in CF epithelia, and this change may exacerbate the Na+hyperabsorption and surface liquid dehydration observed in CF airway epithelia.
Robert Boyle's 'Designe about Natural History'
Authors: Hunter Michael , Anstey Peter
Published: February 2008

This paper provides an analysis of Robert Boyle's most detailed discussion of the Baconian method of natural history. In a long letter to Henry Oldenburg dated 13 June 1666 and in ancillary manuscript material, Boyle spells out the method or 'Designe' by which he believes experimental programs in natural philosophy should be written up. The 'Designe' is enormously important in giving a clear statement of the precise contours of Boyle's Baconian methodology and providing a key to understanding the rationale, composition and format for many of his published histories. Works such as New Experiments and Considerations Touching Cold, Memoirs for the Natural History of Human Blood and even his more speculative writings such as An Introduction to the History of Particular Qualities are best understood in terms of Boyle's overall method as articulated in his “Designe about Natural History”.
Dielectrophoretic Deposition of Graphite Oxide Soot Particles
Authors: Hong Seunghyun , Jung Sehun , Kang Sunjung , Kim Youngjin , Chen Xinqi , Stankovich Sasha , Ruoff S. Rodney , Baik Seunghyun
Published: February 2008

Alternating current dielectrophoresis in water was used to position graphite oxide soot (GO-soot) particles generated by rapid thermal expansion of graphite oxide under inert gas. The dielectrophoretic deposition was carried out at a frequency of 10 MHz and a peak-to-peak voltage of
10 V, and the deposited particles were analyzed using scanning electron microscopy. The vertical cross section, obtained by focused ion beam cutting, shows the wrinkled layers of the GO-soot particles and cavities between the layers. The electrical transport measurements show typical characteristics
of metal-like pathways. The improved electrical contact between electrodes and GO-soot, probably due to the thin platelet structure of GO-soot, makes the material favorable for electrical device applications. The results demonstrate that AC dielectrophoresis can be used to selectively deposit
graphite oxide soot particles at desired locations.
Supermax Prisons in the Consciousness of Prisoners
Authors: Hartman Kenneth E.
Published: February 2008

Prison administrators claim that supermax prisons are essential tools in the management of unruly prisoners and that these facilities act as a deterrent to prisoners contemplating violence. Pelican Bay, known in prison jargon as “Up North,” is California's supermax prison. This article states that not only does Pelican Bay not deter, it has created a new standard of toughness, an upper class of prisoners whose survival at Pelican Bay affords them high status. This status allows them great influence on daily events in the prison. Instead of isolating the most negative elements, the supermax has simply raised the bar on toughness. California now has a new breed of prisoners who have survived the trial by fire and are afraid of nothing. Suggestions are offered to ease the conversation away from the supermax and toward programs that might actually work on the ground. Ask the prisoners what they think will work. Include prisoners in designing and implementing smarter programs.
Social Representations of Barriers to Care Early in the Careers of Caregivers of Persons With Alzheimer's Disease
Authors: Carpentier Normand , Ducharme Francine , Kergoat Marie-Jeanne , Bergman Howard
Published: February 2008

The first signs of cognitive impairment in the elderly generally elicit much concern among family members. Reactions run from denial to the active search for information. Some families manage to set up relatively well-organized networks of informal support to help both caregivers and elderly relatives. However, little is known about the processes underlying the different pathways that families follow at the onset of Alzheimer-type dementia in elderly relatives. To gain a better understanding of barriers to care early in the caregiving career, from the first signs of illness to diagnosis, the authors conducted interviews with 52 caregivers recruited at two cognition clinics. Barriers to help resources were analyzed from the viewpoint of social representations. This approach allowed the consideration of a broad range of individual and group phenomena capable of fashioning caregivers' representations of this period. The results confirmed the importance of the symbolic dimension of experience in steering social practice.
Reflective judgment as world disclosure
Authors: Pía Lara María
Published: February 2008

In this article I deal with Kant's concept of reflective judgment, and recover it through its links to the aesthetic dimension as its fundamental scenario. Then I go on to explain why Hannah Arendt understood this important Kantian connection, and why she thought it would allow her to develop it through a political dimension. Last, having reviewed both Kant and Arendt's contributions to the concept of reflective judgment, I recover my own input to the concept by showing its linguistic dimension based on the Heideggerian notion of world-disclosure. With this in mind, I show how the concept of reflective judgment is the most suitable to analyze evil actions.
Comparative and Correlative Neuroanatomy for the Toxicologic Pathologist
Authors: Bolon Brad
Published: February 2008

