bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple copies of MATE elements support autonomous plasmid replication in Aspergillus nidulans
Authors: Aleksenko Alexei , Gems David , Clutterbuck John
Published: October 2006

The AMA1 sequence is an efficient plasmid replicator and transformation enhancer in Aspergillus nidulans. It comprises two long perfect inverted repeats (MATE elements) flanking a short, unique, central spacer. Subclone analysis indicates that the complete inverted duplication, but not the unique central spacer, is necessary for efficient plasmid replication. The smallest fragments able to affect transformation efficiency lie within the AT‐rich portions of the inverted repeats. We demonstrate that two or more copies of the repeat in any relative orientation are able to perform the replicator function. A single copy of a MATE element increases transformation frequency to a modest extent but leads to multiple rearrangement, unstable integration or concatenation of vector molecules. Multimeric concatenates generated during this process are more stable mitotically, and when reisolated, transform the fungus at a much higher frequency than the original monomeric vector. Selection for multiple copies leads to the accumulation of multimeric products which resemble amplified DNA in various eukaryotic systems.
Depletion in the levels of the release factor eRF1 causes a reduction in the efficiency of translation termination in yeast
Authors: Stansfield Ian , Akhmaloka Lily Eurwilaichitr , Tuite Mick F.
Published: October 2006

In Saccharomyces cerevisiae, translation termination is mediated by a complex of two proteins, eRF1 and eRF3, encoded by the SUP45and SUP35 genes, respectively. Mutations in the SUP45 gene were selected which enhanced suppression by the weak ochre (UAA) suppressor tRNASerSUQ5. In each of four such allo‐suppressor alleles examined, an in‐frame ochre (TAA) mutation was present in the SUP45 coding region; therefore each allele encoded both a truncated eRF1 protein and a full‐length eRF1 polypeptide containing a serine missense substitution at the premature UAA codon. The full‐length eRF1 generated by UAA read‐through was present at sub‐wild‐type levels. In an suq5+ (i.e. non‐suppressor) background none of the truncated eRF1 polypeptides were able to support cell viability, with the loss of only 27 amino acids from the C‐terminus being lethal. The reduced eRF1 levels in these sup45 mutants did not lead to a proportional reduction in the levels of ribosome‐bound eRF3, indicating that eRF3 can bind the ribosome independently of eRF1. A serine codon inserted in place of the premature stop codon at codon 46 in the sup45–22 allele did not generate an allosuppressor pheno‐type, thereby ruling out this‘missense’mutation as the cause of the allosuppressor phenotype. These data indicate that the cellular levels of eRF1 are important for ensuring efficient translation termination in yeast.
Effect of the pufQ‐pufB intercistronic region on puf mRNA stability in Rhodobacter capsulatus
Authors: Heck Claudia , Rothfuchs Rüdiger , Jäger Andreas , Rauhut Reinhard , Klug Gabriele
Published: October 2006

Differential expression of genes localized within the polycistronic puf operon of Rhodobacter capsulatus is partly due to altered stabilities of individual mRNA segments. We show that the 5’untranslated region (UTR) of pufB contributes to the unusual longevity of the 0.5 kb light‐harvesting (LH) I specific pufBA mRNA and of the 2.7 kb pufBALMX mRNA. Three stem‐loop structures have been identified within the pufQ‐pufB intercistronic region by means of RNA secondary‐structure analysis in vitro and in vivo. Deletion analysis of the pufB 5’UTR indicates that the complete set of secondary structures is required to maintain wild‐type levels of pufBA mRNA stability. A phylogenetic comparison of pufB 5’UTRs of other photosynthetic bacteria reveals an evolutionary conservation of the base‐pairing potential despite sequence divergence. Comparison of puf mRNA decay in Escherichia coli strains with or without endoribonuclease E (RNase E) activity suggests that the pufB 5′ secondary structures protect the downstream mRNA segment against degradation by RNase E. Removal of the 117‐nucleotide pufQ‐pufB intercistronic region results in loss of stability for the pufBA and pufBALMX mRNAs with concomitant stabilization of the full‐length puf primary transcript (QBALMX). We therefore conclude that the deleted sequence functions both as a stabilizing element for pufBALMX and pufBA segments and as a target site for initial rate‐limiting decay of the unstable pufQBALMX mRNA.
Multimers of the precursor of a type IV pilin‐like component of the general secretory pathway are unrelated to pili
Authors: Pugsley Anthony P.
Published: October 2006

Both the mature and precursor forms of PulG, a type IV pilin‐like component of the general secretory pathway of Klebsiella oxytoca, can be chemically cross‐linked into multimers similar to those obtained by cross‐linking the components of type IV pili. To explore the possibility that the PulG precursor could form a pilus‐like structure, the PulG sequence was altered in a variety of ways, including (i) replacement of the characteristic hydrophobic region, which is required for the assembly of type IV pilins by the MalE signal peptide, or (ii) fusion of β‐lactamase (βlaM) to the C‐terminus. Neither of these changes affected multimerization. PulG precursor could be post‐translationally processed by pre‐pilin peptidase (PulO), indicating that the N‐terminus of pre PulG remains on the cytoplasmic side of the cytoplasmic membrane where it is accessible to the catalytic site of this enzyme. Finally, precursor and mature forms of PulG could be efficiently cross‐linked in a mixed dimer, indicating that at least a subpopu‐lation of the two forms of the protein are probably located in clusters in the cytoplasmic membrane. These results provide further evidence that the cross‐linked multimers of the precursor form of PulG are unrelated to type IV pilus‐like structures. It is still unclear whether a subpopulation of processed PulG can be assembled into a rudimentary pilus‐like structure.
The orlA gene from Aspergillus nidulans encodes a trehalose‐6‐phosphate phosphatase necessary for normal growth and chitin synthesis at elevated temperatures
Authors: Borgia Peter T. , Miao Yihong , Dodge Carol L.
Published: October 2006

