bluuebunny/crossref_metadata_2025 huggingface dataset:
Monitoring Apoptosis with Fluorescent Zn
2+
-Indicators
Authors: Kimura Eiichi , Takasawa Ryoko , Tanuma Sei-ichi , Aoki Shin
Published: March 2004

Apoptosis, a mechanism of programmed cell death that removes superfluous and harmful cells, is important both during development and in tissue homeostasis. Although Zn
2+
is believed to be critical in apoptosis, the precise details of its role have yet to be elucidated. The macrocyclic Zn
2+
ligand dansylamidoethylcyclen [L
1
•(HCl)
4
•(H
2
O)
2
], which is found primarily in a diprotonated form (H
2
L
1
), is cell-permeable and forms a strongly fluorescent 1:1 Zn
2+
complex when Zn
2+
entry into cells is facilitated by the Zn
2+
ionophore pyrithione. H
2
L
1
can be used to readily identify HeLa cells undergoing the early stages of etoposide-induced apoptosis because of the increased level of free Zn
2+
that occurs at this time. The selectivity of H
2
L
1
for the detection of apoptotic cells was verified by a conventional probe for apoptosis, annexin V-Cy3. Here, we describe methods for detecting apoptotic cells with H
2
L
1
and for comparing detection of apoptosis with H
2
L
1
to detection with annexin V-Cy3 and Zinquin.
Rapid Depletion of Budding Yeast Proteins by Fusion to a Heat-Inducible Degron
Authors: Sanchez-Diaz Alberto , Kanemaki Masato , Marchesi Vanessa , Labib Karim
Published: March 2004

One effective way to study the biological function of a protein in vivo is to inactivate it and see what happens to the cell. For proteins that are dispensable for cell viability, the corresponding gene can simply be deleted from its chromosomal locus. The study of essential proteins is more challenging, however, because the function of the protein must be inactivated conditionally. Here, we describe a method that allows the target protein to be depleted rapidly and conditionally, so that the immediate effects on the cell can be examined. The chromosomal locus of a budding yeast gene is modified so that a "heat-inducible degron cassette" is added to the N terminus of the encoded protein, causing it to be degraded by a specific ubiquitin-mediated pathway when cells are shifted from 24° to 37°C. Degradation requires recognition of the degron cassette by the evolutionarily conserved Ubr1 protein, which is associated with a ubiquitin-conjugating enzyme. To promote rapid and conditional depletion of the target protein, we use a yeast strain in which expression of the
UBR1
gene can be either repressed or strongly induced. Degron strains are constructed by a simple "one-step" approach using the polymerase chain reaction.
The LspB Protein Is Involved in the Secretion of the LspA1 and LspA2 Proteins byHaemophilus ducreyi
Authors: Ward Christine K. , Mock Jason R. , Hansen Eric J.
Published: March 2004

The LspA1 and LspA2 proteins ofHaemophilus ducreyi35000 are two very large macromolecules that can be detected in concentrated culture supernatant fluid. Both of these proteins exhibit homology with the N-terminal region of theBordetella pertussisfilamentous hemagglutinin (FHA), which is involved in secretion of the latter macromolecule. ThelspA2open reading frame is flanked upstream by a gene,lspB, that encodes a predicted protein with homology to theB. pertussisFhaC outer membrane protein that is involved in secretion of FHA across the outer membrane. TheH. ducreyi lspBgene encodes a protein with a predicted molecular mass of 66,573 Da. Reverse transcription-PCR analysis suggested that thelspBgene was transcribed together with thelspA2gene on a single mRNA transcript. PolyclonalH. ducreyiLspB antiserum reacted with a 64-kDa antigen present in the Sarkosyl-insoluble cell envelope fraction ofH. ducreyi35000, which indicated that the LspB protein is likely an outer membrane protein. Concentrated culture supernatant fluids fromH. ducreyi lspBandlspA1 lspBmutants did not contain detectable LspA1 and detectable LspA2, respectively. However, complementation of thelspBmutant with the wild-typelspBgene on a plasmid restored LspB protein expression and resulted in release of detectable amounts of the LspA1 protein into culture supernatant fluid. When evaluated in the temperature-dependent rabbit model of infection, thelspBmutant was attenuated in the ability to cause lesions and was never recovered in a viable form from lesions. These results indicated that theH. ducreyiLspB protein is involved in secretion of the LspA1 and LspA2 proteins across the outer membrane.
GNA33 of
Neisseria meningitidis
Is a Lipoprotein Required for Cell Separation, Membrane Architecture, and Virulence
Authors: Adu-Bobie Jeannette , Lupetti Pietro , Brunelli Brunella , Granoff Dan , Norais Nathalie , Ferrari Germano , Grandi Guido , Rappuoli Rino , Pizza Mariagrazia
Published: March 2004

GNA33 is a membrane-bound lipoprotein with murein hydrolase activity that is present in all
Neisseria
species and well conserved in different meningococcal isolates. The protein shows 33% identity to a lytic transglycolase (MltA) from
Escherichia coli
and has been shown to be involved in the degradation of both insoluble murein sacculi and unsubstituted glycan strands. To study the function of the gene and its role in pathogenesis and virulence, a knockout mutant of a
Neisseria meningitidis
serogroup B strain was generated. The mutant exhibited retarded growth in vitro. Transmission electron microscopy revealed that the mutant grows in clusters which are connected by a continuous outer membrane, suggesting a failure in the separation of daughter cells. Moreover, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatant revealed that the mutant releases several proteins in the medium. The five most abundant proteins, identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, belong to the outer membrane protein family. Finally, the mutant showed an attenuated phenotype, since it was not able to cause bacteremia in the infant rat model. We conclude that GNA33 is a highly conserved lipoprotein which plays an important role in peptidoglycan metabolism, cell separation, membrane architecture, and virulence.
Aeromonas
Flagella (Polar and Lateral) Are Enterocyte Adhesins That Contribute to Biofilm Formation on Surfaces
Authors: Kirov Sylvia M. , Castrisios Marika , Shaw Jonathan G.
Published: March 2004

