bluuebunny/crossref_metadata_2025 huggingface dataset:
Otter (Lutra lutra) predation on stocked brown trout (Salmo trutta) in two Danish lowland rivers
Authors: Jacobsen L.
Published: February 2005

Abstract – This study aimed to evaluate otter predation on stocked trout. Large hatchery‐reared trout (16–30 cm) were stocked into two Danish rivers with different fish populations. Otter diet before and after trout stocking was determined by analysing 685 spraints, collected regularly during the 35‐day study period. Fish composition in the rivers before stocking was assessed by electrofishing. In River Trend, a typical trout river, the proportion of trout in the otter diet increased from 8% before stocking to 33% a few days after stocking. Moreover, trout lengths in the diet changed significantly towards the lengths of stocked trout, indicating that newly stocked trout were preferred to wild trout. In River Skals, dominated by cyprinids, there was no change in otter diet after stocking of hatchery trout, i.e., these were ignored by otter. Otter predation should be taken into account together with fish and bird predation, when stocking is used as a measure for conserving endangered salmonid populations.
Can feeding of European catfish prevent cyprinids from reaching a size refuge?
Authors: Wysujack K. , Mehner T.
Published: February 2005

Abstract – The size structure of a predator population can cause differences in the relative survival of different prey length classes whereby the biggest prey may reach a safe size because of the size‐dependent character of predation. In the present study, the diet of European catfish was investigated to examine if catfish feeding can prevent cyprinids from reaching such a size refuge. In the lake studied, catfish was stocked for biomanipulation purposes to reduce unwanted roach and bream populations. Crayfish and roach were the most important prey items of catfish. If only species composition in the diet was considered, no clear changes were recorded in relation to catfish size. However, the length of roach as prey significantly increased with catfish length. Catfish were significantly larger than the other piscivorous fish in the lake, but took relatively smaller roach in comparison with similar‐sized pike or pikeperch. Nevertheless, because of the high mean length of catfish, roach cannot reach a size refuge. For unknown reasons, the expected and intended predation on bream was not observed. Catfish took smaller prey than could be expected from mouth gape data. By extending the relationship between catfish as predator and roach as prey beyond the predator length range currently found in the lake, it could be shown that even catfish of 150 cm length will probably feed upon only 65% of all available bream length classes. Therefore, stocking with catfish cannot prevent a size refuge for the bream.
A comparative study of juvenile salmon density in 20 streams throughout a very large river system in northern Norway
Authors: Johansen M. , Elliott J. M. , Klemetsen A.
Published: February 2005

Abstract – The objective was to compare juvenile salmon density in 20 streams throughout the very large River Tana, northern Norway, and to relate variation in density to a suite of environmental factors. Four sampling sites were electrofished in each stream (one at the mouth of the stream and three within the stream) in August and October 2000, 2001, 2002. 0+ salmon parr were absent from seven streams, present at the mouth of 11 streams, and present within only two streams, both of which were probably spawning streams. Older parr migrated upstream into most streams and their highest densities were usually found in streams flowing directly into the spawning habitat in the three largest tributaries of the Tana or the river itself. Juvenile salmon were sparse or absent in streams flowing into smaller tributaries. Most streams with high parr densities were those of dense riparian vegetation that provided terrestrial invertebrates as drift food for the salmon parr, cover for fish, cooler stream temperatures in summer, and food for benthic stream invertebrates that were also a source of food for the parr.
An Unconventional NLS is Critical for the Nuclear Import of the Influenza A Virus Nucleoprotein and Ribonucleoprotein
Authors: Cros Jerome F. , García‐Sastre Adolfo , Palese Peter
Published: February 2005

Replication of the RNAs of influenza virus occurs in the nucleus of infected cells. The nucleoprotein (NP) has been shown to be important for the import of the viral RNA into the nucleus and has been proposed to contain at least three different nuclear localization signals (NLSs). Here, an import assay in digitonin‐permeabilized cells was used to further define the contribution of these NLSs. Mutation of the unconventional NLS impaired the nuclear import of the NP. A peptide bearing the unconventional NLS could inhibit the nuclear import of the NP in this import assay and prevent the NP–karyopherin α interaction in a binding assay confirming the crucial role of this signal. Interestingly, a peptide containing the SV40 T antigen NLS was unable to inhibit the nuclear import of NP or the NP–karyopherin α interaction, suggesting that the NP and the SV40 T antigen do not share a common binding site on karyopherin α. We also investigated the question of which NLS(s) is/are necessary for the viral ribonucleoprotein complex to enter the nucleus. We found that the peptide containing the unconventional NLS efficiently inhibited the nuclear import of the ribonucleoprotein complexes. This finding suggests that the unconventional NLS is the major signal necessary not only for the nuclear transport of free NP but also for the import of the ribonucleoprotein complexes. Finally, viral replication could be specifically inhibited by a membrane‐permeable peptide containing the unconventional NLS, confirming the crucial role of this signal during the replicative cycle of the virus.
The Nanopore Connection to Cell Membrane Unitary Permeability
Authors: Peters Reiner
Published: February 2005

