Influence of different culture conditions on sarcoplasmic reticular calcium transport in isolated neonatal rat cardiomyocytes

This study investigates sarcoplasmic reticulum (SR) calcium-(Ca²⁺) transport ATPase (SERCA2a) and phospholamban (PLB) in cultured spontaneously contracting neonatal rat cardiomyocytes (CM) to ascertain the function of both SR proteins under various culture conditions. The two major SR proteins were readily detectable in cultured CM by immunofluorescent microscopy using specific anti-SERCA2 and anti-PLB antibodies. Double labeling technique revealed that PLB-positive CM also labeled with anti-SERCA2. Coexpression of SERCA2 and PLB in CM was supported by measurement of cell homogenate oxalate-supported Ca²⁺ uptake which was completely inhibited by thapsigargin and stimulated by protein kinase A-catalyzed phosphorylation. Under serum-free conditions, incubation of CM with the SERCA2a expression modulator 3,3,5-triiodo-L-thyronine (100 nM, 72 h) resulted in elevated Ca²⁺ uptake of +33%. Specific Ca²⁺ uptake activity was not altered if insulin was omitted from the serum-free culture medium but total SR Ca²⁺ transport activity was reduced under this culture condition. The results indicate that primary culture of spontaneously contracting neonatal rat CM can be employed as a useful model system for investigating both short- and long-term mechanisms determining the Ca²⁺ re-uptake function of the SR under defined culture conditions.     

Brain-computer interfaces for control of neuroprostheses: from synchronous to asynchronous mode of operation.

Transferring a brain-computer interface (BCI) from the laboratory environment into real world applications is directly related to the problem of identifying user intentions from brain signals without any additional information in real time. From the perspective of signal processing, the BCI has to have an uncued or asynchronous design. Based on the results of two clinical applications, where 'thought' control of neuroprostheses based on movement imagery in tetraplegic patients with a high spinal cord injury has been established, the general steps from a synchronous or cue-guided BCI to an internally driven asynchronous brain-switch are discussed. The future potential of BCI methods for various control purposes, especially for functional rehabilitation of tetraplegics using neuroprosthetics, is outlined.     

Tyr-716 in the platelet-derived growth factor beta-receptor kinase insert is involved in GRB2 binding and Ras activation.

Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.     

Reactions of D-penicillamine with copper in Wilson's disease

X-ray photoelectron spectroscopy proved very appropriate to determine the genuine oxidation state of copper in copper(D-penicillamine) chelates. The present data corroborate the ‘reductive chelation’ mechanism proposed byPeisach and Blumberg (Mol.Pharmacol. 5,200–209,1969) for the mobilization of Cu(II) with D-penicillamine (RSH). Moreover, it was concluded that the resulting complex [RSCu(I)]_n is a suitable form for urinary excretion of copper.     

alphavbeta3 integrin-dependent endothelial cell dynamics in vivo

A major challenge confronting developmental cell biologists is to understand how individual cell behaviors lead to global tissue organization. Taking advantage of an endothelial cell-specific marker and scanning time-lapse microscopy, we have examined the formation of the primary vascular pattern during avian vasculogenesis. Five types of distinguishable endothelial cell motion are observed during formation of a vascular plexus: (1) global tissue deformations that passively convect endothelial cells; (2) vascular drift, a sheet-like medial translocation of the entire vascular plexus; (3) structural rearrangements, such as vascular fusion; (4) individual cell migration along existing endothelial structures; and (5) cell process extension into avascular areas, resulting in new links within the plexus. The last four types of motion are quantified and found to be reduced in the presence of an alphavbeta3 integrin inhibitor. These dynamic cell motility data result in new hypotheses regarding primordial endothelial cell behavior during embryonic vasculogenesis.     

Alveolar epithelial cells type II are major target cells for C. pneumoniae in chronic but not in acute respiratory infection

Pulmonary presence of Chlamydia pneumoniae is associated with acute and chronic infections. We show that unapparent chlamydial infection in four out of 31 chronic obstructive pulmonary disease (COPD) patients (12.9%) is characterized by a significant increase in infected alveolar epithelial cells type II (18.2 +/- 3.5% vs. 2.3 +/- 0.9; IHC/ISH) compared to a newly established model of acute chlamydial infection (ACIM) in vital lung specimens from pulmonary lobectomy. Expression of cHSP60 demonstrated pathogen viability and virulence in the ACIM. We conclude that target cells differ in acute and chronic chlamydial infection and suggest the ACIM as a novel tool to analyze the host-pathogen-interactions in acute respiratory infections.     