Aeromonas
spp. (gram-negative, aquatic bacteria which include enteropathogenic strains) have two distinct flagellar systems, namely a polar flagellum for swimming in liquid and multiple lateral flagella for swarming over surfaces. Only ∼60% of mesophilic strains can produce lateral flagella. To evaluate flagellar contributions to
Aeromonas
intestinal colonization, we compared polar and lateral flagellar mutant strains of a diarrheal isolate of
Aeromonas caviae
for the ability to adhere to the intestinal cell lines Henle 407 and Caco-2, which have the characteristic features of human intestinal enterocytes. Strains lacking polar flagella were virtually nonadherent to these cell lines, while loss of the lateral flagellum decreased adherence by ∼60% in comparison to the wild-type level. Motility mutants (unable to swim or swarm in agar assays) had adhesion levels of ∼50% of wild-type values, irrespective of their flagellar expression. Flagellar mutant strains were also evaluated for the ability to form biofilms in a borosilicate glass tube model which was optimized for
Aeromonas
spp. (broth inoculum, with a 16- to 20-h incubation at 37°C). All flagellar mutants showed a decreased ability to form biofilms (at least 30% lower than the wild type). For the chemotactic motility mutant
cheA
, biofilm formation decreased >80% from the wild-type level. The complementation of flagellar phenotypes (polar flagellar mutants) restored biofilms to wild-type levels. We concluded that both flagellar types are enterocyte adhesins and need to be fully functional for optimal biofilm formation.
Salivary Secretory Leukocyte Protease Inhibitor and Oral Candidiasis in Human Immunodeficiency Virus Type 1-Infected Persons
Authors: Chattopadhyay Amit , Gray Laurie R. , Patton Lauren L. , Caplan Daniel J. , Slade Gary D. , Tien Hsaio-Chuan , Shugars Diane C.
Published: March 2004

Oropharyngeal candidiasis, typically caused by
Candida albicans
, is the most common oral disease associated with human immunodeficiency virus type 1 (HIV-1) infection. Secretory leukocyte protease inhibitor (SLPI), a 12-kDa antiprotease, suppresses the growth of
C. albicans
in vitro. To determine whether the mucosal protein plays a role in protecting oral tissues against fungal infection, we conducted a cross-sectional study investigating the oral and systemic health and salivary SLPI levels in 91 dentate HIV-1-infected adults receiving medical care in the southeastern United States. Participants with a self-reported history of clinical oropharyngeal candidiasis during the previous 2 years constituted the test group (
n
= 52), while the comparison group (
n
= 39) had no oropharyngeal candidiasis during that period. Data collected from medical records, oral examination, and SLPI enzyme-linked immunosorbent assay quantitation of whole saliva were analyzed by
t
test, analysis of variance, linear regression, and unconditional logistic regression. The test group had a significantly higher mean salivary SLPI level than the comparison group (1.9 μg/ml versus 1.1 μg/ml,
P
< 0.05). Linear regression modeling identified CD4 cell count and history of oropharyngeal candidiasis as key predictors of salivary SLPI and revealed a significant interaction (
P
< 0.05) between immunosuppression (CD4 cell count below 200 cells/μl) and positive history of oropharyngeal candidiasis in predicting salivary SLPI level. By logistic regression modeling, a salivary SLPI level exceeding 2.1 μg/ml, low CD4 count, antiretroviral monotherapy, and smoking were key predictors of oropharyngeal candidiasis. These data support a key role for SLPI in the oral mucosal defense against
C. albicans
. The antimicrobial mucosal protein may serve as an indicator of previous oropharyngeal candidiasis infection among immunosuppressed persons.
An Abundance of
Escherichia coli
Is Harbored by the Mucosa- Associated Bacterial Flora of Interleukin-2-Deficient Mice
Authors: Schuppler M. , Lötzsch K. , Waidmann M. , Autenrieth I. B.
Published: March 2004

Mice deficient in interleukin-2 are well suited for use as an animal model for inflammatory bowel disease. Raised under specific-pathogen-free conditions, interleukin-2-deficient mice develop an inflammatory bowel disease resembling ulcerative colitis in humans. The finding that colitis was attenuated when the mice were kept under germfree conditions implies that the resident intestinal flora is involved in the pathogenesis of colitis. The present study addresses the composition of the mucosa-associated bacterial flora in colon samples from interleukin-2-deficient mice that developed colitis. This was investigated by comparative 16S ribosomal DNA (rDNA) sequence analysis and fluorescence in situ hybridization using rRNA-targeted fluorescent probes to quantify the bacterial populations of the mucosa-associated flora. The investigations revealed distinct differences in the bacterial composition of the mucosa-associated flora between interleukin-2-deficient mice and healthy controls. Fluorescence in situ hybridization identified up to 10% of the mucosa-associated flora in interleukin-2-deficient mice as
Escherichia coli
, whereas no
E. coli
was detected in the mucosa from healthy wild-type mice. This finding was consistent with the results from comparative 16S rDNA analysis. About one-third of the clones analyzed from 16S rDNA libraries of interleukin-2-deficient mice represented
Enterobacteriaceae
, whereas none of the clones analyzed from the healthy controls harbored 16S rDNA from
Enterobacteriaceae
. The abundance of
E. coli
in the colonic mucosa of interleukin-2-deficient mice strongly suggests a participation in the pathogenesis of colitis in the interleukin-2-deficient mouse model for inflammatory bowel disease.
Mycobacterium tuberculosisLipomannan Induces Apoptosis and Interleukin-12 Production in Macrophages
Authors: Dao D. N. , Kremer L. , Guérardel Y. , Molano A. , Jacobs W. R. , Porcelli S. A. , Briken V.
Published: March 2004

