bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel model of sensitization and oral tolerance to peanut protein
Authors: Strid Jessica , Thomson Melanie , Hourihane Jonathan , Kimber Ian , Strobel Stephan
Published: October 2004

The prevalence of food allergic diseases is rising and poses an increasing clinical problem. Peanut allergy affects around 1% of the population and is a common food allergy associated with severe clinical manifestations. The exact route of primary sensitization is unknown although the gastrointestinal immune system is likely to play an important role. Exposure of the gastrointestinal tract to soluble antigens normally leads to a state of antigen‐specific systemic hyporesponsiveness (oral tolerance). A deviation from this process is thought to be responsible for food‐allergic diseases. In this study, we have developed a murine model to investigate immunoregulatory processes after ingestion of peanut protein and compared this to a model of oral tolerance to chicken egg ovalbumin (OVA). We demonstrate that oral tolerance induction is highly dose dependent and differs for the allergenic proteins peanut and OVA. Tolerance to peanut requires a significantly higher oral dose than tolerance to OVA. Low doses of peanut are more likely to induce oral sensitization and increased production of interleukin‐4 and specific immunoglobulin E upon challenge. When tolerance is induced both T helper 1 and 2 responses are suppressed. These results show that oral tolerance to peanut can be induced experimentally but that peanut proteins have a potent sensitizing effect. This model can now be used to define regulatory mechanisms following oral exposure to allergenic proteins on local, mucosal and systemic immunity and to investigate the immunomodulating effects of non‐oral routes of allergen exposure on the development of allergic sensitization to peanut and other food allergens.
Immunoassays: their history, development and current place in food science and technology
Authors: Bonwick Graham A. , Smith Christopher J.
Published: October 2004

The purpose of this paper is to introduce the reader to immunoassays. This paper is the first in a themed issue of the Journal in which a number of papers have been brought together in order to demonstrate the types and variety of immunoassays, which are currently available. Indeed it might be said that all an analyst needs to do is to name a molecule and somewhere there will now be an immunoassay for the detection of that molecule. This obviously is not entirely accurate, however immunoassays do provide a powerful tool, which can be used in the analysis and quality control of food materials. For both the novice and the experienced worker the specialist terminology of a subject presents an initial barrier, which must be overcome before full understanding is achieved. In this paper an attempt is made to introduce the important terms with which the reader should be familiar and to try to set the various technologies in context. The various basic methods are described and the theoretical and practical basis of more sophisticated assays now being devised are introduced.
Fluorescence polarization immunoassay based on a monoclonal antibody for the detection of ochratoxin A
Authors: Shim Won‐Bo , Kolosova Anna Yu. , Kim Yoon‐Jung , Yang Zheng‐You , Park Seon‐Ja , Eremin Sergei A. , Lee In‐Seon , Chung Duck‐Hwa
Published: October 2004

A fluorescence polarization immunoassay (FPIA) based on a monoclonal antibody for the determination of ochratoxin A (OTA) was developed. Fluorescein‐labelled OTA derivative (tracer) was synthesized and purified by thin‐layer chromatography. The optimized OTA FPIA had a dynamic range from 5 to 200 ng mL−1 with IC50 value of 30 ng mL−1 and a detection limit of 3 ng mL−1. The method developed was characterized by high specificity and reproducibility. Cross‐reactivity with other mycotoxins (zearalenone, aflatoxins, patulin and T‐2 toxin) was negligible (<0.1%). Methanol extracts of barley samples were used for the analysis. The results of OTA determination in barley were compared with those determined by indirect competitive enzyme‐linked immunosorbent assay (ELISA). Recoveries for the samples spiked at 50, 100 and 500 ng g−1 levels were 91, 90 and 97%, respectively, for FPIA, and 98, 98 and 102%, for ELISA. Naturally contaminated barley samples were analysed by these methods but some disagreement was observed between the results. The FPIA method can be applied for screening of food samples for OTA residues without a complicated clean‐up.
Species identification of meat products by ELISA
Authors: Giovannacci Inès , Guizard Cécile , Carlier Martine , Duval Valérie , Martin Jean‐Luc , Demeulemester Claude
Published: October 2004

ELISA methods used in this study are proved to detect low contents of animal species (pork, beef, sheep and poultry), even in highly processed foods. They present the advantages of being robust, cheap and easy to perform. Nevertheless, F factors, determining the threshold values of the test, need to be validated for each species.
Application of immunological methods for the detection of species adulteration in dairy products
Authors: Hurley Ian P. , Elyse Ireland H. , Coleman Robert C. , Williams John H. H.
Published: October 2004

A number of enzyme‐linked immunosorbent assays (ELISAs) have been developed for the detection of milk adulteration in dairy products. Target antigens have been caseins, lactoglobulins, immunoglobulins and other whey proteins. Polyclonal and monoclonal antibodies have been used in a variety of formats including direct, indirect, competitive and sandwich ELISAs. ELISAs have been successfully applied to the detection of cows' milk adulteration of sheep, goat and buffalo milk. Goat milk adulteration of sheep milk has also been detected. A number of ELISAs have also been applied to cheese. It is recommended that ELISA should be used in combination with PCR to ensure compliance with current legislation.
Pharmacokinetic and pharmacodynamic evaluation of intravenous hydromorphone in cats1
Authors: Wegner K. , Robertson S. A. , Kollias‐Baker C. , Sams R. A. , Muir W. W.
Published: October 2004

