bluuebunny/crossref_metadata_2025 huggingface dataset:
Pre‐endoscopic proton pump inhibitor therapy reduces recurrent adverse gastrointestinal outcomes in patients with acute non‐variceal upper gastrointestinal bleeding
Authors: KEYVANI L. , MURTHY S. , LEESON S. , TARGOWNIK L. E.
Published: September 2006

Background

Proton pump inhibitors (PPIs) following endoscopic haemostasis reduce rebleeding rates in patients with high‐risk acute non‐variceal upper gastrointestinal bleeding. Many advocate the use of PPIs prior to endoscopy, although its incremental benefit is unproven.

Aim

To determine if providing PPIs before endoscopy reduces adverse gastrointestinal outcomes in acute non‐variceal upper gastrointestinal bleeding patients.

Methods

We performed a retrospective review to identify patients presenting to two tertiary care centres with acute non‐variceal upper gastrointestinal bleeding between 1999 and 2004. Subjects receiving PPI therapy before endoscopy were compared with those not receiving pre‐endoscopic PPI therapy. The primary outcome measure was the development of any adverse bleeding outcome (rebleeding, surgery for control of bleeding, in‐hospital mortality, readmission within 30 days for acute non‐variceal upper gastrointestinal bleeding).

Results

385 patients were included in our study [132 (12 intravenous/120 po) pre‐endoscopic PPI vs. 253 no pre‐endoscopic PPI]. Patients receiving pre‐endoscopic PPI therapy were significantly less likely to develop adverse outcomes compared with those not given pre‐endoscopic PPIs (25% vs. 13%, P = 0.005). Rebleeding, upper gastrointestinal surgery, mortality and length of hospital stay were also significantly lower in patients receiving pre‐endoscopic PPI.

Conclusions

The use of PPIs before endoscopy significantly reduces the risk of developing adverse gastrointestinal outcomes in patients with acute non‐variceal upper gastrointestinal bleeding. Future studies are required to better characterize this relationship.
Tolerability and pharmacokinetics of inhaled bimosiamose disodium in healthy males
Authors: Meyer Michael , Beeh Kai‐Michael , Beier Jutta , Beyer Diana , Aydt Ewald , Zahlten Rainer , Jilma Bernd , Wolff Gerhard
Published: September 2006

Aims

The aim of these first‐in‐human studies was to investigate the tolerability and the pharmacokinetics of bimosiamose disodium (TBC1269Z) administered by inhalation.

Methods

Two randomized, double‐blind, placebo‐controlled Phase I trials were performed in healthy males. In a single‐dose escalating study 48 subjects received doses of 2–140 mg bimosiamose disodium by inhalation and in a multiple‐dose study 32 subjects received 8–70 mg bimosiamose disodium twice daily. In both studies 4 ml of the drug solution was administered via nebulizer over 15 min. Adverse events, vital signs, ECG, clinical laboratory parameters and forced expiratory volume in 1 s (FEV1) data were recorded and nasopharyngeal examinations were performed to address the safety and tolerability. Blood was collected for the determination of plasma concentrations of bimosiamose.

Results

All subjects completed the study. No deaths or severe adverse events occurred. Eleven mild adverse events occurred in the dose‐escalation study and 34 in the multiple‐dose study after inhalation of bimosiamose disodium. Adverse events were more frequent at the highest dose (140 mg) of the dose‐escalation study. For placebo treatment one moderate adverse event was observed in the dose‐escalation study after placebo treatment, eight mild and three moderate adverse events occurred in the multiple‐dose study. Bimosiamose was detected in plasma (maximum concentration 64 ng ml−1) only at doses ≥50 mg given twice daily and 105 mg once daily. For the highest dose a median value of 5746 h ng ml−1 was determined for the AUC over the entire period of treatment of the multiple‐dose study.

Conclusion

The results suggest that single and multiple inhalation of bimosiamose disodium up to 70 mg is well tolerated in healthy males. Systemic bioavailability after inhalation is low.
A simple device to rapidly prepare whole mounts of murine intestine
Authors: Rudling R. , Hassan A. B. , Kitau J. , Mandir N. , Goodlad R. A.
Published: September 2006

Abstract.  Many mouse models of neoplasia and pre‐neoplasia require the examination of whole mounts of the gastrointestinal tract. A simple device has been produced to facilitate the rapid preparation of mouse intestines for subsequent quantification of tumours and pre‐neoplastic lesions such as aberrant crypt foci. The device greatly speeds up the production of whole mounts and also provides far more consistent and better‐quality preparations.
Intestinal crypt properties fit a model that incorporates replicative ageing and deep and proximate stem cells
Authors: Lobachevsky P. N. , Radford I. R.
Published: September 2006

Abstract.  A model of intestinal crypt organization is suggested based on the assumption that stem cells have a finite replicative life span. The model assumes the existence in a crypt of a quiescent (‘deep’) stem cell and a few more actively cycling (‘proximate’) stem cells. Monte Carlo computer simulation of published intestinal crypt mutagenesis data is used to test the model. The results of the simulation indicate that stabilization of the crypt mutant phenotype following treatment with external mutagen is consistent with a stem cell replicative life span of about 40 divisions for mouse colon and 90–100 divisions for mouse small intestine, corresponding to a deep stem cell cycle time of about 3.9 and 8.5 weeks for colon and small intestine, respectively. Simulation of the data obtained for human colorectal crypts suggests that the proximate stem cell cycle time is about 80 h, assuming a replicative life span of 50–150 divisions, and that the deep stem cell divides approximately every 30 weeks.
The reversion to diploid cells from established triploid V79 cells
Authors: Miyagoshi M. , Fujikawa‐Yamamoto K.
Published: September 2006