The mycobacterial cell wall component lipoarabinomannan (LAM) has been described as a virulence factor ofMycobacterium tuberculosis, and modification of the terminal arabinan residues of this compound with mannose caps (producing mannosyl-capped LAM [ManLAM]) inM. tuberculosisor with phosphoinositol caps (producing phosphoinositol-capped LAM [PILAM]) inMycobacterium smegmatishas been implicated in various functions associated with these lipoglycans. A structure-function analysis was performed by using LAMs and their biosynthetic precursor lipomannans (LMs) isolated from different mycobacterial species on the basis of their capacity to induce the production of interleukin-12 (IL-12) and/or apoptosis of macrophage cell lines. Independent of the mycobacterial species, ManLAMs did not induce IL-12 gene expression or apoptosis of macrophages, whereas PILAMs induced IL-12 secretion and apoptosis. Interestingly, uncapped LAM purified fromMycobacterium chelonaedid not induce IL-12 secretion or apoptosis. Furthermore, LMs, independent of their mycobacterial origins, were potent inducers of IL-12 and apoptosis. The precursor of LM, phosphatidyl-myo-inositol dimannoside, had no activity, suggesting that the mannan core of LM was required for the activity of LM. The specific interaction of LM with Toll-like receptor 2 (TLR-2) but not with TLR-4 suggested that these responses were mediated via the TLR-2 signaling pathway. Our experiments revealed an important immunostimulatory activity of the biosynthetic LAM precursor LM. The ratio of LAM to LM in the cell wall of mycobacteria may be an important determinant of virulence, and enzymes that modify LM could provide targets for development of antituberculosis drugs and for derivation of attenuated strains ofM. tuberculosis.
Reactive Oxygen and Nitrogen Species Differentially Regulate Toll-Like Receptor 4-Mediated Activation of NF-κB and Interleukin-8 Expression
Authors: Ryan Kieran A. , Smith Michael F. , Sanders Michael K. , Ernst Peter B.
Published: March 2004

Toll-like receptor 4 (TLR4) has been identified as a transmembrane protein involved in the host innate immune response to gram-negative bacterial lipopolysaccharide (LPS). Upon activation by LPS recognition, the TIR domain of TLR4 signals through MyD88 to activate the nuclear factor κB (NF-κB) pathway, a critical regulator of many proinflammatory genes, including interleukin-8 (IL-8). Emerging evidence suggests that reactive oxygen species (ROS) can contribute to diverse signaling pathways, including the LPS-induced cascade. In the present study we investigated the role of ROS in TLR-mediated signaling. PurifiedEscherichia coliLPS, a highly specific TLR4 agonist, elicited an oxidative burst in the monocyte-like cell line THP-1 in a time- and dose-dependent manner. This oxidative burst was shown to be dependent on the presence of TLR4 through transfection studies in HEK cells, which do not normally express this protein, and with bone marrow-derived macrophages from C3H/HeJ mice, which express a mutated TLR4 protein. LPS-stimulated IL-8 expression could be blocked by the antioxidantsN-acetyl-l-cysteine and dimethyl sulfoxide at both the protein and mRNA levels. These antioxidants also blocked LPS-induced IL-8 promoter transactivation as well as the nuclear translocation of NF-κB. These data provide evidence that ROS regulate immune signaling through TLR4 via their effects on NF-κB activation.
Rapid Expression of Chemokines and Proinflammatory Cytokines in Newly Hatched Chickens Infected withSalmonella entericaSerovar Typhimurium
Authors: Withanage G. S. K. , Kaiser Pete , Wigley Paul , Powers Claire , Mastroeni Pietro , Brooks Heather , Barrow Paul , Smith Adrian , Maskell Duncan , McConnell Ian
Published: March 2004

Poultry meat and eggs contaminated withSalmonella entericaserovar Enteritidis orSalmonella entericaserovar Typhimurium are common sources of acute gastroenteritis in humans. However, the exact nature of the immune mechanisms protective againstSalmonellainfection in chickens has not been characterized at the molecular level. In the present study, bacterial colonization, development of pathological lesions, and proinflammatory cytokine and chemokine gene expression were investigated in the liver, spleen, jejunum, ileum, and cecal tonsils in newly hatched chickens 6, 12, 24, and 48 h after oral infection withSalmonellaserovar Typhimurium. Very high bacterial counts were found in the ileum and cecal contents throughout the experiment, whereasSalmonellastarted to appear in the liver only from 24 h postinfection. Large numbers of heterophils, equivalent to neutrophils in mammals, and inflammatory edema could be seen in the lamina propria of the intestinal villi and in the liver. Interleukin 8 (IL-8), K60 (a CXC chemokine), macrophage inflammatory protein 1 β, and IL-1β levels were significantly upregulated in the intestinal tissues and in the livers of the infected birds. However, the spleens of the infected birds show little or no change in the expression levels of these cytokines and chemokines. Increased expression of the proinflammatory cytokines and chemokines (up to several hundred-fold) correlated with the presence of inflammatory signs in those tissues. This is the first description of in vivo expression of chemokines and proinflammatory cytokines in response to oral infection withSalmonellain newly hatched chickens.
Role of Biofilm-Associated Protein Bap in the Pathogenesis of Bovine
Staphylococcus aureus
Authors: Cucarella Carme , Tormo M. Ángeles , Úbeda Carles , Trotonda M. Pilar , Monzón Marta , Peris Critòfol , Amorena Beatriz , Lasa Íñigo , Penadés José R.
Published: March 2004