A cosmid carrying the orIA gene from Aspergillus nidulans was identified by complementation of an orlA1 mutant strain with DNA from the pKBY2 cosmid library. An orlA1 complementing fragment from the cosmid was sequenced. orlA encodes a predicted polypeptide of 227 amino acids (26 360 Da) that is homologous to a 211‐amino‐acid domain from the polypeptide encoded by the Saccharomyces cerevisiae TPS2 gene and to almost the entire Escherichia coli of otsB‐encoded polypeptide. TPS2 and otsB each specify a trehalose‐6‐phosphate phosphatase, an enzyme that is necessary for trehalose synthesis. orlA disruptants accumulate trehalose‐6‐phosphate and have reduced trehalose‐6‐phosphatate phosphatase levels, indicating that the gene encodes a tre‐halose‐6‐phosphatate phosphatase. Disruptants have a nearly‐wild‐type morphology at 32°C. When germinated at 42°C, the conidia and hyphae from disruptants are chitin deficient, swell excessively, and lyse. The lysis is almost completely remedied by osmotic stabilizers and is partially remedied by N‐acetylglucosamine (GlcNAc). The activity of glutamine:fructose‐6‐phosphate amido‐transferase (GFAT), the first enzyme unique to aminosugar synthesis, is reduced and is labile in orIA disruption strains. The findings are consistent with the hypothesis that trehalose‐6‐phosphate reduces the temperature stability of GFAT and other enzymes of chitin metabolism at elevated temperatures. The results extend to filamentous organisms the observation that mutations in fungal trehalose synthesis are highly pleiotropic and affect aspects of carbohydrate metabolism that are not directly related to trehalose synthesis.
An unorthodox sensor protein (TorS) mediates the induction of the tor structural genes in response to trimethylamine N‐oxide in Escherichia coli
Authors: Jourlin Cécile , Bengrine Abderrahmane , Chippaux Marc , Méjean Vincent
Published: October 2006

We isolated and characterized three spontaneous mutations leading to trimethylamine N‐oxide (TMAO)‐independent expression of the tor operon encoding the TMAO‐reductase anaerobic respiratory system in Escherichia coli. The mutations lie in a new tor regulatory gene, the torS gene, which probably encodes a sensor protein of a two‐component regulatory system. One mutation, which leads to full TMAO‐constitutive expression, is a 3‐amino‐acid deletion within the potential N‐terminal periplasmic region, suggesting that this region contains the TMAO‐detector site. For the other two mutations, a further induction of the tor operon is observed when TMAO is added. Both are single substitutions and affect the linker region located between the detector and the conserved transmitter domains. Thus, as proposed for other sensors, the TorS linker region might play an essential role in propagating conformational changes between the detector and the cytoplasmic signalling regions. The TorR histidine kinase is an unorthodox sensor that contains a receiver and a C‐terminal alternative transmitter domain in addition to the domains found in most sensors. Previously, we showed that TMAO induction of the tor operon requires the TorR response regulator and the TorT periplasmic protein. Additional genetic data confirm that torS encodes the sensor partner of TorR and TorT. First, insertion within torS abolishes tor operon expression whatever the growth conditions. Second, overexpressed TorR bypasses the requirement for torS, whereas the torT gene product is dispensable for tor operon expression in a torS constitutive mutant. This supports a signal‐transduction cascade from TorT to TorR via TorS.
Identification of two regions of Klebsiella oxytoca pullulanase that together are capable of promoting β‐lactamase secretion by the general secretory pathway
Authors: Sauvonnet Nathalie , Pugsley Anthony P.
Published: October 2006

Pullulanase (PulA) is a 116kDa amylolytic lipoprotein secreted by the Gram‐negative bacterium Klebsiella oxytoca via the general secretory pathway. A deletion strategy was used in an attempt to determine the nature and the location of the secretion signal(s) in PulA presumed to be necessary for its specific secretion. The starting material was a gene fusion coding for an efficiently secreted PulA‐β‐lactamase hybrid protein. Successive series of exonuclease III‐generated deletions were used to remove internal segments of PulA from this hybrid. A simple plate test allowed the identification of truncated hybrids that retained β‐lactamase activity and that were secreted. Two non‐adjacent regions, A and B (78 and 80 amino acids, respectively), were together necessary and sufficient to promote β‐lactamase translocation across the outer membrane. Secretion of PulA itself was markedly reduced when either of these regions was deleted, and was completely abolished when both regions were eliminated.
The AngR protein and the siderophore anguibactin positively regulate the expression of iron‐transport genes in Vibrio anguillarum
Authors: Chen Qian , Wertheimer Anne M. , Tolmasky Marcelo E. , Crosa Jorge H.
Published: October 2006

Vibrio anguillarum virulence is associated with the presence of a piasmid‐mediated iron‐uptake system expressed under iron‐limiting conditions, which consists of the siderophore anguibactin and specific iron‐transport proteins. This system is maximally expressed under iron‐limiting conditions and requires the AngR protein that acts as a positive regulator of anguibactin biosynthesis and also possess an EntE‐like enzymatic function that may play a role in anguibactin biosynthesis. In this work, we demonstrate that in addition to possessing these functions related to anguibactin production, AngR also positively regulated transcription of the iron‐transport genes fatA and fatB. We also show that transcription of angR is repressed by Fur under iron‐rich conditions. In addition, we present evidence that anguibactin itself enhanced transcription of the iron‐transport genes fatA and fatB, independently of AngR and the frans‐acting factor (TAF) product(s). The presence of either AngR (together with the TAF product(s)) or anguibactin alone led to a partial level of expression of the iron‐transport genes fatA and fatB, while full expression is achieved when AngR, the TAF products and anguibactin are all present.
Fimbrial biogenesis genes of Pseudomonas aeruginosa: pilW and pilX increase the similarity of type 4 fimbriae to the GSP protein‐secretion systems and pilY1 encodes a gonococcal PilC homologue
Authors: Aim Richard A. , Hallinan James P. , Watson Alison A. , Mattick John S.
Published: October 2006