Abstract.  The triploid V79 cells are stable under usual culture conditions, and do not revert to being diploid. Here, the triploid–diploid transition of triploid V79 cells has been successfully induced in suspension culture in culture dishes with untreated surfaces. The diploid cells began to appear in a population of triploid V79 cells cultured under these conditions for 4 weeks. All of the triploid cells were transformed to diploid through subsequent monolayer culture for 5 weeks. It was confirmed that the revertant diploid cells had the same characteristics as original diploid V79 cells, with respect to DNA histograms, cell volume and chromosome number. Thus, it seems that suspension culturing is an important factor that induces the triploid–diploid transition.
Effect of over‐expression of neuropathy target esterase on mammalian cell proliferation
Authors: Chang P.‐A. , Liu Ch.‐Y. , Chen R. , Wu Y.‐J.
Published: September 2006

Abstract.  Neuropathy target esterase (NTE), the human homologue of a protein required for brain development in Drosophila, is expressed primarily in neural cells but is also detected in non‐neural cells. Although NTE has been proposed to play a role in neurite outgrowth and process elongation during neurodifferentiation, the function of NTE has not been defined in neural cells. In this study we have investigated the possible role of NTE in neuroblastoma cells and non‐neural cells using an over‐expression strategy. Over‐expression of NTE in human neuroblastoma SH‐SY5Y cells and monkey kidney COS7 cells led to an accumulation of NTE on the cytoplasmic surface of the endoplasmic reticulum and inhibition of cell proliferation. In particular, high levels of NTE arrested COS7 cells at G2/M stage yet was not associated with arrest at a particular phase of the cell cycle in SH‐SY5Y cells. Moreover, over‐expression of NTE did not induce apoptosis in two kinds of cell lines as assessed by flow cytometry. These results suggest that the role of NTE over‐expression in cell proliferation is associated with different mechanisms in different cells.
Mutational analysis of the PTH 3′‐untranslated region in parathyroid disorders
Authors: Costa‐Guda Jessica , Lauter Kelly , Naveh‐Many Tally , Silver Justin , Arnold Andrew
Published: September 2006

Objective  Sequence alterations in untranslated regions (UTRs) of genes are important contributors to human diseases, including hereditary thrombophilia, hereditary hyperferritinaemia‐cataract and fragile X mental retardation syndromes. Recently, functional studies of the 3′‐UTR of the PTH gene, encoding parathyroid hormone, have highlighted it as a potential target for pathogenic mutations in patients with parathyroid dysfunction. Regulation of PTH gene expression occurs in part through protein binding to a specific 26 nucleotide instability element in the 3′‐UTR of PTH mRNA, in a sequence‐dependent manner. Thus, the PTH 3′‐UTR has emerged as an important potential contributor to parathyroid dysfunction. Therefore, we sought to rigorously examine the PTH 3′‐UTR in patients with primary and secondary parathyroid disorders, including primary parathyroid hyperplasia, secondary parathyroid hyperplasia, sporadic parathyroid adenoma and familial hypoparathyroidism of unknown genetic basis.

Patients and design  Twenty‐one parathyroid glands from 14 patients with primary parathyroid hyperplasia, 40 sporadic parathyroid adenomas from 40 patients, 42 parathyroid glands from 29 patients with secondary parathyroid hyperplasia and peripheral blood leucocytes from 24 affected members of eight kindreds with familial hypoparathyroidism of unknown genetic basis were examined for mutations in the 3′‐UTR of the PTH gene.

Results  No alterations from the normal sequence were detected in any of the 127 samples examined.

Conclusions  Based on the absence of identifiable DNA sequence alterations in these forms of parathyroid dysfunction, it is unlikely that mutation of the PTH 3′‐UTR contributes frequently to their pathogenesis.
Trends in residential policies and services for people with intellectual disabilities in Taiwan
Authors: Chou Y. C. , Schalock R. L.
Published: September 2006

Background  In Taiwan, 92–95% of people with an intellectual disability (ID) live with their families, with the remaining residing in residential facilities. Instead of funding community‐living alternatives, the Taiwanese Government funds only registered facilities as part of its residential policies and services. The purpose of this study was to evaluate current policies and services trends regarding people with an ID in Taiwan.

Methods  Both documentary research, such as an analysis of policies, services programmes, official statistics, surveys, reports and funding provision reports, and a mail survey, were conducted to examine current trends and characteristics of the 96 residential settings available for people with an ID in Taiwan.

Results  During the 1990s in Taiwan, residential programmes for people with an ID showed the biggest growth since 1952. Since the first ‘Community Home’ was launched in 1990, the number of smaller scale residential settings with a unit size of less than 30 has grown significantly, particularly since 2000. However, the rate of institutionalization of people with an ID and who live in the institutions has also risen.