This study describes the pharmacokinetics of intravenous hydromorphone in cats and the simultaneous measurement of antinociceptive pharmacodynamic effects using a thermal threshold testing system. Following establishment of a baseline thermal threshold, six adult cats were administered 0.1 mg/kg of hydromorphone intravenously. Thermal threshold testing and blood collection were conducted simultaneously at predetermined time points. Plasma hydromorphone concentrations were determined by a liquid chromatographic‐mass spectral method and pharmacokinetic analysis was performed by nonlinear least squares regression analysis. Plasma hydromorphone concentrations declined rapidly over time, and were below the limit of quantification of the assay (LOQ = 1.0 ng/mL) by 360 min. In contrast, thermal thresholds rose from a pretreatment value of 40.9 ± 0.65 °C (mean ± SEM) to instrument cut‐out (55 °C) within 15 min and remained significantly elevated from 15–450 min after treatment. Inspection of the data revealed no direct correlation between plasma hydromorphone concentrations and the antinociceptive effect of this drug in cats. These findings support the importance of conducting pharmacokinetic studies in parallel with objective measurements of drug effect.
Evaluation of dexamethasone for the treatment of intracerebral hemorrhage using a collagenase‐induced intracerebral hematoma model in rats
Authors: Lema P. P. , Girard C. , Vachon P.
Published: October 2004

Dexamethasone was evaluated for the treatment of intracerebral hemorrhage using a rat model of cerebral hematoma induced by intracerebral injection of collagenase. The treatment group consisted of hematoma rats receiving dexamethasone 1 mg/kg intraperitoneal (i.p.) at 1 and 24 h following surgery. Controls included hematoma rats receiving saline i.p. and sham‐operated animals receiving saline i.p. Each animal was evaluated neurologically prior to, as well as 24 and 48 h following surgery. After the last neurological evaluation, animals were deeply anesthetized and the brain was removed following perfusion for microscopic examination and glial fibrillary acidic protein immunohistochemistry. Behavioral scores were significantly improved in the treated group (P < 0.0001). The hematoma volume was significantly smaller (P < 0.02). Neutrophils and astrocytes were less numerous in the hematoma of dexamethasone‐treated animals (P < 0.001), however the number of necrotic neurons in the penumbra was not changed by the treatment. The number of necrotic neurons in the cerebral cortex was less in treated than in nontreated animals (P < 0.01). Controls had many vascular changes including necrotic endothelium and fibrin deposits compared with treated animals. In conclusion, dexamethasone administered shortly after an intracerebral hematoma appears beneficial for the treatment of this condition.
Meta‐analysis of pharmacokinetic data of veterinary drugs using the Food Animal Residue Avoidance Databank: oxytetracycline and procaine penicillin G
Authors: Craigmill A. L. , Miller G. R. , Gehring R. , Pierce A. N. , Riviere J. E.
Published: October 2004

Investigators frequently face the quandary of how to interpret the oftentimes disparate pharmacokinetic parameter values reported in the literature. Combining of data from multiple studies (meta‐analysis) is a useful tool in pharmacokinetics. Few studies have explored the use of meta‐analysis for veterinary species. Even fewer studies have explored the potential strengths and weaknesses of the various methods of performing a meta‐analysis. Therefore, in this study we performed a meta‐analysis for oxytetracycline (OTC) and procaine penicillin G (PPG) given intramuscularly to cattle. The analysis included 28 individual data sets from 18 published papers for PPG (288 data points), and 41 individual data sets from 25 published papers for OTC (489 data points). Three methods were used to calculate the parameters. The first was a simple statistical analysis of the parameter values reported in each paper. The second method was a standard Two‐Stage Method (TSM) using the mean concentration vs. time data extracted from each paper. The third method was the use of nonlinear mixed effect modeling (NMEM) of the concentration vs. time data reported in the various papers, treating the mean data as if each set came from an individual animal. The results of this evaluation indicate that all three methods generate comparable mean parameter estimates for OTC and PPG. The only significant difference noted was for OTC absorption half‐lives taken from the published literature, a difference attributable to the use of an alternative method of parameter calculation. The NMEM procedure offers the possibility of including covariates such as dose, age, and weight. In this study the covariates did not influence the derived parameters. A combination approach to meta‐analysis of published mean data is recommended, where the TSM is the first step, followed by the NMEM approach.
Multiple oral dosing of ketoconazole influences pharmacokinetics of quinidine after intravenous and oral administration in beagle dogs
Authors: Kuroha M. , Shirai Y. , Shimoda M.
Published: October 2004

In this study, we investigated the effect of multiple oral dosing of ketoconazole (KTZ) on pharmacokinetics of quinidine (QN), a CYP3A substrate with low hepatic clearance, after i.v. and oral administration in beagle dogs. Four dogs were given p.o. KTZ for 20 days (200 mg, b.i.d.). QN was administered either i.v. (1 mg/kg) or p.o. (100 mg) 10 and 20 days before the KTZ treatment and 10 and 20 days after start of KTZ treatment. Multiple oral dosing of KTZ decreased significantlyαandβ, whereas increasedt1/2β, V1, andka. The KTZ treatment also decreased significantly both total body clearance (Cltot) and oral clearance (Cloral). No significant change in bioavailability was observed in the presence of KTZ. Co‐administration of KTZ increasedCmaxof QN to about 1.5‐fold. Mean resident time after i.v. administration (MRTi.v.), and after oral administration (MRTp.o.) of QN were prolonged to about twofold, whereas mean absorption time (MAT) was decreased to 50%. Volume of distribution at steady state (Vd(ss)) of QN was unchanged in the presence of KTZ. These alterations may be because of a decrease in metabolism of QN by inhibition of KTZ on hepatic CYP3A activity. In conclusion, multiple oral dosing of KTZ affected largely pharmacokinetics of QN after i.v. and oral administration in beagle dogs. Therefore, KTZ at a clinical dosing regimen may markedly change the pharmacokinetics of drugs primarily metabolized by CYP3A with low hepatic clearance in dogs. In clinical use, much attention should be paid to concomitant administration of KTZ with the drug when given either p.o. or i.v.
Pharmacokinetics of orally administered pirfenidone in male and female beagles
Authors: Bruss M. L. , Margolin S. B. , Giri S. N.
Published: October 2004

