Disturbances in glucose-tolerance, insulin-release, and stress-induced hyperglycemia upon disruption of the Ca(v)2.3 (alpha 1E) subunit of voltage-gated Ca(2+) channels

Multiple types of voltage-activated Ca(2+) channels (T, L, N, P, Q, R type) coordinate Ca(2+)-dependent processes in neurons and neuroendocrine cells. Expressional and functional data have suggested a role for Ca(v)2.3 Ca(2+) channels in endocrine processes. To verify its role in vivo, Ca(v)2.3(-/-) mutant mice were generated, thus deficient in alpha 1E/R-type Ca(2+) channel. Intraperitoneal injection of D-glucose showed that glucose tolerance was markedly reduced, and insulin release into plasma was impaired in Ca(v)2.3-deficient mice. In isolated islets of Langerhans from these animals, no glucose-induced insulin release was detected. Further, in stressed Ca(v)2.3-deficient mice, the rate of glucose release into the blood was only 29% of that observed for wild-type animals. Thus, the deletion of Ca(v)2.3 causes deficits not only in insulin release but also in stress-induced hyperglycemia. The complex phenotype of Ca(v)2.3-deficient mice has dual components related to endocrine and neurological defects. The present findings provide direct evidence of a functional role for the Ca(v)2.3 subunit in hormone secretion and glucose homeostasis.     

The adrenomedullin receptor acts as clearance receptor in pulmonary circulation

Adrenomedullin (AM) is a powerful pulmonary vasodilator with antimitogenic properties. We investigated the role of the AM receptor (AMR) and the calcitonin gene-related peptide type-1 receptor (CGRP1R) in regulating pulmonary vascular AM levels. The AMR antagonist hAM(22-52) (120 nmol/L) significantly elevated AM release compared with controls to 250% after 2 h in isolated rat lungs and to 830% after 4 h in pulmonary artery endothelial cells (PAEC). CGRP1R blockade had no effect. AMR blockade did not influence prepro-AM mRNA levels nor did inhibition of protein synthesis by cycloheximide (0.01 mg/mL) abolish the effect of the AMR antagonist. Radioligand-binding studies with PAEC membranes revealed a decrease by 44% of the AMR density in response to AMR antagonism. Altogether, the pulmonary vascular AMR represents not only a functionally active, but also a clearance receptor; its expression is constitutively stimulated by basal AM. This identifies a novel mechanism for controlling pulmonary AM levels.     

Potential of embryonic and adult stem cells in vitro

Recent developments in the field of stem cell research indicate their enormous potential as a source of tissue for regenerative therapies. The success of such applications will depend on the precise properties and potentials of stem cells isolated either from embryonic, fetal or adult tissues. Embryonic stem cells established from the inner cell mass of early mouse embryos are characterized by nearly unlimited proliferation, and the capacity to differentiate into derivatives of essentially all lineages. The recent isolation and culture of human embryonic stem cell lines presents new opportunities for reconstructive medicine. However, important problems remain; first, the derivation of human embryonic stem cells from in vitro fertilized blastocysts creates ethical problems, and second, the current techniques for the directed differentiation into somatic cell populations yield impure products with tumorigenic potential. Recent studies have also suggested an unexpectedly wide developmental potential of adult tissue-specific stem cells. Here too, many questions remain concerning the nature and status of adult stem cells both in vivo and in vitro and their proliferation and differentiation/transdifferentiation capacity. This review focuses on those issues of embryonic and adult stem cell biology most relevant to their in vitro propagation and differentiation. Questions and problems related to the use of human embryonic and adult stem cells in tissue regeneration and transplantation are discussed.     