Conclusions  In Taiwan, unlike in Western societies, residential services for people with an ID provided by formal care systems are tending to grow in number, and these include both large and smaller residential settings.
Co‐cladogenesis spanning three phyla: leafhoppers (Insecta: Hemiptera: Cicadellidae) and their dual bacterial symbionts
Authors: TAKIYA DANIELA M. , TRAN PHAT L. , DIETRICH CHRISTOPHER H. , MORAN NANCY A.
Published: September 2006

Endosymbioses are a major form of biological complexity affecting the ecological and evolutionary diversification of many eukaryotic groups. These associations are exemplified by nutritional symbioses of insects for which phylogenetic studies have demonstrated numerous cases of long‐term codiversification between a bacterial and a host lineage. Some insects, including most leafhoppers (Insecta: Hemiptera: Cicadellidae), have more than one bacterial symbiont within specialized host cells, raising questions regarding the patterns of codiversification of these multiple partners and the evolutionary persistence of complex symbiotic systems. Previous studies reported the presence of two dominant symbiont types in a member of the leafhopper subfamily Cicadellinae (sharpshooters). In this study, 16S rRNA sequences were obtained and used to examine the occurrence and evolutionary relationships of the two dominant symbiont types across 29 leafhopper species. Candidatus Sulcia muelleri (Bacteroidetes) was detected in all leafhopper species examined, a finding that is consistent with a previous report of its ancient association with the Auchenorrhyncha (a grouping that includes leafhoppers, treehoppers, cicadas, planthoppers, and spittlebugs). Baumannia cicadellinicola (Proteobacteria), previously known from only five sharpshooter species, was found only in the sharpshooter tribes Cicadellini and Proconiini, as well as in the subfamily Phereurhininae. Mitochondrial and nuclear gene sequences were obtained and used to reconstruct host phylogenies. Analyses of host and symbiont data sets support a congruent evolutionary history between sharpshooters, Sulcia and Baumannia and thus provide the first strong evidence for long‐term co‐inheritance of multiple symbionts during the diversification of a eukaryotic host. Sulcia shows a fivefold lower rate of 16S rDNA sequence divergence than does Baumannia for the same host pairs. The term ‘coprimary’ symbiont is proposed for such cases.
Brooding crustaceans in a highly fragmented habitat: the genetic structure of Mediterranean marine cave‐dwelling mysid populations
Authors: LEJEUSNE C. , CHEVALDONNÉ P.
Published: September 2006

Habitat fragmentation and climate change are two major threats on biodiversity. Fragmentation limits the number of patches and their decreased connectivity cannot always maintain populations at dynamic equilibrium. The natural extreme fragmentation of marine cave habitats represents an opportunity to understand how these processes interact. The hypothesis of a low gene flow among populations due to a high level of fragmentation was tested by analysing sequence variation in a fragment of the mitochondrial gene of the cytochrome oxidase subunit I in 170 individuals (23 localities across the NW Mediterranean) of two marine cave‐dwelling mysids of the genus Hemimysis. The species Hemimysis margalefi recently replaced its congener Hemimysis speluncola, a species shift that could be related to the warming of the Mediterranean Sea and to differences in their thermal tolerances. There were too few H. speluncola samples to further discuss their genetic structuring, but for H. margalefi, the present study revealed high levels of genetic diversity and genetic structuring, as shown by the eight genetically distinct groups identified. The Croatian group might constitute a sibling species due to a strong divergence (15%). Nevertheless, these groups present reduced but orientated gene flow according to the general circulation in the Mediterranean, and fit a stepping‐stone model. At local scale (Marseille area, France), gene flow among caves is dependent on unexpected local hydrodynamic barriers, that determine metapopulation sizes. Through the example of mysid species inhabiting marine caves, the present work confirms the strong influence of habitat disjunction (natural fragmentation) on population structure, and stresses the importance of coastal geomorphological features in inducing complex interactions between the circulation of water masses and the circulation of genes.
Convergence, constraint and the role of gene expression during adaptive radiation: floral anthocyanins in Aquilegia
Authors: WHITTALL JUSTEN B. , VOELCKEL CLAUDIA , KLIEBENSTEIN DANIEL J. , HODGES SCOTT A.
Published: September 2006

Convergent phenotypes are testament to the role of natural selection in evolution. However, little is known about whether convergence in phenotype extends to convergence at the molecular level. We use the independent losses of floral anthocyanins in columbines (Aquilegia) to determine the degree of molecular convergence in gene expression across the anthocyanin biosynthetic pathway (ABP). Using a phylogeny of the North American Aquilegia clade, we inferred six independent losses of floral anthocyanins. Via semiquantitative reverse transcriptase–polymerase chain reaction (RT–PCR), we monitored developmental and tissue‐specific variation in expression of the six major structural ABP loci in three Aquilegia species, two that produce anthocyanins (A+) and one that does not (A−). We then compared ABP expression in petals of old‐bud and pre‐anthesis flowers of 13 Aquilegia species, eight wild species and two horticultural lines representing seven independent A− lineages as well as three wild A+ species. We only found evidence of down‐regulation of ABP loci in A− lineages and losses of expression were significantly more prevalent for genes late in the pathway. Independent contrast analysis indicates that changes in expression of dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS) are strongly phylogenetically correlated consistent with the multilocus targets of trans‐regulatory elements in the ABP of other systems. Our findings strongly suggest that pleiotropy constrains the evolution of loss of floral anthocyanins to mutations affecting genes late in the ABP mostly through convergent changes in regulatory genes. These patterns support the hypothesis that rapid evolutionary change occurs largely through regulatory rather than structural mutations.
The small untranslated RNA SR1 from the Bacillus subtilis genome is involved in the regulation of arginine catabolism
Authors: Heidrich Nadja , Chinali Alberto , Gerth Ulf , Brantl Sabine
Published: September 2006