Type 4 fimbriae of Pseudomonas aeruginosa are surface filaments involved in host colonization. They mediate both attachment to host epithelial cells and flagella‐independent twitching motility. Four additional genes, pilW, pilX, pilY1 and pilY2, are located on Spel fragment E in the 5 kb intergenic region between the previously characterized genes pilV and pilE, which encode prepilin‐like proteins involved in type 4 fimbrial biogenesis. The phenotypes of a transposon insertion and other mutations constructed by allelic exchange show that these genes are involved in the assembly of type 4 fimbriae. The PilW and PilX proteins are membrane located, possess the hydrophobic N‐terminus characteristic of prepilin‐like proteins, and appear to belong to the GspJ and GspK group of proteins that are required for protein secretion in a wide range of Gram‐negative bacteria. These findings increase the similarities between the fimbrial biogenesis and the Gsp‐based protein‐secretion super‐systems. PilY1 is a large protein with C‐terminal homology to the PilC2 protein of Neisseria gonorrhoeae, thought to be a fimbrial tip‐associated adhesin, and which, like PilY1, is involved in fimbrial assembly. PilY1 appears to be located in both the membrane and the external fimbrial fractions. PilY2 is a small protein that appears to play a subtle role In fimbrial biogenesis and represents a new class of protein.
RNase E: still a wonderfully mysterious enzyme
Authors: Cohen Stanley N. , McDowall Kenneth J.
Published: October 2006

Ribonuclease E (RNase E), which is encoded by an essential Escherichia coli gene known variously as rne, ams, and hmp, was discovered initially as an rRNA‐processing enzyme but is now known to have a general role in RNA decay. Multiple functions, including the ability to cleave RNA endonucleolyticaliy in AU‐rich single‐strand regions, RNA‐binding capabilities, and the ability to interact with polynucleotide phosphorylase and other proteins implicated in the processing and degradation of RNA, are encoded by its 1061 amino acid residues. The presence of homologues and functional analogues of the rne gene in a variety of prokaryotic and eukaryotic species suggests that its functions have been highly conserved during evolution. While much has been learned in recent years about the structure and functions of RNase E, there is continuing mystery about possible additional activities and molecular interactions of this enzyme.
Chemical information from the fungus Amylostereum areolatum and host‐foraging behaviour in the parasitoid Ibalia leucospoides
Authors: MARTÍNEZ ANDRÉS S. , FERNÁNDEZ‐ARHEX VALERIA , CORLEY JUAN C.
Published: October 2006

Abstract Parasitoids locate hosts using reliable and predictable cues such as smells derived from host plants or from the hosts themselves. For host species that live with mutualistic organisms, such as several wood boring insects, cues derived from the symbionts are likely to be exploited by specific parasitoids. Through a set of bioassays, the behaviour of the parasitoid Ibalia leucospoides Hochenwarth (Hymenoptera: Ibaliidae) is studied in response to the fungus Amylostereum areolatum Boidin (Basidiomycotina: Corticiaceae), a symbiont of its host, the wood wasp Sirex noctilio Fabricius (Hymenoptera: Siricidae). The results show that parasitoids are attracted to the fungus when growing naturally within pine logs, and also when growing on an artificial medium. Fungal volatiles also elicit increased parasitoid activity and may provide information on relative densities of hosts available for parasitization. It is speculated that the the chemical information derived from the host fungal symbiont comprises reliable and detectable host‐locating cues used by parasitoids to search for concealed hosts.
Influence of High‐Fat Feeding on Both Naive and Antigen‐Experienced T‐Cell Immune Response in DO10.11 Mice
Authors: Verwaerde C. , Delanoye A. , Macia L. , Tailleux A. , Wolowczuk I.
Published: October 2006

Obesity is becoming one of the most serious public health problems in industrialized societies, due to the profound changes in lifestyle, and notably in nutrition. Beside diabetes, cardiovascular diseases or hypertension, increased susceptibility to infection is one of the pathological consequences of being overweight. In this paper, we have assessed the influence of a high‐fat diet (HFD) rich in saturated fatty acids on the immune system of DO11.10 mice, which are transgenic for a T‐cell receptor specifically recognizing a peptide of ovalbumin. We showed that the specific T‐cell immune response was impaired by high‐fat feeding, and that the expression of this defect is different depending on whether T cells are naive or Ag experienced. Indeed, on in vitro ovalbumin stimulation, spleen T cells from naive HFD‐fed transgenic mice showed proliferation similar to that of cells from standard diet (SD)‐fed mice, but exhibited a strong inflammatory profile as shown by the markedly increased IFN‐γ/IL‐4 ratio. Inversely, spleen T cells from ovalbumin‐immunized HFD mice were impaired in their Ag‐dependent proliferation compared to cells from SD mice. By co‐culture experiments, we showed that both T cells and antigen‐presenting cells were involved in this impairment. Moreover, in ovalbumin‐immunized HFD animals, a trend towards Th2 response was noted, compared to immunized SD mice. This data implies that naive T cells could participate actively in the low‐grade systemic inflammation observed in overweight patients. Moreover, the impaired activity of Ag‐experienced T cells could have major consequences both in defence against infection and/or in vaccination protocols.
Chimaeric Protein Improved Immunogenicity Compared with Fusion Protein of Ag85B and ESAT‐6 Antigens of Mycobacterium tuberculosis
Authors: Xu Y. , Wang B. , Chen J. , Wang Q. , Zhu B. , Shen H. , Qie Y. , Wang J. , Wang H.
Published: October 2006