Artificial nanopores have recently emerged as versatile tools for analyzing and sorting single molecules at high speed. However, the biological cell has already developed a large set of sophisticated protein nanopores that are able to selectively translocate all types of molecules through membranes. Therefore, hybrid devices combining artifical solid‐state with biomimetic protein nanopores appear to us as a particularly promising approach to the creation of powerful diagnostic, preparative and therapeutic devices. Here, we discuss a technique, optical single‐transporter recording (OSTR), in which arrays of artificial micropores and nanopores are employed to analyze protein nanopores of cellular membranes. After briefly summarizing some salient features of OSTR, the technique is compared with the electrical patch clamp method and the first results of our efforts to amalgamate optical and electrical recording are described. Finally, prospects for combining OSTR with 4Pi microscopy, single‐molecule fluorescence spectroscopy and fluorescence correlation spectroscopy are discussed.
Regulation and Signal Transduction of Toll‐Like Receptors in Human Chorioncarcinoma Cell Lines
Authors: Klaffenbach Daniela , Rascher Wolfgang , Röllinghoff Martin , Dötsch Jörg , Meißner Udo , Schnare Markus
Published: February 2005

Problem:  Toll‐like receptors (TLRs) are expressed on placental cells. The aim of this study is to analyze signaling components activated in placenta cells after TLR ligand engagement.

Methods of study:  In chorioncarcimoma cell lines the regulation of TLRs was determined by real time polymerase chain reaction as well as by fluorescence‐activated cell sorter analysis. Activation of NF‐κB was determined in a reporter assay system and the activation of the mitogen‐activated protein kinase signaling pathways by immunoblot analysis.

Results:  Both lipopolysaccharide (LPS) and DNA oligonucleotides containing unmethylated CpG motifs (CpG) induced the enhanced expression of TLR2 mRNA as well as a TLR2 surface protein expression. Functionally, incubation of JAR cells with microbial stimuli such as LPS activated NF‐κB, as well as the phosphorylation of ERK1/2 and p38 MAP kinases and secretion of interleukin‐8.

Conclusion:  The functional expression of TLRs on placental cells may play an important role in the initiation of an immune response in the developing fetus.
A Case–Control Study in IL6 and TGFB1 Gene Polymorphisms and Recurrent Spontaneous Abortion in Southern Brazilian Patients
Authors: von Linsingen Renate , Bompeixe Eni Picchioni , da Graca Bicalho Maria
Published: February 2005

Problem:  A high proportion of recurrent spontaneous abortions (RSA) remains unexplained. Cytokine genotyping has been investigated. We studied the relationship between unexplained RSA, IL6 (−174 G→C) and TGFB1 (+869, T→C; +915, G→C) gene polymorphisms.

Method of Study:  The case–control study composed of 57 south Brazilian women, with unexplained RSA and 74 controls carefully matched was performed. Cytokine genotyping was performed by the polymerase chain reaction–sequence specific primer method, using the ‘Cytokine Genotyping Tray’.

Results:  The results showed that the genotypic frequencies did not differ from samples for TGFB1 gene. In relation to IL6 gene polymorphism there was a statistical difference in genotypic distribution between samples (P ≤ 0.025). The frequency of the C/C genotype was increased in women with RSA in comparison with the frequency observed in controls: 18% versus 4% (P = 0.01).

Conclusions:  This result strengthened the importance of IL6 genotypes in the pathogenesis of RSA of unknown cause in the south Brazilian population.
Endometriosis and Systemic Lupus Erythematosus: A Comparative Evaluation of Clinical Manifestations and Serological Autoimmune Phenomena
Authors: Pasoto Sandra G. , Abrao Mauricio S. , Viana Vilma S.T. , Bueno Cleonice , Leon Elaine P. , Bonfa Eloisa
Published: February 2005

Problem:  In view of evidences suggesting association between endometriosis (EM) and systemic lupus erythematosus (SLE), we have performed a comparative evaluation of clinical and humoral immunologic abnormalities in both diseases.

Method of study:  Forty‐five women (18–40 years) with histologically confirmed pelvic EM, 21 healthy‐women and 15 female SLE‐patients (18–40 years) without surgically confirmed EM were prospectively evaluated. Immunologic investigations were performed by blinded researchers.

Results:  None of the EM‐patients fulfilled criteria for SLE. However, EM‐patients presented higher frequencies of arthralgia (62%) and generalized myalgia (18%) superior than normal‐controls (24%, P = 0.004/0%, P = 0.048) but comparable with SLE‐patients (33%, P = 0.052/27%, P = 0.5). Similarly to SLE (7%), 9% of EM‐patients presented fibromyalgia. Antinuclear antibodies (ANA) were detected in 18% of EM‐sera, as compared with healthy‐women (0%, P = 0.014) and SLE‐patients (93%, P = 0.0005). In contrast with SLE, antibodies to dsDNA, Sm and U1RNP were negative in EM‐sera. Anti‐Ro and anticardiolipin antibodies were more often in SLE (40%, 33%) than in EM‐patients (2%, P < 0.001/9%, P = 0.04). Elevated immune‐complexes and low total complement were more frequent in SLE (40%, 13%) compared with EM‐sera (7%, P = 0.005/0%, P = 0.01).

