Physical mapping of the human carbonic anhydrase gene cluster on chromosome 8

A cluster of genes encoding the three cytoplasmic carbonic anhydrase isozymes CAI, CAII, and CAIII lie on the long arm of chromosome 8 (8q22) in humans. These genes have been mapped using pulsed-field gel electrophoresis. The genes lie in the order CA2, CA3, CA1. CA2 and CA3 are separated by 20 kb and are transcribed in the same direction, away from CA1. CA1 is separated from CA3 by over 80 kb and is transcribed in the direction opposite to CA2 and CA3. The arrangement of the genes is consistent with proposals that the duplication event which gave rise to CA1 predated the duplication which gave rise to CA2 and CA3. The order of these three genes differs from that suggested for the mouse based on recombination frequency.     

Allometric and Group Differences in the Xenarthran Femur

The aim of this study is to analyze shape variation in the xenarthran femur to gain insights into their behavior and locomotion. Specimens of both Cingulata (armadillos and glyptodonts) and Pilosa (anteaters and sloths) were studied and within each group body mass varies by several orders of magnitude. The main focus of the analysis was allometric variation in femoral shape in the three groups studied, armadillos, glyptodonts, and pilosans. Three dimensional coordinates were recorded for 40 homologous landmarks on each of 51 xenarthran femurs. The data were analyzed by geometric morphometric methods, and form space analysis was used to identify the allometric variation in each of the three groups. Across all groups, larger specimens tended to have larger articular surfaces, more robust femora generally, and the shape of the femoral condyles was more suited to extended postures. In addition, in larger specimens the medial condyle was much larger than the lateral condyle and the third trochanter was located more distally. The larger armadillo femora had a greater trochanter located considerably proximal to the femoral head and this is thought to improve femoral extension, but in glyptodonts and pilosans the larger specimens had a greater trochanter that was far lateral to the femoral head and this is interpreted as enhancing femoral rotation.     

Mammalian Fbh1 is important to restore normal mitotic progression following decatenation stress

We have addressed the role of the F-box helicase 1 (Fbh1) protein during genome maintenance in mammalian cells. For this, we generated two mouse embryonic stem cell lines deficient for Fbh1: one with a homozygous deletion of the N-terminal F-box domain (Fbh1^f/f), and the other with a homozygous disruption (Fbh1^?/?). Consistent with previous reports of Fbh1-deficiency in vertebrate cells, we found that Fbh1^?/? cells show a moderate increase in Rad51 localization to DNA damage, but no clear defect in chromosome break repair. In contrast, we found that Fbh1^f/f cells show a decrease in Rad51 localization to DNA damage and increased cytoplasmic localization of Rad51. However, these Fbh1^f/f cells show no clear defects in chromosome break repair. Since some Rad51 partners and F-box-associated proteins (Skp1-Cul1) have been implicated in progression through mitosis, we considered whether Fbh1 might play a role in this process. To test this hypothesis, we disrupted mitosis using catalytic topoisomerase II inhibitors (bisdioxopiperazines), which inhibit chromosome decatenation. We found that both Fbh1^f/f and Fbh1^?/? cells show hypersensitivity to topoisomerase II catalytic inhibitors, even though the degree of decatenation stress was not affected. Furthermore, following topoisomerase II catalytic inhibition, both Fbh1-deficient cell lines show substantial defects in anaphase separation of chromosomes. These results indicate that Fbh1 is important for restoration of normal mitotic progression following decatenation stress.     

What happens in Vegas,better stay in Vegas: <Emphasis Type="Italic">Phragmites australis</Emphasis> hybrids in the Las Vegas Wash

While hybridization between Native and Introduced Phragmites australis has not been documented across much of North America, it poses an ongoing threat to Native P. australis across its range. This is especially true for native populations in the biologically rich, but sparsely distributed wetlands of the southwest United States, which are among the most imperiled systems in North America. We identified multiple Hybrid P. australis stands in the Las Vegas Wash watershed, NV, a key regional link to the Colorado River basin. Rapid urbanization in this watershed has caused striking changes in water and nutrient inputs and the distribution of wetland habitats has also changed, with urban wetlands expanding but an overall reduction in wetland habitats regionally. Native P. australis has likely been present in the Wash wetland community in low abundance for thousands of years, but today Hybrid and Native plants dominate the shoreline along much of the Wash. In contrast, Introduced P. australis is rare, suggesting that opportunities for novel hybridization events remain uncommon. Hybrid crosses derived from both the native and introduced maternal lineages are widespread, although the conditions that precluded their establishment are unknown and we did not find evidence for backcrossing. Spread of Hybrid plants is likely associated with flooding events as well as restoration activities, including revegetation efforts and construction for erosion control, that have redistributed sediments containing P. australis rhizomes. Downstream escape of Hybrid plants to Lake Mead and wetlands throughout the lower Colorado River basin is of management concern as these Hybrids appear vigorous and could spread rapidly.     