Dual role of CCR2 during initiation and progression of collagen-induced arthritis: evidence for regulatory activity of CCR2+ T cells

Chemokines play an important role in the recruitment of leukocytes and have recently been shown to also attract regulatory T cells. Using blocking mAbs, we analyzed the role of the chemokine receptor CCR2 during initiation and progression of collagen-induced arthritis in mice. Blockade of CCR2 from days 0 to 15 markedly improved clinical signs of arthritis and histological scores measuring leukocyte infiltration, synovial hyperplasia, and bone and cartilage erosion. CCR2 blockade during disease initiation significantly reduced plasma titers of collagen Abs in vivo. In vitro CCR2 blockade also interfered with collagen-specific activation and proliferation of T cells. Surprisingly, CCR2 blockade from days 21 to 36 markedly aggravated clinical and histological signs of arthritis and increased the humoral immune response against collagen. We show that CCR2 is expressed on regulatory T cells. Purified CCR2+ T cells are fully anergic toward polyclonal and collagen-specific activation and potently suppress activation of other T and B cells. The subpopulation of CCR2+ CD25+ regulatory T cells increases approximately 5-fold in the progression phase, while CCR2 expression on other leukocyte populations remains unchanged. These findings identify CCR2+ T cells as regulatory T cells and indicate that CCR2 also plays an important role in down-modulating an inflammatory response.     

Monovalent cation conductance in Xenopus laevis oocytes expressing hCAT-3

hCAT-3 (human cationic amino acid transporter type three) was investigated with both the two-electrode voltage clamp method and tracer experiments. Oocytes expressing hCAT-3 displayed less negative membrane potentials and larger voltage-dependent currents than native or water-injected oocytes did. Ion substitution experiments in hCAT-3-expressing oocytes revealed a large conductance for Na+ and K+. In the presence of L-Arg, voltage-dependent inward and outward currents were observed. At symmetrical (inside/outside) concentrations of L-Arg, the conductance of the transporter increased monoexponentially with the L-Arg concentrations; the calculated Vmax and KM values amounted to 8.3 microS and 0.36 mM, respectively. The time constants of influx and efflux of [3H]L-Arg, at symmetrically inside/outside L-Arg concentrations (1 mM), amounted to 79 and 77 min, respectively. The flux data and electrophysiological experiments suggest that the transport of L-Arg through hCAT-3 is symmetric, when the steady state of L-Arg flux has been reached. It is concluded that hCAT-3 is a passive transport system that conducts monovalent cations including L-Arg. The particular role of hCAT-3 in the diverse tissues remains to be elucidated.     

Voltage dependence of L-arginine transport by hCAT-2A and hCAT-2B expressed in oocytes from Xenopus laevis

Membrane potential and currents were investigated with thetwo-electrode voltage-clamp technique in Xenopus laevisoocytes expressing hCAT-2A or hCAT-2B, the splice variants of the human cationic amino acid transporter hCAT-2. Both hCAT-2A- andhCAT-2B-expressing oocytes exhibited a negative extracellularL-arginine concentration ([L-Arg]_o)-sensitive membrane potential,additive to the K⁺ diffusion potential, when cells wereincubated in Leibovitz medium (containing 1.45 mM L-Arg and0.25 mM L-lysine). The two carrier proteins produced inwardand outward currents, which were dependent on the L-Arggradient and membrane potential. Ion substitution experiments showedthat the hCAT-induced currents were independent of externalNa⁺, K⁺, Ca²⁺, or Mg²⁺.The apparent Michaelis-Menten constant values at 60 mV, obtained fromplots of L-Arg-induced currents against[L-Arg]_o, were 0.97 and 0.13 mM in oocytesexpressing hCAT-2A and hCAT-2B, respectively; maximal currentsamounted to 194 ± 8 and 84 ± 2 nA, respectively. Atsaturating [L-Arg]_o, the current-voltagerelationships of hCAT-2A-expressing oocytes became steeper, yielding anadditional conductance up to 2 µS/oocyte, whereas those ofhCAT-2B-expressing oocytes were simply shifted to the right, resultingin voltage-independent difference currents. The distinctelectrochemical properties of the two isoforms of hCAT-2 are assumed tocontribute differentially to the membrane transport and the maintenanceof cationic amino acids in various tissues.