Staphylococcus aureus
is a common cause of intramammary infections, which frequently become chronic, associated with the ability of the bacteria to produce biofilm. Here, we report a relationship between the ability to produce chronic bovine mastitis and biofilm formation. We have classified bovine mastitis
S. aureus
isolates into three groups based on the presence of particular genetic elements required for biofilm formation: group 1 (
ica
+
bap
+
), group 2 (
ica
+
,
bap
negative), and group 3 (
ica
negative,
bap
negative). Overall, animals naturally infected with group 1 and 2 isolates had a lower milk somatic cell count than those infected with isolates of group 3. In addition, Bap-positive isolates were significantly more able to colonize and persist in the bovine mammary gland in vivo and were less susceptible to antibiotic treatments when forming biofilms in vitro. Analysis of the structural
bap
gene revealed the existence of alternate forms of expression of the Bap protein in
S. aureus
isolates obtained under field conditions throughout the animal's life. The presence of anti-Bap antibodies in serum samples taken from animals with confirmed
S. aureus
infections indicated the production of Bap during infection. Furthermore, disruption of the
ica
operon in a
bap
-positive strain had no effect on in vitro biofilm formation, a finding which strongly suggested that Bap could compensate for the deficiency of the PIA/PNAG product (a biofilm matrix polysaccharide). Altogether, these results demonstrate that, in the bovine intramammary gland, the presence of Bap may facilitate a biofilm formation connected with the persistence of
S. aureus
.
Localization of BmpA on the Exposed Outer Membrane ofBorrelia burgdorferiby Monospecific Anti-Recombinant BmpA Rabbit Antibodies
Authors: Shin Junghee J. , Bryksin Anton V. , Godfrey Henry P. , Cabello Felipe C.
Published: March 2004

BmpA (P39) is an immunodominant chromosomally encodedBorrelia burgdorferiprotein. The potential strong cross-reactivity of anti-BmpA antibodies with the other members of this paralogous protein family and the previous use of antibodies whose reactivity to the other Bmp proteins was uncharacterized have resulted in continued controversy over its localization inB. burgdorferi. In an effort to provide a definitive demonstration of the localization of BmpA, rabbit antibodies raised to recombinant BmpA (rBmpA) were rendered monospecific by absorption with rBmpB. This reagent did not react with rBmpB, rBmpC, or rBmpD in dot immunobinding, detected only a single 39-kDa band and a single 39-kDa, pI 5.0 spot on one- and two-dimensional immunoblots ofB. burgdorferilysates, respectively, and immunoprecipitated a single 39-kDa protein from these lysates. It detected BmpA in the Triton X-114-soluble and -insoluble fractions ofB. burgdorferi, suggesting association with both inner and outer bacterial cell membranes. Treatment of intactB. burgdorferiwith proteinase K partially digested BmpA, consistent with a limited surface exposure on the outer bacterial membrane, a suggestion confirmed by immunofluorescence of unfixedB. burgdorfericultured in vitro and in vivo. Anti-rBmpA antibody was bacteriostatic forB. burgdorferiB31 in culture, again suggesting localization of BmpA on the exposed spirochetal outer surface. Surface localization of BmpA, growth inhibition by anti-rBmpA antibodies, and the previously reported conservation ofbmpAin differentB. burgdorferisensu lato strains may indicate that BmpA plays an essential role inB. burgdorferibiology.
Identification of a Novel
Citrobacter rodentium
Type III Secreted Protein, EspI, and Roles of This and Other Secreted Proteins in Infection
Authors: Mundy Rosanna , Petrovska Liljana , Smollett Katherine , Simpson Nandi , Wilson Rebecca K. , Yu Jun , Tu Xuanlin , Rosenshine Ilan , Clare Simon , Dougan Gordon , Frankel Gad
Published: March 2004

Citrobacter rodentium
is a member of a group of pathogens that colonize the lumen of the host gastrointestinal tract via attaching and effacing (A/E) lesion formation.
C. rodentium
, which causes transmissible colonic hyperplasia in mice, is used as an in vivo model system for the clinically significant A/E pathogens enterohemorrhagic and enteropathogenic
Escherichia coli
. These bacteria all contain a pathogenicity island called the locus of enterocyte effacement (LEE), which encodes a type III secretion system that is designed to deliver effector proteins into eukaryotic host cells. These effectors are involved in the subversion of host eukaryotic cell functions to the benefit of the bacterium. In this study we used mutant strains to determine the effects of the
C. rodentium
LEE-encoded effectors EspF, EspG, EspH, and Map on virulence in the mouse model. In addition, we identified a novel secreted protein, EspI encoded outside the LEE, whose secretion is also dependent on a functional type III secretion system. Mutant strains with each of the effectors investigated were found to be outcompeted by wild-type bacteria in mixed-infection experiments in vivo, although the effects of EspF and EspH were only subtle. In single-infection experiments, we found that EspF, EspG, and EspH are not required for efficient colonization of the mouse colon or for the production of hyperplasia. In contrast, strains producing EspI and Map had significant colonization defects and resulted in dramatically reduced levels of hyperplasia, and they exhibited very different growth dynamics in mice than the wild-type strain exhibited.
Porphyromonas gingivalis
-Specific Immunoglobulin G Prevents
P. gingivalis
-Elicited Oral Bone Loss in a Murine Model
Authors: Gibson Frank C. , Gonzalez Dario A. , Wong Jodie , Genco Caroline Attardo
Published: March 2004