Whereas about 70 small non‐coding RNAs have been found in the Escherichia coli genome, relatively little is known about regulatory RNAs from Gram‐positive bacteria. Here, we demonstrate that the recently identified small untranslated RNA SR1 from the Bacillus subtilis genome is a regulatory RNA involved in fine‐tuning of arginine catabolism. 2D protein gel electrophoresis indicated three possible SR1 targets that are regulated by the transcriptional activator AhrC, which was shown to be the primary target of SR1. In vitro pairing studies and an in vivo reporter gene test demonstrated a specific interaction between SR1 and ahrC mRNA. This interaction did not lead to degradation of ahrC mRNA, but inhibited translation at a post‐initiation stage. Our data show that the Hfq chaperone was not required for the stabilization of SR1 in vivo. The amount of SR1 was increased upon addition of l‐arginine and l‐ornithine, but not l‐citrulline or l‐proline.
Two higBA loci in the Vibrio cholerae superintegron encode mRNA cleaving enzymes and can stabilize plasmids
Authors: Christensen‐Dalsgaard Mikkel , Gerdes Kenn
Published: September 2006

Vibrio cholerae codes for 13 toxin–antitoxin (TA) loci all located within the superintegron on chromosome II. We show here that the two higBA TA loci of V. cholerae encode functional toxins, HigB‐1 and HigB‐2, whose ectopic expression inhibits cell growth of Escherichia coli, and functional antitoxins, HigA‐1 and HigA‐2, which counteract the toxicity of the cognate toxins. Three hours of ectopic expression of the HigB toxins resulted in bacteriostasis without any detectable loss of cell viability. The HigB toxins inhibited translation by cleavage of mRNA. Efficient mRNA cleavage occurred preferentially within the translated part of a model mRNA and only when the mRNA was translatable. Promoter analysis in V. cholerae and E. coli showed that the two higBA loci are both transcribed into bi‐cistronic mRNAs and that the higBA‐2 mRNA is leaderless. Transcription of the two higBA loci was strongly induced by amino acid (aa) starvation in V. cholerae and E. coli, indicating that the regulatory mechanisms of transcriptional induction are conserved across the two species. Both higBA loci stabilized a test‐plasmid very efficiently in E. coli, raising the possibility that the loci contribute to maintain genetic stability of the V. cholerae superintegron. Based on these results we discuss the possible biological functions of the TA loci of V. cholerae.
The HD‐GYP domain of RpfG mediates a direct linkage between the Rpf quorum‐sensing pathway and a subset of diguanylate cyclase proteins in the phytopathogen Xanthomonas axonopodis pv citri
Authors: Andrade Maxuel O. , Alegria Marcos Castanheira , Guzzo Cristiane R. , Docena Cassia , Pareda Rosa Maria Claudia , Ramos Carlos H. I. , Farah Chuck S.
Published: September 2006

Bacteria use extracellular levels of small diffusible autoinducers to estimate local cell‐density (quorum‐sensing) and to regulate complex physiological processes. The quorum‐sensing signal transduction pathway of Xanthomonas spp. phytopathogens has special features that distinguish it from that of other pathogens. This pathway consists of RpfF, necessary for the production of the unique autoinducer ‘diffusible signalling factor’ (DSF), and RpfC and RpfG, a two‐component system necessary for the DSF‐dependent production of extracellular pathogenicity factors and cellular dispersion. Yeast two‐hybrid and direct in vitro assays were used to identify interactions involving the Rpf group of proteins. We show that RpfC, a protein consisting of N‐terminal transmembrane, histidine kinase, response‐regulator and C‐terminal histidine phosphotransfer domains interacts with both RpfG, a protein consisting of an N‐terminal response regulator domain and a C‐terminal HD‐GYP domain, and with RpfF. We also show that RpfC interacts with the only known homologue of ‘conditioned medium factor’, which is involved in quorum‐sensing in Dictyostelium discoideum under conditions of nutritional stress. Furthermore, RpfCG is shown to interact with a second two‐component system made up of NtrB and NtrC homologues. Finally we show that the recently characterized HD‐GYP phosphodiesterase domain of RpfG interacts directly with diguanylate cyclase GGDEF domain‐containing proteins coded by the Xanthomonas axonopodis pv. citri genome, which in other bacteria produce cyclic diGMP, an important second messenger involved in the regulation of complex bacterial processes including biofilm production, virulence and motility. These results demonstrate a direct physical linkage between quorum‐sensing and cyclic diGMP signalling pathways in bacteria.
YpdC determines site‐1 degradation in regulated intramembrane proteolysis of the RsiW anti‐sigma factor of Bacillus subtilis
Authors: Heinrich Janine , Wiegert Thomas
Published: September 2006

