SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.     

Substantial Metabolic Activity of Human Brown Adipose Tissue during Warm Conditions and Cold-Induced Lipolysis of Local Triglycerides

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Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression

CD82, also known as KAI1, was recently identified as a prostate cancer metastasis suppressor gene on human chromosome 11p1.2 (ref. 1). The product of CD82 is KAI1, a 40- to 75-kDa tetraspanin cell-surface protein also known as the leukocyte cell-surface marker CD82 (refs. 1,2). Downregulation of KAI1 has been found to be clinically associated with metastatic progression in a variety of cancers, whereas overexpression of CD82 specifically suppresses tumor metastasis in various animal models. To define the mechanism of action of KAI1, we used a yeast two-hybrid screen and identified an endothelial cell-surface protein, DARC (also known as gp-Fy), as an interacting partner of KAI1. Our results indicate that the cancer cells expressing KAI1 attach to vascular endothelial cells through direct interaction between KAI1 and DARC, and that this interaction leads to inhibition of tumor cell proliferation and induction of senescence by modulating the expression of TBX2 and p21. Furthermore, the metastasis-suppression activity of KAI1 was significantly compromised in DARC knockout mice, whereas KAI1 completely abrogated pulmonary metastasis in wild-type and heterozygous littermates. These results provide direct evidence that DARC is essential for the function of CD82 as a suppressor of metastasis.     

Local expression of apolipoprotein A-I gene and a possible role for HDL in primary defence in the carp skin

Antimicrobial proteins and peptides play an important role in the primary defence of epithelial barriers in vertebrates and invertebrates. Here we report the detection of the apolipoproteins A-I and A-II in the epidermis and epidermal mucus of the carp (Cyprinus carpio L.) by immunohistochemistry and Western blot analysis. Both apolipoproteins are major constituents of high density lipoprotein and have been shown to display antiviral and antimicrobial activity in mammals. Therefore the aim of this study was to evaluate if they could be part of the innate immune system of teleost fish. A cDNA clone containing most of the coding region for carp apoA-I was isolated and used as a probe to demonstrate the expression of apoA-I gene in the skin. In addition, mucus apoA-I was shown to be associated to small particles that could correspond to nascent HDL. Finally, affinity purified plasma HDL displayed bactericidal activity in vitro against a non-pathogenic Escherichia coli strain, suggesting a defensive role for HDL and its associated proteins in the carp epidermis and mucus.     

Multiparametric magnetic resonance imaging-guided salvage radiotherapy in prostate cancer

AimTo analyse the efficacy and toxicity of postprostatectomy SRT in patients with a BCR evaluated with mpMRI.BackgroundMultiparametric magnetic resonance imaging (mpMRI) has the ability to detect the site of pelvic recurrence in patients with biochemical recurrence (BCR) after radical prostatectomy (RP). However, we do not know the oncological outcomes of mpMRI-guided savage radiotherapy (SRT).ResultsLocal, lymph node, and pelvic bone recurrence was observed in 13, 4 and 2 patients, respectively. PSA levels were significantly lower in patients with negative mpMRI (0.4 ng/mL [0.4]) vs. positive mpMRI (2.2 ng/mL [4.1], p = 0.003). Median planning target volume doses in patients with visible vs. non-visible recurrences were 76 Gy vs. 70 Gy. Overall, mean follow-up was 41 months (6–81). Biochemical relapse-free survival (bRFS) at 3 years was 82.3% and 82.5%, respectively, for the negative and positive mpMRI groups (p = 0.800). Three-year rates of late grade ≥2 urinary and rectal toxicity were 14.8% and 1.9%, respectively; all but one patient recovered without sequelae.ConclusionSRT to the macroscopic recurrence identified by mpMRI is a feasible and well-tolerated option. In this study, there were no differences in bRFS between MRI-positive and MRI-negative patients, indicating effective targeting of MRI-positive lesions.     

