An analysis of astrocytic cell lines with different abilities to promote axon growth

The adult mammalian central nervous system (CNS) lacks the capacity to support axonal regeneration. There is increasing evidence to suggest that astrocytes, the major glial population in the CNS, may possess both axon-growth promoting and axon-growth inhibitory properties and the latter may contribute to the poor regenerative capacity of the CNS. In order to examine the molecular differences between axon-growth permissive and axon-growth inhibitory astrocytes, a panel of astrocyte cell lines exhibiting a range of axon-growth promoting properties was generated and analysed. No clear correlation was found between the axon-growth promoting properties of these astrocyte cell lines with: (i) the expression of known neurite-outgrowth promoting molecules such as laminin, fibronectin andN-cadherin; (ii) the expression of known inhibitory molecules such tenascin and chondroitin sulphate proteoglycan; (iii) plasminogen activator and plasminogen activator inhibitor activity; and (iv) growth cone collapsing activity. EM studies on aggregates formed from astrocyte cell lines, however, revealed the presence of an abundance of extracellular matrix material associated with the more inhibitory astrocyte cell lines. When matrix deposited by astrocyte cell lines was assessed for axon-growth promoting activity, matrix from permissive lines was found to be a good substrate, whereas matrix from the inhibitory astrocyte lines was a poor substrate for neuritic growth. Our findings, taken together, suggest that the functional differences between the permissive and the inhibitory astrocyte cell lines reside largely with the ECM.     

Stereoselectivity of actions of the calcium sensitizer [+]-EMD 60263 and its enantiomer [-]-EMD 60264

The thiadiazinone derivative [+]-EMD 60263 ((+)-5-(1-(α-ethylimino-3,4-dimethoxybenzyl)-1,2,3,4- tetrahydroquinoline-6-yl)-6-methyl-3,6-dihydro-2H-1,3,4 -thiadiazine-2-on) is a Ca²⁺-sensitizing agent with only minor phosphodiesterase inhibitory activity. Our aim was to characterize the inotropic and electrophysiological effects of [+]-EMD 60263 and its enantiomer [-]-EMD 60264 in several cardiac muscle preparations. The Ca²⁺-sensitizing activity resided in the [+]-enantiomer only. [+]-EMD 60263 (3 μM) shifted the EC₅₀ of Ca²⁺ for contractile activation of skinned fibers of pig heart from 2.41 μM to 0.73 μM, whereas [-]-EMD 60264 (30 μM) was ineffective. In Langendorff-perfused guinea pig hearts, [+]-EMD 60263 and [-]-EMD 60264 induced concentration-dependent positive and negative inotropic effects, respectively; both enantiomers reduced spontaneous heart rate but did not influence perfusion pressure. The maximum increase in force of human atrial trabeculae was 35 % of pre-drug control with [+]-EMD 60263 in comparison to 113 % with forskolin. In guinea-pig papillary muscles, [+]-EMD 60263 and [-]-EMD 60264 had opposite inotropic responses, however, both agents similarly prolonged action potential duration. Both enantiomers concentration-dependently blocked the rapidly activating component I_Kr of the delayed rectifier in guinea-pig myocytes. The block saturated at potentials positive to +30 mV, closely resembling the effects of the antiarrhythmic agent E-4031 which had been originally used to define I_Kr. It is concluded, that the positive inotropic action of [+]-EMD 60263 can be explained by prevalence of the Ca²⁺-sensitizing effect. The accompanying prolongation in action potential duration is caused by block of the I_Kr component of the delayed rectifier. While the inotropic effects are stereoselective, most of the electrophysiological actions are clearly independent of sterical configuration. The combination of Ca²⁺-sensitizing with class-III antiarrhythmic action may provide an interesting pharmacological profile of potential therapeutic use. Received: 7 January 1997 / Accepted: 25 February 1997     

Tumor induction by ras and myc oncogenes in fetal and neonatal brain: modulating effects of developmental stage and retroviral dose

Introduction into fetal rat brain cells of a replication-defective retroviral vector harboring v-Ha-ras and v-gag-myc rapidly causes the induction of highly malignant undifferentiated neuroectodermal tumors following transplantation into the brains of syngeneic hosts [Wiestler, et al. (1992) Cancer Res. 52: 3760–3767]. In the present study, we have investigated the modulating effect of the developmental stage of neural target cells and of the dose of the retroviral vector used in the grafting experiments. Exposure of fetal cells from embryonic day (E)12 or E14 produced a 100% incidence of malignant neuroectodermal tumors which led to the death of recipient animals after a median latency period of 32 days. A 100-fold reduction of the virus dose from 2.062×10⁶ to 2.062×10⁴ focus-forming units/ml resulted in a lower tumor incidence of 25%. Of six neural grafts exposed to v-Ha-ras and v-myc at E16, only one showed evidence of tumorigenesis (low-grade astrocytoma and hemangioma). All other transplants were morphologically normal for observation periods of 26 weeks, indicating a marked loss of transforming activity of ras and myc in more advanced stages of brain development. In retrovirus-exposed donor cells which caused the development of neural tumors in recipient rats, malignant transformation was also evident during culture in vitro, usually after 9–12 days. Oncogene complementation was also studied in the newborn rat brain. After microinjection of the retroviral vector into the brain at postnatal day (P)0, P1 and P3, 5 out of 20 animals (25%) developed a total of seven brain tumors. Histopathologically, three of these neoplasms were malignant neuroectodermal tumors which, in contrast to those induced in fetal brain transplants showed evidence of focal glial and/or neuronal differentiation. In addition, we observed one oligodendroglioma, two hemangiomas and a malignant hemangioendothelioma. These data indicate that neural precursor cells and endothelia of the rat brain represent the major target cells for the complementary action of ras and myc and that the use of target cells from later developmental stages (E16 and postnatal) leads to the induction of both primitive and more differentiated neoplasms.These studies were supported by the Fonds zur Förderung der wissenschaftlichen Forschung in Österreich (Erwin Schrödinger fellowship, JO501-MED), by the Swiss National Science Foundation and by the Cancer League of the Kanton of Zürich     