Xenobiotic-induced neuroanatomic alterations are always regarded as adverse and are commonly used to define reference doses to manage neurotoxic risk. Thus, the neuropathologist plays an essential role in evaluating potential neurotoxicants. The pathologist must be able to recognize the morphologic differences that exist among species, strains, and ages or between genders (comparative neuroanatomy) and to grasp the impact of structural damage on neural function (correlative neuroanatomy). Brain anatomy and function may be used to group the mammals used in neurotoxicity bioassays into 3 classes: rodent, carnivore, and primate. Neural function may or may not be affected by the structural divergence. Rodents are preferred for neurotoxicity assays because their reduced body size allows optimal perfusion at little cost and their smaller brain size permits screening of multiple regions using few sections. However, care must be exercised when interpreting rodent neuropathology data because the rodent paleocortex does not recapitulate the sophisticated neocortical circuitry and functions of carnivores and primates. Knowledge of the neuroanatomic variations that exist among test species assists the neuropathologist in defining the relevance of structural alterations, the potential clinical sequelae of such findings, and the possible significance of similar changes in humans.
Microglial Response to Brain Injury: A Brief Synopsis
Authors: Streit Wolfgang J.
Published: February 2008

In addition to astrocytes and oligodendrocytes, microglia represent the third major population of glial cells within the central nervous system (CNS). Microglia are distributed ubiquitously throughout the brain and spinal cord, and one of their main functions is to monitor and sustain neuronal health. Microglial cells are quite sensitive to even minor disturbances in CNS homeostasis, and they become readily activated during most neuropathologic conditions, including peripheral nerve injury, trauma and stroke, inflammatory disease, and neurotoxicant-induced neuronal injury. During activation, microglia display conspicuous functional plasticity, which involves changes in cell morphology, cell number, cell surface receptor expression, and production of growth factors and cytokines. The many changes occurring in activated cells reflect the altered functional states of microglia that are induced by signals arising from injured neurons. Thus, neuronal-microglial signaling plays a fundamental role in understanding how the CNS responds to injury. Reactive microgliosis should be viewed as a cellular effort to initiate ameliorative and reparative measures in the injured brain.
Synaptic Clefts Are Made to Be Crossed: Neurotransmitter Signaling in the Central Nervous System
Authors: Patrick Robert L.
Published: February 2008

The primary means of communication between neurons in the mammalian central nervous system (CNS) is via release of chemical transmitters. Although the first transmitters to be discovered were the biogenic amines, such as acetylcholine and norepinephrine, involved in transmission in the autonomic nervous system, the contribution of other types of transmitters, such as amino acids and peptides, to CNS transmission has been the subject of recent study. Part of this interest stems from the relatively large percentage of neuronal connections that make use of amino acid transmitters such as gamma-aminobutyric acid and glutamate and also from the tremendous diversity possible when peptides are used as transmitters. Several disorders of CNS transmission are related to the degeneration of neuronal pathways in the brain. Two of the most prevalent neurologic disorders that result from degeneration are Alzheimer's and Parkinson's diseases. Aspects of these disorders related to chemical neurotransmission are discussed, along with implications with regard to therapeutic strategies. Functions of and possible abnormalities in amino acid transmission that may be associated with CNS disorders are examined. Several peptides are postulated to play a role in neurotransmission and concepts regarding the significance of the coexistence and release of biogenic amines and peptides at the same neuronal terminals are presented.
Mechanisms of Toxic Injury in the Peripheral Nervous System: Neuropathologic Considerations
Authors: Jortner Bernard S.
Published: February 2008

The anatomical distribution and organization of the peripheral nervous system as well as its frequent ability to reflect neurotoxic injury make it useful for the study of nerve fiber and ganglionic lesions. Contemporary neuropathologic techniques provide sections with excellent light-microscopic resolution for use in making such assessments. The histopathologist examining such peripheral nerve samples may see several patterns of neurotoxic injury. Most common are axonopathies, conditions in which axonal alterations are noted; these axonopathies often progress toward the Wallerian-like degeneration of affected fibers. These are usually more severe in distal regions of the neurite, and they affect both peripheral and central fibers. Examples of such distal axonopathies are organophosphorous ester-induced delayed neuropathy, hexacarbon neuropathy, and p-bromophenylacetylurea intoxication. These axonopathies may have varying pathologic features and sometimes have incompletely understood toxic mechanisms. In such neuropathies with fiber degeneration, peripheral nerve axons may regenerate, which can complicate pathologic interpretation of neurotoxicity. On occasion neurotoxins elicit more severe injury in proximal regions of the fiber (not included in this review). Axonal pathology is also a feature of the neuronopathies, toxic states in which the primary injuries are found in neuronal cell bodies. This is exemplified by pyridoxine neurotoxicity, where there is sublethal or lethal damage to larger cytons in the sensory ganglia, with failure of such neurons to maintain their axons. Lastly, one may encounter myelinopathies, conditions in which the toxic effect is on the myelin-forming cell or sheath. An example of this is tellurium intoxication, where demyelination noted in young animals is coincident with toxin-induced interference of cholesterol synthesis by Schwann cells. In this paper, the above-noted examples of toxic neuropathy are discussed, with emphasis on mechanistic and morphologic considerations.
Application of Silver Degeneration Stains for Neurotoxicity Testing
Authors: Switzer Robert C.
Published: February 2008

