Expression analysis of the novel gene collagen triple helix repeat containing-1 (Cthrc1)

We recently identified collagen triple helix repeat containing-1 (Cthrc1) as a novel gene induced in adventitial fibroblasts after arterial injury. Cthrc1 is a 30 kDa secreted protein that has the ability to inhibit collagen matrix synthesis. Cthrc1 is also glycosylated and retains a signal sequence consistent with the presence of Cthrc1 in the extracellular space. In injured arteries and skin wounds, we have found Cthrc1 expression to be associated with myofibroblasts and sites of collagen matrix deposition. Furthermore, we demonstrated that Cthrc1 inhibits collagen matrix deposition in vitro. Using in situ hybridization and immunohistochemistry, we characterized the expression domains of Cthrc1 during murine embryonic development and in postnatal tissues. In mouse embryos, Cthrc1 was expressed in the visceral endoderm, notochord, neural tube, developing kidney, and heart. Abundant expression of Cthrc1 was observed in the developing skeleton, i.e., in cartilage primordia, in growth plate cartilage with exclusion of the hypertrophic zone, in the bone matrix and periostium. Bones from adults showed expression of Cthrc1 only in the bone matrix and periostium while the articular cartilage lacked expression. Cthrc1 is typically expressed at epithelial-mesenchymal interfaces that include the epidermis and dermis, basal corneal epithelium, airway epithelium, esophagus epithelium, choroid plexus epithelium, and meninges. In the adult kidney, collecting ducts and distal tubuli expressed Cthrc1. Collectively, the sites of Cthrc1 expression overlap considerably with those reported for TGF-beta family members and interstitial collagens. The present study provides useful information towards the understanding of potential Cthrc1 functions.     

Caveola-dependent endocytic entry of amphotropic murine leukemia virus

Early results suggested that the amphotropic murine leukemia virus (A-MLV) does not enter cells via endocytosis through clathrin-coated pits and this gammaretrovirus has therefore been anticipated to fuse directly with the plasma membrane. However, here we present data implicating a caveola-mediated endocytic entry route for A-MLV via its receptor Pit2. Caveolae belong to the cholesterol-rich microdomains characterized by resistance to nonionic detergents such as Triton X-100. Extraction of murine fibroblastic NIH 3T3 cells in cold Triton X-100 showed the presence of the A-MLV receptor Pit2 in detergent-insoluble microdomains. Using coimmunoprecipitation of cell extracts, we were able to demonstrate direct association of Pit2 with caveolin-1, the structural protein of caveolae. Other investigations revealed that A-MLV infection in contrast to vesicular stomatitis virus infection is a slow process (t(1/2) approximately 5 h), which is dependent on plasma membrane cholesterol but independent of NH4Cl treatment of cells; NH4Cl impairs entry via clathrin-coated pits. Furthermore, expression of dominant-negative caveolin-1 decreased the susceptibility to infection via Pit2 by approximately 70%. These results show that A-MLV can enter cells via a caveola-dependent entry route. Moreover, increase in A-MLV infection by treatment with okadaic acid as well as entry of fusion-defective fluorescent A-MLV virions in NIH 3T3 cells further confirmed our findings and show that A-MLV can enter mouse fibroblasts via an endocytic entry route involving caveolae. Finally, we also found colocalization of fusion-defective fluorescent A-MLV virions with caveolin-1 in NIH 3T3 cells. This is the first time substantial evidence has been presented implicating the existence of a caveola-dependent endocytic entry pathway for a retrovirus.     

Microphytobenthos of Arctic Kongsfjorden (Svalbard, Norway): biomass and potential primary production along the shore line

