To move or not to move? Semaphorin signalling in cell migration

Semaphorins were discovered 11 years ago as molecular cues for axon guidance that are conserved from invertebrates to humans. More than 20 semaphorin genes have been identified in mammals and their protein products are now known to be involved in a range of processes from the guidance of cell migration to the regulation of the immune response, angiogenesis and cancer. Plexins, either alone or in association with neuropilins, constitute high-affinity semaphorin receptors. However, other transmembrane molecules have been implicated in semaphorin receptor complexes, and interactions between plexins and a range of intracellular effectors have been reported. These data indicate that semaphorins might be able to elicit responses through more than one signalling pathway. Interestingly, according to recent findings, the semaphorin-dependent control of cell migration crucially involves integrin-based adhesive structures through which polarized cell-membrane protrusion is coupled to cytoskeletal dynamics. This review focuses on the mechanisms whereby semaphorins are thought to regulate cell migration.     

Redox state and carbonic anhydrase isozyme IX expression in human renal cell carcinoma: biochemical and morphological investigations

Clear renal cell carcinomas (RCC) frequently express carbonic anydrase IX (CA IX) because of non-functional mutation of von Hippel Lindau (VHL) tumor suppressor gene. CA IX is a tumor-associated transmembrane antigen, which catalyzes the extracellular, reversible hydration of carbon dioxide to bicarbonate and proton and thereby contributes to acidification of extracellular milieu. Extracellular acidic pH facilitates tumor growth and progression. CA IX expression is upregulated by Hypoxia Inducible Factor-1 (HIF-1), which is negatively controlled by oxygen via wild type VHL protein and is also regulated by the cell redox state. We investigated the immunohistochemical pattern of distribution of CA IX in a small series (14 cases) of RCCs. CA IX expression was matched with the redox state of RCC, stratifying our series in relation to clinical and histopathological parameters, such as Fuhrman grade, staging, proliferation markers expression, and particularly, the presence of necrosis. Our results show for the first time the existence of a perivascular pattern of CA IX distribution in RCC. We also found a significant relationship between CA IX expression and the presence of necrosis. Tumors with higher CA IX expression exhibited higher degree of necrosis (p < 0.05). Notably, an almost significant relationship between the redox state and CA IX expression was detected in RCC patients with 5 years disease-free survival, most of them showing organ-confined disease. Tumors with lower redox state showed an algebraically higher degree of CA IX expression. On the contrary, tumors with higher redox state exhibited an algebraically lower CA IX expression (p = 0.057). The observed relationship of CA IX expression and necrosis suggests a role for CA IX in RCC. Further investigations are necessary to further establish the role of the redox state in regulation of CA IX expression in RCC.     

Structure and function of the Gondwanian hemoglobin of Pseudaphritis urvillii, a primitive notothenioid fish of temperate latitudes

The suborder Notothenioidei dominates the Antarctic ichthyofauna. The non-Antarctic monotypic family Pseudaphritidae is one of the most primitive families. The characterization of the oxygen-transport system of euryhaline Pseudaphritis urvillii is herewith reported. Similar to most Antarctic notothenioids, this temperate species has a single major hemoglobin (Hb 1, over 95% of the total). Hb 1 has strong Bohr and Root effects. It shows two very uncommon features in oxygen binding: At high pH values, the oxygen affinity is exceptionally high compared to other notothenioids, and subunit cooperativity is modulated by pH in an unusual way, namely the curve of the Hill coefficient is bell-shaped, with values approaching 1 at both extremes of pH. Molecular modeling, electronic absorption and resonance Raman spectra have been used to characterize the heme environment of Hb 1 in an attempt to explain these features, particularly in view of some potentially important nonconservative replacements found in the primary structure. Compared to human HbA, no major changes were found in the structure of the proximal cavity of the alpha-chain of Hb 1, although an altered distal histidyl and heme position was identified in the models of the beta-chain, possibly facilitated by a more open heme pocket due to reduced steric constraints on the vinyl substituent groups. This conformation may lead to the hemichrome form identified by spectroscopy in the Met state, which likely fulfils a potentially important physiological role.     