Silver staining procedures have been used in numerous ways to render a variety of physical and biological features visible. In biological tissue, histologic protocols use silver to visualize diverse structures or features, such as reticulin, melanin, fungi, chromosome bands, nucleolar organizing regions, and different features in the nervous system. A comparison of the specific steps in these protocols indicates that the silver is "directed" to stain any given feature by the type of fixation, the pretreatment ("mordanting"), the composition of the silver-containing solution(s), and the form of development (reduction). Since the mechanisms of staining have not been understood historically (nor are they now), each method was developed by trial and error. Keystone methods such as those of Bodian and Bielschowsky exploit the nervous system's affinity for silver (argyrophilia). The beginning of a new era in brain research came with the recognition that distinct silver-impregnated morphologic changes occurring in damaged axons could be used for tracing axon pathways in experimental animals with specifically placed lesions. Improvements in staining methods used to selectively impregnate the disintegrating axons but to leave normal axons unstained were achieved by Nauta and Gygax (early workers with these procedures) and spawned a host of method variations known as the "Nauta" methods. Of these, the Fink-Heimer and de Olmos cupric-silver methods were able to unambiguously demonstrate disintegrating synaptic terminals, thereby allowing complete tracing of axon pathways. The late 1970s and 1980s witnessed innovative applications of these techniques. The silver methods once used to trace axon pathways became indicators of the extreme endpoint of neurotoxicity: disintegrative degeneration of neurons induced by neurotoxic chemicals that were administered systemically. The hallmark of neurotoxic substances is the selectivity with which each destroys specific populations or subpopulations of neurons. The high contrast and sensitivity of the silver degeneration stains greatly facilitate the screening process to detect these affected populations, especially when there is no basis for knowing where in the brain to look for damage. More recently, in addition to expanded use in screening for neurotoxic effects, the silver degeneration stains are being used to chart the neuron populations undergoing programmed cell death in the developing brain. Other newly developed silver methods have been refined to show nondisintegrative degeneration, such as the plaques and tangles of Alzheimer's disease.
Stress Proteins as Molecular Markers of Neurotoxicity
Authors: Rajdev Sunita , Sharp Frank R.
Published: February 2008

In response to many environmental and pathophysiologic stressful stimuli, cells undergo a stress response characterized by induction of a variety of proteins, including the heat shock protein family. The inducible heat shock protein 70 (hsp70) is believed to participate in an array of cellular activities, including cytoprotection. Normal brain cells have little detectable hsp70 RNA or protein. However, following a stressful condition hsp70 mRNA and protein are induced in different cell types depending on the severity and the nature of the stimulus. The induction of hsp70 protein correlates with the regional and cellular vulnerability to a particular injury as identified by standard histologic methods. The pattern of hsp70 expression differs in response to various neurotoxic stimuli, including hyperthermia, ischemia, seizures, hemorrhage, and N-methyl-D-aspartate receptor antagonist administration. Hsp70 expression is a useful marker of cellular injury and may help to identify previously unrecognized areas of vulnerability in the nervous system after a neurotoxic stimulus. Hsp70 may also play a neuroprotective role in the brain.
Teased-Fiber Technique for Peripheral Myelinated Nerves: Methodology and Interpretation
Authors: Krinke Georg J. , Vidotto Nella , Weber Edgar
Published: February 2008

Teased-fiber technique is the best approach for studying peripheral myelinated nerve fibers in their continuity. It enables the assessment of size of myelin segments formed by Schwann cells and characterization of pathologic changes affecting the internodia, the paranodal regions, and the invested axons. Fiber teasing is performed on prestained proximodistally oriented portions of peripheral nerves. Specimens about 10 mm long are stained for 24-48 hours in Sudan black and then transferred to glycerin, where, using a pair of fine forceps and a stereomicroscope, they are separated into smaller fiber bundles from which single fibers are isolated. The work is performed on a glass slide with an adhesive surface (albuminized or "superfrost"), on which the fibers are placed in strict proximodistal orientation. Following drying in an oven, the slides are mounted with glycerin-gelatine (same as used for frozen sections). The changes, when present, can usually be recognized during the preparation, but fibers are reexamined and changes confirmed in mounted slides. Photographic reconstruction of the fibers facilitates their assessment and enables the documentation of findings. The teased-fiber technique is auxiliary to histopathology, and to limit the workload and save costs, it can be performed on only a few specimens selected for better characterization of changes recognized or suspected in tissue sections. In particular, segmental demyelination and early stages of Wallerian or secondary axonal degeneration can be recognized in teased fibers. Segmental demyelination is characterized by loss of fully myelinated segments and their replacement by newly formed short and thin segments, remyelinating the preserved axon. The early stage of secondary axonal degeneration is recognized by formation of ovoidal fiber fragments in the midinternodal region.