Active immunization with
Porphyromonas gingivalis
whole-cell preparations has been shown to prevent
P. gingivalis
infection and oral bone loss. Employing passive antibody transfer and opsonization, we demonstrate with this study that immunization-elicited
P. gingivalis
-specific immunoglobulin G facilitates clearance of
P. gingivalis
in a subcutaneous chamber model and prevents
P. gingivalis
-elicited oral bone loss.
Protein Serine/Threonine Kinase StkP Positively Controls Virulence and Competence in
Streptococcus pneumoniae
Authors: Echenique Jose , Kadioglu Aras , Romao Susana , Andrew Peter W. , Trombe Marie-Claude
Published: March 2004

In the
Streptococcus pneumoniae
genome,
stkP
, encoding a membrane-associated serine/threonine kinase, is not redundant (L. Novakova, S. Romao, J. Echenique, P. Branny, and M.-C. Trombe, unpublished results). The data presented here demonstrate that StkP belongs to the signaling network involved in competence triggering in vitro and lung infection and bloodstream invasion in vivo. In competence, functional StkP is required for activation of
comCDE
upstream of the autoregulated ring orchestrated by the competence-stimulating peptide. This is the first description of positive regulation of
comCDE
transcription in balance with its repression by CiaRH.
Monitoring of DNA Adducts in Humans and 32P-Postlabelling Methods. A Review
Authors: Stiborová Marie , Rupertová Martina , Hodek Petr , Frei Eva , Schmeiser Heinz H.
Published: March 2004

DNA adduct formation in humans is a promising biomarker for elucidating the molecular epidemiology of cancer. For detection of DNA adducts, the most widely used methods include mass spectroscopy, fluorescence spectroscopy, immunoassays and 32P-postlabelling. Among them, the 32P-postlabelling method appears to meet best the criteria of sensitivity and amount of DNA needed, and, therefore, is one of the most appropriate methods for biomonitoring of human DNA adducts. Most classes of carcinogens have been subjected to 32P-postlabelling analysis, ranging from bulky and/or aromatic compounds to small and/or aliphatic compounds; it has also been used, with modifications, to detect apurinic sites in DNA, oxidative damage to DNA, UV-induced photodimers and, to a lesser extent, DNA damage caused by cytotoxic drugs. It has been used in human biomonitoring studies to detect DNA damage from occupational exposure to carcinogens, and also from environmental (i.e. non-occupational) exposures. It has also led to the discovery of the presence of numerous modifications in DNA arising from endogenous processes. The principle of the method is the enzymatic digestion of DNA to nucleotides, 5'-labelling of these nucleotides with an isotopically labelled phosphate group, and the resolution, detection and quantitation of the labelled products. Since the development of the original procedure in the early 1980s, many methods have been developed to increase the sensitivity by enrichment of modified nucleotides prior to labelling. The review presents the individual 32P-postlabelling techniques (standard procedure, enrichment methods) and a critical evaluation of these assays, besides reviewing the applications of the method to different DNA modifications, and its utilization in human biomonitoring studies. A review with 179 references.
Carbohydrate and Non-Carbohydrate Ligands for the C-Type Lectin-Like Receptors of Natural Killer Cells. A Review
Authors: Bezouška Karel
Published: March 2004

The superfamily of C-type animal lectins is defined by a sequence motif of the carbohydrate- recognition domains (CRDs) and comprises seven groups of molecules. The soluble proteins are group I proteoglycans, group III collectins, and group VII containing the isolated CRDs. Type I membrane proteins include group IV selectins and group VI macrophage receptors and related molecules. Type II membrane proteins are group II hepatic lectins and group V natural killer cell receptors. The latter group has recently attracted considerable attention of the biomedical community. These receptors are arranged at the surface of lymphocytes as homo- or heterodimers composed of two polypeptides consisting of N-terminal peptide tails responsible for signaling, transmembrane domain, neck regions of varying length, and C-terminal lectin-like domains (CTLDs). Since this group is evolutionarily most distant from the rest of C-type animal lectins, the sequence of the C-terminal ligand-binding domain has diversified to accommodate other ligands than calcium or carbohydrates. These domains are referred to as natural killer domains (NKDs) forming a large percentage of CTLDs in vertebrates. Here are summarized the data indicating that calcium, carbohydrates, peptides, and large proteins such as major histocompatibility complex (MHC) class I can all be ligands for NKDs. The wide range of ligands that can be recognized by NKDs includes some new, unexpected compounds such as signal peptide-derived fragments, heat shock proteins, or oxidized lipids. The biological importance of this extended range of recognition abilities is also discussed. A review with 134 references.
Is risk of fast bowling injury in cricketers greatest in those who bowl most? A cohort of young English fast bowlers
Authors: Gregory P L , Batt M E , Wallace W A
Published: March 2004

Objectives:To determine whether young fast bowlers are exceeding directives limiting bowling and how incidence of fast bowling injury varies with amount of bowling.