Genes of Bacillus subtilis controlled by the alternative extracytoplasmic function family sigma factor σW constitute an antibiosis regulon. Its activity is modulated by RsiW, a transmembrane anti‐sigma factor that sequesters and inactivates σW. Upon a stress signal, RsiW is degraded by a mechanism of regulated intramembrane proteolysis. To identify genes which influence RsiW degradation, a transposon screen with a reporter fusion of the green fluorescent protein to RsiW was performed. Among several gene loci identified, the ypdC (prsW) gene displayed a strong effect on RsiW stability. In a ypdC null mutant, induction of σW‐controlled genes is abolished and site‐1 proteolysis of RsiW is completely blocked. Transcriptional analysis revealed that ypdC is a monocistronic gene, and the defect of σW induction of the null mutant was complemented by ectopically integrated ypdC under xylose control. Orthologues of YpdC can be found in a variety of different bacteria. Its membrane topology was analysed by alkaline phosphatase fusions, revealing that YpdC contains five transmembrane segments and two larger extracytoplasmic loops. In the first loop, two invariantly conserved glutamate residues can be found. In an Escherichia coli system, the cloned ypdC is the only determinant of efficient degradation of RsiW; however, YpdC does not display plain similarities to known proteases, suggesting that it either controls the activity of site‐1 proteolysis of RsiW or represents a new type of protease.
Excision and transfer of the Mesorhizobium loti R7A symbiosis island requires an integrase IntS, a novel recombination directionality factor RdfS, and a putative relaxase RlxS
Authors: Ramsay Joshua P. , Sullivan John T. , Stuart Gabriella S. , Lamont Iain L. , Ronson Clive W.
Published: September 2006

The Mesorhizobium loti strain R7A symbiosis island is an Integrative Conjugative Element (ICE), herein termed ICEMlSymR7A, which integrates into a phetRNA gene. Integration reconstructs the phetRNA gene at one junction with the core chromosome, and a direct repeat of the 3‐prime 17 bp of the gene is formed at the other junction. We show that the ICEMlSymR7AintS gene, which encodes an integrase of the phage P4 family, is required for integration and excision of the island. Excision also depended on a novel recombination directionality factor encoded by msi109 (rdfS). Constitutive expression of rdfS resulted in curing of ICEMlSymR7A. The rdfS gene is part of an operon with genes required for conjugative transfer, allowing co‐ordinate regulation of ICEMlSymR7A excision and transfer. The excised form of ICEMlSymR7A was detectable during exponential growth but occurred at higher frequency during stationary phase. ICEMlSymR7A encodes homologues of the traR and traI genes of Agrobacterium tumefaciens that regulate Ti plasmid transfer via quorum sensing. The presence of a plasmid with cloned island traR traI2 genes resulted in excision of ICEMlSymR7A in all cells regardless of culture density, indicating that excision may be similarly regulated. Maintenance of ICEMlSymR7A in these cells depended on msi106 (rlxS) that encodes a putative relaxase. Transfer of the island to non‐symbiotic mesorhizobia required intS, rlxS and rdfS. The rdfS and rlxS genes are conserved across a diverse range of α‐, β‐ and γ‐proteobacteria and identify a large family of genomic islands with a common transfer mechanism.
Regulated intramembrane proteolysis of FtsL protein and the control of cell division in Bacillus subtilis
Authors: Bramkamp Marc , Weston Louise , Daniel Richard A. , Errington Jeff
Published: September 2006

The small bitopic division protein FtsL is an essential part of the division machinery (divisome) in most eubacteria. In Bacillus subtilis FtsL is a highly unstable protein and the turnover has been implicated in regulation of division in response to DNA damage. N‐terminal deletions and a domain swap experiment identified the short cytoplasmic domain of FtsL as being required for instability. We then identified a zinc metalloprotease, YluC, required for turnover, and likely sequence motifs involved in substrate recognition. YluC belongs to the site‐2‐protease (S2P) family of proteases involved in regulated intramembrane proteolysis (RIP), which plays a role in diverse regulatory phenomena from bacteria to man. The yluC mutant, and strains with N‐terminal truncations of ftsL have a short cell phenotype, indicating that that FtsL is normally rate‐limiting for division. Coexpression experiments of FtsL and YluC in Escherichia coli corroborated a model in which FtsL is directly cleaved by the membrane metalloprotease. The results shed new light on the regulation of cell division in B. subtilis and identify a novel class of targets for RIP.
Measurement of anti‐CD154 monoclonal antibody in primate sera by competitive inhibition ELISA
Authors: Tai Hao‐Chih , Campanile Nathalie , Ezzelarab Mohamed , Cooper David K. C. , Phelps Carol
Published: September 2006

Abstract:  Background:  Anti‐human CD154 monoclonal antibody (mAb)‐based regimens have been demonstrated to prevent T cell‐dependent elicited antibody response in baboon recipients of pig hematopoietic progenitor cells, organs and islets. Monitoring of anti‐CD154 mAb in serum is important to ensure maintenance of adequate levels and for adjusting dosage of the anti‐CD154 mAb. We describe a method for measuring the level in primate sera.

Methods:  The anti‐CD154 mAb level in primate serum was measured with a competitive inhibition enzyme linked immunosorbent assay in which the extent of inhibition of binding by anti‐CD154 mAb conjugated to horseradish peroxidase (anti‐CD154‐HRP) to soluble CD154 was used to determine the serum level. Briefly, a 96‐well maxisorb plate coated with soluble human CD154, and blocked with bovine serum albumin, was loaded with graded doses of anti‐CD154 mAb or primate sera containing anti‐CD154 mAb. Both were mixed with a known dosage of anti‐CD154‐HRP before loading. Bound anti‐CD154‐HRP was detected by color developed using 3,3′,5,5′ tetramethyl‐benzidine as substrate. Absorbance was measured in a SynergyTM HT Multi‐Detection Microplate Reader at a wavelength of 450 nm. Data analysis was carried out using BioTek's KC4TM Data Analysis Software. The standard curve was generated from the wells loaded with the mixture of anti‐CD154 mAb and anti‐CD154‐HRP.

