Endoglin expression regulates basal and TGF-beta1-induced extracellular matrix synthesis in cultured L6E9 myoblasts.

BACKGROUND/AIMS: TGF-beta1 plays a major role in extracellular matrix (ECM) accumulation in tissue fibrosis. Connective tissue growth factor appears to play a critical role in this effect. Endoglin is a component of the transforming growth factor b (TGF-beta) receptor complex. Endoglin is upregulated by TGF-beta1, but its functional role in ECM regulation is unknown. Using rat myoblasts as a model system, we have assessed the role of endoglin on regulating CTGF expression and ECM synthesis and accumulation in the presence or absence of TGF-beta1. METHODS: L6E9 myoblast cell line was transfected with human endoglin, and collagen, fibronectin and CTGF production was assessed by Western blot and by proline incorporation to collagen proteins. RESULTS: Northern blot analysis revealed that parental rat myoblasts L6E9 do not express endogenous endoglin. Upon endoglin transfection, endoglin-expressing cells displayed a decreased CTGF expression and decreased collagen and fibronectin accumulation respect to mock transfectants. Northern blot analysis also revealed a decreased alpha2 (I) procollagen mRNA expression in endoglin transfectants. TGF-beta1 treatment induced an increase in CTGF expression and collagen synthesis and accumulation in L6E9 myoblasts. This effect was significantly lower in endoglin-transfected than in mock-transfected cells. CONCLUSION: These results demonstrate that endoglin expression negatively regulates basal and TGF-beta1-induced CTGF and collagen expression and synthesis.     

A CULTURE STUDY OF SALINITY RESPONSES IN ECOTYPES OF TWO ESTUARINE RED ALGAE1

Laboratory culture studies on the euryhalinity of Bostrychia radicans Montagne and Caloglossa leprieurii (Montagne) J. Agardh from the mouth and head of the Mullica River estuary, New Jersey, revealed both species probably have ecotypes whose growth patterns correlate with the salinity regime of their habitat in nature. Significant growth differences of tetrasporelings were determined in response to four salinities (5, 15, 25, 35%c) even after acclimation periods of the tetrasporophytes from 6 mo–2 yr in laboratory culture. However, one isolate of Bostrychia and both isolates of Caloglossa also demonstrated some capability for physiological adaptation to salinity changes although this was less significant statistically than their ecotypic response. It thus appears that certain euryhaline algae may consist of ecotypes, each of which has some capacity for physiological adaptation to salinity variations.     

Determination of oxytetracycline by flow injection with chemiluminescence detection.

A flow-injection chemiluminescent method for the determination of oxytetracycline was developed. The method is based on an enhancement by oxytetracycline of the chemiluminescence light emission of tris(2,2'-bipyridine) ruthenium (II), generated by the continuous oxidation of tris(2,2'-bipyridine) ruthenium (II) by cerium (IV) sulphate in sulphuric acid. Under the optimum conditions, the calibration curve was linear over the range 1.0 x 10(-7)-1.0 x 10(-5) g/mL for oxytetracycline with the linear equation: DeltaINT = 148.77 x C + 0.6637 (R2 = 0.9994). The detection limit was 4.52 x 10(-8) g/mL. The proposed method was also successfully used to determine oxytetracycline in pharmaceutical formulations. The mean recovery of determination of oxytetracycline was 92.73%. A mechanism for the chemiluminescence enhancement by oxytetracycline of tris(2,2'-bipyridine)-ruthenium (II) and cerium (IV) sulphate system is also proposed.     

Natural killer cells: detectors of stress

Natural killer (NK) cells are a key component of the innate immune system, as they are able to detect microbe-infected cells, tumors as well as allogeneic cells, without specific sensitization. NK cell effector functions (cytotoxicity, cytokine secretion) are regulated by a wide array of inhibitory and activating receptors. MHC class I molecules are the ligands of most inhibitory receptors, while activating receptors recognize either pathogen-encoded molecules, or self-proteins whose expression is up-regulated upon microbial infection or tumor development. Upon integration of these negative and positive signals, Natural Killer cells can discriminate between healthy "self" (tolerance) and autologous cells undergoing different types of cellular stress or allogeneic cells (immunosurveillance). The knowledge of the different mechanisms of target cell recognition is thus crucial to dissect NK cell involvement in homeostatic and disease conditions as well as to develop novel alternative therapeutic approaches based on NK cell manipulation.     