Conclusions:  Our data indicate differences of ANA antigenic specificity and complement consumption between EM and SLE. The high prevalence of generalized musculoskeletal complaints in EM justifies a multidisciplinary approach.
Jimmy Carter: The Re‐emergence of Faith‐Based Politics and the Abortion Rights Issue
Authors: FLINT ANDREW R. , PORTER JOY
Published: February 2005

This article will extend the current re‐evaluation of the Carter presidency through a detailed examination of the enduring impact of his evangelical Christian faith upon modern American political discourse. Carter successfully reawakened faith‐based politics but, because his faith did not exactly mirror the religious and political agenda of the disparate groups that make up the religious conservative movement within the United States, that newly awakened force within American politics ultimately used its power to replace him with Ronald Reagan, a president who more carefully articulated their agenda. As this article will show, the key issue that marked the intrusion of highly contentious religious‐cultural issues into the political debate was abortion. This issue was emblematic of both the engagement of religious conservatives in political life in this period and of the limitations of Carter as their authentic political agent.
The Institutionalization of Regulatory Review: Organizational Stability and Responsive Competence at OIRA
Authors: WEST WILLIAM F.
Published: February 2005

Scholars have argued that presidents’ need for responsiveness induces them to adopt ad hoc, politicized management strategies. Most have been critical of this tendency, contending that presidents are better served by established organizations and civil servants within the executive office. Their premise is that the neutral competence brought to bear through these institutional resources will faithfully promote the interests of whoever occupies the White House. Yet others have argued that political responsiveness is essential to the accomplishment of presidential goals, and that this can only be obtained through personalized management techniques that bypass the bureaucracy.

This study examines these assertions in the context of regulatory review. Centralized executive oversight of agency policy making has been implemented through a stable organization staffed almost exclusively by career bureaucrats, but it has also reflected the agendas of different presidents. An analysis of the structure and the implementation of regulatory review is thus inconsistent with popular descriptions of executive management. Although it supports the prescriptive argument that established institutions can serve presidents effectively, it does not suggest that such responsiveness is likely to occur through a cultural medium of neutral competence. Regulatory review has promoted executive interests across administrations precisely because the process has internalized incumbents’ political preferences.
The Contemporary Presidency: Do Nebraska and Maine Have the Right Idea? The Political and Partisan Implications of the District System
Authors: TURNER ROBERT C.
Published: February 2005

After the 2000 presidential election, twenty‐one states considered legislation to award their presidential electors using the district system, wherein one elector is awarded to the popular vote winner of each congressional district and the two state electors are awarded to the popular vote winner statewide. Historically, the district system is viewed as the politically feasible alternative to direct elections because it removes the distortions of the winner‐take‐all system and can be enacted by state law rather than constitutional amendment. However, the ultimate desirability of the district system lies in how it would change the conduct of presidential campaigns, not the counting of electoral college votes. Under the district system, presidential campaigns would shift their priorities from battleground states to battleground districts. The complexity and uncertainty in targeting these districts would force candidates to contest a larger and more geographically diverse percentage of the population than the current system. Moreover, the changes in presidential campaigns would increase the likelihood of presidential coattails and the prospects for unified government by increasing the competitiveness of congressional elections in swing presidential districts. Finally, the district system has a conservative bias because the swing battleground districts favor Republican presidential candidates and have fewer minority residents and recipients of public assistance than the nation as a whole.
Elections: Turnout in the 2004 Presidential Election
Authors: WATTENBERG MARTIN P.
Published: February 2005

Throughout the United States in 2004 turnout rates were up substantially. Contributing to this positive development were increased interest in the campaign and improvements in vote recording systems. Yet, reports of record turnout need to be tempered. The percentage of citizens who went to the polls fell quite short of the levels found in 1960 in most non‐southern states. Barring further extraordinary events such as those experienced during George W. Bush's first term, it is likely that the turnout increase of 2004 will prove to be a blip rather than the start of an upward trend.
GLOBALIZATION LABOR AND JUSTICE
Authors: Fletcher Bill
Published: February 2005

Organized labor in the U.S. has had difficulty interacting with the global justice movement not so much because these are different labor market sectors with different traditions—although that is certainly a factor—but because there is no strategic agreement on the nature of the enemy. While there are many critical remarks one can make about the global justice movement, this article focuses on the challenges facing organized labor in addressing not simply the global justice movement, but also the issue of global justice as such by building international working‐class solidarity.
A LONG JOBLESS RECOVERY: INFORMATION TECHNOLOGY LABOR MARKETS AFTER THE BURSTING OF THE HIGH‐TECH BUBBLE
Authors: Srivastava Snigdha , Theodore Nik
Published: February 2005

This article assesses the employment situation in the U.S. information technology (IT) industry, examining recent job growth trends and the underlying factors, such as flexible staffing arrangements and offshore outsourcing, which are responsible for falling IT employment. The IT industry, which was at the center of the 1990s boom, was caught in the midst of the 2001 recession and the ensuing jobless recovery. Employment has fallen dramatically between March 2001 and March 2004 and the IT industry lost approximately 403,000 jobs, more than half of which were eliminated during the economic recovery. While cyclical factors are partially responsible for these employment declines, underlying weaknesses in IT labor markets remain as U.S. corporations continue to pursue restructuring tactics aimed at achieving immediate reductions in labor costs.
Interaction among Btn1p, Btn2p, and Ist2p Reveals Potential Interplay among the Vacuole, Amino Acid Levels, and Ion Homeostasis in the Yeast
Saccharomyces cerevisiae
Authors: Kim Yoojin , Chattopadhyay Subrata , Locke Sarahjane , Pearce David A.
Published: February 2005