Results and conclusions:  The assay has been used successfully to measure anti‐CD154 mAb levels in the serum of both baboons and monkeys.
Protective effects of volatile agents against acetylcholine‐induced bronchoconstriction in isolated perfused rat lungs
Authors: Lele E. , Petak F. , Fontao F. , Morel D. R. , Habre W.
Published: September 2006

Background:  Bronchoactive properties of volatile agents against lung constriction are well established. The purpose of this study was to investigate the ability of halothane (Hal), isoflurane (Iso), sevoflurane (Sev) and desflurane (Des) to alter the lung mechanics in the absence of an airway tone and during acetylcholine (Ach)‐induced bronchoconstriction.

Methods:  Low‐frequency pulmonary impedance data (ZL) were collected from isolated, normo‐perfused rat lungs under baseline conditions and following the injection of Ach (0.1 mg/kg) into the pulmonary artery. Measurements were performed without the administration of any anaesthetic agent in the first phase of the experiments and during inhalation without any volatile agent (control group, n = 6) or during inhalation of Hal (n = 6), Iso (n = 9), Sev (n = 6) or Des (n = 8) at 1 minimum alveolar concentration (MAC). The airway resistance (Raw) and parenchymal damping and elastance were estimated from the ZL data by model fitting.

Results:  Under baseline conditions, the basic value of Raw was significantly decreased by Des (− 31.2 ± 3.8%) and Sev (− 18.0 ± 4.5%) administration, whereas Hal and Iso did not have a statistically significant effect on Raw (− 3.3 ± 5.1% and − 8.6 ± 2.4%, respectively). Moreover, all four inhalation anaesthetics prevented the increase in Raw following Ach administration, the findings ranging between − 14.3 ± 11.4% for Hal and − 37.5 ± 10.9% for Sev.

Conclusions:  Our results on a denervated isolated perfused lung model demonstrate the potential of Des and Sev to decrease the basal airway tone, whereas Iso and Hal are ineffective in this regard. All of these volatile agents markedly protect against Ach‐induced bronchoconstriction.
Effects of two different hydroxyethyl starch solutions (HES200/0.5 vs. HES130/0.4) on the expression of platelet membrane glycoprotein
Authors: Chen G. , Yan M. , Lu Q. H. , Gong M.
Published: September 2006

Background:  There are various hydroxyethyl starch (HES) solutions with different degrees of hydroxylation and different molecular weights. HES200/0.5 solution is most commonly used. HES130/0.4 is a new HES solution and is the ‘state‐of‐the‐art’ in volume substitution. However, the mechanism of the observed anticoagulation action of HES has not been fully delineated. The objective of this study was to further investigate the effect of HES200/0.5 and HES130/0.4 on platelet coagulation.

Methods:  Sixty ASA I–II patients undergoing elective minor surgery were randomly allocated to receive an intravenous infusion (20 ml/kg) of lactated Ringer’s solution (group L), HES200/0.5 (group H) or HES130/0.4 (group V) after the induction of anesthesia. The expression of CD42b, CD41/61 and CD62p in vivo was assessed on non‐stimulated platelets and adenosine diphosphate (ADP) agonist‐activated platelets using flow cytometry.

Results:  Resting glycoprotein expression of the non‐stimulated platelets was observed. HES200/0.5 and HES130/0.4 reduced the CD42b, CD41/61 and CD62p expression of ADP‐agonist‐activated platelets at 15 min after intravenous infusion. At 6 h after intravenous infusion, the trend of decreasing expression of activated CD42b, CD41/61 and CD62p was maintained in group H. However, CD42b, CD41/61 and CD62p expression returned to the pre‐operative level in group V.

Conclusion:  This study showed that both HES200/0.5 and HES130/0.4 can inhibit platelet coagulation. Platelet dysfunction experienced a faster recovery after the infusion of HES130/0.4 than after HES200/0.5. Liquid resuscitation with HES130/0.4 may decrease the risk of hemorrhage in the operative period.
Can We Increase the Estimative Value of Semen Assessment?*
Authors: Rodríguez‐Martínez H
Published: September 2006

Contents

Estimating the fertility of a semen sample or of the male from where it has been collected by simple assessment of in vitro sperm characteristics is still difficult, owing to the variable correlations that laboratory results achieve with in vivo fertility. A major reason behind these variations is the fact that the ejaculate and the artificial insemination (AI)‐doses it generates are composed of a diverse sperm population. Such heterogeneity is reflected both in differences of intactness of attributes needed for fertilization, such as motility or morphology, but also in the relative ability of spermatozoa to prevail fertile over time, handling and exposure to different stimuli, all of which account for innate variations in fertilizing ability among doses, ejaculates and sires. However, methods are already available to select sub‐populations of intact spermatozoa which can be tested for their degree of competence for fertilization and whose estimative power is promising, allowing the elimination of cases of sub‐fertility, particularly in bovine. Examples of these methods are the separation of viable spermatozoa by swim‐up or discontinuous gradient centrifugation, followed by testing the ability of the selected spermatozoa to dose‐response/time sustain capacitation and acrosome reaction induction. Finding how large a sperm population with non‐compensable attributes for fertilization and ability to display and sustain stimuli is, perhaps by a quick screening of membrane integrity and stability by multi‐parametric methods, would allow, provided the particular male produces this sub‐population in a repeatable manner, for a better estimation of fertility.
Mammalian Sperm Energy Resources Management and Survival during Conservation in Refrigeration
Authors: Rodriguez‐Gil JE
Published: September 2006

