Conserved sequences in enzymes of the UDP-GlcNAc/MurNAc family are essential in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase

The UDP-GlcNAc/MurNAc family of eukaryotic and prokaryotic enzymes use UDP-GlcNAc or UDP-MurNAc-pentapeptide as donors, dolichol-P or polyprenol-P as acceptors, and generate sugar-P-P-polyisoprenols. A series of six conserved sequences, designated A through F and ranging from 5 to 13 amino acid residues, has been identified in this family. To determine whether these conserved sequences are required for enzyme function, various mutations were examined in hamster UDP- GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT). Scramble mutations of sequences B-F, generated by scrambling the residues within each sequence, demonstrated that each is important in GPT. While E and F scrambles appeared to prevent stable expression of GPT, scrambling of B- D resulted in GPT mutants that could be stably expressed and bound tunicamycin, but lacked enzymatic activity. Further, the C and D scramble mutants had an unexpected sorting defect. Replacement of sequences B-F with prokaryotic counterparts from either the B.subtilis mraY or E.coli rfe genes also affected GPT by preventing expression of the mutant protein (B, F) or inhibiting its enzymatic activity (C-E). For the C-E replacements, no acquisition of acceptor activity for polyprenol-P, the fully unsaturated natural bacterial acceptor, was detected. These studies show that the conserved sequences of the UDP- GlcNAc/MurNAc family are important, and that the eukaryotic and prokaryotic counterparts are not freely interchangeable. Since several mutants were efficiently expressed and bound tunicamycin, yet lacked enzymatic activity, the data are consistent with these sequences having a direct role in product formation.      

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Dissecting mechanisms of immunodominance to the common tuberculosis antigens ESAT-6, CFP10, Rv2031c (hspX), Rv2654c (TB7.7), and Rv1038c (EsxJ)

Diagnosis of tuberculosis often relies on the ex vivo IFN-γ release assays QuantiFERON-TB Gold In-Tube and T-SPOT.TB. However, understanding of the immunological mechanisms underlying their diagnostic use is still incomplete. Accordingly, we investigated T cell responses for the TB Ags included in the these assays and other commonly studied Ags: early secreted antigenic target 6 kDa, culture filtrate protein 10 kDa, Rv2031c, Rv2654c, and Rv1038c. PBMC from latently infected individuals were tested in ex vivo ELISPOT assays with overlapping peptides spanning the entirety of these Ags. We found striking variations in prevalence and magnitude of ex vivo reactivity, with culture filtrate protein 10 kDa being most dominant, followed by early secreted antigenic target 6 kDa and Rv2654c being virtually inactive. Rv2031c and Rv1038c were associated with intermediate patterns of reactivity. Further studies showed that low reactivity was not due to lack of HLA binding peptides, and high reactivity was associated with recognition of a few discrete dominant antigenic regions. Different donors recognized the same core sequence in a given epitope. In some cases, the identified epitopes were restricted by a single specific common HLA molecule (selective restriction), whereas in other cases, promiscuous restriction of the same epitope by multiple HLA molecules was apparent. Definition of the specific restricting HLA allowed to produce tetrameric reagents and showed that epitope-specific T cells recognizing either selectively or promiscuously restricted epitopes were predominantly T effector memory. In conclusion, these results highlight the feasibility of more clearly defined TB diagnostic reagent.     

Substrate specificity of Staphylococcus aureus cysteine proteases--Staphopains A, B and C

Human strains of Staphylococcus aureus secrete two papain-like proteases, staphopain A and B. Avian strains produce another homologous enzyme, staphopain C. Animal studies suggest that staphopains B and C contribute to bacterial virulence, in contrast to staphopain A, which seems to have a virulence unrelated function. Here we present a detailed study of substrate preferences of all three proteases. The specificity of staphopain A, B and C substrate-binding subsites was mapped using different synthetic substrate libraries, inhibitor libraries and a protein substrate combinatorial library. The analysis demonstrated that the most efficiently hydrolyzed sites, using Schechter and Berger nomenclature, comprise a P2–Gly↓Ala(Ser) sequence motif, where P2 distinguishes the specificity of staphopain A (Leu) from that of both staphopains B and C (Phe/Tyr). However, we show that at the same time the overall specificity of staphopains is relaxed, insofar as multiple substrates that diverge from the sequences described above are also efficiently hydrolyzed.     

Islet-specific glucose-6-phosphatase catalytic subunit-related protein-reactive CD4+ T cells in human subjects

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is recognized as a major autoantigen for autoimmune type 1 diabetes (T1D) in the NOD mouse model. This study was undertaken to examine CD4+ T cell responses toward IGRP in human subjects. The tetramer-guided epitope mapping approach was used to identify IGRP-specific CD4+ T cell epitopes. IGRP(23-35) and IGRP(247-259) were identified as DRA1*0101/DRB1*0401-restricted epitopes. IGRP(13-25) and IGRP(226-238) were identified as DRA1*0101/DRB1*0301-restricted epitopes. IGRP-specific tetramers were used to evaluate the prevalence of IGRP-reactive T cells in healthy and T1D subjects. More than 80% of subjects with either DRB1*0401 or DRB1*0301 haplotype have IGRP-specific CD4+ T cell responses for at least one IGRP epitope. IGRP-specific T cells from both healthy and T1D groups produce both gamma-IFN and IL-10. DRA1*0101/DRB1*0401 IGRP(247-259)-restricted T cells also show cross-reactivity to an epitope derived from liver/kidney glucose-6-phosphatase. The detection of IGRP-reactive T cells in both type 1 diabetic subjects and healthy subjects and recent reports of other autoreactive T cells detected in healthy subjects underscore the prevalence of potentially autoreactive T cells in the peripheral immune system of the general population.     