During summer 2007, Arctic microphytobenthic potential primary production was measured at several stations around the coastline of Kongsfjorden (Svalbard, Norway) at ≤5 m water depth and at two stations at five different water depths (5, 10, 15, 20, 30 m). Oxygen planar optode sensor spots were used ex situ to determine oxygen exchange in the overlying water of intact sediment cores under controlled light (ca. 100 μmol photons m⁻² s⁻¹) and temperature (2–4°C) conditions. Patches of microalgae (mainly diatoms) covering sandy sediments at water depths down to 30 m showed high biomass of up to 317 mg chl a m⁻². In spite of increasing water depth, no significant trend in “photoautotrophic active biomass” (chl a, ratio living/dead cells, cell sizes) and, thus, in primary production was measured at both stations. All sites from ≤5 to 30 m water depth exhibited variable rates of net production from −19 to +40 mg O₂ m⁻² h⁻¹ (−168 to +360 mg C m⁻² day⁻¹) and gross production of about 2–62 mg O₂ m⁻² h⁻¹ (17–554 mg C m⁻² day⁻¹), which is comparable to other polar as well as temperate regions. No relation between photoautotrophic biomass and gross/net production values was found. Microphytobenthos demonstrated significant rates of primary production that is comparable to pelagic production of Kongsfjorden and, hence, emphasised the importance as C source for the zoobenthos.     

Efficient Pre-mRNA Cleavage Prevents Replication-Stress-Associated Genome Instability

1. Download high-res image (215KB)
2. Download full-size image

     

Tolerance induction in composite facial allograft transplantation in the rat model

Clinical application of composite tissue allograft transplants opened discussion on the restoration of facial deformities by allotransplantation. The authors introduce a hemifacial allograft transplant model to investigate the rationale for the development of functional tolerance across the major histocompatibility complex barrier. Eighteen rats in three groups were studied. The composite hemifacial allotransplantations including the ear and scalp were performed between Lewis-Brown Norway (RT1l+n) and Lewis (RT1l) rats and isotransplantations were performed between Lewis rats. Isograft controls (n = 6) and allograft controls (n = 6) did not receive treatment. Allografts in treatment group (n = 6) were treated with cyclosporine A 16 mg/kg/day during the first week; this dose was tapered to 2 mg/kg/day over 4 weeks and maintained at this level thereafter. Functional tolerance to face allografts was evaluated clinically and histologically. Donor-specific chimerism was assessed at days 21 and 63 by flow cytometry. In vitro evaluation of donor-specific tolerance was performed by mixed lymphocyte reaction at day 160 after transplantation. Isograft controls survived indefinitely. All nontreated allografts were rejected within 5 to 7 days after transplantation, as confirmed by histopathologic analysis. Five of six face allografts under the cyclosporine A protocol showed no signs of rejection for up to 240 days and remained alive and under evaluation, whereas one animal showed signs of rejection at day 140. This was reversed by adjustment of the cyclosporine A dose. At day 21 after transplantation, flow cytometric analysis of the donor-specific chimerism showed 1.11 percent of double-positive CD4FITC/RT1Ac-Cy7 and 1.43 percent of double-positive CD8PE/RT1Ac-Cy7 T-cell populations in the peripheral blood of hemiface allotransplant recipients. The chimerism level of double-positive CD4FITC/RT1Ac-Cy7 T cells increased to 3.39 percent, whereas it remained stable for the double-positive CD8PE/RT1Ac-Cy7 T-cell population at day 63 after transplantation (1.00 percent). The mixed lymphocyte reaction assay at day 160 after transplantation revealed donor-specific tolerance to donor (Lewis-Brown Norway) antigens and strong reactivity to the third-party (ACI) alloantigens. In this study, donor-specific chimerism and functional tolerance were induced in hemifacial allograft transplants across the major histocompatibility complex barrier under cyclosporine A monotherapy protocol. This model will allow further studies on tolerance induction protocols.     

Guanylate Cyclase-Activating Protein-2 Undergoes Structural Changes upon Binding to Detergent Micelles and Bicelles

