A chitotetrose specific lectin from Luffa acutangula :Physico-chemical properties and the assignment of orientation of sugars in the lectin binding site

A chitooligosaccharide specific lectin (Luffa acutangula agglutinin) has been purified from the exudate of ridge gourd fruits by affinity chromatography on soybean agglutininglycopeptides coupled to Sepharose-6B. The affinity purified lectin was found homogeneous by polyacrylamide gel electrophoresis, in sodium dodecyl sulphate-polyacrylamide gels, by gel filtration on Sephadex G-100 and by sedimentation velocity experiments. The relative molecular weight of this lectin is determined to be 48,000 ± 1,000 by gel chromatography and sedimentation equilibrium experiments. The sedimentation coefficient (S20, w) was obtained to be 4·06 S. The Stokes’ radius of the protein was found to be 2·9 nm by gel filtration. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis the lectin gave a molecular weight of 24,000 in the presence as well as absence of 2-mercaptoethanol. The subunits in this dimeric lectin are therefore held by non-covalent interactions alone. The lectin is not a glycoprotein and circular dichroism spectral studies indicate that this lectin has 31% α-helix and no ß-sheet. The lectin is found to bind specifically to chitooligosaccharides and the affinity of the lectin increases with increasing oligosaccharide chain length as monitored by near ultra-violetcircular dichroism and intrinsic fluorescence titration. The values of ΔG, ΔΗ and ΔS for the binding process showed a pronounced dependence on the size of the oligosaccharide. The values for both ΔΗ and ΔS show a significant increase with increase in the oligosaccharide chain length showing that the binding of higher oligomers is progressively more favoured thermodynamically than chitobiose itself. The thermodynamic data is consistent with an extended binding site in the lectin which accommodates a tetrasaccharide. Based on the thermodynamic data, blue shifts and fluorescence enhancement, spatial orientation of chitooligosaccharides in the combining site of the lectin is assigned.

Plasticity in the primary binding site of galactose/N-acetylgalactosamine-specific lectins - Implication of the C-H center dot center dot center dot O hydrogen bond at the specificity-determining C-4 locus of the saccharide in 4-methoxygalactose recognition by jacalin and winged bean (basic) agglutinin I

It is currently believed that an unsubstituted axial hydroxyl at the specificity-determining C-4 locus of galactose is indispensable for recognition by galactose/N-acetylgalactosamine-specific lectins. Titration calorimetry demonstrates that 4-methoxygalactose retains binding allegiance to the Moraceae lectin jacalin and the Leguminosae lectin, winged bean (basic) agglutinin (WBA I). The binding reactions were driven by dominant favorable enthalpic contributions and exhibited significant enthalpy-entropy compensation. Proton NMR titration of C-methoxygalactose with jacalin and WBA I resulted in broadening of the sugar resonances without any change in chemical shift. The alpha-and beta-anomers of 4-methoxygalactose were found to be in slow exchange with free and lectin-bound states. Both the anomers experience magnetically equivalent environments at the respective binding sites. The binding constants derived from the dependence of NMR line widths on 4-methoxygalactose concentration agreed well with those obtained from titration calorimetry. The results unequivocally demonstrate that the loci corresponding to the axially oriented C-4 hydroxyl group of galactose within the primary binding site of these lectins exhibit plasticity. These analyses suggest, for the first time, the existence of C-H ... O-type hydrogen-bond(s) in protein-carbohydrate interactions in general and between the C-4 locus of galactose derivative and the lectins jacalin and WBA I in particular.

HBHA-specific IFN-{gamma} Synthesis at the Site of Infection during Active Tuberculosis in Humans.