The Xmrk oncogene can escape nonfunctionalization in a highly unstable subtelomeric region of the genome of the fish Xiphophorus

The Xmrk oncogene involved in melanoma formation in the fish Xiphophorus was formed relatively recently by duplication of the epidermal growth factor co-orthologue egfrb. In the platyfish X. maculatus, Xmrk is located close to the major sex-determining locus in a subtelomeric region of the X and Y sex chromosomes that frequently undergoes duplications and other rearrangements. This region accumulates repetitive sequences: more than 80% of the 33-kb region 3' of Xmrk is constituted by retrotransposable elements. The high degree of nucleotide identity between X- and Y-linked sequences and the rarity of gonosome-specific rearrangements indicated that the instability observed was not a manifestation of gonosome-specific degeneration. Seven other duplicated genes were found, all corresponding, in contrast to Xmrk, to pseudogenes (nonfunctionalization). Functional persistence of Xmrk in a highly unstable region in divergent Xiphophorus species suggests a beneficial function under certain conditions for this dispensable and potentially injurious gene.     

Development of Agrobacterium tumefaciens C58-induced plant tumors and impact on host shoots are controlled by a cascade of jasmonic acid,auxin, cytokinin,ethylene and abscisic acid

The development of Agrobacterium tumefaciens-induced plant tumors primarily depends on the excessive production of auxin and cytokinin by enzymes encoded on T-DNA genes integrated into the plant genome. The aim of the present study was to investigate the involvement of additional phytohormone signals in the vascularization required for rapid tumor proliferation. In stem tumors of Ricinus communis L., free auxin and zeatin riboside concentrations increased within 2 weeks to 15-fold the concentrations in control stem tissue. Auxin and cytokinin immunolocalization revealed the highest concentrations within and around tumor vascular bundles with concentration gradients. The time-course of changes in free auxin concentration in roots was inversely correlated with that in the tumors. The high ethylene emission induced by increased auxin- and cytokinin correlated with a 36-fold accumulation of abscisic acid in tumors. Ethylene emitted from tumors and exogenously applied ethylene caused an increase in abscisic acid concentrations also in the host leaves, with a diminution in leaf water vapor conductance. Jasmonic acid concentration reached a maximum already within the first week of bacterial infection. A wound effect could be excluded. The results demonstrate the concerted interaction of a cascade of transiently induced, non-T-DNA-encoded phytohormones jasmonic acid, ethylene and abscisic acid with T-DNA-encoded auxin and zeatin riboside plus trans-zeatin, all of which are required for successful plant tumor vascularization and development together with inhibition of host plant growth.     

GacS sensor domains pertinent to the regulation of exoproduct formation and to the biocontrol potential of Pseudomonas fluorescens CHA0

In the root-colonizing biocontrol strain CHA0 of Pseudomonas fluorescens, cell density-dependent synthesis of extracellular, plant-beneficial secondary metabolites and enzymes is positively regulated by the GacS/GacA two-component system. Mutational analysis of the GacS sensor kinase using improved single-copy vectors showed that inactivation of each of the three conserved phosphate acceptor sites caused an exoproduct null phenotype (GacS-), whereas deletion of the periplasmic loop domain had no significant effect on the expression of exoproduct genes. Strain CHA0 is known to synthesize a solvent-extractable extracellular signal that advances and enhances the expression of exoproduct genes during the transition from exponential to stationary growth phase when maximal exoproduct formation occurs. Mutational inactivation of either GacS or its cognate response regulator GacA abolished the strain's response to added signal. Deletion of the linker domain of the GacS sensor kinase caused signal-independent, strongly elevated expression of exoproduct genes at low cell densities. In contrast to the wild-type strain CHA0, the gacS linker mutant and a gacS null mutant were unable to protect tomato plants from crown and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici in a soil-less microcosm, indicating that, at least in this plant-pathogen system, there is no advantage in using a signal-independent biocontrol strain.     

Functional analysis of barley RAC/ROP G-protein family members in susceptibility to the powdery mildew fungus