Antigen 85B (Ag85B) and ESAT‐6 are important immunodominant antigens of Mycobacterium tuberculosis, and both are very promising vaccine candidate molecules. In this study, we relied on the T‐cell epitopes of Ag85B and ESAT‐6 to design a chimaeric protein by inserting ESAT‐6 into Ag85B from the amino acids 167–182. We found the ratio of IgG2b/IgG1 and the secretion of interferon (IFN)‐γ in the mice vaccinated with the new protein with adjuvant MPL and TDM were higher than the mice immunized with fusion protein Ag85B‐ESAT‐6, which have been reported and could induce levels of protective immunity similar to BCG in the mouse model of tuberculosis (TB) infection. These results suggest that the chimaeric protein Ag85BN‐ESAT‐6‐Ag85BC is a strong candidate for further study and the T‐cell epitopes of the antigens should be considered when we design the subunit vaccine.
Dysregulation of TNF/TNFR Superfamily Members: A Systemic Link Between Intra‐ and Extrathyroidal Manifestations in Graves’ Disease
Authors: Quadbeck B. , Stucke M. , Eckstein A. K. , Heise D. J. , Mann K. , Gieseler R. K.
Published: October 2006

Graves’ disease (GD) coincides with the occurrence of disease‐associated intrathyroidal dendritic cells (DC) and intraorbital inflammatory macrophages (Mφ). Physiologically, tumour necrosis factor‐α (TNF‐α) strongly affects the differentiation of DC and Mφ from monocytic precursors; we thus hypothesized that dysregulation of the TNF/TNFR superfamilies may provide a systemic pathogenic link in GD. In patients without eye symptoms, percentages of TNF‐α‐stimulated blood monocytes were highly significantly (P < 0.001) elevated, corresponding to both intrathyroidal DC maturation as well as increases in mature blood DC (MHC‐IIhi/CD40+/RFD1hi) and B cells (CD20hi/CD40+). GD patients also displaying eye symptoms revealed a striking reduction in blood monocytes, yet significantly (P < 0.05) increased CD40hi and TNF‐αhi leucocytes. These findings suggest for GD that excess TNF‐α induces monocytes to differentiate into hyperactivated thyroidal DC that, once emigrated, initiate systemic humoral autoimmunity associated with CD40/TNF‐α upregulation. Such overexpression may instigate differentiation of periorbital inflammatory Mφ from CD14hi/CD16+ monocytes as a likely precursor subset. These results indicate that dysregulation of TNF/TNFR superfamily members provides a systemic pathogenic link in GD in that hyperactivated circulating monocytic precursors give rise to locally restricted, disease‐associated DC and Mφ. Monocytes, therefore, may serve as a suitable target to therapeutically address the common precursor of key promoters of GD.
Frequency Analysis of HBsAg‐specific B Lymphocytes in High‐responder Individuals to Recombinant Hepatitis B Vaccine: Comparison of LDA and ELISPOT Assays
Authors: Shokrgozar M. A. , Sam M. R. , Amirkhani A. , Shokri F.
Published: October 2006

The determination of the frequency of antigen‐specific lymphocytes may provide invaluable information for the evaluation of the immune response to different antigens and pathogens. Different methods are employed to determine the frequency of specific B lymphocytes in peripheral blood. In this study, the frequency of hepatitis B surface antigen (HBsAg)‐specific B lymphocytes was determined by a limiting dilution assay (LDA) and an enzyme‐linked immunospot assay (ELISPOT) in seven healthy adult high responders to recombinant HBsAg. Peripheral blood mononuclear cells isolated at different time intervals (1, 2, 4, 8 and 16 weeks) following administration of a booster dose were either transformed with Epstein–Barr virus (LDA) or stimulated with Pokeweed mitogen (ELISPOT). In an LDA, anti‐HBs positive wells were screened by a sandwich ELISA and the frequency of specific B lymphocytes was estimated based on the Poisson statistical analysis. In an ELISPOT, coloured spots representing specific B lymphocytes were finally enumerated by stereomicroscope. Our results showed a significant increase in the number of specific B lymphocytes in the first week by an ELISPOT compared with an LDA (1:190 versus 1:13,462) (P < 0.001). No significant differences were observed at other time intervals. A significant correlation was observed between the serum titer of anti‐HBs antibody and frequency of HBsAg‐specific B cells obtained by LDA and ELISPOT methods at different time intervals. The highest correlation was found at fourth week in LDA (r = 0.83, P < 0.01) and ELISPOT (r = 0.85, P < 0.01) assays. Furthermore, a significant correlation was observed between an LDA and ELISPOT at different time intervals (highest correlation in second week, r = 0.88, P < 0.008). These findings suggest that in addition to technical advantages, such as speed and simplicity, an ELISPOT is a more sensitive assay, compared with an LDA.
Regulatory Activity of Activated Murine Peripheral CD4+CD25− T Cells: A Possible Mechanism of Feedback Regulation on Adaptive Immunity
Authors: Lu Y. , Xu W. , Zhou Y. , Lv P. , Gao X.‐M.
Published: October 2006

