RadA protein from Archaeoglobus fulgidus forms rings, nucleoprotein filaments and catalyses homologous recombination.

Proteins that catalyse homologous recombination have been identified in all living organisms and are essential for the repair of damaged DNA as well as for the generation of genetic diversity. In bacteria homologous recombination is performed by the RecA protein, whereas in the eukarya a related protein called Rad51 is required to catalyse recombination and repair. More recently, archaeal homologues of RecA/Rad51 (RadA) have been identified and isolated. In this work we have cloned and purified the RadA protein from the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus and characterised its in vitro activities. We show that (i) RadA protein forms ring structures in solution and binds single- but not double-stranded DNA to form nucleoprotein filaments, (ii) RadA is a single-stranded DNA-dependent ATPase at elevated temperatures, and (iii) RadA catalyses efficient D-loop formation and strand exchange at temperatures of 60-70 degrees C. Finally, we have used electron microscopy to visualise RadA-mediated joint molecules, the intermediates of homologous recombination. Intriguingly, RadA shares properties of both the bacterial RecA and eukaryotic Rad51 recombinases.

Identification of proteoglycans as the APRIL-specific binding partners.

B cell activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferation-inducing ligand (APRIL) are closely related ligands within the TNF superfamily that play important roles in B lymphocyte biology. Both ligands share two receptors--transmembrane activator and calcium signal--modulating cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA)--that are predominantly expressed on B cells. In addition, BAFF specifically binds BAFF receptor, whereas the nature of a postulated APRIL-specific receptor remains elusive. We show that the TNF homology domain of APRIL binds BCMA and TACI, whereas a basic amino acid sequence (QKQKKQ) close to the NH2 terminus of the mature protein is required for binding to the APRIL-specific "receptor." This interactor was identified as negatively charged sulfated glycosaminoglycan side chains of proteoglycans. Although T cell lines bound little APRIL, the ectopic expression of glycosaminoglycan-rich syndecans or glypicans conferred on these cells a high binding capacity that was completely dependent on APRIL's basic sequence. Moreover, syndecan-1-positive plasma cells and proteoglycan-rich nonhematopoietic cells displayed high specific, heparin-sensitive binding to APRIL. Inhibition of BAFF and APRIL, but not BAFF alone, prevented the survival and/or the migration of newly formed plasma cells to the bone marrow. In addition, costimulation of B cell proliferation by APRIL was only effective upon APRIL oligomerization. Therefore, we propose a model whereby APRIL binding to the extracellular matrix or to proteoglycan-positive cells induces APRIL oligomerization, which is the prerequisite for the triggering of TACI- and/or BCMA-mediated activation, migration, or survival signals.

A phenotypical study of vascular smooth muscle cells in human arterial and venous stenosis