Degradation of the Retinoblastoma Tumor Suppressor by the Human Papillomavirus Type 16 E7 Oncoprotein Is Important for Functional Inactivation and Is Separable from Proteasomal Degradation of E7

The steady-state level and metabolic half-life of retinoblastoma tumor suppressor protein pRB are decreased in cells that express high-risk human papillomavirus (HPV) E7 proteins. Here we show that pRB degradation is a direct activity of E7 and does not reflect a property of cell lines acquired during the selection process for E7 expression. An amino-terminal domain of E7 that does not directly contribute to pRB binding but is required for transformation is also necessary for E7-mediated pRB degradation. Treatment with inhibitors of the 26S proteasome not only blocks E7-mediated pRB degradation but also causes the stabilization of E7. Mutagenic analyses, however, reveal that the processes of proteasomal degradation of E7 and pRB are not linked processes. HPV type 16 E7 also targets the pRB-related proteins p107 and p130 for destabilization by a proteasome-dependent mechanism. Using the SAOS2 flat-cell assay as a biological indicator for pRB function, we demonstrate that pRB degradation, not solely binding, is important for the E7-induced inactivation of pRB.     

Eimeria tenella ROP kinase EtROP1 induces G0/G1 cell cycle arrest and inhibits host cell apoptosis

Coccidia are obligate intracellular protozoan parasites responsible for human and veterinary diseases. Eimeria tenella, the aetiologic agent of caecal coccidiosis, is a major pathogen of chickens. In Toxoplasma gondii, some kinases from the rhoptry compartment (ROP) are key virulence factors. ROP kinases hijack and modulate many cellular functions and pathways, allowing T. gondii survival and development. E. tenella's kinome comprises 28 putative members of the ROP kinase family; most of them are predicted, as pseudokinases and their functions have never been characterised. One of the predicted kinase, EtROP1, was identified in the rhoptry proteome of E. tenella sporozoites. Here, we demonstrated that EtROP1 is active, and the N‐terminal extension is necessary for its catalytic kinase activity. Ectopic expression of EtROP1 followed by co‐immunoprecipitation identified cellular p53 as EtROP1 partner. Further characterisation confirmed the interaction and the phosphorylation of p53 by EtROP1. E. tenella infection or overexpression of EtROP1 resulted both in inhibition of host cell apoptosis and G0/G1 cell cycle arrest. This work functionally described the first ROP kinase from E. tenella and its noncanonical structure. Our study provides the first mechanistic insight into host cell apoptosis inhibition by E. tenella. EtROP1 appears as a new candidate for coccidiosis control.     

Onset of Spermatogenesis in Corriedale Ram Lambs under Extensive Rearing Conditions in Uruguay

The present study was undertaken to estimate the time for the attainment of spermatogenesis in spring-born Corriedale ram lambs under conditions of extensive grazing systems in Uruguay. Clinical (live weight, scrotal circumference, penile/preputial separation), morphologi-cal(light and electron microscopy) and endocrinological (testosterone levels) parameters were examined. Two experiments in 2 consecutive years were carried out. In Exp. I, 40 ram lambs were clinically examined, weighed, and blood-sampled at 2 week-intervals between 78 and 216 days of age. Sixteeen were castrated in 3 selected periods: 132-162 (n: 2), 145–175 (n: 6) and 186–216 days (n: 8). In Exp. II, 40 ram lambs appertaining to the next generation of the same flock were examined as above at 180–210 days of age, and castrated for morphological studies. The time for attainment of complete spermatogenesis correlated significantly with most corporal parameters (i.e. scrotal circumference (r = 0.52); testicular weight (r = 0.61), epididymal weight (r = 0.60), penile/preputial separation (r = 0.75). The age of castrated ram lambs correlated with the degree of spermatogenesis (r = 0.83), although no significant correlations were found with live weight or with levels of circulating testosterone. The histology showed major variations in the degree of development of the seminiferous epithelium. Cells undergoing degeneration were a common finding through the initial stages of spermatogenesis, coincident to the presence of sperm abnormalities and foreign cells in semen. By day 180 and onwards, both histology and seminal picture normalized. It is concluded that, under these rearing conditions, the onset of puberty (expressed as morphologically established spermatogenesis) in Corriedale ram lambs is attained at 180-216 days of age when they reach 23 cm of scrotal circumference and 191 g of testis weight. The finding of a high correlation between these parameters (r: 0.93) confirms the usefulness of the measurement of scrotal circumference during clinical examination of ram lambs in this breed.