Establishment of ionic channels and signalling cascades in the embryonic stem cell-derived primitive endoderm and cardiovascular system

The first organ system to be established in early embryogenesis is the cardiovascular system which develops upon interaction with hypoblastic cells of the primitive endoderm. Here we focus on recent work on embryoid bodies derived from pluripotent embryonic stem (ES) cells. Ca(2+) oscillations and Ca(2+) signalling pathways during the differentiation of primitive endodermal cell layers are reported. Furthermore, the development-dependent expression of ion channels and the buildup of signalling cascades involved in the modulation of voltage-dependent L-type Ca(2+) channels during early cardiomyogenesis and the formation of functional vascular structures in the process of vasculogenesis and angiogenesis are reviewed. We also report on the use of green fluorescent protein reporter gene expression under the control of cardiac-specific promoters, e.g. the human cardiac alpha-actin promoter, which enables the identification and in vivo characterization of cardiomyocytes at very early stages of cardiomyogenesis.     

Cell-associated, non-replicating strand(+) hepatitis C virus-RNA shedding in cervicovaginal secretions from chronically HCV-infected women.

BACKGROUND: Lack of mucosal hepatitis C virus (HCV) transmission may be due to fairly low infectivity of body fluids in HCV-infected individuals in association with yet unknown innate or acquired resistance factors in individuals exposed to the virus. OBJECTIVE: To evaluate HCV excretion patterns in cervicovaginal secretions obtained from chronically HCV-infected women. STUDY DESIGN: Fifteen chronically HCV-infected women of childbearing age hospitalized for chronic hepatitis were prospectively recruited. Cervicovaginal secretions were obtained by vaginal washing with 3 ml phosphate-buffered saline (PBS). All cervicovaginal secretions were free of hemoglobin traces and also free of semen traces. Free HCV-RNA and cell-associated HCV-RNA were examined in acellular part and cellular part of the cervicovaginal secretions, respectively, by in-house qualitative PCR for 5'-HCV-non-coding region (NCR). Negative strand HCV-RNA, a marker of HCV replication, was searched by using tag-RT-nested PCR (tag-RT-NPCR). RESULTS: HCV-RNA could not be detected in the acellular fractions of the 15 evaluated cervicovaginal secretions. In contrast, HCV-RNA could be detected in the cellular fractions of four of 15 (27%) cervicovaginal secretions. None of the cervicovaginal secretions, including the four positive cell-associated HCV-RNA, contained negative strand, replicating HCV-RNA. CONCLUSIONS: Our results suggest that positive strand HCV-RNA may be present outside the menstruation periods as cell-associated virus in the cervicovaginal secretions of a minority of untreated HCV-seropositive, HCV-RNA-viremic women, and that the lower female genital tract does not constitute a reservoir where HCV replicates. These observations thus provide the basis for the low risk of female-to-male sexual transmission of HCV infection.     

Topographical study of the neurons containing hpGRF immunoreactivity in monkey hypothalamus

Hypothalamic neurons producing growth hormone-releasing factor (GRF) have been characterized by immunohistochemistry in monkey hypothalamus, using an antiserum raised against hpGRF1-40, a peptide with GRF activity isolated from a human pancreatic tumor. Cell bodies with hpGRF immunoreactivity were found in arcuate and ventromedial nuclei. From these neurons, bundles of fibers innervate median eminence and appear to terminate in contact with portal vessels. In addition to median eminence, hpGRF immunoreactive fibers were found mostly in the anterior hypothalamus and the arcuate and ventromedial nuclei where they give perineuronal endings. These results correlate with earlier physiological data on hypothalamic control of growth hormone secretion and suggest that GRF is also involved in interneuronal relationships related or unrelated to neurohumoral control of pituitary secretions.     

Obscurin: a multitasking muscle giant

Obscurin (~800 kDa) is the third member of a family of giant proteins expressed in vertebrate striated muscle, along with titin (3–3.7 MDa) and nebulin (~800 kDa). Like its predecessors, it is a multidomain protein composed of tandem adhesion modules and signaling domains. Unlike titin and nebulin, which are integral components of sarcomeres, obscurin is concentrated at the peripheries of Z-disks and M-lines, where it is appropriately positioned to communicate with the surrounding myoplasm. This unique distribution allows obscurin to bind small ankyrin 1, an integral component of the sarcoplasmic reticulum (SR) membrane. Obscurin also associates with the contractile apparatus through its binding to titin, sarcomeric myosin and perhaps other proteins of the contractile apparatus. Overexpression of the COOH-terminus of obscurin in primary myotubes has a dramatic and specific effect on the organization of sarcomeric myosin, indicating a role in the organization and regular assembly of A-bands. Given its ability to associate tightly, selectively and periodically with the periphery of the myofibril, its high affinity for an integral membrane protein of the SR and its close association with thick filaments, we speculate that obscurin is ideally suited to play key roles in modulating the organization and assembly of both the myofibril and the SR.     