Methods:A prospective cohort study of injuries sustained by 70 fast bowlers (mean (SD) age 15.3 (2.4) years) was undertaken. Bowlers were recruited from the Centres of Excellence of three “First Class” counties in England in January 1998. Details of injuries were collected by telephone questionnaire every six weeks for six months from each bowler. The number of balls bowled in matches and practices by each bowler was recorded. The cohort was divided into groups according to the number of balls bowled in the study period, and bowling injury incidences were calculated for each group.

Main outcome measures:All injuries caused by bowling and interfering with bowling.

Results:Telephone follow up was achieved when planned on 97.9% of occasions. There were 23 bowling injuries reported in the study period that met the inclusion criteria. The overall incidence of bowling injury in the study period was 32.8 per 100 fast bowlers. The incidence of bowling injury for the 15 bowlers who bowled less than 1000 balls in the study period was 20.0 per 100 fast bowlers. The incidence for the 32 who bowled 1000–2000 balls was 37.5 per 100 fast bowlers. The incidence for the 14 who bowled 2000–3000 balls was 35.7 per 100. The incidence for the nine who bowled more than 3000 balls was 33.3 per 100.

Conclusions:The expected increased incidence of bowling injury in young fast bowlers who bowl most was not observed, although more than 12% exceeded the recommended limit.
Hormonal responses during prolonged exercise are influenced by a selective DA/NA reuptake inhibitor
Authors: Piacentini M F , Meeusen R , Buyse L , De Schutter G , De Meirleir K
Published: March 2004

Objective:A decrease in dopamine activity is thought to lead to a reduction in motivation and arousal and therefore to the “central” component of fatigue. The purpose of the present study was to investigate the effects of a dopamine (DA) noradrenaline (NA) reuptake inhibitor, bupropion (Zyban™), on exercise performance and on the hormonal response to exercise.

Methods:Eight healthy well trained male cyclists (Wattmax397±15 W) participated in the study. Subjects completed one maximal exercise test (to determine maximal power output Wattmax), and two endurance performance tests (time trials) in a double blind randomised cross-over design. Subjects took either placebo capsules (lactose) or 2×300 mg bupropion (BUP). Blood samples were collected for adrenocorticotropin (ACTH), prolactin, cortisol, growth hormone, beta-endorphins, and catecholamines.

Results:Performance was not influenced by BUP (placebo: 89±1 min; BUP 2×300 mg: 89±0.7 min). All hormones increased during exercise in all trials. Cortisol plasma concentrations were significantly higher in the BUP trial at rest, at min 60, and at the end of exercise, while beta-endorphins were higher in the BUP trial at the end of exercise and during recovery, and ACTH at the end of exercise.

Conclusion:From the present results, we can conclude that bupropion had a more marked central noradrenergic effect (compared to dopaminergic) on the hormonal response to exercise, but no effect on the outcome of performance.
Non-participation in sports injury research: why football players choose not to be involved: Table 1
Authors: Braham R , Finch C , McCrory P
Published: March 2004

Objective:To ascertain the reasons behind players not participating in a sports safety research project.

Methods:During the preseason, 10 Australian football clubs volunteered 23 teams to participate in a protective equipment randomised controlled trial, the Australian Football Injury Prevention Project (AFIPP). All players from these teams were invited to participate. Players who did not agree to participate in AFIPP were surveyed about their reasons for non-involvement.

Results:110 football players (response rate 63.6%) completed the non-responder survey and cited the two main reasons behind non-involvement in the project as “I did not know about the project” (39.4%) and “I was not at training when the research team visited” (36.5%).

Conclusions and implications:Preseason may not be the best time for maximal player recruitment in community based sports safety research. Enhanced communication between researchers and players at community level football clubs during the recruitment phase is likely to improve response rates.
Revalidation for general practitioners: randomised comparison of two revalidation models
Authors: Bruce David , Phillips Katie , Reid Ross , Snadden David , Harden Ronald
Published: March 2004

Objective To compare two models of revalidation for general practitioners.

Design Randomised comparison of two revalidation models.

Setting Primary care in Tayside, Scotland.

Participants 66 Tayside general practitioners (principals and non-principals), 53 of whom completed the revalidation folders.

Interventions Two revalidation models: a minimum criterion based model with revalidation as the primary purpose, and an educational outcome model with emphasis on combining revalidation with continuing professional development.

Main outcome measures Feasibility and acceptability of each approach and effect on the doctor's continuing professional development. The ability to make a summative judgment on completed models and whether either model would allow patient groups to have confidence in the revalidation process.

Results The criterion model was preferred by general practitioners. For both models doctors reported making changes to their practice and felt a positive effect on their continuing professional development. Summative assessment of the folders showed reasonable inter-rater reliability.

Conclusions The criterion model provides a practical and acceptable model for general practitioners to use when preparing for revalidation.
SCATTERING OF MASSLESS DIRAC FIELDS BY A KERR BLACK HOLE
Authors: HÄFNER DIETRICH , NICOLAS JEAN-PHILIPPE
Published: March 2004

For the massless Dirac equation outside a slow Kerr black hole, we prove asymptotic completeness. We introduce a new Newman–Penrose tetrad in which the expression of the equation contains no artificial long-range perturbations. The main technique used is then a Mourre estimate. The geometry near the horizon requires us to apply a unitary transformation before we find ourselves in a situation where the generator of dilations is a good conjugate operator. The results are eventually re-interpreted geometrically to provide the solution to a Goursat problem on the Penrose compactified exterior.
PARTICLE PRODUCTION BY THE THICK-WALLED BUBBLE
Authors: MAZIASHVILI MICHAEL
Published: March 2004