GCAPs are neuronal Ca^2 +-sensors playing a central role in light adaptation. GCAPs are N-terminally myristoylated membrane-associated proteins. Although, the myristoylation of GCAPs plays an important role in light adaptation its structural and physiological roles are not yet clearly understood. The crystal-structure of GCAP-1 shows the myristoyl moiety inside the hydrophobic core of the protein, stabilizing the protein structure; but ²H-solid-state NMR investigations on the deuterated myristoyl moiety of GCAP-2 in the presence of liposomes showed that it is inserted into the lipid bilayer. In this study, we address the question of the localization of the myristoyl group of Ca^2 +-bound GCAP-2, and the influence of CHAPS-, DPC-micelles and DMPC/DHPC-bicelles on the structure, and on the localization of the myristoyl group, of GCAP-2 by solution-state NMR. We also carried out the backbone assignment. Characteristic chemical shift differences have been observed between the myristoylated and the non-myristoylated forms of the protein. Our results support the view that in the absence of membrane forming substances the myristoyl moiety is buried inside a hydrophobic pocket of GCAP-2 similar to the crystal structure of GCAP-1. Addition of CHAPS-micelles and DMPC/DHPC-bicelles cause specific structural changes localized in and around the myristoyl binding pocket. We interpret these changes as an indication for the extrusion of the myristoyl moiety from its binding pocket and its insertion into the hydrophobic interior of the membrane mimic. On the basis of the backbone chemical shifts, we propose a structural model of myristoylated GCAP-2 in the presence of Ca^2 + and membrane mimetics.     

Sexually selected male weapon is associated with lower inbreeding load but higher sex load in the bulb mite

Elaborate sexually selected ornaments and armaments are costly but increase the reproductive success of their bearers (usually males). It has been postulated that high-quality males can invest disproportionately more in such traits, making those traits honest signals of genetic quality. However, genes associated with such traits may have sexually antagonistic effects on fitness. Here, using a bulb mite Rhizoglyphus robini, a species in which a distinct dimorphism exists between males in the expression of a sexually selected weapon, we compare inbreeding and gender load between lines derived from armed fighters and unarmed scramblers. After four generations of sib-mating, inbreeding depression for female fitness was significantly lower in fighter-derived lines compared to scrambler-derived lines, suggesting that fighter males had significantly higher genetic quality. However, outbred females from fighter-derived lines had significantly lower fitness compared to outbred females from scrambler-derived lines, demonstrating significant gender load associated with the presence of a sexually selected male weapon. Our results imply that under outbreeding, genetic benefits of mating with bearers of elaborate sexually selected traits might be swamped by the costs of decreased fitness of female progeny due to sexually antagonistic effects.     

Prognostic Significance of ESR1 Amplification and ESR1 PvuII,CYP2C19*2, UGT2B15*2 Polymorphisms in Breast Cancer Patients

Introduction

Amplification of the ESR1 gene, coding for estrogen receptor alpha, was shown to predict responsiveness to tamoxifen, however its prognostic impact in breast cancer patients has not been thoroughly investigated. Other factors that could contribute to responsiveness to tamoxifen treatment are polymorphisms in ESR1 gene and genes involved in tamoxifen metabolism.The aim of this study was to assess the prognostic role of ESR1 gene dosage in a consecutive group of breast cancer patients and to correlate this feature with clinico-pathological factors. Additionally, ESR1 PvuII, CYP2C19*2 and UGT2B15*2 polymorphisms were analyzed in the tamoxifen-treated subgroup of patients.

Materials and Methods

Primary tumor samples from 281 stage I-III consecutive breast cancer patients were analyzed for ESR1 gene dosage using real-time PCR with locked nucleic acids hydrolysis probes. In the tamoxifen-treated subgroup of patients, ESR1 PvuII, CYP2C19*2 and UGT2B15*2 polymorphism in leukocytes genomic DNA were analyzed. Results were correlated with clinico-pathological factors and with disease-free survival (DFS) and overall survival (OS).

Results

ESR1 amplification (with a cut-off level of 2.0) was found in 12% of the entire group of breast cancer patients, and in 18% of the ER-negative subgroup. This feature was associated with decreased DFS both in the entire group (P=0.007) and in the ER-negative subgroup (P=0.03), but not in the tamoxifen-treated patients.Patients with ESR1 PvuII wt/wt genotype and at least one UGT2B15 wt allele had a worse DFS (P=0.03) and showed a trend towards decreased Os (P=0.08) in comparison to patients with ESR1 PvuII wt/vt or vt/vt genotype and UGT2B15 *2/*2 genotype.

Conclusions

ESR1 amplification can occur in ER-negative tumors and may carry poor prognosis. In the tamoxifen-treated subgroup, poor prognosis was related to the combined presence of ESR1 PvuII wt/wt and UGT2B15wt/wt or wt/*2 genotype.