Btn2p, a novel cytosolic coiled-coil protein in
Saccharomyces cerevisiae
, was previously shown to interact with and to be necessary for the correct localization of Rhb1p, a regulator of arginine uptake, and Yif1p, a Golgi protein. We now report the biochemical and physical interactions of Btn2p with Ist2p, a plasma membrane protein that is thought to have a function in salt tolerance. A deletion in Btn2p (
btn2Δ
strains) results in a failure to correctly localize Ist2p, and strains lacking Btn2p and Ist2p (
btn2Δ ist2Δ
strains) are unable to grow in the presence of 0.5 or 1.0 M NaCl. Btn2p was originally identified as being up-regulated in a
btn1Δ
strain, which lacks the vacuolar-lysosomal membrane protein, Btn1p, and serves as a model for Batten disease. This up-regulation of Btn2p was shown to contribute to the maintenance of a stable vacuolar pH in the
btn1Δ
strain. Btn1p was subsequently shown to be required for the optimal transport of arginine into the vacuole. Interestingly,
btn1Δ ist2Δ
strains are also unable to grow in the presence of 0.5 or 1.0 M NaCl, and
ist2Δ
suppresses the vacuolar arginine transport defect in
btn1Δ
strains. Although further investigation is required, we speculate that altered vacuolar arginine transport in
btn1Δ
strains represents a mechanism for maintaining or balancing cellular ion homeostasis. Btn2p interacts with at least three proteins that are seemingly involved in different biological functions in different subcellular locations. Due to these multiple interactions, we conclude that Btn2p may play a regulatory role across the cell in response to alterations in the intracellular environment that may be caused by changes in amino acid levels or pH, a disruption in protein trafficking, or imbalances in ion homeostasis resulting from either genetic or environmental manipulation.
Role for Hsp70 Chaperone in
Saccharomyces cerevisiae
Prion Seed Replication
Authors: Song Youtao , Wu Yue-xuan , Jung Giman , Tutar Yusuf , Eisenberg Evan , Greene Lois E. , Masison Daniel C.
Published: February 2005

The
Saccharomyces cerevisiae
[
PSI

+

] prion is a misfolded form of Sup35p that propagates as self-replicating cytoplasmic aggregates. Replication is believed to occur through breakage of transmissible [
PSI

+

] prion particles, or seeds, into more numerous pieces. In [
PSI

+

] cells, large Sup35p aggregates are formed by coalescence of smaller sodium dodecyl sulfate-insoluble polymers. It is uncertain if polymers or higher-order aggregates or both act as prion seeds. A mutant Hsp70 chaperone, Ssa1-21p, reduces the number of transmissible [
PSI

+

] seeds per cell by 10-fold but the overall amount of aggregated Sup35p by only two- to threefold. This discrepancy could be explained if, in
SSA1-21
cells, [
PSI

+

] seeds are larger or more of the aggregated Sup35p does not function as a seed. To visualize differences in aggregate size, we constructed a Sup35-green fluorescent protein (GFP) fusion (NGMC) that has normal Sup35p function and can propagate like [
PSI

+

]. Unlike GFP fusions lacking Sup35p's essential C-terminal domain, NGMC did not form fluorescent foci in log-phase [
PSI

+

] cells. However, using fluorescence recovery after photobleaching and size fractionation techniques, we find evidence that NGMC is aggregated in these cells. Furthermore, the aggregates were larger in
SSA1-21
cells, but the size of NGMC polymers was unchanged. Possibly, NGMC aggregates are bigger in
SSA1-21
cells because they contain more polymers. Our data suggest that Ssa1-21p interferes with disruption of large Sup35p aggregates, which lack or have limited capacity to function as seed, into polymers that function more efficiently as [
PSI

+

] seeds.
Strains and Strategies for Large-Scale Gene Deletion Studies of the Diploid Human Fungal Pathogen
Candida albicans
Authors: Noble Suzanne M. , Johnson Alexander D.
Published: February 2005

Candida albicans
is the most common human fungal pathogen and causes significant morbidity and mortality worldwide. Nevertheless, the basic principles of
C. albicans
pathogenesis remain poorly understood. Of central importance to the study of this organism is the ability to generate homozygous knockout mutants and to analyze them in a mammalian model of pathogenesis.
C. albicans
is diploid, and current strategies for gene deletion typically involve repeated use of the
URA3
selectable marker. These procedures are often time-consuming and inefficient. Moreover,
URA3
expression levels—which are susceptible to chromosome position effects—can themselves affect virulence, thereby complicating analysis of strains constructed with
URA3
as a selectable marker. Here, we describe a set of newly developed reference strains (
leu2Δ/leu2Δ
,
his1Δ/his1Δ
;
arg4Δ/arg4Δ
,
his1Δ/his1Δ
; and
arg4Δ/arg4Δ
,
leu2Δ/leu2Δ
,
his1Δ/his1Δ
) that exhibit wild-type or nearly wild-type virulence in a mouse model. We also describe new disruption marker cassettes and a fusion PCR protocol that permit rapid and highly efficient generation of homozygous knockout mutations in the new
C. albicans
strains. We demonstrate these procedures for two well-studied genes,
TUP1
and
EFG1
, as well as a novel gene,
RBD1
. These tools should permit large-scale genetic analysis of this important human pathogen.
Extrachromosomal Telomeric Circles Contribute to Rad52-, Rad50-, and Polymerase δ-Mediated Telomere-Telomere Recombination in
Saccharomyces cerevisiae
Authors: Lin Chi-Ying , Chang Hsih-Hsuan , Wu Kou-Juey , Tseng Shun-Fu , Lin Chuan-Chuan , Lin Chao-Po , Teng Shu-Chun
Published: February 2005