203Pb-labeled alpha-melanocyte-stimulating hormone peptide as an imaging probe for melanoma detection.

Peptide-targeted alpha-therapy with 7.4 MBq of (212)Pb-[1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-ReO-[Cys(3,4,10),d-Phe(7),Arg(11)]alpha-MSH(3-13) ((212)Pb-DOTA-Re(Arg(11))CCMSH) cured 45% of B16/F1 murine melanoma-bearing C57 mice in a 120-d study, highlighting its melanoma treatment potential. However, there is a need to develop an imaging surrogate for patient-specific dosimetry and to monitor the tumor response to (212)Pb-DOTA-Re(Arg(11))CCMSH therapy. The purpose of this study was to evaluate the potential of (203)Pb-DOTA-Re(Arg(11))CCMSH as a matched-pair SPECT agent for (212)Pb-DOTA-Re(Arg(11))CCMSH. METHODS: DOTA-Re(Arg(11))CCMSH was labeled with (203)Pb in 0.5 M NH(4)OAc buffer at pH 5.4. The internalization and efflux of (203)Pb-DOTA-Re(Arg(11))CCMSH were determined in B16/F1 melanoma cells. The pharmacokinetics of (203)Pb-DOTA-Re(Arg(11))CCMSH was examined in B16/F1 melanoma-bearing C57 mice. A micro-SPECT/CT study was performed with (203)Pb-DOTA-Re(Arg(11))CCMSH in a B16/F1 melanoma-bearing C57 mouse at 2 h after injection. RESULTS: (203)Pb-DOTA-Re(Arg(11))CCMSH was easily prepared in NH(4)OAc buffer and completely separated from the excess nonradiolabeled peptide by reversed-phase high-performance liquid chromatography (RP-HPLC). (203)Pb-DOTA-Re(Arg(11))CCMSH displayed fast internalization and extended retention in B16/F1 cells. Approximately 73% of (203)Pb-DOTA-Re(Arg(11))CCMSH activity internalized after a 20-min incubation at 25 degrees C. After incubation of the cells in culture medium for 20 min, 78% of internalized activity remained in the cells. (203)Pb-DOTA-Re(Arg(11))CCMSH exhibited a biodistribution pattern similar to that of (212)Pb-DOTA-Re(Arg(11))CCMSH in B16/F1 melanoma-bearing mice. (203)Pb-DOTA-Re(Arg(11))CCMSH exhibited a peak tumor uptake of 12.00+/-3.20 percentage injected dose per gram (%ID/g) at 1 h after injection. The tumor uptake gradually decreased to 3.43+/-1.12 %ID/g at 48 h after injection. (203)Pb-DOTA-Re(Arg(11))CCMSH exhibited a peak tumor-to-kidney uptake ratio of 1.53 at 2 h after injection. The absorbed doses to the tumor and kidneys were 4.32 and 4.35 Gy, respectively, per 37 MBq. Whole-body clearance of (203)Pb-DOTA-Re(Arg(11))CCMSH was fast, with approximately 89% of the injected activity cleared through the urinary system by 2 h after injection. (203)Pb showed 1.6-mm SPECT resolution, which was comparable to (99m)Tc. Melanoma lesions were visualized through SPECT/CT images of (203)Pb-DOTA-Re(Arg(11))CCMSH at 2 h after injection. CONCLUSION: (203)Pb-DOTA-Re(Arg(11))CCMSH exhibited favorable pharmacokinetic and tumor imaging properties, highlighting its potential as a matched-pair SPECT agent for (212)Pb-DOTA-Re(Arg(11))CCMSH melanoma treatment.     

A novel assay for factor XIII based on cross-linking of synthetic peptides: analysis of different substrates.

A novel assay for factor XIII is described that utilizes exclusively small synthetic peptides as substrates for the cross-linking reaction catalyzed by activated factor XIII (FXIIIa). The acyl donor substrate (selection peptide) is immobilized on a microplate via biotin while the acyl acceptor substrate (detection peptide) is labeled with the fluorochrome Oregon green to allow sensitive detection without the need for secondary enzyme systems for signal amplification. Starting with an amino acid sequence from the fibrin gamma-chain (GQQHHLGGAKQAGDV) as a prototype peptide, the influence of amino acid exchanges were investigated with respect to their impact on the FXIIIa-catalyzed reaction. It was found that FXIIIa readily accepts a broad range of substrate peptides, with a proline neighboring the essential lysine having the most detrimental effect. The assay appears to be valuable for the molecular characterization of factor XIII and may be used for a deeper investigation into the substrate requirements of this final enzyme of wound repair, and eventually also for the characterization of other transglutaminases.