Preparation and some properties of homogeneous Neurospora crassa assimilatory NADPH-nitrite reductase.

The Neurospora crassa assimilatory NADPH-nitrite reductase (NAD(P)H: nitrite oxidoreductase, EC 1.6.6.4), which catalyzes the NADPH-dependent formation of ammonia from nitrite, has been purified to homogeneity as judged by polyacrylamide gel electrophoresis. The specific activity of the purified enzyme is 26.9 mumol nitrite reduced/min per mg protein, which corresponds to a turnover number of 7800 min(-1). The enzyme also has associated NADH-nitrite reductase, NADPH-hydroxylamine reductase and NADH-hydroxylamine reductase activities. The stoichiometry of 3 mol NADPH oxidized per mol nitrite reduced and ammonia formed has been confirmed. The visible absorption spectrum of the nitrite reductase reveals maxima at 280,390 (Soret) and 580 (alpha) nm. The latter bands are indicative of the occurrence of siroheme as a prosthetic group. The A280nm/A390nm ratio of 7.0 and the Soret/alpha ratio of 3.8 are compatible with values reported for other purified siroheme-containing enzymes. These results are discussed in terms of the comparative biochemistry of various enzymes involved in nitrite, hydroxylamine and sulfite metabolism in Neurospora crassa and other organisms.     

The modifying effect of manganese on the enzymic profiles and pathways of carbohydrate metabolism in rat liver and adipose tissue during development

The effect of Mn²⁺ on the pattern of emergence of enzymes in rat liver and adipose tissue was studied in weaned rats given a milk diet (high fat) or sucrose-casein diet (high carbohydrate) for three weeks. Addition of Mn²⁺ to the high fat diet was associated with induction of key glycolytic, lipogenic and pentose pathway enzymes in both liver and adipose tissue; parallel increases were found in the incorporation of [1-¹⁴C] glucose into lipid and CO₂. Mn²⁺ induced a change in the profile of enzyme activity similar in pattern to that found in rats given a high sucrose diet or that produced by insulin treatment. Mn²⁺ appears partially to overcome the regulatory feed-back mechanisms of the high fat diet and to provide a signal for the coordinated increase of glucose catabolic and lipogenic processes.     

African Herald snakes,Crotaphopeltis, show population structure for a widespread generalist but deep genetic divergence for forest specialists

The African colubrid snake genus Crotaphopeltis currently comprises six species and occurs throughout sub-Saharan Africa. The most widespread of these, Crotaphopeltis hotamboeia, inhabits most biomes, aside from rainforest and hyper-arid regions, and its catholic niche has presumably facilitated substantial gene flow. Despite this, the geographical range is large enough that ecological or physical barriers might exist, facilitating allopatric diversification. In contrast, most of the other species are habitat specialists with limited distributions (e.g., Crotaphopeltis tornieri) and would be expected to show strong genetic structure. We therefore examined species boundaries within Crotaphopeltis in a phylogenetic context using five markers (16S, cyt b, ND4, c-mos, and RAG-1) for four of the six species. Species delimitation methods included two coalescent-based and one barcoding approach. Widespread geographical sampling of C. hotamboeia allowed examination of genetic structuring across its range. The species status of Crotaphopeltis barotseensis, C. degeni, and C. hotamboeia was confirmed, whereas the Afromontane species C. tornieri comprised two candidate species. Crotaphopeltis hotamboeia did not show cryptic speciation, although its phylogeographic structure corresponded with the spatiotemporal pattern of the African savanna. Our results show how the heterogeneous African environment could influence genetic partitioning of habitat specialist and generalist species at broad geographical scales.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Cryptic species,taxonomic inflation,or a bit of both? New species phenomenon in Sri Lanka as suggested by a phylogeny of dwarf geckos (Reptilia,Squamata, Gekkonidae,Cnemaspis)

We evaluated the status of 16 of 22 recognized Sri Lankan Cnemaspis Strauch species, and flagged overlooked diversity with two mitochondrial (cyt b & ND2) and two nuclear markers (RAG1 & PDC) totalling 2829 base pairs. A fossil-calibrated timetree and sampling of other South Asian Cnemaspis provide insights into the diversification of the genus in peninsular India and Sri Lanka. Phylogenetic analyses consistently inferred two broad clades within South Asian Cnemaspis, with Sri Lankan species in two clades, which we call the podihuna and kandiana clades. Each Sri Lankan clade as a whole is sister to Indian taxa and nested within Indian lineages. Cnemaspis modigliani Das from Indonesia is a member of the kandiana clade. This suggests a minimum of two dispersal events between India and Sri Lanka and one between Sri Lanka/India and South-east Asia. South Asian Cnemaspis date back to at least the Eocene, in Sri Lanka to the early Miocene, with late Miocene diversification in the kandiana clade. All but one of the named species we sampled is likely to be valid, and 10 divergent unnamed lineages may warrant specific recognition. A resolution of Sri Lankan Cnemaspis taxonomy will require thorough sampling and the use of both morphological and molecular data.     