This study was undertaken to investigate the possibility that peripheral CD4+CD25− T cells, once fully activated, transiently obtain suppressive function against other T cells. CD4+CD25− T cells, isolated from splenocytes of BALB/c mice, were stimulated with anti‐CD3 MoAb in the presence of feeder cells for 72 h. The activated CD4+ T cells (Tact) thus obtained were able to inhibit the activation and proliferation of bystander CD4+ T cells in a non‐MHC‐restricted manner. Tact‐mediated suppression was cell contact dependent, reversible by exogenous IL‐2 as well as anti‐GITR antibody. Furthermore, adoptive transfer of Tact cells significantly downregulated humoral response of BALB/c mice to s.c. immunization with ovalbumin. We argue that suppression mediated by activated CD4+CD25− T cells may play an important role in maintaining homeostasis of the immune system and preventing excessive T‐cell responses in vivo.
Changing indications for red cell transfusion from 2000 to 2004 in the North of England
Authors: Wallis J. P. , Wells A. W. , Chapman C. E.
Published: October 2006

summary Falling donor numbers and the threat of transfusion‐transmitted variant Creutzfeldt‐Jakob disease may lead to shortages in the national blood supply. Knowledge of current patterns of transfusion and trends in usage will help predict future change in blood use. Our previous survey identified medical indications as the major reason for transfusion, but detailed information within this category was limited.

We performed prospective surveys of indications for red cell transfusion for two 14‐day periods in 2004 in the North of England, concentrating on medical reasons for transfusion. Data were obtained for transfusion indications of 9003 units, which accounted for over 99% of red cell issues from the regional blood centre during the study. In 2004, medical patients received 62% (5558 units) of all transfused units, surgical patients 33% (3001 units) and Obstetric & Gynaecology patients 5% (444 units). These figures compare with 52, 41 and 6% for Medicine, Surgery, and Obstetrics & Gynaecology in 1999/2000.

The three largest uses of blood within the medical category were for patients with primary haematological disorders (18·2% of all transfused blood), for management of gastrointestinal haemorrhage (13·8%) and for patients with nonhaematological malignancies (8·8%).

There has been a significant reduction in use of blood for surgical indications over the last 5 years, but an absolute increase in use of blood for medical indications. Lower transfusion triggers, education, use of cell salvage, the increasing price of a unit of red cells and changing population demographics may all have contributed to the reduction in surgical blood use. Promotion of good transfusion practice and alternatives to allogeneic transfusion should now focus on medical and surgical use of blood transfusion.
An updated mutation spectrum in an Australian series of PJS patients provides further evidence for only one gene locus
Authors: Chow E , Meldrum CJ , Crooks R , Macrae F , Spigelman AD , Scott RJ
Published: October 2006

The genetic predisposition Peutz–Jeghers Syndrome (PJS) has been shown to be associated with mutations in the serine threonine kinase 11 (STK11) gene but only a proportion of probands have been shown to harbour changes in the gene. The remaining patients were proposed to be either associated with a second PJS gene or they harboured more cryptic mutations within the STK11 gene itself. With the introduction of the multiplex ligation probe amplification (MLPA) assay, large sequence losses or gains can be more readily identified. In this report we have screened 33 PJS patients from unrelated families, employing a combination of denaturing high‐performance liquid chromatography, direct DNA sequencing and the MLPA assay to identify deleterious changes in the STK11 gene. The results revealed that 24 (73%) of patients diagnosed with PJS‐harboured pathogenic mutations in the STK11 gene, including 10 (36%) with exonic or whole‐gene deletions. No phenotypic differences were identified in patients harbouring large deletions in the STK11 gene compared to patients harbouring missense or nonsense mutations. Mutation analysis in PJS should include techniques such as MLPA to identify large exonic or whole‐gene deletions and rearrangements. The high proportion of families with identifiable mutations in the STK11 gene using this range of techniques suggests that most, if not all PJS, is attributable to mutations in the STK11 gene, perhaps including as yet undiscovered changes in promoter or enhancer sequences or other cryptic changes.
Significant morbidity associated with RSV infection in immunosuppressed children following liver transplantation: Case report and discussion regarding need of routine prophylaxis
Authors: Blanchard Samra S. , Gerrek Marla , Siegel C. , Czinn Steven J.
Published: October 2006

Abstract:  Respiratory syncytial virus (RSV) is an important cause of lower respiratory tract infection in infants and young children. In immunocompromised children, RSV infection poses a serious health threat with significantly increased and prolonged virus shedding and the development of severe respiratory disease. We report two patients, eight months and 20 months of age, who were admitted with severe RSV infection two months and 10 months post‐transplant respectively. Major risk factors for severe infection is the degree of immunosuppression and the age of the patient (<24 months). Based on the significant morbidity associated with RSV infection in these patients, we recommend randomized trials in larger pediatric solid organ transplant centers to evaluate the use of palivizumab prophylaxis is efficacious to prevent morbidity in patients under the age of 24 months, while we emphasize good hygienic practices to prevent RSV nosocomial infection.
Restrictive hemodynamics are present at the time of diagnosis of allograft coronary artery disease in children
Authors: Law Yuk , Boyle Gerard , Miller Susan , Clendaniel James , Ettedgui Jose , Beerman Lee , Counihan Peter , Webber Steven
Published: October 2006

