Antioxidants for bone marrow cryopreservation

Addition of antioxidants into the preservation solution improved the cryoprotection of human bone marrow cells. The viability was studied by the growth of GM-CFC in agar culture before and after storage at -196 degrees C. All used antioxidative drugs (selenomethionine, methionine, tocopherol, penicillin/Fe++) increased the tolerance of the stem cells to freezing and thawing and elevated the number of surviving GM-CFC up to the twofold in comparison with that of controls. More immature colony forming cells were especially protected.     

Pharmacoresistant Cav2·3 (E‐type/R‐type) voltage‐gated calcium channels influence heart rate dynamics and may contribute to cardiac impulse conduction

Voltage‐gated Ca²⁺ channels regulate cardiac automaticity, rhythmicity and excitation–contraction coupling. Whereas L‐type (Ca_v1·2, Ca_v1·3) and T‐type (Ca_v3·1, Ca_v3·2) channels are widely accepted for their functional relevance in the heart, the role of Ca_v2·3 Ca²⁺ channels expressing R‐type currents remains to be elucidated. We have investigated heart rate dynamics in control and Ca_v2·3‐deficient mice using implantable electrocardiogram radiotelemetry and pharmacological injection experiments. Autonomic block revealed that the intrinsic heart rate does not differ between both genotypes. Systemic administration of isoproterenol resulted in a significant reduction in interbeat interval in both genotypes. It remained unaffected after administering propranolol in Ca_v2·3(?|?) mice. Heart rate from isolated hearts as well as atrioventricular conduction for both genotypes differed significantly. Additionally, we identified and analysed the developmental expression of two splice variants, i.e. Ca_v2·3c and Ca_v2·3e. Using patch clamp technology, R‐type currents could be detected in isolated prenatal cardiomyocytes and be related to R‐type Ca²⁺ channels. Our results indicate that on the systemic level, the pharmacologically inducible heart rate range and heart rate reserve are impaired in Ca_v2·3 (?|?) mice. In addition, experiments on Langendorff perfused hearts elucidate differences in basic properties between both genotypes. Thus, Ca_v2·3 does not only contribute to the cardiac autonomous nervous system but also to intrinsic rhythm propagation. Copyright © 2012 John Wiley & Sons, Ltd.     

Expression of the ribosome-inactivating protein JIP60 from barley in transgenic tobacco leads to an abnormal phenotype and alterations on the level of translation

In this paper we report the in-planta activity of the ribosome-inactivating protein JIP60, a 60-kDa jasmonate-induced protein from barley (Hordeum vulgare L.), in transgenic tobacco (Nicotiana tabacum L.) plants. All plants expressing the complete JIP60 cDNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited conspicuous and similar phenotypic alterations, such as slower growth, shorter internodes, lanceolate leaves, reduced root development, and premature senescence of leaves. Microscopic inspection of developing leaves showed a loss of residual meristems and higher degree of vacuolation of mesophyll cells as compared to the wild type. When probed with an antiserum which was immunoreactive against both the N- and the C-terminal half of JIP60, a polypeptide with a molecular mass of about 30 kDa, most probably a processed JIP60 product, could be detected. Phenotypic alterations could be correlated with the differences in the detectable amount of the JIP60 mRNA and processed JIP60 protein. The protein biosynthesis of the transformants was characterized by an increased polysome/monosome ratio but a decreased in-vivo translation activity. These findings suggest that JIP60 perturbs the translation machinery in planta. An immunohistological analysis using the JIP60 antiserum indicated that the immunoreactive polypeptide(s) are located mainly in the nucleus of transgenic tobacco leaf cells and to a minor extent in the cytoplasm. Received: 31 July 1996 / Accepted: 18 February 1997     

Single-Channel Resolution of the Interaction between C-Terminal CaV1.3 Isoforms and Calmodulin

Voltage-dependent calcium (Ca_V) 1.3 channels are involved in the control of cellular excitability and pacemaking in neuronal, cardiac, and sensory cells. Various proteins interact with the alternatively spliced channel C-terminus regulating gating of Ca_V1.3 channels. Binding of a regulatory calcium-binding protein calmodulin (CaM) to the proximal C-terminus leads to the boosting of channel activity and promotes calcium-dependent inactivation (CDI). The C-terminal modulator domain (CTM) of Ca_V1.3 channels can interfere with the CaM binding, thereby inhibiting channel activity and CDI. Here, we compared single-channel gating behavior of two natural Ca_V1.3 splice isoforms: the long Ca_V1.3₄₂ with the full-length CTM and the short Ca_V1.3_42A with the C-terminus truncated before the CTM. We found that CaM regulation of Ca_V1.3 channels is dynamic on a minute timescale. We observed that at equilibrium, single Ca_V1.3₄₂ channels occasionally switched from low to high open probability, which perhaps reflects occasional binding of CaM despite the presence of CTM. Similarly, when the amount of the available CaM in the cell was reduced, the short Ca_V1.3_42A isoform showed patterns of the low channel activity. CDI also underwent periodic changes with corresponding kinetics in both isoforms. Our results suggest that the competition between CTM and CaM is influenced by calcium, allowing further fine-tuning of Ca_V1.3 channel activity for particular cellular needs.