RATIONALE: Tuberculosis (TB) remains a major cause of mortality. A better understanding of the immune responses to mycobacterial antigens may be helpful to develop improved vaccines and diagnostics. OBJECTIVE: The mycobacterial antigen heparin-binding-hemagglutinin (HBHA) induces strong interferon-gamma (IFN-gamma) responses by circulating lymphocytes from Mycobacterium tuberculosis latently infected subjects, and low responses associated with CD4(+) regulatory T (Treg) cells in TB patients. Here, we investigated HBHA-specific IFN-gamma responses at the site of the TB disease. METHODS: Bronchoalveolar lavages, pleural fluids and blood were prospectively collected from 61 patients with a possible diagnosis of pulmonary and/or pleural TB. HBHA-specific IFN-gamma production was analyzed by flow cytometry and ELISA. The suppressive effect of pleural Treg cells was investigated by depletion experiments. MEASUREMENTS AND MAIN RESULTS: The percentages of HBHA-induced IFN-gamma(+) alveolar and pleural lymphocytes were higher for pulmonary (P<0.0001) and for pleural (P<0.01) TB than for non-TB controls. Local CD4(+) and CD8(+) T cells produced the HBHA-specific IFN-gamma. This local secretion was not suppressed by Treg lymphocytes, contrasting with previously reported data on circulating lymphocytes. CONCLUSION: TB patients display differential effector and regulatory T cell responses to HBHA in local and circulating lymphocytes with a predominant effector CD4(+) and CD8(+) response locally, compared to a predominant Treg response among circulating lymphocytes. These findings may be helpful for the design of new vaccines against TB, and the detection of HBHA-specific T cells at the site of the infection may be a promising tool for the rapid diagnosis of active TB.

DNA-dependent Protein Kinase-mediated Phosphorylation of Protein Kinase B Requires a Specific Recognition Sequence in the C-terminal Hydrophobic Motif

DNA-dependent protein kinase (DNA-PK) has been implicated in a variety of nuclear processes including DNA double strand break repair, V(D)J recombination, and transcription. A recent study showed that DNA-PK is responsible for Ser-473 phosphorylation in the hydrophobic motif of protein kinase B (PKB/Akt) in genotoxic-stressed cells, suggesting a novel role for DNA-PK in cell signaling. Here, we report that DNA-PK activity toward PKB peptides is impaired in DNA-PK knock-out mouse embryonic fibroblast cells when compared with wild type. In addition, human glioblastoma cells expressing a mutant form of DNA-PK (M059J) displayed a lower DNA-PK activity when compared with glioblastoma cells expressing wild-type DNA- PK (M059K) when PKB peptide substrates were tested. DNA- PK preferentially phosphorylated PKB on Ser-473 when compared with its known in vitro substrate, p53. A consensus hydrophobic amino acid surrounding the Ser-473 phospho-acceptor site in PKB containing amino acids Phe at position +1 and +4 and Tyr at position -1 are critical for DNA- PK activity. Thus, these data define the specificity of DNA- PK action as a Ser-473 kinase for PKB in DNA repair signaling.

Organization specific predictors of job satisfaction: findings from a Canadian multi-site quality of work life cross-sectional survey

Background: Organizational features can affect how staff view their quality of work life. Determining staff perceptions about quality of work life is an important consideration for employers interested in improving employee job satisfaction. The purpose of this study was to identify organization specific predictors of job satisfaction within a health care system that consisted of six independent health care organizations.

Methods: 5,486 full, part and causal time (non-physician) staff on active payroll within six organizations (2 community hospitals, 1 community hospital/long-term care facility, 1 long-term care facility, 1 tertiary care/community health centre, and 1 visiting nursing agency) located in five communities in Central West Ontario, Canada were asked to complete a 65-item quality of work life survey. The self-administered questionnaires collected staff perceptions of: co-worker and supervisor support; teamwork and communication; job demands and decision authority; organization characteristics; patient/resident care; compensation and benefits; staff training and development; and impressions of the organization. Socio-demographic data were also collected.