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the telomerase reverse transcriptase. In both tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative recombination mechanism. By using an in vivo inducible Cre-
loxP
system to generate and trace the fate of marked telomeric DNA-containing rings, the efficiency of telomere-telomere recombination can be determined quantitatively. We show that the telomeric loci are the primary sites at which a marked telomeric ring-containing DNA is observed among wild-type and surviving cells lacking telomerase. Marked telomeric DNAs can be transferred to telomeres and form tandem arrays through Rad52-, Rad50-, and polymerase δ-mediated recombination. Moreover, increases of extrachromosomal telomeric and Y′ rings were observed in telomerase-deficient cells. These results imply that telomeres can use looped-out telomeric rings to promote telomere-telomere recombination in telomerase-deficient
Saccharomyces cerevisiae
.
Yarrowia lipolytica
Mutants Devoid of Pyruvate Carboxylase Activity Show an Unusual Growth Phenotype
Authors: Flores Carmen-Lisset , Gancedo Carlos
Published: February 2005

We have cloned and characterized the gene
PYC1
, encoding the unique pyruvate carboxylase in the dimorphic yeast
Yarrowia lipolytica
. The protein putatively encoded by the cDNA has a length of 1,192 amino acids and shows around 70% identity with pyruvate carboxylases from other organisms. The corresponding genomic DNA possesses an intron of 269 bp located 133 bp downstream of the starting ATG. In the branch motif of the intron, the sequence CCCTAAC, not previously found at this place in spliceosomal introns of
Y. lipolytica
, was uncovered. Disruption of the
PYC1
gene from
Y. lipolytica
did not abolish growth in glucose-ammonium medium, as is the case in other eukaryotic microorganisms. This unusual growth phenotype was due to an incomplete glucose repression of the function of the glyoxylate cycle, as shown by the lack of growth in that medium of double
pyc1 icl1
mutants lacking both pyruvate carboxylase and isocitrate lyase activity. These mutants grew when glutamate, aspartate, or Casamino Acids were added to the glucose-ammonium medium. The cDNA from the
Y. lipolytica PYC1
gene complemented the growth defect of a
Saccharomyces cerevisiae pyc1 pyc2
mutant, but introduction of either the
S. cerevisiae PYC1
or
PYC2
gene into
Y. lipolytica
did not result in detectable pyruvate carboxylase activity or in growth on glucose-ammonium of a
Y. lipolytica pyc1 icl1
double mutant.
The Heterotrimeric G-Protein Subunits GNG-1 and GNB-1 Form a Gβγ Dimer Required for Normal Female Fertility, Asexual Development, and Gα Protein Levels in
Neurospora crassa
Authors: Krystofova Svetlana , Borkovich Katherine A.
Published: February 2005

We have identified a gene encoding a heterotrimeric G protein γ subunit,
gng-1
, from the filamentous fungus
Neurospora crassa. gng-1
possesses a gene structure similar to that of mammalian Gγ genes, consisting of three exons and two introns, with introns present in both the open reading frame and 5′-untranslated region. The GNG-1 amino acid sequence displays high identity to predicted Gγ subunits from other filamentous fungi, including
Giberella zeae
,
Cryphonectria parasitica
,
Trichoderma harzianum
, and
Magnaporthe grisea
. Deletion of
gng-1
leads to developmental defects similar to those previously characterized for Δ
gnb-1
(Gβ) mutants. Δ
gng-1
, Δ
gnb-1
, and Δ
gng-1
Δ
gnb-1
strains conidiate inappropriately in submerged cultures and are female sterile, producing aberrant female reproductive structures. Similar to previous results obtained with Δ
gnb-1
mutants, loss of
gng-1
negatively influences levels of Gα proteins (GNA-1, GNA-2, and GNA-3) in plasma membrane fractions isolated from various tissues of
N. crassa
and leads to a significant reduction in the amount of intracellular cyclic AMP. In addition, we show that GNB-1 is essential for maintenance of normal steady-state levels of GNG-1, suggesting a functional interaction between GNB-1 and GNG-1. Direct evidence for a physical association between GNB-1 and GNG-1 in vivo was provided by coimmunoprecipitation.
Under-recognised paradox of neuropathy from rapid glycaemic control
Authors: Leow M K S , Wyckoff J
Published: February 2005

Insulin induced neuropathy has been reported previously in people with diabetes treated with insulin, and subsequently reported in patients with insulinomas. However, neuropathy caused by rapid glycaemic control in patients with poorly controlled diabetes with chronic hyperglycaemia is not a widely recognised entity among clinicians worldwide. It is expected that this phenomenon of paradoxical complication of neuropathy in the face of drastic decreases in glycosylated haemoglobin concentrations will assume greater importance with clinicians achieving glycaemic targets at a faster pace than before.
Current dilemmas in overseas doctors’ training
Authors: Sandhu D P S
Published: February 2005

International medical graduates (IMGs) are a remarkably successful professional group in the United Kingdom making up to 30% of the NHS work force. Their very success and media publicity about general practice and consultant shortages, has led to a large influx of inexperienced doctors seeking training opportunities in competitive specialties. In 2003 a record 15 549 doctors joined the medical register of which 9336 doctors were non-European Economic Area citizens. The number of candidates sitting PLAB part 1 and part 2 in 2003 rose by 267% and 283% respectively compared with 2001. Changes to Department of Health, Home Office, and deanery regulations with expansion of medical schools, implementation of European Working Time Directive, Modernising Medical Careers, and the future role of the Postgraduate Medical Education and Training Board, will have an important impact on IMGs’ training. Dissemination of realistic information about postgraduate training opportunities is important as the NHS for some time will continue to rely on IMGs.
PROLINE HYDROXYLATION AND GENE EXPRESSION
Authors: Kaelin William G.
Published: February 2005

