bluuebunny/crossref_metadata_2025_split huggingface dataset:
A 38 kDa precursor protein of aqualysin I (a thermophilic subtilisin‐type protease) with a C‐terminal extended sequence: its purification and in vitro processing
Authors: Kurosaka Keisuke , Ohta Takahisa , Matsuzawa Hiroshi 
Published: October 2006

SummaryThe precursor of aqualysin I, an extracellular subtilisin‐type protease produced by Thermus aquaticus, consists of four domains: an N‐terminal signal peptide, an N‐terminal pro‐sequence, a protease domain, and a C‐terminal extended sequence. In an Escherichia coli expression system for the aqualysin I gene, a 38 kDa precursor protein consisting of the protease domain and the C‐terminal extended sequence is accumulated in the membrane fraction and processed to a 28 kDa mature enzyme upon heat treatment at 65°C. The 38 kDa precursor protein is separated as a soluble form from denatured E. coli proteins after heat treatment. Accordingly, purification of the 38 kDa proaqualysin I was performed using chromatography. The purified precursor protein gave a single band on SDS‐polyacrylamide gels. The precursor protein exhibited proteolytic activity comparable to that of the mature enzyme. The purified precursor protein was processed to the mature enzyme upon heat treatment. The processing was inhibited by diisopropyl fluorophosphate. The processing rate increased upon either the addition of mature aqualysin I or upon an increase in the concentration of the precursor, suggesting that the cleavage of the C‐terminal extended sequence occurs through an intermolecular self‐processing mechanism.
Autoregulation of luxR: the Vibrio harveyi lux‐operon activator functions as a repressor
Authors: Chatterjee Jaidip , Miyamoto Carol M. , Meighen Edward A. 
Published: October 2006

SummaryMobility‐shift assays have been used to demonstrate that the activator of the Vibrio harveyi lux operon, LuxR, binds independently, and with similar affinity, to two sites upstream of its own open reading frame. One site was located between 52 and 107 bp upstream of, and the other site in a region 25 bp downstream of, the transcriptional start site. The luxR promoter, in a transcriptional fusion with the chloramphenicol acetyl transferase (cat) gene, could readily be expressed in Escherichia coli as well as V. harveyi in the absence of LuxR. In both species, the presence of the luxR gene product resulted in repression of luxR promotion. These results show that LuxR directly regulates its own expression by functioning as an autorepressor. A mechanism for this repression is suggested by evidence showing that LuxR has a negative effect on RNA polymerase binding to the luxR promoter. In light of the fact that LuxR is also part of a regulatory family of repressors, the mechanism by which LuxR functions as a transcriptional activator of the lux operon has been re‐examined.
Multiple copies of MATE elements support autonomous plasmid replication in Aspergillus nidulans
Authors: Aleksenko Alexei , Gems David , Clutterbuck John 
Published: October 2006

SummaryThe 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

SummaryIn 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

SummaryDifferential 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

SummaryBoth 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

SummaryA 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

SummaryWe 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

SummaryPullulanase (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

Summary 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

Summary 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

SummaryRibonuclease 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.
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

AbstractObesity 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

AbstractAntigen 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

AbstractGraves’ 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.