Abstract:  Clinical recognition of allograft coronary artery disease (ACAD) is challenging. We examined whether right heart hemodynamics can aid its diagnosis in pediatric recipients. We retrospective analyzed hemodynamic data of recipients with ACAD versus age and date‐of‐transplant matched controls. From 1982–2001, 18 cases fulfilled study entry criteria. Median age at transplant was 12 years for subjects and 8 years for controls. Median time to diagnosis of ACAD was 65 months (14.5–124 months) and 67 months (16–140 months) to arteriography for controls. The median right ventricular end‐diastolic pressure (RVEDP) at diagnosis was 11.0 vs. 6.0 mmHg for controls (p = 0.003). Pulmonary capillary wedge pressure (PCWP) at diagnosis was 14.0 vs. 8.0 mmHg for controls (p = 0.001). When subdivided by severity of ACAD, the difference was greater in the moderate/severe group. Compared to the previous catheterization (median interval 10 months for subjects, 12.0 for controls ), there was an increase of 4.0 mmHg in RVEDP in ACAD subjects (n = 13, p = .003) versus 0 mmHg in controls (p = 0.042), and an increase in PCWP of 5.5 in subjects (p = .002) versus 0 mmHg in controls (p = 0.066). The presence of elevated filling pressures plus an interim increase should alert to the presence of ACAD and help guide further investigation.
ANOTHER LOOK AT THE PRESUMED‐VERSUS‐INFORMED CONSENT DICHOTOMY IN POSTMORTEM ORGAN PROCUREMENT
Authors: JACOB MARIE‐ANDRÉE
Published: October 2006

In this paper I problematise quite a simple assertion: that the two major frameworks used in assessing consent to post‐mortem organ donation, presumed consent and informed consent, are procedurally similar in that both are ‘default rules.’ Because of their procedural common characteristic, both rules do exclude marginalized groups from consent schemes. Yet this connection is often overlooked. Contract theory on default rules, better than bioethical arguments, can assist in choosing between these two rules. Applying contract theory to the question of post‐mortem organ donation suggests that the default rule should be one that goes against the wishes of the stronger party in consent decisions.
CLONING, PARENTHOOD, AND GENETIC RELATEDNESS
Authors: SPARROW ROBERT
Published: October 2006

In this paper I examine what I take to be the best case for reproductive human cloning, as a medical procedure designed to overcome infertility, and argue that it founders on an irresolvable tension in the attitude towards the importance of being ‘genetically related’ to our children implied in the desire to clone. Except in the case where couples are cloning a child they have previously conceived naturally, cloning is unable to establish the right sort of genetic relation to make couples the parents of their cloned child. If anybody is the genetic parent of a cloned child it is the natural parent(s) of the DNA donor. Paradoxically, in order to resist the claims of the parents of the donor to the cloned child, the argument for human reproductive cloning must place more weight on the intention to parent a child, than we do in cases of ordinary reproduction. It must insist that the parental relation is established by the intentions of the couple who bring a clone into the world and not by their genetic relation to the child. The emphasis placed on intention as establishing the parental relationship works to undermine the justification for cloning in the first place. For cloning to play a useful role as a reproductive technology, it must allow couples to become parents who could do so no other way. However, to the extent that intention is sufficient to establish parenthood, adoption or surrogacy, which are existing alternatives to cloning, will serve equally well to allow couples to become parents.
THE CLAIM FROM ADOPTION REVISITED
Authors: RIVERA‐LÓPEZ EDUARDO
Published: October 2006

In a recent paper published in this journal, Thomas S. Petersen makes a qualified defense of what he calls ‘the Claim from Adoption’, according to which, ‘instead of expending resources on bringing new children [in developed countries] into the world using reproductive technology and caring for these children, we ought to devote these resources to the adoption and care of existing destitute children’. My purpose in this paper is not to discuss Petersen’s argument in favor of that claim. Rather, I want to show that, even if the Claim from Adoption might be, other things being equal, true, it is not true all things considered. What is, all things considered, true is a much more complex set of statements, including a general prima facie obligation to adopt, as well as an obligation by the state of developed countries to discourage procreation and promote adoption of destitute children.
MORTUARY DISPLAY AND CULTURAL CONTACT: A CEMETERY AT KASTRI ON THASOS
Authors: OWEN SARA
Published: October 2006

Summary. This article explores how the patterns of development within the local populations prior to Greek colonization, or even Greek contact, can elucidate the process of Greek colonization. Focusing upon the Thracian Early Iron Age cemetery of Kastri on Thasos, it suggests that past interpretations of such cemeteries as undifferentiated is due to the imposition of modern ideas of value. This article instead uses the criterion of diversity to suggest that the cemetery in fact has clear patterns of social differentiation in the first and last periods of use. Furthermore imports are restricted to graves of highest diversity in the last period of use (the early seventh century BC). This pattern is repeated over Early Iron Age Thrace, and is indicative of a social change within Thrace prior to Greek colonization which saw nascent Thracian elites seeking out imports from many areas in order to bolster their status.
THE ‘PERSEUS DANCE’ VASE REVISITED
Authors: HUGHES ALAN
Published: October 2006

Summary. For 70 years the image of an apparent comic actor performing on a simple stage has been known primarily from the drawing by Gilliéron. This paper is founded on the first direct study of the Vlasto chous since 1935. The author compares the vase with the drawing, showing that while scholars ought to have been sceptical of Gilliéron, he was reasonably accurate. Interpretations of the imagery from Caputo, who first studied the vase (1935), toSchmidt (1995) explain it variously as a scene of comedy or a manslaughter trial on a ship. The paper evaluates these views in the light of comparable iconography and fresh study of the vase, and concludes that it is indeed theatrical. However, some aspects of the imagery remain difficult to explain.
Cerebral Metabolism of 6–[18F]Fluoro‐l‐3,4‐Dihydroxyphenylalanine in the Primate
Authors: Firnau G. , Sood S. , Chirakal R. , Nahmias C. , Garnett E. S.
Published: October 2006