The spectrum of created particles during the tunneling process, leading to the decay of a false vacuum state, is studied numerically in the thick-wall approximation. It is shown that in this case the particle production is very intensive for small momenta. The number of created particles is nearly constant n(p)≈1 for 4≤p≤500.
QUANTUM HALL SKYRMIONS IN THE FRAMEWORK OF O(4) NONLINEAR SIGMA MODEL
Authors: BASU B. , DHAR S. , BANDYOPADHYAY P.
Published: March 2004

A new framework for quantum Hall skyrmions in O(4) nonlinear sigma model is studied here. The size and energy of the skyrmions are determined incorporating the quartic stability term in the Lagrangian. Moreover, the introduction of a θ-term determines the spin and statistics of these skyrmions.
SOME EXACT FORMULAE ON LONG-RANGE CORRELATION FUNCTIONS OF THE RECTANGULAR ISING LATTICE
Authors: CHANG SHU-CHIUAN , SUZUKI MASUO
Published: March 2004

We study long-range correlation functions of the rectangular Ising lattice with cyclic boundary conditions. Specifically, we consider the situation in which two spins are on the same column, and at least one spin is on or near free boundaries. The low-temperature series expansions of the correlation functions are presented when the spin–spin couplings are the same in both directions. The exact correlation functions can be obtained by D log Padé for the cases with simple algebraic resultant expressions. The present results show that when the two spins are infinitely far from each other, the correlation function is equal to the product of the row magnetizations of the corresponding spins, as expected. In terms of low-temperature series expansions, the approach of this m th row correlation function to the bulk correlation function for increasing m can be understood from the observation that the dominant terms of their series expansions are the same successively in the above two correlation functions. The number of these dominant terms increases monotonically as m increases.
SUPER-SYNERGY IN THE BRAIN: THE GRANDMOTHER PERCEPT IS MANIFESTED BY MULTIPLE OSCILLATIONS
Authors: BAŞAR EROL , ÖZGÖREN MURAT , KARAKAŞ SIREL , BAŞAR-EROĞLU CANAN
Published: March 2004

The present report describes the dynamic foundations of long-standing experimental work in the field of oscillatory dynamics in the human and animal brain. It aims to show the role of multiple oscillations in the integrative brain function, memory, and complex perception by a recently introduced conceptional framework: the super-synergy in the whole brain. Results of recent experiments related to the percept of the grandmother-face support our concept of super-synergy in the whole brain in order to explain manifestation of Gestalts and Memory-Stages. This report may also provide new research avenues in macrodynamics of the brain.
DISTANCES OF TIME SERIES COMPONENTS BY MEANS OF SYMBOLIC DYNAMICS
Authors: KELLER KARSTEN , WITTFELD KATHARINA
Published: March 2004

In this note we describe a simple method for visualizing time-dependent similarities and dissimilarities between the components of a high-dimensional time series. On the base of symbolic dynamics, the time series is turned into a series of matrices whose rows quantify pattern types in the components of the original series. For different scales we introduce distances between the components via the obtained pattern type distributions and approximate them in a one-dimensional manner. The method is illustrated for 19-channel EEG data.
HOW AND WHY BRAINS CREATE MEANING FROM SENSORY INFORMATION
Authors: FREEMAN WALTER J.
Published: March 2004

Semantics is the essence of human communication. It concerns the manufacture and use of symbols as representations to exchange meanings. Information technology is faced with the problem of using intelligent machines as intermediaries for interpersonal communication. The problem of designing such semantic machines has been intractable because brains and machines work on very different principles. Machines process information that is fed to them. Brains construct hypotheses and test them by acting and sensing. Brains do not process information because the intake through the senses is infinite. Brains sample information, hold it briefly, construct meaning, and then discard the information. A solution to the problem of communication with machines is to simulate how brains create meaning and express it as information by making a symbol to represent the meaning to another brain in pairwise communication. An understanding of the neurodynamics by which brains create meaning and represent it may enable engineers to build devices with which they can communicate pairwise, as they do now with colleagues.
CORRELATES OF PERCEPTIVE INSTABILITIES IN EVENT-RELATED POTENTIALS
Authors: KORNMEIER JÜRGEN , BACH MICHAEL , ATMANSPACHER HARALD
Published: March 2004

The study of instabilities in perception has attracted much interest in recent decades. The investigations presented here focus on electrophysiological correlates of orientation reversals of both ambiguous visual stimuli and alternating nonambiguous stimuli, representing the two options of the ambiguous version. Based on a refined experimental setup, significant features in the event-related potentials associated with the perception of orientation reversal were found in both cases. Their occipital location, their early occurrence (200–250 ms), and their latency difference (50 ms) offer interesting perspectives for an understanding of unstable brain states in terms of basic concepts of dynamical systems.
AN APPROACH TO MULTIVARIATE PHASE SYNCHRONIZATION ANALYSIS AND ITS APPLICATION TO EVENT-RELATED POTENTIALS
Authors: ALLEFELD CARSTEN , KURTHS JÜRGEN
Published: March 2004

A method for the multivariate analysis of statistical phase synchronization phenomena in empirical data is presented. A first statistical approach is complemented by a stochastic dynamic model, to result in a data analysis algorithm which can in a specific sense be shown to be a generic multivariate statistical phase synchronization analysis. The method is applied to EEG data from a psychological experiment, obtaining results which indicate the relevance of this method in the context of cognitive science as well as in other fields.
PARALLELIZING GRAMMATICAL FUNCTIONS: P600 AND P345 REFLECT DIFFERENT COST OF REANALYSIS
Authors: FRISCH STEFAN , BEIM GRABEN PETER , SCHLESEWSKY MATTHIAS
Published: March 2004