Contents

The present review has as its main aim to present an overview regarding the mechanisms utilized by mammalian sperm to manage its intracellular energy levels. This management will strongly influence the sperm's ability to maintain its overall function during its entire life span. Thus, the precise knowledge of these mechanisms will be of the utmost interest to optimize the systems utilized to conserve mammalian sperm for a medium‐to‐long time‐lapse. Briefly, utilization of hexoses as energy substrates by mammalian sperm is very finely regulated from the very first step of its metabolization. Furthermore, the equilibrium among the separate, monosaccharide metabolization pathways in mammalian sperm depends on many factors. This prevents the possibility to draw a general vision of sperm energy utilization, which explains the results of all mammalian species in all points of the sperm life‐cycle. To complicate the matter further, there are separate energy phenotypes among mammalian spermatozoa. The precise knowledge of these phenotypes is of the greatest importance in order to optimize the design of new extenders for sperm conservation in refrigerated conditions. Moreover, sugars can act on sperm not only as passive metabolic substrates, but also as direct function activators through mechanisms like specific changes in the tyrosine phosphorylation status of distinct proteins. Finally, mammalian sperm utilizes non‐glucidic substrates like citrate and lactate to obtain energy in a regular form. This utilization is also finely regulated and of importance to maintain overall sperm function. This implies that the exact proportion of glucidic and non‐glucidic energy substrates could be very important to optimize the survival ability of these cells in conservation.
Developmental Consequences of Sexual Dimorphism During Pre‐implantation Embryonic Development
Authors: Gutiérrez‐Adán A , Perez‐Crespo M , Fernandez‐Gonzalez R , Ramirez MA , Moreira P , Pintado B , Lonergan P , Rizos D
Published: September 2006

Contents

Abnormalities of development potential arising from pre‐implantation environment are not limited to in vitro culture (IVC) (for, i.e. in ruminants the large offspring syndrome produced by IVC), they may also be consequence of specific stress conditions experienced in vivo, like maternal diet, toxins, etc. A complex group of mechanisms (gene expression, epigenetic, metabolic, etc.) may operate to link early embryo environment with future health. Furthermore, during the pre‐implantation period, in vitro produced male embryos have a higher metabolic rate, they grow faster than females, and they also have differential gene transcription of genes located in the Y‐, X‐, or in autosomal‐chromosomes. As a consequence of these differences embryos may be affected differentially by natural or artificial environmental conditions, depending on their gender. It has been suggested that under some stress conditions male embryos are more vulnerable than females; however the biological fragility of male embryos is poorly understood. Evidences suggest that epigenetic differences produced by the presence of one or two X‐chromosomes are the principal cause of the male and female pre‐implantation differences, and we put forward the possible role of these early sex differences to control sex ratio of the offspring under different environmental conditions in Nature. By following the differences between male and female early embryos not only may be possible to manipulate sex ratio in farm animals, we can also gain further insight into aspects of early embryo development, X inactivation, and epigenetic and genetic processes related with early development that may have a long‐term effect on the offspring.
Inbreeding and Reproduction in Endangered Ungulates: Preservation of Genetic Variation through the Organization of Genetic Resource Banks
Authors: Roldan ERS , Gomendio M , Garde JJ , Espeso G , Ledda S , Berlinguer F , Del Olmo A , Soler AJ , Arregui L , Crespo C , González R
Published: September 2006

Contents

There is a constant increase in the number of species suffering marked reductions in population size. This reduction in size and the lack of genetic flow may lead to a decrease in genetic variability and to matings between close relatives (i.e. inbreeding) with an ensuing reduction in fitness. It is thus important to understand the mechanism underlying the deleterious effects of inbreeding and to develop reproductive biotechnologies that will allow the reduction of inbreeding depression by facilitating gene exchange between populations. The study of three endangered species of gazelles, Cuvier's gazelle (Gazella cuvieri), Mohor gazelle (Gazella dama mhorr) and dorcas gazelle (Gazella dorcas neglecta) has revealed that inbreeding negatively affects several semen parameters (motility, sperm morphology, acrosome integrity). Semen cryopreservation has been achieved in the three species but success varies depending on the diluent employed and the level of inbreeding. Artificial insemination of Mohor gazelles have led to the birth of the first gazelle born using frozen‐thawed semen but improvements are needed before this technology can be applied on a routine basis for the genetic management of the populations. Collection of oocytes after ovarian stimulation, followed by in vitro maturation, fertilization and culture has met with some initial success in the Mohor gazelle. These, together with other reproductive technologies, will offer an invaluable help in preserving the maximum of genetic diversity of these and related endangered ungulate species.
OC1 Effects of Cryopreservation on the Expression of Glut‐3, Glut‐5 and As‐A Proteins in Iberian Boar Sperm Membranes
Authors: Sancho S , Casas I , Pinart E , Briz M , Garcia‐Gil N , Bassols J , Pruneda A , Bussalleu E , Yeste M , Flores E , Rodríguez‐Gil JE , Ekwall H. , Rodríguez‐Martínez H. , Bonet S
Published: September 2006