Abstract: The tracers 6‐[18F]fluoro‐1‐3,4‐dihydroxyphenyl‐alanine (6–[18F]fluoro‐l‐DOPA) and 1‐[14C]DOPA were injected simultaneously into rhesus monkeys, and the time course of their metabolites was measured in the striatum and in the occipital and frontal cortices. In the striatum, 6‐[18F]fluoro‐L‐DOPA was metabolized to 6–[18F]fluorodo‐pamine, 3,4‐dihydroxy‐6–[18F]fluorophenylaceticacid, and 6–[18F]fluorohomovanillic acid. The metabolite pattern was qualitatively similar to that of 1‐[14C]DOPA. 6–[18F]Fluorodopamine was synthesized faster than [14C]do‐pamine. In the frontal cortex, the major metabolite was also 6–[18F]fluorodopamine or [14C]dopamine. In the occipital cortex, the major metabolite was 3‐O‐methyl‐6–[18F]fluoro‐L‐DOPA. On the basis of these data, the images obtained with 6–[l8F]fluoro‐l‐DOPA and positron emission tomography in humans can now be interpreted in neurochemical terms.
Comparison of Tryptic Peptides of Benzodiazepine Binding Proteins Photolabeled with [3H]Flunitrazepam or [3H]Ro 15–4513
Authors: Sieghart W. , Eichinger A. , Zezula J.
Published: October 2006

Abstract: When rat brain membranes were incubated with the benzodiazepine agonist [3H]flunitrazepam or the partial inverse benzodiazepine agonist [3H]Ro 15–4513 in the presence of ultraviolet light one protein (P51) was specifically and irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. After digestion of the membranes with trypsin, protein P51 was degraded into several peptides. When P51 was photolabeled with [3H]Ro 15–4513, four peptides with apparent molecular weights of 39,000, 29,000, 21,000, and 17,000 were observed. When P51 was labeled with [3H]flunitrazepam, only two peptides with apparent molecular weights of 39,000 and 25,000 were obtained. Protein P55 was only partially degraded by trypsin, and whether it was labeled with [3H]‐flunitrazepam or [3H]Ro 15–4513 it yielded the same two proteolytic peptides with apparent molecular weights of 42,000 and 45,000. These results support the existence of at least two different benzodiazepine receptor subtypes associated with proteins P51 and P55. The different receptors seem to be differentially protected against treatment with trypsin. In addition, these results indicate that in the benzodiazepine receptor subtype associated with P51 benzodiazepine agonists and partial inverse benzodiazepine agonists irreversibly bind to different parts of the molecule.
Influence of Na+, K+, and Ca2+ on Glutamine Synthesis and Distribution in Rat Brain Cortex Slices: A Possible Linkage of Glutamine Synthetase with Cerebral Transport Processes and Energetics in the Astrocytes
Authors: Benjamin A. M.
Published: October 2006

Abstract: The ouabain‐induced suppression of glutamine synthesis and retention in incubating rat brain cortex slices was found to be mimicked by changes in the cationic content of the incubation medium, which cause an increase in the intracellular [Na+] and a decrease in the intracellular [K+]. The suppression of glutamine synthesis (and fixation of ammonia) was also found to take place when Ca2+ was omitted from the incubation medium. This occurred whether endogenous or exogenous glutamate was the substrate for glutamine synthesis. The suppressions cannot be due solely to an effect on glutamate uptake, because the uptake is not markedly affected by these conditions. The results show that Na+, K+, and Ca2+ influence the synthesis and distribution of glutamine in the brain. They suggest that Ca2+ and the Na+, K+ pump may serve a role in regulating the activity of ATP‐dependent glutamine synthetase, a key enzyme of the glutamate‐glutamine cycle, located in the astrocytes. This may be mediated via a direct effect on the enzyme or through an effect on the production of ATP.
Purification and Properties of Calmodulin‐Lysine N‐Methyltransferase from Rat Brain Cytosol
Authors: Morino Hideo , Kawamoto Tatsuo , Miyake Masaharu , Kakimoto Yasuo
Published: October 2006

Abstract: A S‐adenosylmethionineiprotein‐lysine N‐methyltransferase (EC 2.1.1.43) has been purified from rat brain cytosol 7,080‐fold with a yield of 8%, using octopus calmodulin as a substrate. It contains a lysine residue that is not fully methylated. The enzyme was purified by ammonium sulfate fractionation, Sephacryl S‐200 gel filtration, and phosphocellulose and octopus calmodulin‐Sepharose affinity chromatographies. Among protein substrates, it was highly specific toward octupus calmodulin. The Km values for octopus calmodulin and S‐adenosyl‐L‐methionine were found to be 2.2 × 10−8M and 0.8 × 10−6M, respectively. The molecular weight was estimated to be 57,000 by gel filtration and the pH optimum was between 7.5 and 8.5. The enzyme was stimulated in the presence of 10−7M Mn2+ and 10−4M Ca2+. HPLC of the acid hydrolysate of methyl‐3H‐labeled calmodulin showed the formation of ε‐N‐mono‐, ε‐N‐di‐, and ε‐N‐trimethyllysine. Reverse‐phase HPLC of tryptic peptides of the methyl‐3H‐labeled calmodulin demonstrated that the labeled N‐methyllysine lies in the 107–126 peptide. These findings suggest that this enzyme methylated a specific lysine residue of octopus calmodulin.
[3H]Dipyridamole Binding to Guinea Pig Brain Membranes: Possible Heterogeneity of Central Adenosine Uptake Sites
Authors: Marangos Paul J. , Deckert Jurgen
Published: October 2006