Abstract Introduction The primary function of the contractile vascular smooth muscle cells (cVSMCs) is the regulation of the vascular contractility which means the adaptation of the vascular tonus in response to the modulation of the blood pressure and blood flow. The cVSMCs are essentially quiescent, and therefore their synthesis rate is very limited. They are characterized by the expression of contractile proteins specific to the muscular tissue including myosin, h-­208;caldesmon and <-­208;smooth muscle actin (
2296;-­208;SMA). These contractile cells are strongly represented in the media layer of the arterial wall and, in a smaller proportion, of the vein wall. Their typical stretched-­208;out morphology allows recognizing them by a histological analysis. They do not produce any extracellular matrix (ECM), and do not migrate through the different layers of the vessel wall, and are not directly involved in the development of intimal hyperplasia (IH). Neointimal formation occurs after endothelial disruption leading to complex molecular and biological mechanisms. The de-­208;differentiation of cVSMCs into synthetic VSMCs (sVSMCs) is mentioned as a key element. These non mature cells are able to proliferate and produce ECM. The characterization of the vascular smooth muscle cells (VSMCs) from healthy and stenosed vascular tissues will contribue to the understanding of the different biological processes leading to IH and will be useful for the development of new therapies to interfere with the cVSMCs growth and migration. The aim of our research was to quantify the proportion of cVSMCs and sVSMCs into the healthy and pathologic human blood vessel wall and to characterize their phenotype. Methods We selected 23 specimens of arterial and venous segments from 18 patients. All these specimens were stored in the biobank from the thoracic and vascular surgery departement. 4 groups were designed (group 1 :arteries without lesions (n=3) ;group 2 : veins without lesions (n=1); group 3: arteries with stenosis (n=9); group 4: veins with stenosis (n=10)). Histology: 5µm-­208;sections were made from each sample embedded in paraffin wax and further stained with hematoxylin & eosin (HE), Van Gieson's stain (VGEL) and Masson's Trichrome (TMB). Pathologic tissues were defined using the label that was given to the macroscopic samples by the surgeon and also, based on the histological analysis with HE and VGEL evaluating the presence of a thickened intima. The same was done to the control samples evaluating the absence of thickening. Immunohistochemistry : The primary antibodies were used :
2296;-­208;SMA, vimentin, h-­208; caldesmon, calponin, smooth muscle-myosin heavy chain (SM-­208;MHC), tropomyosin-­208;4, retinol binding protein-­208;1 (RBP-­208;1), nonmuscle-­208;myosin heavy chain-­208;B (NM-­208;MHC-­208;B), Von Willebrand factor (VWF). A semi-­208;quantitative assessment of the intensity of each sample stained was performed. Western Blot : Segments of arteries and veins were analyzed using the following primary antibodies :
2296;-­208;SMA, Calponin, SM-­208;MHC, NM-­208;MHC-­208;B. The given results were then normalized with tubulin. Results Our data showed that, when using immunohistochemistry analysis we found that
2296;-­208;SMA was mostly expressed in control arteries, whereas NM-­208;MHC-­208;B in the pathologic ones. Using SM-­208;MHC, calponin, vimentin and caldesmon we found no significative differences in the expression of these proteins in the control and in the pathologic samples. Western Blot analysis showed an inverse correlation between healthy and pathological samples as <-­208; SMA was more expressed in the pathological samples, while NM-­208;MHC-­208;B in the control group; SM-­208;MHC and calponin were mostly expressed in the pathologic samples. Conclusion Our study showed no clear differences between stenotic and control arterial and venous segments using semi-­208;quantitative assessement by immunohistochemistry. Western Blot showed a significant increased expression of
2296;-­208;SMA, calponin and SM-­208;MHC in the arteries with stenosis, while NM-­208;MHC-­208;B was mostly expressed in the arteries without lesions. Further studies are needed to track the lineage of VSMCs to understand the mechanisms leading toIH.

Improving binding affinity and stability of peptide ligands by substituting glycines with D-amino acids.

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer-related protease urokinase-type plasminogen activator (uPA), we observed a glycine residue that has a positive ϕ dihedral angle when bound to the target. We hypothesized that replacing it with a D-amino acid, which favors positive ϕ angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D-serine in the bicyclic peptide indeed improved inhibitory activity (1.75-fold) and stability (fourfold). X-ray-structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ϕ angles are found in many protein-bound peptides. These results suggest that the glycine-to-D-amino acid mutagenesis strategy could be broadly applied.

Cellular origin of T-cell lymphomas.

In this issue of Blood, Iqbal et al, having compiled gene expression profiles from >300 peripheral T-cell lymphomas, expand previous findings on the diagnostic value of molecular signatures that correlate with different histological types of T-cell lymphomas. They report the discovery of 2 molecular subgroups of peripheral T-cell lymphomas, not otherwise specified (PTCL, NOS), characterized by high expression of either GATA-binding protein 3 (GATA-3) or t-box 21 (TBX21) transcription factors and corresponding target genes, with the GATA3 subgroup being associated with distinctly worse prognosis. In an independent study, Wang et al(2) also show that GATA3 expression in a subset of PTCL, NOS identifies a subgroup of patients with inferior survival.