The Importance of Ventilation in Exercise-Induced Asthma

The degree of post treadmill-running decrease in pulmonary function (Exercise-Induced Asthma) in 11 adult asthmatics was compared with the decrease in pulmonary function followed by resting isocapnic hyperventilation. It was checked that ventilation during the hyperventilation was kept identical to the ventilation during treadmill-running by continuous recording of respiratory frequency, minute ventilation, tidal volume and accumulated ventilation. The temperature of the inspired air was identical in the two situations and the relative humidity was 40% during treadmill-running and 15% during hyperventilation. The average accumulated ventilation during treadmill-running and hyperventilation was 411 1/6 min in both events. The decrease in peak expiratory flow after treadmill-running was 25% and after isocapnic hyperventilation 24%. It is concluded that the ventilation is of more importance for the decrease in pulmonary function after exercise, than the work load.     

The actin binding domain of ACF7 binds directly to the tetratricopeptide repeat domains of rapsyn

Formation of the neuromuscular junction requires the release of agrin from the presynaptic terminal of motor neurons. Clustering of acetylcholine receptors (AChRs) on the postsynaptic sarcolemma is initiated by agrin-dependent activation of the muscle-specific kinase. While the postsynaptic scaffolding protein rapsyn is vital for high density AChR aggregation, little is known about the mechanism through which AChRs are immobilized on the postsynaptic membrane. Ultrastructural and immunohistochemical studies of rat skeletal muscle have suggested that AChRs are anchored to a membrane-associated cytoskeleton that contains spectrin-like proteins and is thus similar to that of the human erythrocyte [Bloch RJ, Bezakova G, Ursitti JA, Zhou D, Pumplin DW (1997) A membrane skeleton that clusters nicotinic acetylcholine receptors in muscle. Soc Gen Physiol Ser 52:177–195]. We are studying a protein of the spectrin superfamily, ACF7 (also known as MACF), as a postsynaptic cytoskeletal component of the neuromuscular junction. ACF7 has multiple cytoskeleton-binding domains, including an N-terminal actin-binding domain that, we postulate, may interact with rapsyn, the scaffolding protein that binds directly to AChRs. To test this hypothesis, we co-expressed fragments of these molecules in cultured fibroblasts and assessed their co-distribution and interaction using confocal microscopy and co-immunoprecipitation. We demonstrate that the actin-binding domain of ACF7 specifically interacts with the tetratricopeptide repeat domains of rapsyn. Furthermore, we show using surface plasmon resonance and blot overlay that the actin-binding domain of ACF7 binds directly to rapsyn. These results suggest that, in mammalian skeletal muscle, AChRs are immobilized in the membrane through rapsyn-mediated anchoring to an ACF7-containing network that in turn is linked to the actin cytoskeleton.     

Action du clopamide et de l'acide ethacrynique sur la sécrétion de rénine chez le chien

Summary Renin secretion was studied in anesthetized dogs (Pentobarbital), after an injection of 5 mg/kg i.v. clopamide (10 animals) or ethacrynic acid (10 animals). Renin activity in venous renal blood (Boucher's method), renal blood flow (electromagnetic flowmeter), blood pressure, diuresis and natriuresis have been measured simultaneously. Basic renin secretion was 22,4±8,2 ng/g of kidney/min. During the 8 min following the ethacrynic acid injection renin secretion increased to 74,4±41,1 ng/g of kidney/min (p<0.005). Within the same time it dropped to 11.1±6.8 ng/g of kidney/min (p<0.005) after clopamide.These variations of renin secretion are related to the diuretic's action on the nephron, and depend on an endorenal mechanism. Ethacrynic acid suppresses the corticopapillary gradient while clopamide does not. We have not found any correlation between renin secretion and either natriuresis (mg/min) or diuresis (ml/min). But there is a connection (r=0.7367) between renin secretion after ethacrynic acid injection and sodium concentration in urine.

     

Dose properties of x-ray beams produced by laser-wakefield-accelerated electrons

Given that laser wakefield acceleration (LWFA) has been demonstrated experimentally to accelerate electron beams to energies beyond 25 MeV, it is reasonable to assess the ability of existing LWFA technology to compete with conventional radiofrequency linear accelerators in producing electron and x-ray beams for external-beam radiotherapy. We present calculations of the dose distributions (off-axis dose profiles and central-axis depth dose) and dose rates of x-ray beams that can be produced from electron beams that are generated using state-of-the-art LWFA. Subsets of an LWFA electron energy distribution were propagated through the treatment head elements (presuming an existing design for an x-ray production target and flattening filter) implemented within the EGSnrc Monte Carlo code. Three x-ray energy configurations (6 MV, 10 MV and 18 MV) were studied, and the energy width deltaE of the electron-beam subsets varied from 0.5 MeV to 12.5 MeV. As deltaE increased from 0.5 MeV to 4.5 MeV, we found that the off-axis and central-axis dose profiles for x-rays were minimally affected (to within about 3%), a result slightly different from prior calculations of electron beams broadened by scattering foils. For deltaE of the order of 12 MeV, the effect on the off-axis profile was of the order of 10%, but the central-axis depth dose was affected by less than 2% for depths in excess of about 5 cm beyond d(max). Although increasing deltaE beyond 6.5 MeV increased the dose rate at d(max) by more than 10 times, the absolute dose rates were about 3 orders of magnitude below those observed for LWFA-based electron beams at comparable energies. For a practical LWFA-based x-ray device, the beam current must be increased by about 4-5 orders of magnitude.