Abstract: The binding of [3H]dipyridamole ([3H]DPR) to guinea pig brain membranes is described and compared to that of [3H]nitrobenzylthioinosine ([3H]NBI). The binding of [3H]DPR is saturable, reversible, and specific with phar‐macologic evidence indicating that this ligand is binding to the adenosine uptake site. Compared to [3H]NBI the binding of [3H]DPR is of higher capacity (Bmax= 208 ±16 fmol/ mg protein for [3H]NBI and 530 ± 40 fmol/mg protein for [3H]DPR) and lower affinity (KD= 0.35 ± 0.02 nM for [3H]NBI and 7.6 ± 0.7 nM for [3H]DPR). The adenosine uptake inhibitors are the most potent inhibitors of binding (Ki of 10−8‐10−7M) whereas adenosine receptor ligands such as cyclohexyladenosine, 2‐chloroadenosine, and various methylxanthines are several orders of magnitude less potent (Ki 10−5‐10−2). The inhibition of [3H]DPR binding by NBI is biphasic, with only 40% of binding being susceptible to inhibition by NBI concentrations < 10−5M. The tissue distribution of [3H]DPR binding parallels that of [3H]NBI although in most cases significantly more sites are observed with [3H]DPR. Calcium channel blocking agents such as nifedipine, nimodipine, and verapamil are also inhibitors of [3H]DPR binding with potencies in the micromolar range. The data are consistent with [3H]DPR being a useful additional ligand for the adenosine uptake site and provide evidence that multiple uptake binding sites exist of which only about 40% are NBI‐sensitive.
Ganglioside GM1 Antibodies and B‐Cholera Toxin Bind Specifically to Embryonic Chick Dorsal Root Ganglion Neurons but Do Not Modulate Neurite Regeneration
Authors: Doherty Patrick , Walsh Frank S.
Published: October 2006

Abstract: Polyclonal antibodies to ganglioside Gmi have been prepared and characterised by direct and competitive enzyme‐linked immunoassay. An immunoglobulin fraction was prepared from a rabbit antisera showing high specificity and antibody titre for GMI relative to the other major brain gangliosides. The anti‐GMI immunoglobulin fraction and B‐cholera toxin specifically labelled neurons in primary cultures of embryonic chick dorsal root ganglia and there was a good correlation between the relative increase in binding of anti‐GMI immunoglobulin and B‐cholera toxin following neuraminidase treatment of a variety of cell types. At antibody concentrations that show saturable binding to endogenous ganglioside in the neuronal membrane, the anti‐GM1 immunoglobulin fraction did not interfere with the nerve growth factor (NGF)‐mediated fibre outgrowth and neuronal survival as indexed by measurement of neu‐rofilament protein levels. Similarly, at levels in excess of those shown to stimulate thymocyte proliferation, B‐cholera toxin was also without effect. These data are not consistent with GMI in the neuronal membrane functioning as a receptor molecule for NGF and/or other differentiation factors present in the tissue culture media.
Brain Endo‐Oligopeptidase A, a Putative Enkephalin Converting Enzyme
Authors: Camargo Antonio C. M. , Oliveira Eduardo B. , Toffoletto Odaly , Metters Kathleen M. , Rossier Jean
Published: October 2006

Abstract: Endo‐oligopeptidase A, highly purified from the cytosol fraction of bovine brain by immunoaffinity chroma‐tography, has been characterized as a thiol endopeptidase. This enzyme, known to hydrolyze the Phe5‐Ser6 bond of bradykinin and the Arg8‐Arg9 bond of neurotensin, has been shown to produce, by a single cleavage, Leu5‐enkephalin or Met5‐enkephalin from small enkephalin‐containing pep‐tides. Enkephalin formation could be inhibited in a concentration‐dependent manner by the alternative substrate bradykinin. The optimal substrate size was found to be eight to 13 amino acids, with enkephalin the only product released from precursors in which this sequence is immediately followed by a pair of basic residues. However, the specificity constants (Kcat/Km) obtained for endo‐oligopeptidase A hydrolysis of bradykinin, neurotensin, and dynorphin B are of the same order, a result indicating that the substrate amino acid sequence is not the only factor determining the cleavage site of this enzyme.
Phenylalanine Transport Across the Blood‐Brain Barrier as Studied with the In Situ Brain Perfusion Technique
Authors: Momma Seiji , Aoyagi Masaki , Rapoport Stanley I. , Smith Quentin R.
Published: October 2006

Abstract: Unidirectional L‐phenylalanine transport into six brain regions of pentobarbital‐anesthetized rats was studied using the in situ brain perfusion technique. This technique allows both accurate measurements of cerebrovascular amino acid transport and complete control of perfusate amino acid composition. l‐Phenylalanine influx into the brain was sodium independent and could be described by a model with a saturable and a nonsaturable component. Best‐fit values for the kinetic constants in the parietal cortex equaled 6.9 × 10−4 Mmol/s/g for Vmax, 0.011 μmol/ml for Km, and 1.8 × 10−4 ml/s/g for Kd during perfusion with fluid that did not contain competing amino acids. d‐Phenylala‐nine competitively inhibited l‐phenylalanine transport with a Ki∼ 10‐fold greater than the Km for l‐phenylalanine. There were no significant regional differences in Km,Kd, or Ki, whereas Vmax was significantly greater in the cortical lobes than in the other brain regions. l‐Phenylalanine influx during plasma perfusion was only 30% of that predicted in the absence of competing amino acids. Competitive inhibition increased the apparent Km during plasma perfusion by ∼20‐fold, to 0.21 pmol/ml. These data provide accurate new estimates of the kinetic constants that describe L‐phenylalanine transport across the blood‐brain barrier. In addition, they indicate that the cerebrovascular transfer site affinity (1/Km) for L‐phenylalanine is three‐ to 12‐fold greater than previously estimated in either awake or anesthetized animals.