Pirfenidone, a promising antifibrotic agent, was administered orally to dogs at 0, 40, 140, and 400 mg/kg/day. Serum was collected for pirfenidone assay at 0, 26 and 39 weeks of treatment. From the pirfenidone concentrations, pharmacokinetic parameters were determined for each dog at each treatment interval. The only significant differences because of gender were for concentration maxima. Unsurprisingly, there were many significant differences because of dose in concentration maximum and area under curve (AUC), and significant, positive linear correlations of both parameters with dose. There were few significant differences in time of maximal concentration and no correlation with dose. The mean ± SE clearances were 1.99 ± 0.13, 1.64 ± 0.13 and 1.78 ± 0.14 L/h/kg for doses of 40, 140, and 400 mg/kg, respectively, with no significant differences attributable to dose. There was an unexplained pattern in maximal concentration and AUC with regard to duration of treatment, with the parameters being highest at week 0, lowest at week 26, and intermediate at week 39. Clearance had the reverse pattern; time of maximal concentration had no pattern.
Treatment of patients with HCV infection with or without liver biopsy
Authors: Andriulli A. , Persico M. , Iacobellis A. , Maio G. , Di Salvo D. , Spadaccini A. , Bacca D. , Leandro G. , Ventrella F. , Mangia A.
Published: October 2004

Summary.  Expert consensus recommends liver biopsy before therapy for chronic hepatitis C. A cost effectiveness analysis suggested that the best strategy in the management of patients was to treat without biopsy. We compared therapy in patients who did, or did not undergo biopsy. Hepatitis C virus (HCV)‐positive patients (78) who did not agree to (n = 57) or with contraindications to liver biopsy (n = 21) (group A) were matched for age, sex and genotype with those who consented (group B). Before therapy (interferon/ribavirin for 12 months), a clinical diagnosis of chronic hepatitis, on the basis of standard biochemical and ultrasonographic parameters. The two groups showed similar baseline characteristics. A noninvasive, diagnosis of chronic hepatitis was made in 75.6% of group A, and in 83.3% of group B (P = 0.26). Concordance between clinical and histological diagnosis in group B amounted to 91%. End‐of‐therapy virological response was 52.6% in group A, and 57.7% in group B (P = 0.63). Sustained virological response was 41.0% [95% confidence interval (CI) 30.1–51.9] and 43.6% (95% CI 32.6–54.6) in the two groups (P = 0.87). Predictors of sustained response were noninvasive diagnosis of chronic hepatitis (P = 0.006), lack of portal hypertension (P = 0.037), platelets >105/mm3 (P = 0.007), prothrombin >70% (P = 0.02), and genotype 2 or 3 (P < 0.0001). At multivariate analysis, genotype (P < 0.0001) and platelets (P = 0.004) maintained their predictive power. In most patients with HCV infection, virological clearance after therapy can be achieved irrespective of whatever a liver biopsy might show.
Inhibition of subgenomic hepatitis C virus RNA in Huh‐7 cells: ribavirin induces mutagenesis in HCV RNA
Authors: Kanda T. , Yokosuka O. , Imazeki F. , Tanaka M. , Shino Y. , Shimada H. , Tomonaga T. , Nomura F. , Nagao K. , Ochiai T. , Saisho H.
Published: October 2004

Summary. Hepatitis C virus (HCV) infection is a major problem throughout the world. Combination therapy of interferon (IFN) and ribavirin is the best treatment for eradication at present, but the mechanism is not completely understood. We used the HCV replicon system to investigate this mechanism. The effects of six drugs (UDCA, glycyrrhizin, TJ‐9, bezafibrate, ribavirin, and alpha‐IFN 2b) on HCV subgenomic RNA (genotype 1b, NS5B 415Y) were examined by reverse transcription polymerase chain reaction, cloning and sequencing. The HCV replication was inhibited by alpha‐IFN 2b (7.39–13.2% at 10 U/mL, 3.29–6.12% at 100 U/mL, 1.3–4.86% at 1000 U/mL) and by ribavirin (4.36–13.9% at 100 μg/mL), but not by the other drugs at 24–72 h after treatment. Furthermore, the combination treatment was superior to IFN monotherapy and to ribavirin monotherapy at 72 h post‐treatment. Sequence analyses of the double‐stranded RNA‐activated protein kinase (PKR)‐binding domain and flanking regions within the HCV NS5A region revealed that the total numbers of substitutions caused by ribavirin (n = 36) or combination treatment (n = 57) were more than those of IFN alone (n = 5) and controls (n = 6). The HCV replicon system is the most efficient system for HCV replication and is an excellent choice for testing anti‐HCV drugs and disinfectants. Our results further suggested that the combination of alpha‐IFN 2b and ribavirin might induce mutations, and inhibit HCV RNA synthesis in hepatocytes to a greater extent than ribavirin monotherapy.
HBV core sequence: definition of genotype‐specific variability and correlation with geographical origin
Authors: Jazayeri M. , Basuni A. A. , Sran N. , Gish R. , Cooksley G. , Locarnini S. , Carman W. F.
Published: October 2004