▪ Abstract  Hypoxia-inducible factor (HIF) is a master transcriptional regulator of hypoxia-inducible genes and consists of a labile α subunit (such as HIF1α) and a stable β subunit (such as HIF1β or ARNT). In the presence of oxygen, HIFα family members are hydroxylated on one of two conserved prolyl residues by members of the egg-laying-defective nine (EGLN) family. Prolyl hydroxylation generates a binding site for a ubiquitin ligase complex containing the von Hippel-Lindau (VHL) tumor suppressor protein, which results in HIFα destruction. In addition, the HIFα transcriptional activation function is modulated further by asparagine hydroxylation by FIH (factor-inhibiting HIF), which affects recruitment of the coactivators p300 and CBP. These findings provide new mechanistic insights into oxygen sensing by metazoans and are the first examples of protein hydroxylation being used in intracellular signaling. The existence of three human EGLN family members, as well as other putative hydroxylases, raises the possibility that this signal is used in other contexts by other proteins.
Regulation of Intestinal Phosphate Transport I. Segmental expression and adaptation to low-Pidiet of the type IIb Na+-Picotransporter in mouse small intestine
Authors: Radanovic Tamara , Wagner Carsten A. , Murer Heini , Biber Jürg
Published: February 2005

The Na+-Picotransporter NaPi-IIb (SLC34A2) has been described to be involved in mouse small intestinal absorption of Piand to be regulated by a number of hormones and metabolic factors. However, a possible segmental expression of NaPi-llb in small intestine has not been addressed so far. Here, we describe that the NaPi-IIb cotransporter is highly abundant in the ileum of mouse small intestine, whereas it is almost absent in the duodenum and in the jejunum. Na+-Picotransport studies with isolated brush border membranes confirmed that NaPi-IIb cotransport is highest in the ileum. Upregulation by a low-phosphate diet of NaPi-IIb and NaPi-IIb cotransport was observed both in the jejunum and the ileum. Furthermore, evidence is provided that a low-phosphate diet provokes an increase of the NaPi-IIb mRNA abundance along the entire small intestine. These data suggest that in mouse small intestine, phosphate is absorbed transcellulary by an Na+-dependent pathway in the ileum, whereas in the duodenum and jejunum, this pathway is of minimal importance. Furthermore, we conclude that along the entire mouse small intestine, low-phosphate diet affects transcription and/or the stability of NaPi-IIb mRNA.
Natural resistance to liver cold ischemia-reperfusion injury associated with the hibernation phenotype
Authors: Lindell Susanne L. , Klahn Shawna L. , Piazza Timothy M. , Mangino Martin J. , Torrealba Jose R. , Southard James H. , Carey Hannah V.
Published: February 2005

The success of liver grafts is currently limited by the length of time organs are cold preserved before transplant. Novel insights to improve viability of cold-stored organs may emerge from studies with animals that naturally experience low body temperatures (Tb) for extended periods. In this study, we tested whether livers from hibernating ground squirrels tolerate cold ischemia-warm reperfusion (cold I/R) for longer times and with better quality than livers from rats or summer squirrels. Hibernators were used when torpid (Tb< 10°C) or aroused (Tb= 37°C). Livers were stored at 4°C in University of Wisconsin solution for 0–72 h and then reperfused with 37°C buffer in vitro. Lactate dehydrogenase (LDH) release after 60 min was increased 37-fold in rat livers after 72 h cold I/R but only 10-fold in summer livers and approximately three- to sixfold in torpid and aroused hibernator livers, despite twofold higher total LDH content in livers from hibernators compared with rats or summer squirrels. Reperfusion for up to 240 min had the least effect on LDH release in livers from hibernators and the greatest effect in rats. Compared with rats or summer squirrels, livers from hibernators after 72 h cold I/R showed better maintenance of mitochondrial respiration, bile production, and sinusoidal lining cell viability, as well as lower vascular resistance and Kupffer cell phagocytosis. These results demonstrate that the hibernation phenotype in ground squirrels confers superior resistance to liver cold I/R injury compared with rats and summer squirrels. Because hibernation-induced protection is not dependent on animals being in the torpid state, the mechanisms responsible for this effect may provide new strategies for liver preservation in humans.
Mechanism of TNF-α modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression
Authors: Ma Thomas Y. , Boivin Michel A. , Ye Dongmei , Pedram Ali , Said Hamid M.
Published: February 2005