Benefits,costs and reactivity of inducible defences: an experimental test with rotifers

1. A key aspect of the ecology and evolution of adaptive prey responses to predator risk is the timing by which the former develop a defensive trait in response to inducing signals released by the latter. This property, called reactivity, has been shown to affect population stability and persistence. 2. Theoretically, the minimal predator density required by prey to exhibit induced defences is expected to increase with the effectiveness of the defence and decrease with its cost. Likewise, the time required for the prey population to exhibit an induced defence is expected to increase together with cost. 3. The freshwater rotifers Brachionus calyciflorus and B. havanaensis and their predator Asplanchna brightwelli were used to test the hypothesis that prey species exhibiting defences that offer a larger fitness benefit and lower fitness cost are more reactive to predator signals, in terms of requiring shorter exposure time and lower signal concentration to trigger a morphological defence reaction. 4. Our results showed that both prey species exhibited costly and effective defences after induction by predator infochemicals. Faster reactions were observed at higher levels of predator cues. Nevertheless, the observed relationship between reactivity and benefit/cost of defences did not agree with our expectations. 5. To our knowledge, this is the first study in which the timing of induction of morphological defences is experimentally assessed over a gradient of risk signals. We propose new research directions to disentangle the mechanisms and project the consequences of prey decisions at the morphological level.     

Cathepsin V, a novel and potent elastolytic activity expressed in activated macrophages

Atherosclerosis is characterized by a thickening and loss of elasticity of the arterial wall. Loss of elasticity has been attributed to the degradation of the arterial elastin matrix. Cathepsins K and S are papain-like cysteine proteases with known elastolytic activities, and both enzymes have been identified in macrophages present in plaque areas of diseased blood vessels. Here we demonstrate that macrophages express a third elastolytic cysteine protease, cathepsin V, which exhibits the most potent elastase activity yet described among human proteases and that cathepsin V is present in atherosclerotic plaque specimens. Approximately 60% of the total elastolytic activity of macrophages can be attributed to cysteine proteases with cathepsins V, K, and S contributing equally. From this 60%, two-thirds occur extracellularly and one-third intracellularly with the latter credited to cathepsin V. Ubiquitously expressed glycosaminoglycans (GAGs) such as chondroitin sulfate specifically inhibit the elastolytic activities of cathepsins V and K via the formation of specific cathepsin-GAG complexes. In contrast, cathepsin S, which does not form complexes with chondroitin sulfate is not inhibited; thus suggesting a specific regulation of elastolytic activities of cathepsins by GAGs. Because the GAG content is reduced in atherosclerotic plaques, an increase of cathepsins V and K activities may accelerate the destruction of the elastin matrix in diseased arteries.     

GeneCensus: genome comparisons in terms of metabolic pathway activity and protein family sharing

We present a prototype of a new database tool, GeneCensus, which focuses on comparing genomes globally, in terms of the collective properties of many genes, rather than in terms of the attributes of a single gene (e.g. sequence similarity for a particular ortholog). The comparisons are presented in a visual fashion over the web at GeneCensus.org. The system concentrates on two types of comparisons: (i) trees based on the sharing of generalized protein families between genomes, and (ii) whole pathway analysis in terms of activity levels. For the trees, we have developed a module (TreeViewer) that clusters genomes in terms of the folds, superfamilies or orthologs—all can be considered as generalized ‘families’ or ‘protein parts’—they share, and compares the resulting trees side-by-side with those built from sequence similarity of individual genes (e.g. a traditional tree built on ribosomal similarity). We also include comparisons to trees built on whole-genome dinucleotide or codon composition. For pathway comparisons, we have implemented a module (PathwayPainter) that graphically depicts, in selected metabolic pathways, the fluxes or expression levels of the associated enzymes (i.e. generalized ‘activities’). One can, consequently, compare organisms (and organism states) in terms of representations of these systemic quantities. Develop ment of this module involved compiling, calculating and standardizing flux and expression information from many different sources. We illustrate pathway analysis for enzymes involved in central metabolism. We are able to show that, to some degree, flux and expression fluctuations have characteristic values in different sections of the central metabolism and that control points in this system (e.g. hexokinase, pyruvate kinase, phosphofructokinase, isocitrate dehydrogenase and citric synthase) tend to be especially variable in flux and expression. Both the TreeViewer and PathwayPainter modules connect to other information sources related to individual-gene or organism properties (e.g. a single-gene structural annotation viewer).     

Phylogenetic relationships and taxonomic position of Chlorella -like isolates from low pH environments (pH < 3.0)

Background  

Little is known about phytoplankton communities inhabiting low pH environments such as volcanic and geothermal sites or acidic waters. Only specialised organisms are able to tolerate such extreme conditions. There is, thus, low species diversity. We have characterised the previously isolated acid tolerant Chlorella -like microalgae Viridiella fridericiana and Chlorella protothecoides var. acidicola by microscopical and biomolecular methods in order to assess their phylogenetic relationships.