Arf and Rho GAP adapter protein ARAP1 participates in the mobilization of TRAIL-R1/DR4 to the plasma membrane

TRAIL, a ligand of the TNFα family, induces upon binding to its pro-death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 the apoptosis of cancer cells. Activated receptors incite the formation of the Death-Inducing Signaling Complex followed by the activation of the downstream apoptotic signaling. TRAIL-induced apoptosis is regulated at multiple levels, one of them being the presence and relative number of TRAIL pro- and anti-apoptotic receptors on the cytoplasmic membrane. In a yeast two-hybrid search for proteins that interact with the intracellular part (ICP) of DR4, we picked ARAP1, an adapter protein with ArfGAP and RhoGAP activities. In yeast, DR4(ICP) interacts with the alternatively spliced ARAP1 lacking 11 amino acids from the PH5 domain. Transfected ARAP1 co-precipitates with DR4 and co-localizes with it in the endoplasmic reticulum/Golgi, at the cytoplasmic membrane and in early endosomes of TRAIL-treated cells. ARAP1 knockdown significantly compromises the localization of DR4 at the cell surface of several tumor cell lines and slows down their TRAIL-induced death. ARAP1 overexpressed in HEL cells does not affect their TRAIL-induced apoptosis or the membrane localization of DR4, but it enhances the cell-surface presentation of phosphatidyl serine. Our data indicate that ARAP1 is likely involved in the regulation of the cell-specific trafficking of DR4 and might thus affect the efficacy of TRAIL-induced apoptosis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

"One-pot" syntheses of malondialdehyde adducts of nucleosides

Short, "one-pot" syntheses of malondialdehyde adducts of deoxyguanosine, deoxyadenosine, and deoxycytidine are described. These syntheses proceed in improved yield and easier purification than previous syntheses and are well suited for the preparation of isotopically labeled nucleoside adducts for biomarker and metabolic studies.     

A VANADIUM BROMOPEROXIDASE CATALYZES THE FORMATION OF HIGH‐MOLECULAR‐WEIGHT COMPLEXES BETWEEN BROWN ALGAL PHENOLIC SUBSTANCES AND ALGINATES1

The interaction between phenolic substances (PS) and alginates (ALG) has been suggested to play a role in the structure of the cell walls of brown seaweeds. However, no clear evidence for this interaction was reported. Vanadium bromoperoxidase (VBPO) has been proposed as a possible catalyst for the binding of PS to ALG. In this work, we studied the interaction between PS and ALG from brown algae using size exclusion chromatography (SEC) and optical tweezers microscopy. The analysis by SEC revealed that ALG forms a high‐molecular‐weight complex with PS. To study the formation of this molecular complex, we investigated the in vitro interaction of purified ALG from Fucus vesiculosus L. with purified PS from Padina gymnospora (Kütz.) Sond., in the presence or absence of VBPO. The interaction between PS and ALG only occurred when VBPO was added, indicating that the enzyme is essential for the binding process. The interaction of these molecules led to a reduction in ALG viscosity. We propose that VBPO promotes the binding of PS molecules to the ALG uronic acids residues, and we also suggest that PS are components of the brown algal cell walls.     

The Influence of Clinical Information in the Histopathologic Diagnosis of Melanocytic Skin Neoplasms

Background

We tested the relevance of clinical information in the histopathologic evaluation of melanocytic skin neoplasm (MSN).

Methods

Histopathologic specimens from 99 clinically atypical MSN were circulated among ten histopathologists; each case had clinical information available in a database with a five-step procedure (no information; age/sex/location; clinical diagnosis; clinical image; dermoscopic image); each step had a histopathologic diagnosis (D1 through D5); each diagnostic step had a level of diagnostic confidence (LDC) ranging from 1 (no diagnostic certainty) to 5 (absolute diagnostic certainty). The comparison of the LDC was employed with an analysis of variance (ANOVA) for repeated measures.

Findings

In D1 (no information), 36/99 cases (36.3%) had unanimous diagnosis; in D5 (full information available), 51/99 cases (51.5%) had unanimous diagnosis (p for difference between proportions <0.001). The observer agreement expressed as kappa increased significantly from D1 to D5. The mean LDC linearly increased for each observer from D1 through D5 (p for linear trend <0.001). On average, each histopathologist changed his initial diagnosis in 7 cases (range: 2–23). Most diagnostic changes were in D2 (age/sex/location).

Interpretation

The histopathologic criteria for the diagnosis of MSN can work as such, but the final histopathologic diagnosis is a clinically-aided interpretation. Clinical data sometimes reverse the initial histopathologic evaluation.