TNF-α plays a central role in the intestinal inflammation of various inflammatory disorders including Crohn's disease (CD). TNF-α-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed as one of the proinflammatory mechanisms contributing to the intestinal inflammation. The intracellular mechanisms involved in the TNF-α-induced increase in intestinal TJ permeability remain unclear. The purpose of this study was to investigate the possibility that the TNF-α-induced increase in intestinal epithelial TJ permeability was regulated by myosin light-chain kinase (MLCK) protein expression, using an in vitro intestinal epithelial model system consisting of the filter-grown Caco-2 intestinal epithelial monolayers. TNF-α (10 ng/ml) produced a time-dependent increase in Caco-2 MLCK expression. The TNF-α increase in MLCK protein expression paralleled the increase in Caco-2 TJ permeability, and the inhibition of the TNF-α-induced MLCK expression (by cycloheximide) prevented the increase in Caco-2 TJ permeability, suggesting that MLCK expression may be required for the increase in Caco-2 TJ permeability. The TNF-α increase in MLCK protein expression was preceded by an increase in MLCK mRNA expression but not an alteration in MLCK protein degradation. Actinomycin-D prevented the TNF-α increase in MLCK mRNA expression and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability, suggesting that the increase in MLCK mRNA transcription led to the increase in MLCK expression. The TNF-α increase in MLCK protein expression was also associated with an increase in Caco-2 MLCK activity. The cycloheximide inhibition of MLCK protein expression prevented the TNF-α increase in MLCK activity and Caco-2 TJ permeability. Moreover, inhibitors of MLCK, Mg2+-myosin ATPase, and metabolic energy prevented the TNF-α increase in Caco-2 TJ permeability, suggesting that the increase in MLCK activity was required for the TNF-α-induced opening of the Caco-2 TJ barrier. In conclusion, our results indicate for the first time that 1) the TNF-α increase in Caco-2 TJ permeability was mediated by an increase in MLCK protein expression, 2) the increase in MLCK protein expression was regulated by an increase in MLCK mRNA transcription, and 3) the increase in Caco-2 TJ permeability required MLCK protein expression-dependent increase in MLCK activity.
Physiological Basis for Novel Drug Therapies Used to Treat the Inflammatory Bowel Diseases I. Pathophysiological basis and prospects for probiotic therapy in inflammatory bowel disease
Authors: Shanahan Fergus
Published: February 2005

Mechanisms underlying the conditioning influence of the intestinal flora on mucosal homeostasis, including development and function of immune responses, are attracting increasing scientific scrutiny. The intestinal flora is a positive asset to host defense, but some of its components may, in genetically susceptible hosts, become a risk factor for development of inflammatory bowel disease (IBD). It follows that strategies to enhance assets or offset microbial liabilities represent a therapeutic option; therein lies the rationale for manipulation of the flora in IBD. In addition, the diversity of regulatory signalling among the flora and host epithelum, lymphoid tissue, and neuromuscular apparatus is an untapped reservoir from which novel therapeutics may be mined. Moreover, the capacity to engineer food-grade or commensal bacteria to deliver therapeutic molecules to the intestinal mucosa promises to extend the scope of microbial manipulation for the benefit of mankind.
HSP70 binding modulates detachment of Na-K-ATPase following energy deprivation in renal epithelial cells
Authors: Riordan Michael , Sreedharan Rajasree , Wang Shirley , Thulin Gunilla , Mann Andrea , Stankewich Michael , Van Why Scott , Kashgarian Michael , Siegel Norman J.
Published: February 2005

The molecular mechanisms associated with reestablishment of renal epithelial polarity after injury remain incompletely delineated. Stress proteins may act as molecular chaperones, potentially modulating injury or enhancing recovery. We tested whether overexpression of heat shock protein 70 (HSP70) would stabilize Na-K-ATPase attachment to the cytoskeleton, under conditions of ATP depletion, and whether a direct association existed between Na-K-ATPase and HSP70 in cultured renal epithelial cells. LLC-PK1 cells were transfected with a tagged HSP70 (70FLAG) or vector alone (VA). Detachment of Na-K-ATPase was detected in Triton soluble lysate after ATP depletion. 70FLAG cells demonstrated a significant ( P < 0.01) decrease in detachment of Na-K-ATPase after either 2 or 4 h of ATP depletion. Interactions between HSP70 and Na-K-ATPase were determined by coimmunoprecipitation of 70FLAG and Na-K-ATPase, by direct and competitive binding assays and by immunocytochemical localization. Binding of HSP70 and Na-K-ATPase increased dramatically following injury. Interactions were: 1) reversible; 2) reciprocal to changes in the HSP70 binding protein clathrin; and 3) present only when ATP turnover was inhibited in cell lysate, an established characteristic of HSP binding. These studies indicate that 1) overexpression of HSP70 is associated with decreased detachment of Na-K-ATPase from the cytoskeleton following injury; 2) HSP70 binds to Na-K-ATPase; and 3) binding of HSP70 to Na-K-ATPase is dynamic and specific, increasing in response to injury and decreasing during recovery. Interaction between the molecular chaperone HSP70 and damaged or displaced Na-K-ATPase may represent a fundamental cellular mechanism underlying maintenance and recovery of renal tubule polarity following energy deprivation.
Systemic Acyl-CoA:Cholesterol Acyltransferase Inhibition Reduces Inflammation and Improves Vascular Function in Hypercholesterolemia
Authors: Kharbanda Rajesh K. , Wallace Sharon , Walton Benjamin , Donald Ann , Cross Jennifer M. , Deanfield John
Published: February 2005

Background—

Circulating lipids may initiate and progress atherosclerosis by causing vascular inflammation. Monocytes and tissue macrophages are involved and regulate lipid metabolism in the vascular wall through acetylation of cholesterol by acyl-CoA:cholesterol acyltransferase (ACAT). ACAT inhibition reduces atherosclerosis in animal models by mechanisms that may be independent of their effects on circulating lipids. Because endothelial dysfunction is an important factor in atherosclerosis, we tested the hypothesis that systemic ACAT inhibition would improve endothelial function in hypercholesterolemic humans and assessed its effects on circulating lipids and markers of systemic inflammation.