Summary.  There are eight genotypes and nine subtypes of HBV. Small differences in geographical origin are associated with sequence changes in the surface gene. Here, we compared core gene sequences from different genotypes and geographical regions. Specific combinations of 24 amino acid substitutions at nine residues allowed allocation of a sequence to a subtype. Six of these nine residues were located in different T cell epitopes depending on HBV geographical area and/or genotype. Thirty‐seven nucleotide changes were associated uniquely with specific genotypes and subtypes. Unique amino acid and nucleotide variants were found in a majority of sequences from specific countries as well as within subtype ayw2 and adr. Specific nucleotide motifs were defined for Korean, Indian, Chinese, Italian and Pacific region isolates. Finally, we observed amino acid motifs that were common to either South‐east Asian or Western populations, irrespective of subtype. We believe that HBV strains spread within constrained ethnic groups, result in selection pressures that define sequence variability within each subtype. It suggests that particular T cell epitopes are specific for geographical regions, and thus ethnic groups; this may affect the design of immunomodulatory therapies.
Negative correlation between intrahepatic expression of hepatitis C antigens and apoptosis despite high‐level expression of Fas and HLA antigens
Authors: Caronia S. , McGarvey M. J. , Goldin R. D. , Foster G. R.
Published: October 2004

Summary.  The role of virus‐related apoptosis in hepatic injury in chronic HCV is unclear. It is unknown whether HCV induces apoptosis directly or whether cellular injury is immunologically mediated. We studied the relationship between infected hepatocytes, apoptosis and necroinflammation. We established a Fluorescence Activated Cell Sorter (FACS) based intracellular staining technique for the HCV NS3 protein and examined intrahepatic viraemia, disease activity and apoptosis. We also stained infected cells for expression of human leucocyte antigen (HLA) class I and Fas antigens. We examined 34 liver biopsies (24 from patients with HCV) and found marked variation in the proportion of infected cells (2.5–42%). The number of infected cells correlated with serum viraemia but not histology. The number of infected cells was inversely related to the number of apoptotic cells (P < 0.001); infected cells expressed both HLA class I (14 cases) and Fas antigens (12 cases). The number of hepatocytes infected with hepatitis C is variable and does not influence histological activity. In infected patients, the majority of HCV‐positive hepatocytes express target molecules for activated lymphocytes (Fas and HLA class I antigens) but they do not undergo apoptosis, suggesting that hepatitis C may inhibit apoptosis by modulating intracellular pro‐apoptotic signals.
Beneficial effects of ‘lamivudine pulse’ therapy in HBeAg‐positive patients with normal ALT*
Authors: Sarin S. K. , Sandhu B. S. , Sharma B. C. , Jain M. , Singh J. , Malhotra V.
Published: October 2004

Summary.  Currently no therapy is given to patients with chronic hepatitis B virus (HBV) infection who are HBeAg positive with normal alanine aminotransferase (ALT) levels. Steroid priming has been shown to enhance T‐helper‐1 (Th‐1) cell response. Lamivudine may restore immunologic competence against HBV by causing a sudden decline in the level of the virus. We examined the efficacy of lamivudine pulse therapy on the seroconversion from HBeAg to anti‐HBe. This was a prospective single‐blinded trial including 27 patients with chronic hepatitis B, HBeAg positive with ALT ≤1.5 times upper limit of normal (ULN). Lamivudine was administered initially for 4 weeks, then stopped for 2 weeks and later restarted and continued till 3 months after seroconversion or completion of 2 years of therapy. Twenty‐six patients completed the study. Lamivudine withdrawal led to a rise in ALT levels above the ULN in 11 (42.3%) patients at 6 weeks; seven of them (63.6%) lost HBeAg compared with only two of the 15 patients (13.3%), in whom ALT levels did not rise (P = 0.011). As one patient showed a relapse, a total of eight (31%) patients responded to lamivudine pulse therapy over a mean period of 17.3 ± 4.5 months. Responders had a higher serum albumin (P < 0.05), a lower fibrosis score (P < 0.05), and a relatively high baseline serum ALT levels (P = 0.024) than the nonresponders. YMDD mutations developed in three patients and none responded. No patient developed hepatic decompensation. Hence lamivudine pulse therapy has potential in converting HBeAg‐positive, ‘not‐treat‐worthy’ (ALT < 1.5 ULN) patients to treat‐worthy (ALT > 1.5 ULN) in 42%, with sustained HBeAg and HBV DNA loss in 31% patients. The effects are possibly because of a combination of antiviral and immunomodulating activities of lamivudine.
Contribution of novel choline‐binding proteins to adherence, colonization and immunogenicity of Streptococcus pneumoniae
Authors: Rosenow Carsten , Ryan Patricia , Weiser Jeffrey N. , Johnson Syd , Fontan Patricia , Ortqvist Ake , Masure H. Robert
Published: October 2004

The surface of Streptococcus pneumoniae is decorated with a family of choline‐binding proteins (CBPs) that are non‐covalently bound to the phosphorylcholine of the teichoic acid. Two examples (PspA, a protective antigen, and LytA, the major autolysin) have been well characterized. We identified additional CPBs and characterized a new CBP, CbpA, as an adhesin and a determinant of virulence. Using choline immobilized on a solid matrix, a mixture of proteins from a pspA‐deficient strain of pneumococcus was eluted in a choline‐dependent fashion. Antisera to these proteins passively protected mice challenged in the peritoneum with a lethal dose of pneumococci. The predominant component of this mixture, CbpA, is a 75‐kDa surface‐exposed protein that reacts with human convalescent antisera. The deduced sequence from the corresponding gene showed a chimeric architecture with a unique N‐terminal region and a C‐terminal domain consisting of 10 repeated choline‐binding domains nearly identical to PspA. A cbpA‐deficient mutant showed a >50% reduction in adherence to cytokine‐activated human cells and failed to bind to immobilized sialic acid or lacto‐N‐neotetraose, known pneumococcal ligands on eukaryotic cells. Carriage of this mutant in an animal model of nasopharyngeal colonization was reduced 100‐fold. There was no difference between the parent strain and this mutant in an intraperitoneal model of sepsis. These data for CbpA extend the important functions of the CBP family to bacterial adherence and identify a pneumococcal vaccine candidate.
Zinc(II) tolerance in Escherichia coli K‐12: evidence that the zntA gene (o732) encodes a cation transport ATPase
Authors: Beard Steven J. , Hashim Rohani , Membrillo‐Hernández Jorge , Hughes Martin N. , Poole Robert K.
Published: October 2004