In Vivo Evaluation of Blood Based and Reference Tissue Based PET Quantifications of [11C]DASB in the Canine Brain

This first-in-dog study evaluates the use of the PET-radioligand [¹¹C]DASB to image the density and availability of the serotonin transporter (SERT) in the canine brain. Imaging the serotonergic system could improve diagnosis and therapy of multiple canine behavioural disorders. Furthermore, as many similarities are reported between several human neuropsychiatric conditions and naturally occurring canine behavioural disorders, making this tracer available for use in dogs also provide researchers an interesting non-primate animal model to investigate human disorders. Five adult beagles underwent a 90 minutes dynamic PET scan and arterial whole blood was sampled throughout the scan. For each ROI, the distribution volume (V_T), obtained via the one- and two- tissue compartment model (1-TC, 2-TC) and the Logan Plot, was calculated and the goodness-of-fit was evaluated by the Akaike Information Criterion (AIC). For the preferred compartmental model BP_ND values were estimated and compared with those derived by four reference tissue models: 4-parameter RTM, SRTM2, MRTM2 and the Logan reference tissue model. The 2-TC model indicated in 61% of the ROIs a better fit compared to the 1-TC model. The Logan plot produced almost identical V_T values and can be used as an alternative. Compared with the 2-TC model, all investigated reference tissue models showed high correlations but small underestimations of the BP_ND-parameter. The highest correlation was achieved with the Logan reference tissue model (Y = 0.9266 x + 0.0257; R² = 0.9722). Therefore, this model can be put forward as a non-invasive standard model for future PET-experiments with [¹¹C]DASB in dogs.     

Developmental and light-dependent changes of the cytosolic chaperonin containing TCP-1 (CCT) subunits in maize seedlings, and the localization in coleoptiles

The cytosolic chaperonin containing TCP-1 (CCT) is known to keep fold cytoskeletal proteins and is involved in the proper organization of the cytoskeleton. These studies are based on the assumption that growth responses linked to structural rearrangement of the plant cytoskeleton include the action of CCT and the need for newly synthesized tubulin. The presence of the α- and ɛ- subunits of CCT was investigated in soluble fractions of protein extracts from maize mesocotyls and coleoptiles at distinct growth stages. The CCT-subunits, tubulins and actin decreased in the coleoptile in response to far-red light. In addition, independent from light treatment, the amount of CCTɛ abundance declined with age in coleoptiles and mesocotyls between 2 and 4.5 days after sowing. In contrast to CCTɛ, no significant light regulation of CCTα was found in the mesocotyl. In two day old, light-grown rapidly elongating coleoptiles part of the CCTα subunit and the bulk of actin and tubulin was found shifted into fractions of high molecular weight complexes when compared to slowly elongating, dark grown coleoptiles. In 4.5 day old, etiolated and elongating coleoptiles, part of both CCT-subunits and cytoskeleton proteins were found in fractions of high molecular weight. A complete disappearance of these polypeptides was observed in old far-red irradiated growth-arrested coleoptiles. CCTɛ was found to be co-localized to microtubular structures and to the nucleus. We conclude from our data that abundance of CCT-subunits in soluble extracts is dependent on age and light treatment, but independent from the growth stage of mesocotyl and coleoptile.     