Results: Depending on the organization, between 15 and 30 (of the 40 potential predictor) variables were found to be statistically associated with job satisfaction (univariate analyses). Logistic regression analyses identified the best predictors of job satisfaction and these are presented for each of the six organizations and for all organizations combined.

Conclusions: The findings indicate that job satisfaction is a multidimensional construct and although there appear to be some commonalities across organizations, some predictors of job satisfaction appear to be organization and context specific.

GIS on tourism planning: preventing the impacts caused by the developing tourism activity in a specific site

The environment is one of the greatest concerns of humankind. Nowadays, the activities which improve or destroy it must be assessed and controlled by efficient means which should permit the control of environmental impact caused by the development of these activities. This document presents an information system implementation, as a Decision Support system, allowing the Decision Maker to evaluate, foresee and control the future environmental impact of Tourism through consultation, the management and the presentation of decision schemes based on defined measures of a regional tourism planning.

New generation vaccine induces effective melanoma-specific CD8+ T cells in the circulation but not in the tumor site.

Although increasing evidence suggests that CTL are important to fight the development of some cancers, the frequency of detectable tumor-specific T cells is low in cancer patients, and these cells have generally poor functional capacities, compared with virus-specific CD8(+) T cells. The generation with a vaccine of potent CTL responses against tumor Ags therefore remains a major challenge. In the present study, ex vivo analyses of Melan-A-specific CD8(+) T cells following vaccination with Melan-A peptide and CpG oligodeoxynucleotides revealed the successful induction in the circulation of effective melanoma-specific T cells, i.e., with phenotypic and functional characteristics similar to those of CTL specific for immunodominant viral Ags. Nonetheless, the eventual impact on tumor development in vaccinated melanoma donors remained limited. The comprehensive study of vaccinated patient metastasis shows that vaccine-driven tumor-infiltrating lymphocytes, although activated, still differed in functional capacities compared with blood counterparts. This coincided with a significant increase of FoxP3(+) regulatory T cell activity within the tumor. The consistent induction of effective tumor-specific CD8(+) T cells in the circulation with a vaccine represents a major achievement; however, clinical benefit may not be achieved unless the tumor environment can be altered to enable CD8(+) T cell efficacy.

Specific detection of Lettuce mosaic virus isolates belonging to the Most type

Lettuce mosaic virus (LMV)-Most isolates can infect and are seed-borne in cultivars containing the mol gene. A reverse transcription and polymerase chain reaction (RT-PCR)-based test was developed for the specific detection of LMV-Most isolates. Based on the complete genome sequences of three LMV isolates belonging respectively to the Most type, the Common type and neither of these two types, three different assays were compared: (i) presence of a diagnostic restriction site in the region of the genome encoding the variable N-terminus of the capsid protein, in the 3' end of the genome, (ii) RT-PCR using primers designed to amplify a cDNA corresponding to a portion of the P1 coding region, in the 5' end of the genome and (iii) RT-PCR using primers designed to amplify a central region of the genome. The assays were performed against a collection of 21 isolates from different geographical origins and representing the molecular variability of LMV. RT-PCR of the central region of the genome was preferred because its results are expected to be less affected by natural recombination between LMV isolates, and it allows sensitive detection of LMV-Most in situations of single as well as mixed contamination. (C) 2004 Elsevier B.V. All rights reserved.

H19-DMR allele-specific methylation analysis reveals epigenetic heterogeneity of CTCF binding site 6 but not of site 5 in head-and-neck carcinomas: A pilot case-control analysis