A transposon (Tn10dCam) insertion mutant of Escherichia coli K‐12 was isolated that exhibited hypersensitivity to zinc(II) and cadmium(II) and, to a lesser extent, cobalt(II) and nickel (II). The mutated gene, located between 75.5 and 76.2 min on the chromosome, is named zntA (for Zn(II) transport or tolerance). The metal‐sensitive phenotype was complemented by a genomic DNA clone mapping at 3677.90–3684.60 kb on the physical map. Insertion of a kanamycin resistance (KnR) cassette at a Sal I site in a subcloned fragment generated a plasmid that partially complemented the zinc(II)‐sensitive phenotype. DNA sequence analysis revealed that the KnR cassette was located within the putative promoter region of an ORF (o732 or yhhO) predicted to encode a protein of 732 amino acids, similar to cation transport P‐type ATPases in the Cpx‐type family. Inverse PCR and sequence analysis revealed that the Tn10dCam element was located within o732 in the genome of the zinc(II)‐sensitive mutant. The zntA mutant had elevated amounts of intracellular and cell surface‐bound Zn(II), consistent with the view that zntA+ encodes a zinc(II) efflux protein. Exposure of the zntA mutant to cobalt(II) and cadmium(II) also resulted in elevated levels of intracellular and cell surface‐bound metal ions.
A peptidoglycan hydrolase similar to bacteriophage endolysins acts as an autolysin in Neisseria gonorrhoeae
Authors: Dillard Joseph P. , Seifert H. Steven
Published: October 2004

We have identified a gene encoding an autolysin (atlA) from Neisseria gonorrhoeae. The deduced amino acid sequence of AtlA shows significant similarity to the peptidoglycan degrading transglycosylases (endolysins) of bacteriophages lambda and P2, suggesting that the encoded protein also functions in peptidoglycan hydrolysis. An atlA mutant was identical to the wild‐type strain in exponential growth rate, but demonstrated reduced lysis and peptidoglycan turnover in the stationary phase of growth. When transferred into a buffer solution, at a pH non‐permissive for other gonococcal autolysins, an autolytic activity was detectable in the wild‐type strain that was not present in the mutant. The most dramatic phenotype of the mutant occurred after extended time in stationary phase. After approximately 16 h in stationary phase, both strains underwent an apparent replication event, after which the wild‐type strain died rapidly whereas the atlA mutant survived considerably longer. Even after both the wild‐type and mutant cells were dead, many of the mutant cells maintained intact morphology, whereas the wild‐type cells were lysed. These results suggest that AtlA is a peptidoglycan transglycosylase related to bacteriophage endolysins and acts as an autolysin in the stationary phase.
Molecular mechanisms of Nod factor diversity
Authors: Mergaert Peter , Van Montagu Marc , Holsters Marcelle
Published: October 2004

The rhizobia–legume symbiosis is highly specific. Major host specificity determinants are the bacterial Nod factor signals that trigger the nodulation programme in a compatible host. Nod factors are lipo‐chitooligosaccharides (LCOs) varying in the oligosaccharide chain length, the nature of the fatty acids and substitutions on the oligosaccharide. The nod genotype of rhizobia, which forms the genetic basis for this structural variety, includes a set of nodulation genes encoding the enzymes that synthesize LCOs. Allelic and non‐allelic variation in these genes ensures the synthesis of different LCO structures by the different rhizobia. The nod genotypes co‐evolved with host plant divergence in contrast to the rhizobia, which followed a different evolution. Horizontal gene transfer probably played an important role during evolution of symbiosis. The nod genotypes are particularly well equipped for horizontal gene transfer because of their location on transmissible plasmids and/or on ‘symbiosis islands’, which are symbiotic regions associated with movable elements.
Mutational analysis of the RecA protein L1 region identifies this area as a probable part of the co‐protease substrate binding site
Authors: Nastri Horacio G. , Guzzo Angelina , Lange Craig S. , Walker Graham C. , Knight Kendall L.
Published: October 2004

Previous mutational analysis of the L1 region of the RecA protein suggested that Gly‐157 and Glu‐158 are ‘hot‐spots’ for the occurrence of constitutive LexA co‐protease mutants (coprtc). In the present study, we clearly establish that position 157 is a hot‐spot for the occurrence of such mutants, as 12 of 14 and 10 of 14 substitutions result in this phenotype for UmuD and LexA cleavage respectively. The frequency of such mutations at position 158 is somewhat lower, 8 of 13 and 5 of 13 for UmuD and LexA respectively. Comparison of the UmuD vs. LexA co‐protease activity for all single mutants with substitutions at positions 154, 155, 156, 157 and 158 (47 in total) reveals that, although there is good agreement among most mutants regarding their ability to cleave both LexA and UmuD, there are two in particular (Glu‐154→Asp and Glu‐154→Gln) that show a clear preference for cleavage of UmuD. We also show that three second‐site mutations that completely suppress coprtc activity toward LexA have little or no effect on the coprtc activity of the primary mutant toward UmuD. In addition, we observe a high frequency of second‐site suppressor mutations, suggesting a functional interaction among side‐chains in this region. Together, these results support the idea that the L1 region of RecA makes up part of the co‐protease substrate‐binding site.
Assign 2.0: software for the analysis of Phred quality values for quality control of HLA sequencing‐based typing
Authors: Sayer D.C. , Goodridge D.M. , Christiansen F.T.
Published: October 2004