Small monomeric G-proteins of the plant ras (rat sarcome oncogene product) related C3 botulinum toxin substrate (RAC)/Rho of plants (ROP) family are molecular switches in signal transduction of many cellular processes. RAC/ROPs regulate hormone effects, subcellular gradients of Ca2+, the organisation of the actin cytoskeleton and the production of reactive oxygen intermediates. Therefore, we followed a genetic bottom-up strategy to study the role of these proteins during the interaction of barley (Hordeum vulgare L.) with the fungal biotrophic pathogen Blumeria graminis f.sp. hordei (Bgh). We identified six barley RAC/ROP proteins and studied their gene expression. Five out of six Rac/Rop genes were expressed constitutively in the leaf epidermis, which is the site of interaction with Bgh. None of the genes showed enhancement of mRNA abundance after inoculation with Bgh. After microprojectile mediated transformation of single barley epidermal cells with constitutively activated mutant RAC/ROP proteins, we found an RAC/ROP-specific enhancement of pathogen accessibility, tagging HvRACB, HvRAC3 and HvROP6 as host proteins potentially involved in the establishment of susceptibility to Bgh. Confocal laser scanning microscopy (CLSM) of green fluorescent protein (GFP):HvRAC/ROP-transformed cells revealed varying strengths of plasma membrane association of barley RAC/ROPs. The C-terminal CAAX motif for presumable prenylation or the C-terminal hypervariable region (HVR), respectively, were required for membrane association of the RAC/ROPs. Proper intracellular localisation was essential for HvRACB and HvRAC3 function. Together, our data support the view that different paths of host signal transduction via RAC/ROP G-proteins are involved in processes supporting parasitic entry into epidermal host cells.     

Immunohistochemical distribution of galectin-1, galectin-3, and olfactory marker protein in human olfactory epithelium

The expression pattern of galectin-1 and galectin-3 in the human olfactory epithelium was investigated in relation to olfactory marker protein (OMP) using confocal laser immunofluorescence in human specimens and postmortem biopsies. OMP expression was found in olfactory receptor neurons (ORNs) in the olfactory mucosa and in fibers of the olfactory nerve crossing the submucous connective tissue. Galectin-1 was expressed in both the connective tissue of the nasal cavity and in the basal layer of the olfactory epithelium. In contrast, galectin-3 expression was limited to cells of the upper one-third of the olfactory epithelium. Expression of galectin-3 occurred in a subset of OMP-positive cells. However, between areas of galectin-1 and galectin-3 expression in the lower and upper portion of the epithelium, OMP-positive ORNs did not stain for both galectins. Considering the potential role of galectin-1 and galectin-3 in cell differentiation and maturation, the differential localization of galectins in the olfactory epithelium appears to be consistent with a significant role of these molecules in the physiological turnover of ORNs. Accepted: 20 December 1999     

Empirical calculation of the relative free energies of peptide binding to the molecular chaperone DnaK

We describe a methodology to calculate the relative free energies of protein-peptide complex formation. The interaction energy was decomposed into nonpolar, electrostatic and entropic contributions. A free energy-surface area relationship served to calculate the nonpolar free energy term. The electrostatic free energy was calculated with the finite difference Poisson-Boltzmann method and the entropic contribution was estimated from the loss in the conformational entropy of the peptide side chains. We applied this methodology to a series of DnaK*peptide complexes. On the basis of the single known crystal structure of the peptide-binding domain of DnaK with a bound heptapeptide, we modeled ten other DnaK*heptapeptide complexes with experimentally measured K(d) values from 0.06 microM to 11 microM, using molecular dynamics to refine the structures of the complexes. Molecular dynamic trajectories, after equilibration, were used for calculating the energies with greater accuracy. The calculated relative binding free energies were compared with the experimentally determined free energies. Linear scaling of the calculated terms was applied to fit them to the experimental values. The calculated binding free energies were between -7.1 kcal/mol and - 9.4 kcal/mol with a correlation coefficient of 0.86. The calculated nonpolar contributions are mainly due to the central hydrophobic binding pocket of DnaK for three amino acid residues. Negative electrostatic fields generated by the protein increase the binding affinity for basic residues flanking the hydrophobic core of the peptide ligand. Analysis of the individual energy contributions indicated that the nonpolar contributions are predominant compared to the other energy terms even for peptides with low affinity and that inclusion of the change in conformational entropy of the peptide side chains does not improve the discriminative power of the calculation. The method seems to be useful for predicting relative binding energies of peptide ligands of DnaK and might be applicable to other protein-peptide systems, particularly if only the structure of one protein-ligand complex is available.