Natural CD8<Superscript>+</Superscript> T-cell responses against MHC class I epitopes of the HER-2/<Emphasis Type="Italic">neu</Emphasis> oncoprotein in patients with epithelial tumors

HER-2/neu is an immunogenic protein eliciting both humoral and cellular immune responses in patients with HER-2/neu-positive (⁺) tumors. Preexisting cytotoxic T lymphocyte (CTL) immunity to HER-2/neu has so far been mainly evaluated in terms of detection of CTL precursor (CTLp) frequencies to the immunogenic HLA-A2–binding nona-peptide 369-377 (HER-2(9₃₆₉)). In the present study, we examined patients with HER-2/neu⁺ breast, ovarian, lung, colorectal, and prostate cancers for preexisting CTL immunity to four recently described HER-2/neu–derived and HLA-A2–restricted "cytotoxic" peptides and to a novel one spanning amino acids 777–785 also with HLA-A2–binding motif. We utilized enzyme-linked immunosorbent spot (ELISpot) assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubation. CTL reactivity was determined with an interferon (IFN-) ELISpot assay detecting T cells at the single cell level secreting IFN-. CTLp were defined as peptide-specific precursors per 10⁶ peripheral blood mononuclear cells (PBMCs). Patients' PBMCs with increased CTLp were also tested against autologous tumor targets and peptide-pulsed dendritic cells (DCs) in cytotoxicity assays. We also studied patients with HER-2/neu-negative (^-) tumors and healthy individuals. Of the HER-2/neu⁺ patients examined, 31% had increased CTLp to HER-2(9₉₅₂), 19% to HER-2(9₆₆₅), 16% to HER-2(9₆₈₉), and 12.5% HER-2(9₄₃₅), whereas only 2 of 32 patients (6%) responded to HER-2(9₇₇₇). The CTLp recognizing HER-2(9₉₅₂) were extremely high in two patients with breast cancer, one with lung cancer, and one with prostate cancer. None of the HER-2/neu^- patients or healthy donors exhibited increased CTLp to any of these peptides. Besides IFN- production, preexisting CTL immunity to all five HER-2/neu peptides was also shown in cytotoxicity assays where patients' PBMCs with increased CTLp specifically lysed autologous tumor targets and autologous peptide-pulsed DCs. Our results demonstrate for the first time that (1) preexisting immunity to peptides HER-2(9₄₃₅), HER-2(9₉₅₂), HER-2(9₆₈₉), HER-2(9₆₆₅), and HER-2(9₇₇₇) is present in patients with HER-2/neu⁺ tumors of distinct histology, (2) HER-2(9₇₇₇) is a naturally processed peptide expressed on the surface of HER-2/neu⁺ tumors, as are the other four peptides, and (3) HER-2/neu⁺ prostate tumor cells can be recognized and lysed by autologous HER-2 peptide-specific CTL. Our findings broaden the potential application of HER-2/neu-based immunotherapy.     

COMPASS: a tool for comparison of multiple protein alignments with assessment of statistical significance

We present a novel method for the comparison of multiple protein alignments with assessment of statistical significance (COMPASS). The method derives numerical profiles from alignments, constructs optimal local profile-profile alignments and analytically estimates E-values for the detected similarities. The scoring system and E-value calculation are based on a generalization of the PSI-BLAST approach to profile-sequence comparison, which is adapted for the profile-profile case. Tested along with existing methods for profile-sequence (PSI-BLAST) and profile-profile (prof_sim) comparison, COMPASS shows increased abilities for sensitive and selective detection of remote sequence similarities, as well as improved quality of local alignments. The method allows prediction of relationships between protein families in the PFAM database beyond the range of conventional methods. Two predicted relations with high significance are similarities between various Rossmann-type folds and between various helix-turn-helix-containing families. The potential value of COMPASS for structure/function predictions is illustrated by the detection of an intricate homology between the DNA-binding domain of the CTF/NFI family and the MH1 domain of the Smad family.     

Syntenin-1 and Ezrin Proteins Link Activated Leukocyte Cell Adhesion Molecule to the Actin Cytoskeleton

Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both homotypic interactions with other ALCAM molecules and heterotypic interactions with the surface receptor CD6 expressed at the T cell surface. Despite biochemical and biophysical evidence of a dynamic association between ALCAM and the actin cytoskeleton, no detailed information is available about how this association occurs at the molecular level. Here, we exploit a combination of complementary microscopy techniques, including FRET detected by fluorescence lifetime imaging microscopy and single-cell force spectroscopy, and we demonstrate the existence of a preformed ligand-independent supramolecular complex where ALCAM stably interacts with actin by binding to syntenin-1 and ezrin. Interaction with the ligand CD6 further enhances these multiple interactions. Altogether, our results propose a novel biophysical framework to understand the stabilizing role of the ALCAM supramolecular complex engaged to CD6 during dendritic cell-T cell interactions and provide novel information on the molecular players involved in the formation and signaling of the immunological synapse at the dendritic cell side.