It is well-known from psycholinguistic literature that the human language processing system exhibits preferences when sentence constituents are ambiguous with respect to their grammatical function. Generally, many theories assume that an interpretation towards the subject is preferred in such cases. Later disambiguations which contradict such a preference induce enhanced processing difficulty (i.e. reanalysis) which reflects itself in late positive deflections (P345/P600) in event-related brain potentials (ERPs). In the case of phoric elements such as pronouns, a second strategy is known according to which an ambiguous pronoun preferentially receives the grammatical function that its antecedent has (parallel function strategy). In an ERP study, we show that this strategy can in principle override the general subject preference strategy (known for both pronominal and nonpronominal constituents) and induce an object preference, in case that the pronoun's antecedent is itself an object. Interestingly, the revision of a subject preference leads to a P600 component, whereas the revision of an object preference induces an earlier positivity (P345). In order to show that the latter component is indeed a positivity and not an N400-like negativity in the same time range, we apply an additional analysis based on symbolic dynamics which allows to determine the polarity of an ERP effect on purely methodological grounds. With respect to the two positivities, we argue that the latency differences reflect qualitative differences in the reanalysis processes.
SIMULATED ELECTROCORTICAL ACTIVITY AT MICROSCOPIC, MESOSCOPIC AND GLOBAL SCALES
Authors: WRIGHT J. J. , RENNIE C. J. , LEES G. J. , ROBINSON P. A. , BOURKE P. D. , CHAPMAN C. L. , GORDON E. , ROWE D. L.
Published: March 2004

The interaction of large populations of neurons gives rise to electrical events in the brain, which can be observed at several spatial scales. We show that mutually consistent explanations and simulation of experimental data can be achieved for cortical gamma activity, synchronous oscillation, and the main features of the EEG power spectrum including the cerebral rhythms, and evoked potentials. These simulations include consideration of dendritic and synaptic dynamics, AMPA, NMDA and GABA receptors, and intracortical and cortical/subcortical interactions.

The dynamic properties exhibited in the simulations, Hebbian synaptic modification regulated by a limited set of innate "reward" mechanisms, and infomax principles, can be combined to yield an explanation of elementary adaptive learning.
THE IMPACT OF THE CD8+ CELL NON-CYTOTOXIC ANTIVIRAL RESPONSE (CNAR) AND CYTOTOXIC T LYMPHOCYTE (CTL) ACTIVITY IN A CELL-TO-CELL SPREAD MODEL FOR HIV-1 WITH A TIME DELAY
Authors: LOU JIE , MA ZHIEN , LI JIANQUAN , SHAO YIMING , HAN LITAO
Published: March 2004

In this paper, neglecting the effects of free virus, we consider a simple model of cell-to-cell spread of HIV-1. We discuss the impact of the CD8+ cell non-cytotoxic anti-viral response (CNAR) and cytotoxic T lymphocyte (CTL) activity on infection by HIV-1. Two types of models are considered: the ordinary differential equation (ODE) model and the discrete time delay differential equation (DDE) system. The steady states of the ODE model are globally asymptotically stable respectively under two threshold criteria. In the DDE model, the global stability of the infected steady state of the ODE model becomes only ultimately stable. Moreover, at a certain interval of the time delay, the DDE model will produce Hopf bifurcation or periodic solutions. The introduction of CTL and CNAR will change the values of these steady states and induce fluctuations in the cell concentration. It also affects the critical value of the time delay and is of utility in the interpretation of typical HIV-dynamics data resulting from clinical trials. The DDE model produces sustained infective oscillations in some real parameter values, which is different from the result of the similar cell-free viral spread model with delay.
COMPARING SYNONYMOUS CODON USAGE OF ALTERNATIVELY SPLICED GENES WITH NON-ALTERNATIVELY SPLICED GENES IN HUMAN GENOME
Authors: MA FEI , ZHUANG YONGLONG , CHEN LIMING , LIN LUPING , LI YANDA , XU XIAOFENG , CHEN XUEPING
Published: March 2004

It is becoming clear that alternative splicing plays an important role in expanding protein diversity. However, the previous studies on codons usage did not distinguish alternative splicing from non-alternative splicing. Do codon usage patterns hold distinctions between them? Thus, we attempted to systematically compare the differences of synonymous codon usage patterns between alternatively and non-alternatively spliced genes by analyzing the large datasets from human genome. The results indicated:

(1) There are highly significant differences in the average Nc values between non-alternatively spliced genes and the longer isoform genes as well as the shorter isoform genes, and the level of codon usage bias of non-alternatively spliced genes is to some extent higher than that in alternatively spliced genes.

(2) Very extensive heterogeneity of G+C content in silent third codon position (GC3s) was evident among these genes, and it could be also shown there are highly significant differences in the average GC3s values between non-alternatively spliced genes and the longer isoform genes as well as the shorter isoform genes.

(3) The Nc-plots and correspondence analysis reveal that codon usage bias are mainly dominated by mutation bias, and no correlation between gene expression level and synonymous codon biased usage is found in human genes.

(4) Overall codon usage data analysis indicated that the C-ending codons usage has a highly significant differences between the longer isoform genes and non-alternatively spliced genes as well as the shorter isoform genes, it further found out that there is no significant differences of C-ending codons usage between the shorter isoform genes and non-alternatively spliced genes.

Finally, our results seem to imply that alternative splicing gene may originate from non-alternative splicing gene, and may be created by DNA mutation or gene fusion, and be retained through nature selection and adaptive evolution.