Detection of human growth hormone doping in urine: out of competition tests are necessary.

The misuse of human growth hormone (hGH) in sport is deemed to be unethical and dangerous because of various adverse effects. Thus, it has been added to the International Olympic Committee list of banned substances. Until now, the very low concentration of hGH in the urine made its measurement difficult using classical methodology. Indeed, for routine diagnosis, only plasma measurements were available. However, unlike blood samples, urine is generally provided in abundant quantities and is, at present, the only body fluid allowed to be analysed in sport doping controls. A recently developed enzyme-linked immunosorbent assay (Norditest) makes it now possible, without any extraction, to measure urinary hGH (u-hGH) in a dynamic range of 2-50 ng hGH/l. In our protocol, untreated and treated non-athlete volunteers were followed. Some of them received therapeutical doses of recombinant hGH (Norditropin) for one week either intramuscularly (three increasing doses) or subcutaneously (12 i.u. every day). The u-hGH excretion after treatment showed dramatic increases of 50-100 times the basal values and returned to almost the mean normal level after 24 h. u-hGH was also measured in samples provided by the anti-doping controls at major and minor competitions. Depending on the type of efforts made during the competition, the hGH concentration in urine was dramatically increased. Insulin-like growth factor binding proteins and beta 2-microglobulins in urine and/or in blood could be necessary for the correct investigation of any hGH doping test procedure.

SNARE protein expression in synaptic terminals and astrocytes in the adult hippocampus: a comparative analysis.

Several evidences suggest that astrocytes release small transmitter molecules, peptides, and protein factors via regulated exocytosis, implying that they function as specialized neurosecretory cells. However, very little is known about the molecular and functional properties of regulated secretion in astrocytes in the adult brain. Establishing these properties is central to the understanding of the communication mode(s) of these cells and their role(s) in the control of synaptic functions and of cerebral blood flow. In this study, we have set-up a high-resolution confocal microscopy approach to distinguish protein expression in astrocytic structures and neighboring synaptic terminals in adult brain tissue. This approach was applied to investigate the expression pattern of core SNARE proteins for vesicle fusion in the dentate gyrus and CA1 regions of the mouse hippocampus. Our comparative analysis shows that astrocytes abundantly express, in their cell body and main processes, all three protein partners necessary to form an operational SNARE complex but not in the same isoforms expressed in neighbouring synaptic terminals. Thus, SNAP25 and VAMP2 are absent from astrocytic processes and typically concentrated in terminals, while SNAP23 and VAMP3 have the opposite expression pattern. Syntaxin 1 is present in both synaptic terminals and astrocytes. These data support the view that astrocytes in the adult hippocampus can communicate via regulated exocytosis and also indicates that astrocytic exocytosis may differ in its properties from action potential-dependent exocytosis at neuronal synapses, as it relies on a distinctive set of SNARE proteins.

Calbindin-immunoreactive sensory neurons in dissociated dorsal root ganglion cell cultures of chick embryo: role of culture conditions.

Immunoreactivity to calbindin D-28k, a vitamin D-dependent calcium-binding protein, is expressed by neuronal subpopulations of dorsal root ganglia (DRG) in the chick embryo. To determine whether the expression of this phenotypic characteristic is maintained in vitro and controlled by environmental factors, dissociated DRG cell cultures were performed under various conditions. Subpopulations of DRG cells cultured at embryonic day 10 displayed calbindin-immunoreactive cell bodies and neurites in both neuron-enriched or mixed DRG cell cultures. The number of calbindin-immunoreactive ganglion cells increased up to 7-10 days of culture independently of the changes occurring in the whole neuronal population. The presence of non-neuronal cells, which promotes the maturation of the sensory neurons, tended to reduce the percentage of calbindin-immunoreactive cell bodies. Addition of horse serum enhanced both the number of calbindin-positive neurons and the intensity of the immunostaining, but does not prevent the decline of the subpopulation of calbindin-immunoreactive neurons during the second week of culture; on the contrary, the addition of muscular extract to cultures at 10 days maintained the number of calbindin-expressing neurons. While calbindin-immunoreactive cell bodies grown in culture were small- or medium-sized, no correlation was found between cell size and immunostaining density. At the ultrastructural level, the calbindin immunoreaction was distributed throughout the neuroplasm. These results indicate that the expression of calbindin by sensory neurons grown in vitro may be modulated by horse serum-contained factors or interaction with non-neuronal cells. As distinct from horse serum, muscular extract is able to maintain the expression of calbindin by a subpopulation of DRG cells.