Abstract:  As improvements to DNA sequencing technology have resulted in increasing the throughput of DNA sequencing, the bottleneck for high throughput DNA sequencing‐based typing (SBT) has shifted to sequence analysis, genotyping and quality control (QC). Consistent high‐quality DNA sequence is required in order to reduce manual verification and editing of sequence electropherograms. However, identifying systematic changes in quality is difficult to achieve without the aid of sophisticated sequence analysis programs dedicated to this purpose. We describe a computer software program called Assign 2.0, which integrates sequence QC analysis and genotyping in order to facilitate high‐throughput SBT. Assign 2.0 performs an analysis of Phred quality values in order to produce quality scores for a sample and a sequencing run. This enables sample‐to‐sample and run‐to‐run QC monitoring and provides a mechanism for the comparison of sequence quality between various genes, various reagents and various protocols with the aim of improving the overall quality of DNA sequence data. This, in turn, will result in reducing sequence analysis as a bottleneck for high‐throughput SBT.
Molecular analysis of HLA allele frequencies and haplotypes in Baloch of Iran compared with related populations of Pakistan
Authors: Farjadian S. , Naruse T. , Kawata H. , Ghaderi A. , Bahram S. , Inoko H.
Published: October 2004

Abstract:  The extreme polymorphism in different loci of the human leukocyte antigen (HLA) system has been used as an invaluable tool for anthropological studies. Determination of HLA allele and haplotype frequencies in different ethnic groups is useful for population genetic analyses and the study of genetic relationships among them. In the present study, molecular analysis of HLA‐A, ‐B, ‐C, ‐DQA1, ‐DQB1, and ‐DRB1 genes has been used to assign HLA allele and haplotype frequencies in 100 unrelated healthy individuals from the Baloch ethnic group of Iran. The results were compared with Baloch and other ethnic groups in the neighboring Pakistan. The results of this study showed that the most frequent HLA class I alleles were A*02011 (20.2%), B*4006 (11.1%), and C*04011 (28.6%). The most common HLA class II alleles were DQA1*0101/2 (42.5%), DQB1*0201 (32%), and DRB1*0301 (29%). Three‐locus haplotype analysis revealed that A*11011‐B*4006‐C*15021 (5.8%) and DQA1*0501‐DQB1*0201‐DRB1*0301 (22.1%) were the most common HLA class I and II haplotypes, respectively, in this population. Neighbor‐joining tree based on DA genetic distances and correspondence analysis according to HLA‐A, ‐B, ‐DQB1, and ‐DRB1 allele frequencies showed that Baloch of Iran are genetically very close to Baloch and Brahui of Pakistan. This may reflect an admixture of Brahui and Baloch ethnic groups of Pakistan in the Balochistan province of Iran.
Description of a novel HLA‐B allele, B*5613, identified during HLA‐typing using sequence‐specific oligonucleotide hybridization and sequence‐specific amplification*
Authors: Hoppe B. , Heymann G.A. , Schoenemann C. , Nagy M. , Kiesewetter H. , Salama A.
Published: October 2004

Abstract:  Here, we report on the characterization of a novel human leukocyte antigen (HLA)‐B allele, B*5613. The allele was identified in an adult male from North Africa who was suffering from sickle cell anemia. HLA‐B*5613 most closely matches to B*5601 differing only by a substitution of three nucleotides of codon 180. Due to this substitution, low‐resolution HLA‐typing using sequence‐specific oligonucleotide hybridization or amplification using sequence‐specific primers gave inconclusive results. DNA sequencing confirmed a variation of codon 180 (CTG→GAC) resulting in an amino acid substitution Leu156Asp.
Characterization of a novel HLA‐DQB1 allele, DQB1*020102
Authors: Lv F. , Gao J. , Lin J. , Liu Z. , Xu Y. , Ren X. , Zhang H. , Xu A.
Published: October 2004

Abstract:  A novel human leukocyte antigen (HLA)‐DQB1 allele, DQB1*020102, was detected in a 28‐year‐old woman of Han ethnic in Guangzhou, China. Compared with HLA‐DQB1*020101 and HLA‐DQB1*0202, they differed in only one nucleotide at the position 167 (C to T) of exon 2, which was a highly conserved position. This is a synonymous mutation, which does not cause any change in the amino acid sequence of mature protein.
HLA‐B*8102*, a new allele found in an external proficiency testing scheme
Authors: Faé I. , Lau M. , Voorter C. , Mayr W.R. , Fischer G.F.
Published: October 2004

Abstract:  A new human leukocyte allele HLA‐B*8102 has been identified in a cell provided by the UCLA International Cell Exchange scheme. The nucleotide sequences of exons 2 and 3 of the new allele are identical with HLA‐B*8101. Four nucleotide differences, however, are observed in exon 1, three of them representing non‐synonymous base changes.
High frequency of QPY allele and linkage disequilibrium of granzyme‐B in Epstein‐Barr‐virus‐associated hemophagocytic lymphohistiocytosis
Authors: Zaitsu M. , Yamamoto K. , Ishii E. , Teramura T. , Nakadate N. , Sako M. , Sakata N. , Wakiguchi H. , Hirose M. , Imayoshi M. , Ogata Y. , Imashuku S. , Hamasaki Y. , Yasukawa M.
Published: October 2004