Do the different parental 'heteromes' cause genomic shock in newly formed allopolyploids?

Allopolyploidy, the joining of two parental genomes in a polyploid organism with diploid meiosis, is an important mechanism of reticulate evolution. While many successful long-established allopolyploids are known, those formed recently undergo an instability phase whose basis is now being characterized. We describe observations made with the Arabidopsis system that include phenotypic instability, gene silencing and activation, and methylation changes. We present a model based on the epigenetic destabilization of genomic repeats, which in the parents are heterochromatinized and suppressed. We hypothesize that loss of epigenetic suppression of these sequences, here defined as the heterome, results in genomic instability including silencing of single-copy genes.     

Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma

The mechanisms contributing to airway wall remodeling in asthma are under investigation to identify appropriate therapeutic targets. Bronchial myofibroblasts would represent an important target because they play a crucial role in the genesis of subepithelial fibrosis, a characteristic feature of the remodeling process, but their origin is poorly understood. We hypothesized that they originate from fibrocytes, circulating cells with the unique characteristic of expressing the hemopoietic stem cell Ag CD34 and collagen I. In this study we show that allergen exposure induces the accumulation of fibrocyte-like cells in the bronchial mucosa of patients with allergic asthma. These cells are CD34-positive; express collagen I and alpha-smooth muscle actin, a marker of myofibroblasts; and localize to areas of collagen deposition below the epithelium. By tracking labeled circulating fibrocytes in a mouse model of allergic asthma, we provide evidence that fibrocytes are indeed recruited into the bronchial tissue following allergen exposure and differentiate into myofibroblasts. We also show that human circulating fibrocytes acquire the myofibroblast phenotype under in vitro stimulation with fibrogenic cytokines that are produced in exaggerated quantities in asthmatic airways. These results indicate that circulating fibrocytes may function as myofibroblast precursors and may contribute to the genesis of subepithelial fibrosis in asthma.     

The sensitivity of catecholamine release to botulinum toxin C1 and E suggests selective targeting of vesicles set into the readily releasable pool

The impact of syntaxin and SNAP-25 cleavage on [3H]noradrenaline ([3H]NA) and [3H]dopamine ([3H]DA) exocytotic release evoked by different stimuli was studied in superfused rat synaptosomes. The external Ca2+-dependent K+-induced [3H]catecholamine overflows were almost totally abolished by botulinum toxin C1 (BoNT/C1), which hydrolyses syntaxin and SNAP-25, or by botulinum toxin E (BoNT/E), selective for SNAP-25. BoNT/C1 cleaved 25% of total syntaxin and 40% of SNAP-25; BoNT/E cleaved 40% of SNAP-25 but left syntaxin intact. The GABA uptake-induced releases of [3H]NA and [3H]DA were differentially affected: both toxins blocked the former, dependent on external Ca2+, but not the latter, internal Ca2+-dependent. BoNT/C1 or BoNT/E only slightly reduced the ionomycin-evoked [3H]catecholamine release. More precisely, [3H]NA exocytosis induced by ionomycin was sensitive to toxins in the early phase of release but not later. The Ca2+-independent [3H]NA exocytosis evoked by hypertonic sucrose, thought to release from the readily releasable pool (RRP) of vesicles, was significantly reduced by BoNT/C1. Pre-treating synaptosomes with phorbol-12-myristate-13-acetate, to increase the RRP, enhanced the sensitivity to BoNT/C1 of [3H]NA release elicited by sucrose or ionomycin. Accordingly, cleavage of syntaxin was augmented by the phorbol-ester. To conclude, our results suggest that clostridial toxins selectively target exocytosis involving vesicles set into the RRP.     