Access of extracellular cations to their binding sites in Na,K-ATPase: role of the second extracellular loop of the alpha subunit.

Na,K-ATPase, the main active transport system for monovalent cations in animal cells, is responsible for maintaining Na(+) and K(+) gradients across the plasma membrane. During its transport cycle it binds three cytoplasmic Na(+) ions and releases them on the extracellular side of the membrane, and then binds two extracellular K(+) ions and releases them into the cytoplasm. The fourth, fifth, and sixth transmembrane helices of the alpha subunit of Na,K-ATPase are known to be involved in Na(+) and K(+) binding sites, but the gating mechanisms that control the access of these ions to their binding sites are not yet fully understood. We have focused on the second extracellular loop linking transmembrane segments 3 and 4 and attempted to determine its role in gating. We replaced 13 residues of this loop in the rat alpha1 subunit, from E314 to G326, by cysteine, and then studied the function of these mutants using electrophysiological techniques. We analyzed the results using a structural model obtained by homology with SERCA, and ab initio calculations for the second extracellular loop. Four mutants were markedly modified by the sulfhydryl reagent MTSET, and we investigated them in detail. The substituted cysteines were more readily accessible to MTSET in the E1 conformation for the Y315C, W317C, and I322C mutants. Mutations or derivatization of the substituted cysteines in the second extracellular loop resulted in major increases in the apparent affinity for extracellular K(+), and this was associated with a reduction in the maximum activity. The changes produced by the E314C mutation were reversed by MTSET treatment. In the W317C and I322C mutants, MTSET also induced a moderate shift of the E1/E2 equilibrium towards the E1(Na) conformation under Na/Na exchange conditions. These findings indicate that the second extracellular loop must be functionally linked to the gating mechanism that controls the access of K(+) to its binding site.

Up-regulation of placental leptin by human chorionic gonadotropin.

Leptin, the 16,000 molecular weight protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta, in which it was found to be expressed. In the present work, we have found that recombinant human chorionic gonadotropin (hCG) added to BeWo choriocarcinoma cell line showed a stimulatory effect on endogenous leptin expression, when analyzed by Western blot. This effect was time and dose dependent. Maximal effect was achieved at hCG 100 IU/ml. Moreover, hCG treatment enhanced leptin promoter activity up to 12.9 times, evaluated by transient transfection with a plasmid construction containing different promoter regions and the reporter gene luciferase. This effect was dose dependent and evidenced with all the promoter regions analyzed, regardless of length. Similar results were obtained with placental explants, thus indicating physiological relevance. Because hCG signal transduction usually involves cAMP signaling, this pathway was analyzed. Contrarily, we found that dibutyryl cAMP counteracted hCG effect on leptin expression. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor cAMP response element binding protein repressed leptin expression. Thereafter we determined that hCG effect could be partially blocked by pharmacologic inhibition of MAPK pathway with 50 microM PD98059 but not by the inhibition of the phosphatidylinositol 3-kinase pathway with 0.1 microm wortmannin. Moreover, hCG treatment promoted MAPK kinase and ERK1/ERK2 phosphorylation in placental cells. Finally, cotransfection with a dominant-negative mutant of MAPK blocked the hCG-mediated activation of leptin expression. In conclusion, we provide some evidence suggesting that hCG induces leptin expression in trophoblastic cells probably involving the MAPK signal transduction pathway.