Abstract:  Mediation of Epstein‐Barr virus (EBV)‐specific cytotoxicity in T lymphocyte via the perforin/granzyme pathway has been demonstrated; therefore, a study involving cytolytic molecules was essential for the clarification of hemophagocytic lymphohistiocytosis (HLH) pathogenesis. This investigation, which analysed the frequency of three allelic mutations of granzyme‐B (55Q/R, 95P/A and 247Y/H) in patients with EBV‐HLH and infectious mononucleosis, identified the high prevalence of the QPY haplotype in EBV‐HLH patients in comparison with healthy controls. A > G polymorphism was also detected in intron 5; furthermore, nearly complete linkage disequilibrium was observed among these polymorphisms. The recessive role of the QPY haplotype of granzyme‐B might be responsible for the pathogenesis of EBV‐HLH. Cytotoxicity and DNA fragmentation of cytotoxic T lymphocytes did not differ among patients characterized by the QPY/QPY, RAH/RAH and QPY/RAH genotypes. This finding suggested that DNA fragmentation in target cells is mediated not only by granzyme‐B but also by other molecules, including other granzymes or Fas.
Characterization of a new HLA‐DRB1*01 allele (HLA‐DRB1*010203) in a Caucasian Italian family by using sequence‐based typing
Authors: Bontadini A. , Manfroi S. , Delfino L. , Fruet F. , Iannelli S. , Parodi A.M. , Ferrara G.B. , Conte R.
Published: October 2004

Abstract:  We report the identification of an HLA‐DRB1*01 nucleotide sequence variant in three members of a Caucasian Italian family by using sequence ‐based typing. The nucleotide sequence of exon 2 observed in the new allele is identical to that of HLA‐DRB1*010201 except in position 189 (codon 34) where the adenine of the consensus was replaced by a guanine and it was designated officially as HLA‐DRB1*010203* by the WHO Nomenclature Committee. The nucleotide sequence is available at the ebml database under accession number AJ430382.1.
Mortality in patients with diabetic neuropathic osteoarthropathy (Charcot foot)
Authors: Gazis A. , Pound N. , Macfarlane R. , Treece K. , Game F. , Jeffcoate W.
Published: October 2004

Objective  To determine the mortality of a population of patients diagnosed with Charcot neuropathic osteoarthropathy managed by a single specialist unit and to compare the results with a control population.

Methods  We have undertaken a retrospective analysis of all cases of Charcot foot on the comprehensive database which has been maintained at the specialist diabetic foot clinic at the City Hospital, Nottingham since 1982. Survival and the incidence of amputation (major and minor) was compared with a control population referred with uncomplicated neuropathic ulceration. Controls were individually matched for gender, age (±2 years), disease type, disease duration (±2 years) and year of referral (±3 years).

Results  Forty‐seven cases (21 female, 26 male) of Charcot foot were identified, of whom 18 (38.3%) had Type 1 diabetes. Mean age and disease duration at presentation were 59.2 ± 13.4 (sd) and 16.2 ± 11.2 years, compared with 59.7 ± 12.6 and 16.3 ± 11.2 years, respectively, in the controls. Twenty‐one (44.7%) of those with Charcot had died, after a mean interval of 3.7 ± 2.8 years. This compared with 16 (34.0%) after a mean 3.1 ± 2.7 years in the control group. Mean duration of follow‐up in the survivors was 4.7 ± 4.9 years (Charcot) and 5.3 ± 3.9 years (controls). A total of 11 (23.4%) Charcot patients had had a major amputation on the side of the index lesion, compared with five (10.6%) controls. There was no difference between the two groups (P > 0.05, Chi‐square).

Conclusions  The mortality in this group of patients with Charcot foot was higher than expected. Nevertheless, there was no difference between those with Charcot and those with uncomplicated neuropathic ulceration. It is possible that it is neuropathy, rather than Charcot osteoarthropathy, which is independently associated with increased mortality in diabetes. The mechanism underlying any such association is not known. There is a need for a formal, prospective, multicentre study to investigate the life expectancy and cardiovascular risk of those with Charcot osteoarthropathy.
Does autonomic neuropathy play a role in erythropoietin regulation in non‐proteinuric Type 2 diabetic patients?
Authors: Spallone V. , Maiello M. R. , Kurukulasuriya N. , Barini A. , Lovecchio M. , Tartaglione R. , Mennuni G. , Menzinger G.
Published: October 2004

Aims  Erythropoietin (EPO)‐deficient anaemia has been described in Type 1 diabetic patients with both severe autonomic neuropathy (AN) and proteinuria. This study was aimed at distinguishing between the effects of AN and nephropathy on haemoglobin and EPO levels in Type 2 diabetic patients at an early stage of diabetic nephropathy.

Methods  In 64 Type 2 diabetic patients (age 52 ± 10 years, duration 10 ± 9 years) without overt nephropathy and other causes of anaemia or EPO deficit, we assessed cardiovascular tests of AN, 24‐h blood pressure (BP) monitoring, urinary albumin excretion rate (UAE), a full blood count, and serum EPO.

Results  Although the Type 2 diabetic patients with AN did not show differences in haemoglobin and EPO when compared with patients without AN, the presence of haemoglobin < 13 g/dl was associated with the presence of AN (χ2= 3.9, P < 0.05) and of postural hypotension (χ2= 7.8, P < 0.05). In a multiple regression analysis including as independent variables gender, body mass index, duration of diabetes, smoking, creatinine, 24‐h UAE, 24‐h diastolic BP, ferritin, erythrocyte sedimentation rate, and autonomic score, we found that the only variables independently related to haematocrit were autonomic score, ferritin and erythrocyte sedimentation rate. Finally, the physiological inverse relationship between EPO and haemoglobin present in a control group of 42 non‐diabetic non‐anaemic subjects was completely lost in Type 2 diabetic patients. The slopes of the regression lines between EPO and haemoglobin of the control subjects and the Type 2 diabetic patients were significantly different (t = 14.4, P < 0.0001).