Aberrant methylation of seven potential binding sites of the CTCF factor in the differentially methylated region upstream of the H19 gene (H19-DMR) has been suggested as critical for the regulation of IGF2 and H19 imprinted genes. In this study, we analyzed the allele-specific methylation pattern of CTCF binding sites 5 and 6 using methylationsensitive restriction enzyme PCR followed by RFLP analysis in matched tumoral and lymphocyte DNA from head-and-neck squamous cell carcinoma (HNSCC) patients, as well as in lymphocyte DNA from control individuals who were cancer-free. The monoallelic methylation pattern was maintained in CTCF binding site 5 in 22 heterozygous out of 91 samples analyzed. Nevertheless, a biallelic methylation pattern was detected in CTCF binding site 6 in a subgroup of HNSCC patients as a somatic acquired feature of tumor cells. An atypical biallelic methylation was also observed in both tumor and lymphocyte DNA from two patients, and at a high frequency in the control group (29 out of 64 informative controls). Additionally, we found that the C/T transition detected by HhaI RFLP suppressed one dinucleotide CpG in critical CTCF binding site 6, of a mutation showing polymorphic frequencies. Although a heterogeneous methylation pattern was observed after DNA sequencing modified by sodium bisulfite, the biallelic methylation pattern was confirmed in 9 out of 10 HNSCCs. These findings are likely to be relevant in the epigenetic regulation of the DMR, especially in pathological conditions in which the imprinting of IGF2 and H19 genes is disrupted.

Site- and species-specific responses of forest growth to climate across the European continent

Aim To evaluate the climate sensitivity of model-based forest productivity estimates using a continental-scale tree-ring network. Location Europe and North Africa (30–70° N, 10° W–40° E). Methods We compiled close to 1000 annually resolved records of radial tree growth for all major European tree species and quantified changes in growth as a function of historical climatic variation. Sites were grouped using a neural network clustering technique to isolate spatiotemporal and species-specific climate response patterns. The resulting empirical climate sensitivities were compared with the sensitivities of net primary production (NPP) estimates derived from the ORCHIDEE-FM and LPJ-wsl dynamic global vegetation models (DGVMs). Results We found coherent biogeographic patterns in climate response that depend upon (1) phylogenetic controls and (2) ambient environmental conditions delineated by latitudinal/elevational location. Temperature controls dominate forest productivity in high-elevation and high-latitude areas whereas moisture sensitive sites are widespread at low elevation in central and southern Europe. DGVM simulations broadly reproduce the empirical patterns, but show less temperature sensitivity in the boreal zone and stronger precipitation sensitivity towards the mid-latitudes. Main conclusions Large-scale forest productivity is driven by monthly to seasonal climate controls, but our results emphasize species-specific growth patterns under comparable environmental conditions. Furthermore, we demonstrate that carry-over effects from the previous growing season can significantly influence tree growth, particularly in areas with harsh climatic conditions – an element not considered in most current-state DGVMs. Model–data discrepancies suggest that the simulated climate sensitivity of NPP will need refinement before carbon-cycle climate feedbacks can be accurately quantified.

Specific microbial biomarker signatures from Congo fan sediments of ODP site 175-1075, Congo soils, wetland and estuary sediments

Methane (CH4) is a strong greenhouse gas known to have perturbed global climate in the past, especially when released in large quantities over short time periods from continental or marine sources. It is therefore crucial to understand and, if possible, quantify the individual and combined response of these variable methane sources to natural climate variability. However, past changes in the stability of greenhouse gas reservoirs remain uncertain and poorly constrained by geological evidence. Here, we present a record from the Congo fan of a highly specific bacteriohopanepolyol (BHP) biomarker for aerobic methane oxidation (AMO), 35-aminobacteriohopane-30,31,32,33,34-pentol (aminopentol), that identifies discrete periods of increased AMO as far back as 1.2 Ma. Fluctuations in the concentration of aminopentol, and other 35-aminoBHPs, follow a pattern that correlates with late Quaternary glacial-interglacial climate cycles, with highest concentrations during warm periods. We discuss possible sources of aminopentol, and the methane consumed by the precursor methanotrophs, within the context of the Congo River setting, including supply of methane oxidation markers from terrestrial watersheds and/or marine sources (gas hydrate and/or deep subsurface gas reservoir). Compound-specific carbon isotope values of -30 per mil to -40 per mil for BHPs in ODP 1075 and strong similarities between the BHP signature of the core and surface sediments from the Congo estuary and floodplain wetlands from the interior of the Congo River Basin, support a methanotrophic and likely terrigenous origin of the 35-aminoBHPs found in the fan sediments. This new evidence supports a causal connection between marine sediment BHP records of tropical deep sea fans and wetland settings in the feeding river catchments, and thus tropical continental hydrology. Further research is needed to better constrain the different sources and pathways of methane emission. However, this study identifies the large potential of aminoBHPs, in particular aminopentol, to trace and, once better calibrated and understood, quantify past methane sources and fluxes from terrestrial and potentially also marine sources.