Evaluation of local immune response to Fasciola hepatica experimental infection in the liver and hepatic lymph nodes of goats immunized with Sm14 vaccine antigen

Protection against Fasciola hepatica in goats immunized with a synthetic recombinant antigen from Schistosoma mansoni fatty acid-binding protein 14 (rSm14) was investigated by assessing worm burdens, serum levels of hepatic enzymes, faecal egg count and hepatic damage, which was evaluated using gross and microscopic morphometric observation. The nature of the local immune response was assessed by examining the distribution of CD2+, CD4+, CD8+ and γ´+ T lymphocytes along with IgG+, IL-4+ and IFN-γ+ cells in the liver and hepatic lymph nodes (HLN). The goats used consisted of group 1 (unimmunized and uninfected), group 2 [infected control - immunized with Quillaia A (Quil A)] and group 3 (immunized with rSm14 in Quil A and infected), each containing seven animals. Immunization with rSm14 in Quil A adjuvant induced a reduction in gross hepatic lesions of 56.6% (p < 0.001) and reduced hepatic and HLN infiltration of CD2+, CD4+, CD8+ and γ´+ T lymphocytes as well as IL-4+ and IFN-γ+ cells (p < 0.05). This is the first report of caprine immunization against F. hepatica using a complete rSm14 molecule derived from S. mansoni. Immunization reduced hepatic damage and local inflammatory infiltration into the liver and HLN. However, considering that Quil A is not the preferential/first choice adjuvant for Sm14 immunization, further studies will be undertaken using the monophosphoryl lipid A-based family of adjuvants during clinical trials to facilitate anti-Fasciolavaccine development.

Ubiquitylation of voltage-gated sodium channels.

Ion channel proteins are regulated by different types of posttranslational modifications. The focus of this review is the regulation of voltage-gated sodium channels (Navs) upon their ubiquitylation. The amiloride-sensitive epithelial sodium channel (ENaC) was the first ion channel shown to be regulated upon ubiquitylation. This modification results from the binding of ubiquitin ligase from the Nedd4 family to a protein-protein interaction domain, known as the PY motif, in the ENaC subunits. Many of the Navs have similar PY motifs, which have been demonstrated to be targets of Nedd4-dependent ubiquitylation, tagging them for internalization from the cell surface. The role of Nedd4-dependent regulation of the Nav membrane density in physiology and disease remains poorly understood. Two recent studies have provided evidence that Nedd4-2 is downregulated in dorsal root ganglion (DRG) neurons in both rat and mouse models of nerve injury-induced neuropathic pain. Using two different mouse models, one with a specific knockout of Nedd4-2 in sensory neurons and another where Nedd4-2 was overexpressed with the use of viral vectors, it was demonstrated that the neuropathy-linked neuronal hyperexcitability was the result of Nav1.7 and Nav1.8 overexpression due to Nedd4-2 downregulation. These studies provided the first in vivo evidence of the role of Nedd4-2-dependent regulation of Nav channels in a disease state. This ubiquitylation pathway may be involved in the development of symptoms and diseases linked to Nav-dependent hyperexcitability, such as pain, cardiac arrhythmias, epilepsy, migraine, and myotonias.

Improvement in growth after two years of growth hormone therapy in very young children born small for gestational age and without spontaneous catch-up growth: results of a multicenter, controlled, randomized, open clinical trial.

CONTEXT GH treatment is effective in children born small for gestational age (SGA); however, its effectiveness and safety in very young SGA children is unknown. OBJECTIVE The aim was to analyze the outcome of very young SGA children treated with GH and followed for 2 yr. The results after 24 months of treatment, compared with a control group without treatment during 12 months followed by 12 months of treatment, are shown. DESIGN We performed a multicenter, controlled, randomized, open trial. SETTINGS The pediatric endocrinology departments of 14 public hospitals in Spain participated in the study. PATIENTS Seventy-six children, aged 2-5 yr born SGA and without catch-up growth, were studied. INTERVENTION Children received GH at 0.06 mg/kg.d for 2 yr (group I) or were followed for 12 months with no treatment and then treated for 12 months (group II). MAIN OUTCOME MEASURES Age, general health status, pubertal stage, bone age, height, weight, biochemical and hormonal analyses, and adverse side effects were determined at biannual check-ups. RESULTS The mean height sd score gain for chronological age in children treated for 24 months (group I) was 2.10, whereas in those treated only during the last 12 months (group II) was 1.43. In both groups, children under 4 yr of age had the greatest gain in growth velocity. No significant acceleration of bone age or side effects related to treatment was seen. CONCLUSION Very young SGA children without spontaneous catch-up growth could benefit from GH treatment because growth was accelerated and no negative side effects were observed.