Conclusions  This study documents an early abnormality of EPO regulation in Type 2 diabetes before clinical nephropathy and points to a contributory role of AN in EPO dysregulation.
A prospective study of Type 2 diabetes and depressive symptoms in the elderly: The Rancho Bernardo Study
Authors: Palinkas L. A. , Lee P. P. , Barrett‐Connor E.
Published: October 2004

Aims  The association between Type 2 diabetes and depressive symptoms was examined prospectively to assess possible causal relationships between the two diseases.

Methods  A cohort of 971 men and women aged 50 and older from the adult population of Rancho Bernardo, California had an oral glucose tolerance test and completed the Beck Depression Inventory (BDI) at two clinic visits, 1984–87 and 1992–96.

Results  Depressive symptoms at baseline were associated with higher follow‐up levels of non‐fasting plasma glucose (P = 0.001) and an increased risk of developing Type 2 diabetes [odds ratio (OR) = 2.50; 95% confidence interval (CI) = 1.29–4.87], independent of sex, age, exercise and body mass index. Conversely, baseline non‐fasting plasma glucose was not significantly associated with follow‐up depressive symptoms and Type 2 diabetes at baseline was not significantly associated with the onset of BDI scores ≥ 11 by the second visit (OR = 0.73; 95% CI = 0.41–1.30).

Conclusions  Depressed mood is more likely to be a risk factor for Type 2 diabetes in older adults than the reverse.
Long‐term renoprotection by perindopril or nifedipine in non‐hypertensive patients with Type 2 diabetes and microalbuminuria
Authors: Jerums G. , Allen T. J. , Campbell D. J. , Cooper M. E. , Gilbert R. E. , Hammond J. J. , O'Brien R. C. , Raffaele J. , Tsalamandris C. ,   The Melbourne Diabetic Nephropathy Study Group
Published: October 2004

Aims  To assess the efficacy of an angiotensin converting enzyme (ACE) inhibitor (perindopril), a dihydropyridine calcium channel blocker (sustained release nifedipine) and placebo in preventing the progression of albuminuria and decline in glomerular filtration rate (GFR) in patients with Type 2 diabetes and microalbuminaria.

Methods  A prospective, randomized, open, blinded end point study of 77 patients allocated to three treatment groups (23 perindopril, 27 nifedipine, 27 placebo). Drug doses were adjusted to achieve a decrease in diastolic blood pressure (DBP) of 5 mmHg in the first 3 months and additional therapy was given if hypertension developed (supine DBP > 90 mmHg and/or systolic blood pressure (SBP) > 140 mmHg if ≤ 40 years; supine DBP > 90 mmHg and/or SBP > 160 mmHg if > 40 years). Median follow‐up was 66 months, with 37 patients being followed for at least 6 years.

Results  Blood pressure remained within the non‐hypertensive range in 83% of perindopril‐, 95% of nifedipine‐ and 30% of placebo‐treated patients (P < 0.01). In the first 12 months albumin excretion rate (AER) decreased by 47% only in the perindopril group (P = 0.04). From 12 to 72 months, AER gradients increased by 27% per year only in the placebo group (P < 0.01). After 6 years, macroalbuminuria had developed in 7/15 placebo compared with 2/11 in perindopril and 1/11 nifedipine‐treated patients (P = 0.05). GFR did not change in the first 12 months, but thereafter the median GFR gradient (ml/min/1.73 m2 per year) was −2.4 (P < 0.01) for perindopril‐, −1.3 (P = 0.26) for nifedipine‐ and −4.2 (P = 0.01) for placebo‐treated patients. The rate of decline in GFR for the study group as a whole from 12 months to the end of follow‐up correlated negatively with mean arterial pressure (MAP) (r = −0.38, P < 0.01). During a 3‐month treatment pause in 29 patients AER tended to increase only in the perindopril group (P < 0.07).

Conclusions  Long‐term control of blood pressure with perindopril or nifedipine stabilizes AER and attenuates GFR decline in proportion to MAP in non‐hypertensive patients with Type 2 diabetes and microalbuminuria.
Suppression of the biosynthesis of proanthocyanidin in Arabidopsis by a chimeric PAP1 repressor
Authors: Matsui Kyoko , Tanaka Hideo , Ohme‐Takagi Masaru
Published: October 2004

Flavonoids are secondary metabolites that are specific to higher plants. PAP1, a member of the family of MYB domain transcription factors in Arabidopsis, is a positive regulator of the biosynthesis of anthocyanin. We show here that a chimeric PAP1 repressor, in which the EAR‐motif repression domain from SUPERMAN was fused to PAP1, suppressed the expression of four flavonoid biosynthetic genes, namely CHS, DFR, LDOX, and BAN, in siliques, and inhibited the accumulation of proanthocyanidin, even in the presence of an endogenous positive regulator, such as TT2. This suppression resulted in the formation of light yellow seeds in 35S::PAP1SRDX transgenic plants. Our results indicate that PAP1 has the potential ability to regulate the biosynthesis not only of anthocyanin but also of proanthocyanidin. Our gene silencing system, using chimeric repressors, appears to be a useful tool for the manipulation of the biosynthesis of secondary metabolites in plants.
The role of joint attention in the development of infants’ play with objects
Authors: Bigelow Ann E. , MacLean Kim , Proctor Jane
Published: October 2004

Year‐old infants’ play was scored within and outside joint attention with mother and when alone for four levels of maturity: stereotypical, inappropriate relational, appropriate relational, functional. Maternal sensitivity within joint attention was rated on two measures: following infants’ interests and scaffolding infants’ activities. Infants’ play was more advanced with mother than when alone. With mother, infants had more functional and appropriate relational play within joint attention and more stereotypical play outside joint attention, indicating more advanced play within joint attention and more immature play outside joint attention. Functional play within joint attention, but not outside joint attention, correlated with functional play when alone. Mothers’ ability to scaffold infants’ activities within joint attention may be particularly facilitative to infants’ advanced play.