Selective gamma-hydroxybutyric acid receptor ligands increase extracellular glutamate in the hippocampus, but fail to activate G protein and to produce the sedative/hypnotic effect of gamma-hydroxybutyric acid

Two gamma-hydroxybutyric acid (GHB) analogues, trans-gamma-hydroxycrotonic acid (t-HCA) and gamma-(p-methoxybenzyl)-gamma-hydroxybutyric acid (NCS-435) displaced [3H]GHB from GHB receptors with the same affinity as GHB but, unlike GHB, failed to displace [3H]baclofen from GABAB receptors. The effect of the GHB analogues, GHB and baclofen, on G protein activity and hippocampal extracellular glutamate levels was compared. While GHB and baclofen stimulated 5'-O-(3-[35S]thiotriphospate) [35S]GTPgammaS binding both in cortex homogenate and cortical slices, t-HCA and NCS-435 were ineffective up to 1 mm concentration. GHB and baclofen effect was suppressed by the GABAB antagonist CGP 35348 but not by the GHB receptor antagonist NCS-382. Perfused into rat hippocampus, 500 nm and 1 mm GHB increased and decreased extracellular glutamate levels, respectively. GHB stimulation was suppressed by NCS-382, while GHB inhibition by CGP 35348. t-HCA and NCS-435 (0.1-1000 microm) locally perfused into hippocampus increased extracellular glutamate; this effect was inhibited by NCS-382 (10 microm) but not by CGP 35348 (500 microm). The results indicate that GHB-induced G protein activation and reduction of glutamate levels are GABAB-mediated effects, while the increase of glutamate levels is a GHB-mediated effect. Neither t-HCA nor NCS-435 reproduced GHB sedative/hypnotic effect in mice, confirming that this effect is GABAB-mediated. The GHB analogues constitute important tools for understanding the physiological role of endogenous GHB and its receptor.     

A survey of the gastrointestinal nematodes of Spanish ibex (Capra pyrenaica) in a high mountain habitat

We analyzed the content of the abomasum (n = 79) and small intestine (n = 83) of Spanish ibex from Sierra Nevada Natural Park, southern Spain. Fifteen species of trichostrongylid nematodes were identified, 4 of which were found for the first time in this host, i.e., Nematodirus fillicollis, N. oiratianus, Ostertagia lyrata, and O. ostertagi. Teladorsagia circumcincta and Marshallagia marshalli were the most abundant abomasal species, whereas N. abnormalis, N. davtiani, and N. oiratianus were dominant in the small intestine. Counts of both abomasal and intestinal nematodes were generally low (year-round-median = 292 and 94 worms, respectively), and significantly lower numbers of M. marshalli, N. davtiani, and N. oiratianus were found in summer. No sex-related differences in helminth abundance were found, but young ibex harbored significantly more N. davtiani and N. oiratianus than adults. The presence of scabies was not related to increased nematode counts.     

Role of the asialoglycoprotein receptor in binding and entry of hepatitis C virus structural proteins in cultured human hepatocytes

We used a baculovirus-based system to prepare structural proteins of hepatitis C virus (HCV) genotype 1a. Binding of this preparation to cultured human hepatic cells was both dose dependent and saturable. This binding was decreased by calcium depletion and was partially prevented by ligands of the asialoglycoprotein receptor (ASGP-R), thyroglobulin, asialothyroglobulin, and antibody against a peptide in the carbohydrate recognition domain of ASGP-R but not preimmune antibody. Uptake by hepatocytes was observed with both radiolabeled and dye-labeled HCV structural proteins. With hepatocytes expressing the hH1 subunit of the ASGP-R fused to green fluorescent protein, we could show by confocal microscopy that dye stain cointernalized with the fusion protein in an area surrounding the nucleus. Internalization was more efficient with a preparation containing p7 than with one that did not. The two preparations bound to transfected 3T3-L1 cells expressing either both (hH1 and hH2) subunits of the ASGP-R (3T3-22Z cells) or both hH1 and a functionally defective variant of hH2 (3T3-24X cells) but not to parental cells. Additionally, uptake of dye-labeled preparation containing p7 was observed with 3T3-22Z cells but not with 3T3-L1 or 3T3-24X cells or with the preparation lacking p7, suggesting that p7 regulates the internalization properties of HCV structural proteins. Our observations suggest that HCV structural proteins bind to and cointernalize with the ASGP-R in cultured human hepatocytes.