Methods and Results—

We studied 21 hypercholesterolemic subjects in a double-blind, randomized-crossover, placebo-controlled trial with assessments of circulating lipids, markers of inflammation, resistance-vessel endothelial function (with venous occlusion plethysmography), and conduit-vessel vasoreactivity (brachial artery flow-mediated dilation at baseline and after placebo or treatment with avasimibe 750 mg QDS for 8 weeks. There was a small change in total cholesterol with treatment (326±25 to 311±22 mg/dL,
P
=0.04). Circulating tumor necrosis factor-α was significantly reduced (4.0±0.3 to 3.6±0.2 pg/mL,
P
=0.02); resistance vessel responses to acetylcholine, bradykinin, and verapamil were significantly enhanced; and responses to nitroglycerin and conduit-vessel vasoreactivity were unchanged after ACAT inhibition.

Conclusions—

Systemic ACAT inhibition reduces circulating tumor necrosis factor-α levels in hypercholesterolemic subjects and improves resistance-vessel endothelial function, with small effects on circulating cholesterol. This may be a novel therapeutic strategy to target vascular inflammation and endothelial dysfunction in atherosclerosis.
Adenovirus-Mediated Transfer of Human Placental Ectonucleoside Triphosphate Diphosphohydrolase to Vascular Smooth Muscle Cells Suppresses Platelet Aggregation In Vitro and Arterial Thrombus Formation In Vivo
Authors: Furukoji Eiji , Matsumoto Masanori , Yamashita Atsushi , Yagi Hideo , Sakurai Yoshihiko , Marutsuka Kousuke , Hatakeyama Kinta , Morishita Kazuhiro , Fujimura Yoshihiro , Tamura Shozo , Asada Yujiro
Published: February 2005

Background—

Platelet-rich thrombus formation is a critical event in the onset of cardiovascular disease. Because ADP plays a significant role in platelet aggregation, its metabolism is important in the regulation of platelet activation and recruitment. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) is a key enzyme involved in vascular ADP metabolism. We recently isolated 2 isoforms of E-NTPDase from the human placenta. The present study examined whether these isoforms suppress platelet aggregation and thrombus formation after adenovirus-mediated gene transfer to vascular smooth muscle cells (SMCs).

Methods and Results—

We constructed adenovirus vectors expressing human placental E-NTPDase isoforms I (AdPlac I) and II (AdPlac II) or bacterial β-galactosidase (AdLacZ). Vascular SMCs infected with AdPlac I expressed significant NTPDase activity and inhibited the platelet aggregation induced by ADP and collagen in vitro. In contrast, SMCs infected with AdPlac II and AdLacZ did not exert antiplatelet effects. To investigate the antithrombotic and antiproliferative effects of placental E-NTPDase isoform I in vivo, we generated thrombosis in rat carotid arteries by systemically administered rose Bengal and transluminal green light 5 days after gene transfer and examined neointimal growth 3 weeks after thrombus formation. Blood flow in AdLacZ-infected arteries rapidly deteriorated and vanished within 96±18 seconds of occlusive thrombus formation. In contrast, blood flow in AdPlac I–infected arteries was preserved for at least 10 minutes during irradiation. In addition, thrombus formation and subsequent neointimal growth were obviously suppressed.

Conclusions—

The local expression of placental E-NTPDase in injured arteries might prevent arterial thrombosis and subsequent neointimal growth.
Cardiomyocyte Stiffness in Diastolic Heart Failure
Authors: Borbély Attila , van der Velden Jolanda , Papp Zoltán , Bronzwaer Jean G.F. , Edes Istvan , Stienen Ger J.M. , Paulus Walter J.
Published: February 2005

Background—

Heart failure with preserved left ventricular (LV) ejection fraction (EF) is increasingly recognized and usually referred to as diastolic heart failure (DHF). Its pathogenetic mechanism remains unclear, partly because of a lack of myocardial biopsy material. Endomyocardial biopsy samples obtained from DHF patients were therefore analyzed for collagen volume fraction (CVF) and sarcomeric protein composition and compared with control samples. Single cardiomyocytes were isolated from these biopsy samples to assess cellular contractile performance.

Methods and Results—

DHF patients (n=12) had an LVEF of 71±11%, an LV end-diastolic pressure (LVEDP) of 28±4 mm Hg, and no significant coronary artery stenoses. DHF patients had higher CVFs (7.5±4.0%,
P
<0.05) than did controls (n=8, 3.8±2.0%), and no conspicuous changes in sarcomeric protein composition were detected. Cardiomyocytes, mechanically isolated and treated with Triton X-100 to remove all membranes, were stretched to a sarcomere length of 2.2 μm and activated with solutions containing varying [Ca
2+
]. Compared with cardiomyocytes of controls, cardiomyocytes of DHF patients developed a similar total isometric force at maximal [Ca
2+
], but their resting tension (
F
passive
) in the absence of Ca
2+
was almost twice as high (6.6±3.0 versus 3.5±1.7 kN/m
2
,
P
<0.001).
F
passive
and CVF combined yielded stronger correlations with LVEDP than did either alone. Administration of protein kinase A (PKA) to DHF cardiomyocytes lowered
F
passive
to control values.

Conclusions—

DHF patients had stiffer cardiomyocytes, as evident from a higher
F
passive
at the same sarcomere length. Together with CVF,
F
passive
determined in vivo diastolic LV dysfunction. Correction of this high
F
passive
by PKA suggests that reduced phosphorylation of sarcomeric proteins is involved in DHF.