Adipose tissue gene expression of factors related to lipid processing in obesity.

BACKGROUND Adipose tissue lipid storage and processing capacity can be a key factor for obesity-related metabolic disorders such as insulin resistance and diabetes. Lipid uptake is the first step to adipose tissue lipid storage. The aim of this study was to analyze the gene expression of factors involved in lipid uptake and processing in subcutaneous (SAT) and visceral (VAT) adipose tissue according to body mass index (BMI) and the degree of insulin resistance (IR). METHODS AND PRINCIPAL FINDINGS VLDL receptor (VLDLR), lipoprotein lipase (LPL), acylation stimulating protein (ASP), LDL receptor-related protein 1 (LRP1) and fatty acid binding protein 4 (FABP4) gene expression was measured in VAT and SAT from 28 morbidly obese patients with Type 2 Diabetes Mellitus (T2DM) or high IR, 10 morbidly obese patients with low IR, 10 obese patients with low IR and 12 lean healthy controls. LPL, FABP4, LRP1 and ASP expression in VAT was higher in lean controls. In SAT, LPL and FABP4 expression were also higher in lean controls. BMI, plasma insulin levels and HOMA-IR correlated negatively with LPL expression in both VAT and SAT as well as with FABP4 expression in VAT. FABP4 gene expression in SAT correlated inversely with BMI and HOMA-IR. However, multiple regression analysis showed that BMI was the main variable contributing to LPL and FABP4 gene expression in both VAT and SAT. CONCLUSIONS Morbidly obese patients have a lower gene expression of factors related with lipid uptake and processing in comparison with healthy lean persons.

Effect of carbohydrate overfeeding on whole body and adipose tissue metabolism in humans.

OBJECTIVE: To evaluate the effect of a 4-day carbohydrate overfeeding on whole body net de novo lipogenesis and on markers of de novo lipogenesis in subcutaneous adipose tissue of healthy lean humans. RESEARCH METHODS AND PROCEDURES: Nine healthy lean volunteers (five men and four women) were studied after 4 days of either isocaloric feeding or carbohydrate overfeeding. On each occasion, they underwent a metabolic study during which their energy expenditure and net substrate oxidation rates (indirect calorimetry), and the fractional activity of the pentose-phosphate pathway in subcutaneous adipose tissue (subcutaneous microdialysis with 1,6(13)C2,6,6(2)H2 glucose) were assessed before and after administration of glucose. Adipose tissue biopsies were obtained at the end of the experiments to monitor mRNAs of key lipogenic enzymes. RESULTS: Carbohydrate overfeeding increased basal and postglucose energy expenditure and net carbohydrate oxidation. Whole body net de novo lipogenesis after glucose loading was markedly increased at the expense of glycogen synthesis. Carbohydrate overfeeding also increased mRNA levels for the key lipogenic enzymes sterol regulatory element-binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase. The fractional activity of adipose tissue pentose-phosphate pathway was 17% to 22% and was not altered by carbohydrate overfeeding. DISCUSSION: Carbohydrate overfeeding markedly increased net de novo lipogenesis at the expense of glycogen synthesis. An increase in mRNAs coding for key lipogenic enzymes suggests that de novo lipogenesis occurred, at least in part, in adipose tissue. The pentose-phosphate pathway is active in adipose tissue of healthy humans, consistent with an active role of this tissue in de novo lipogenesis.