Properties of MHC Class I Presented Peptides That Enhance Immunogenicity

T-cells have to recognize peptides presented on MHC molecules to be activated and elicit their effector functions. Several studies demonstrate that some peptides are more immunogenic than others and therefore more likely to be T-cell epitopes. We set out to determine which properties cause such differences in immunogenicity. To this end, we collected and analyzed a large set of data describing the immunogenicity of peptides presented on various MHC-I molecules. Two main conclusions could be drawn from this analysis: First, in line with previous observations, we showed that positions P4–6 of a presented peptide are more important for immunogenicity. Second, some amino acids, especially those with large and aromatic side chains, are associated with immunogenicity. This information was combined into a simple model that was used to demonstrate that immunogenicity is, to a certain extent, predictable. This model (made available at http://tools.iedb.org/immunogenicity/) was validated with data from two independent epitope discovery studies. Interestingly, with this model we could show that T-cells are equipped to better recognize viral than human (self) peptides. After the past successful elucidation of different steps in the MHC-I presentation pathway, the identification of variables that influence immunogenicity will be an important next step in the investigation of T-cell epitopes and our understanding of cellular immune responses.     

A Second Tubulin Binding Site on the Kinesin-13 Motor Head Domain Is Important during Mitosis

Kinesin-13s are microtubule (MT) depolymerases different from most other kinesins that move along MTs. Like other kinesins, they have a motor or head domain (HD) containing a tubulin and an ATP binding site. Interestingly, kinesin-13s have an additional binding site (Kin-Tub-2) on the opposite side of the HD that contains several family conserved positively charged residues. The role of this site in kinesin-13 function is not clear. To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A. We show that the Kin-Tub-2 site enhances tubulin cross-linking and MT bundling properties of KLP10A in-vitro. Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells. The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.     

Role of the Dinaric Karst (western Balkans) in shaping the phylogeographic structure of the threatened crayfish Austropotamobius torrentium

1. This study examines phylogeography and phylogeny of the threatened stone crayfish, Austropotamobius torrentium, in order to elucidate the role of the Dinaric Karst geology in shaping the evolutionary history and genetic diversity of aquatic fauna in the western Balkans. Mitochondrial 16S rRNA and COI genes were partially sequenced from 188 and 159 crayfish, respectively, sampled from 70 localities. Phylogenetic relationships were reconstructed using four methods of phylogenetic inference. Divergence times between phylogroups were estimated in a Bayesian framework, and their demographic history was examined using neutrality tests and mismatch distribution analysis. 2. Seven geographically localised phylogroups separated by pronounced genetic gaps were found. Five of them have a distribution range within the northern‐central Dinaric (NCD) region, while the remaining two include populations from the southern Balkans (SB) and central and south‐eastern Europe (CSE). The oldest divergence event separated two NCD lineages from the rest of populations in the Late Miocene or Early Pliocene. Divergences amongst the five NCD phylogroups and SB + CSE occurred in the Pliocene. The most recent split separated SB and CSE phylogroups during the Late Pliocene. For both genes, uncorrected pairwise divergences between most of the phylogroups (4.1–8.7% for COI and 1.6–4.8% for 16S rRNA) were of the same range as, or higher than, some of the interspecific distances previously reported for the genus Austropotamobius. 3. Geographically isolated and deeply divergent cryptic monophyletic phylogroups within A. torrentium in the NCD region arose in the course of intensification of Neotectonic movements during the Pliocene and the beginning of the Pleistocene and the development of karstification that has heavily fragmented the palaeohydrography of the area. The results confirm a gradual north–south expansion of stone crayfish during the pre‐Pleistocene that preceded the rapid northward post‐glacial re/colonisation of central Europe (CSE phylogroup) through the Danube drainage. 4. Austropotamobius torrentium comprises morphologically cryptic but molecularly distinct taxa. Considering the relatively small geographical areas they inhabit, the NCD phylogroups of stone crayfish should be given the highest conservation priority.