In order to determine the injure produced in boar spermatozoa through cryopreservation process, we analyzed the expression of the hexose transporters Glut‐3 and Glut‐5 and the zona pellucida binding protein As‐A (P68) in three different steps of the freezing‐thawed protocol: at 17°C (fresh BTS‐diluted semen, 1 : 2 v/v, step 1), at 5°C (after glycerol addition; step 2), and post‐thawing (step 3). All sperm analyses were carried out with immunogold techniques under electronic microscopy. For this study eight healthy post‐pubertal Iberian boars were submitted to a collection of twice per week through 3 months, evaluating two ejaculates from each boar. Glut‐3 maintains the expression in the acrosome region post‐thawing but not along the tail where is reduced. The expression of Glut‐5 and As‐A is majority located at the post‐acrosome region of the spermatozoa at step 1, but in step 2 and step 3 this expression is relocated to sperm tail area. In conclusion, while cryopreservation affects the localization and the expression of Glut‐3 and Glut‐5, its fertilizing capacity is not significantly reduced. The stabilization of boar semen at 5°C was found to be the most crucial step for sperm survival.
OC5 Filtration Thought Sephadex Columns Does not Alter the Subpopulation Characteristics in Fresh Boar Semen
Authors: Ramió‐Lluch L , Rivera M , Rigau T , Balasch S , Rodríguez‐Gil JE
Published: September 2006

The aim of this study was to analyse the effect of filtration through Sephadex on the subpopulation characteristics of the boar semen. For this purpose 3 ml of 16 commercial doses of fresh diluted boar semen were filtered through a Sephadex G‐15/Polypropylene column. Motility parameters were analysed by a CASA system and statistical study was performed by SAS package using the VARCLUS and the FASTLUST procedures. Statistical study revealed four subpopulations in fresh boar semen, as previously had described (Theriogenology 61: 673–690).Total motility was higher in control than in filtered semen, but there were not statistical differences (65.63 ± 9.65 vs 41.40 ± 9.02). Moreover, the analysis did not show many changes neither in the characteristics nor in the distribution of the four subpopulations. As example although ALHmed of filtered samples were slightly higher, there were only significant differences (p < 0.001) in two subpopulations (subpopulation 2 : 2.2 ± 0.05 in control vs 2.7 ± 0.08 in filtered. Subpopulation 3 : 4.5 ± 0.11 in control vs 5.8 ± 0.23 in filtered semen). HME was also statistically different (p < 0.005) in one subpopulation, showing great values in filtered semen (1.7 ± 0.15 vs 3.0 ± 0.30). In conclusion, the filtration by Sephadex/Polypropylene column does not cause strong changes in subpopulation sperm distribution.
OC6 Scrotal Heat Stress in Mice Affects Viability and DNA Integrity of Sperm, and Sex Ratio of the Offspring
Authors: Pérez‐Crespo M , Pericuesta E , Rey R , Gutiérrez‐Adán A
Published: September 2006

Heat stress causes infertility in farm animals and represents a major source of economic loss. The objective of this work was to examine the impact of scrotal heat stress (42°C for 30 min) on viability of spermatozoa, integrity of their DNA, and the in vivo reproductive performance of CD1 mice. These parameters were assessed in the spermatozoa recovered from epididymis at 0, 7, 14, 21 and 28 days after heat treatment. Heat stressed males were mated with CD1 females, and the number and the sex ratio of Day14 fetuses was analyzed by PCR. Heat stress increases the percentage of TUNEL positive sperm present in the epididymis at the time of heating (Day 0 and 7). Also the damage was significantly higher than the control at Day 14, 21 and 28 indicating that some germ cells, which were present within the testis at the time of heat stress, were not removed by apoptosis and completed their development producing sperm with damaged DNA. Heat stress also affects viability and motility at Day 14, 21 and 28. When the sex ratio of the offspring of the males mated the Day 0 or 7 after the heat stress was assessed, we observed that the percentage of males was significantly lower than expected (p < 0.05). No differences were observed in the sex ratio of the offspring of the other groups. In conclusion, these data demonstrate that the increase in epididymal temperature can compromise the DNA integrity of spermatozoa and the sex ratio of offspring generated with this sperm, and suggest that spermatocytes are most vulnerable than spermatids to DNA damage induced by heat stress.
OC7 Pregnancy‐Associated Glycoprotein (PAG) Cloning and Expression in Pre‐implantational Ovine Conceptuses
Authors: Serrano B , Yániz J , López‐Gatius F , Garbayo JM
Published: September 2006

Pregnancy‐associated glycoproteins (PAGs) are a multigene family, related to aspartic proteinases, expressed in the placenta of artiodactyls. PAGs cloned from ruminants can be classified into two main groups. The PAG I group, a rapidly‐evolving group expressed in the binucleate cells of the trophectoderm, and the PAG II group, with more ancient origin and expressed throughout trophectoderm. The aim of this study was to identify first PAG transcripts to be expressed around implantation in sheep. Day 14 (n = 4) and 17 (n = 12) ovine conceptuses were used for reverse transcription–polymerase chain reaction (RT‐PCR) performed on RNA extracted from conceptuses pooled on each day of recovery. PCR products were analysed by restriction digest and subcloned. Similar sequences were aligned and the consensus sequence was checked for identity/similarity in the Gene Bank. A single PAG was detected on day 14, ovPAG2, the only PAG cloned in ovine from the PAG II group. On day 17, three more PAG were identified, one similar to ovPAG1 and two unknown named as ovPAG10 (DQ377958) and ovPAG11 (DQ377959), which were close to each other (95% nt, 91% aa) and other PAG I group members. Phylogenetic analysis as well as a conserved site for propeptide cleavage situated these new PAGs into the PAG I group. In summary, ovPAG2 is expressed at day 14 of pregnancy and as well at day 17, together with ovPAG1 and two unknown PAG transcripts, probably belonging to the PAGI group. These early PAGs might play an important role at the beginning of implantation.