Specific cleavage of chromosomal and plasmid DNA strands in gram-positive and gram-negative bacteria can be detected with nucleotide resolution

A sensitive and precise in vitro technique for detecting DNA strand discontinuities produced in vivo has been developed. The procedure, a form of runoff DNA synthesis on molecules released from lysed bacterial cells, mapped precisely the position of cleavage of the plasmid pMV158 leading strand origin in Streptococcus pneumoniae and the site of strand scission, nic, at the transfer origins of F and the F-like plasmid R1 in Escherichia coli. When high frequency of recombination strains of E. coli were examined, DNA strand discontinuities at the nic positions of the chromosomally integrated fertility factors were also observed. Detection of DNA strand scission at the nic position of F DNA in the high frequency of recombination strains, as well as in the episomal factors, was dependent on sexual expression from the transmissable element, but was independent of mating. These results imply that not only the transfer origins of extrachromosomal F and F-like fertility factors, but also the origins of stably integrated copies of these plasmids, are subject to an equilibrium of cleavage and ligation in vivo in the absence of DNA transfer.

Position- and orientation-independent activity of the Schizosaccharomyces pombe meiotic recombination hot spot M26

The activity of the M26 meiotic recombination hot spot of Schizosaccharomyces pombe depends on the presence of the heptamer 5′-ATGACGT-3′. Transplacement of DNA fragments containing the ade6-M26 gene to other chromosomal loci has previously demonstrated that the heptamer functions in some, but not all, transplacements, suggesting that hot spot activity depends on chromosomal context. In this study, hot spot activity was tested in the absence of gross DNA changes by using site-directed mutagenesis to create the heptamer sequence at novel locations in the genome. When created by mutagenesis of 1–4 bp in the ade6 and ura4 genes, the heptamer was active as a recombination hot spot, in an orientation-independent manner, at all locations tested. Thus, the heptamer sequence can create an active hot spot in other chromosomal contexts, provided that the gross chromosomal structure is not altered; this result is consistent with the hypothesis that a specific higher-order chromatin structure is required for M26 hot spot activity.

Identification of specific Rp-phosphate oxygens in the tRNA anticodon loop required for ribosomal P-site binding

tRNA binding to the ribosomal P site is dependent not only on correct codon–anticodon interaction but also involves identification of structural elements of tRNA by the ribosome. By using a phosphorothioate substitution–interference approach, we identified specific nonbridging Rp-phosphate oxygens in the anticodon loop of tRNAPhe from Escherichia coli which are required for P-site binding. Stereo-specific involvement of phosphate oxygens at these positions was confirmed by using synthetic anticodon arm analogues at which single Rp- or Sp-phosphorothioates were incorporated. Identical interference results with yeast tRNAPhe and E. coli tRNAfMet indicate a common backbone conformation or common recognition elements in the anticodon loop of tRNAs. N-ethyl-N-nitrosourea modification–interference experiments with natural tRNAs point to the importance of the same phosphates in the loop. Guided by the crystal structure of tRNAPhe, we propose that specific Rp-phosphate oxygens are required for anticodon loop (“U-turn”) stabilization or are involved in interactions with the ribosome on correct tRNA–mRNA complex formation.