Connexin Hemichannels in Astrocytes: An Assessment of Controversies Regarding Their Functional Characteristics

Astrocytes in the mammalian central nervous system are interconnected by gap junctions made from connexins of the subtypes Cx30 and Cx43. These proteins may exist as hemichannels in the plasma membrane in the absence of a ‘docked’ counterpart on the neighboring cell. A variety of stimuli are reported to open the hemichannels and thereby create a permeation pathway through the plasma membrane. Cx30 and Cx43 have, in their hemichannel configuration, been proposed to act as ion channels and membrane pathways for different molecules, such as fluorescent dyes, ATP, prostaglandins, and glutamate. Published studies about astrocyte hemichannel behavior, however, have been highly variable and/or contradictory. The field of connexin hemichannel research has been complicated by great variability in the experimental preparations employed, a lack of highly specific pharmacological inhibitors and by confounding changes associated with genetically modified animal models. This review attempts to critically assess the gating, inhibition and permeability of astrocytic connexin hemichannels and proposes that connexins in their hemichannel configuration act as gated pores with isoform-specific permeant selectivity. We expect that some, or all, of the controversies discussed here will be resolved by future research and sincerely hope that this review serves to motivate such clarifying investigations.     

Responses of an American eel brain endothelial-like cell line to selenium deprivation and to selenite,selenate, and selenomethionine additions in different exposure media

The effect of selenium deprivation and addition on the American eel brain endothelial cell line (eelB) was studied in three exposure media: complete growth medium (L15/FBS), serum-free medium (L15), and minimal medium (L15/ex). L15/ex contains only galactose and pyruvate and allowed the deprivation of selenium on cells to be studied. In L15/ex, without any obvious source of selenium, eelB cells survived for at least 7 d, formed capillary-like structures (CLS) on Matrigel, and migrated to heal wounds. Three selenium compounds were added to cultures: selenite, selenate, and selenomethionine (SeMet). Adding selenite or selenate to eelB cell cultures for 24 h caused dose-dependent declines in cell viability, regardless of the exposure media. Although varying with exposure media and viability end point, selenite was approximately 70-fold more cytotoxic than selenate. By contrast, 24 h exposures to either dl- or l-SeMet in the three media caused little or no cytotoxicity. However for 7 d exposures in L15/ex, dl- and l-SeMet were very cytotoxic, even at the lowest tested concentration of 31 μM. By contrast in L15 and L15/FBS, cytotoxicity was only observed with 500 and 1000 μM l-SeMet. In L15/FBS, eelB continued to migrate and form CLS in the presence of SeMet but at 500 μM, cell migration appeared stimulated. As judged from a colony-forming assay over 14 d in L15/FBS, 500 and 1000 μM dl- and l-SeMet inhibited cell proliferation. Overall, the responses of eel cells to selenium depended on the selenium form, concentration, and exposure media, with responses to SeMet being most dependent on exposure media.     

Monomolecular multimodal fluorescence-radioisotope imaging agents

Diagnosis of diseases by different imaging methods can provide complementary information about the functional status of diseased tissues or organs. To overcome the current difficulties in coregistering images from different imaging modalities with a high degree of accuracy, we prepared near-infrared (NIR) monomolecular multimodal imaging agents (MOMIAs) consisting of a heptamethine carbocyanine and 111In-DOTA chelate that served as antennae for optical and scintigraphic imaging, respectively. Their spectral properties clearly show that coordination of indium to MOMIA increased the fluorescence intensity of the compounds. The MOMIAs are exceptionally stable in biological media and serum up to 24 h at 37 degrees C. Biodistribution of the compounds in mice obtained by fluorescence photon and gamma-counts demonstrated a similar distribution trend of the molecular probe in different tissues, suggesting that the detected fluorescence and gamma-emissions emanated from the same source (MOMIA). At 24 h postinjection, the MOMIAs were excreted by the renal and hepatobiliary systems and the blood level of a representative MOMIA was very low, thereby reducing background noise caused by circulating molecular probes. These findings demonstrate the feasibility of preparing single molecules with the capacity to emit discernible and diagnostic fluorescent and gamma-radiations for optical and nuclear imaging of living organisms.     

Synthesis and in vitro characterization of organometallic rhenium and technetium glucose complexes against Glut 1 and hexokinase

A series of nine organometallic technetium-99m and rhenium complexes of glucose are presented and characterized in vitro regarding their potential as surrogates of [18F]-2-fluoro-desoxy glucose ([18F]-FDG). The glucose derivatives are functionalized at positions C-1, C-2, C-3, and C-6. Different spacer lengths and chelating systems have been introduced at these sites. For the (radio)labeling, the organometallic precursors [99mTc(H2O)3(CO)3]+ and [ReBr3(CO)3](2-) respectively have been used. The resulting complexes have been characterized chemically and radiochemically. The formation of uniform products has been observed on the macroscopic (Re) and no-carrier-added level (99mTc). The Tc-99m complexes revealed good inertness against ligand exchange (Cys and His) and excellent stability in physiological buffered saline as well as in human plasma over a period of 24 h at 37 degrees C. The rhenium complexes have been tested for competitive inhibition of the (yeast) hexokinase. Only for C-2 derivatized glucose complexes with extended spacer functionalities Ki values in the millimolar and sub-millimolar range have been observed. In silico molecular docking experiments supported these experimental findings. However, the competitive inhibitors are not recognized as a pseudosubstrate of hexokinase. The cellular uptake of all 99mTc-complexes into HT-29 colon carcinoma cells via Glut1 was generally low and unspecific independent of the position at the hexose ring, the chelating systems, or the overall charge of the corresponding metal complexes. The current results seem to preclude the use of these compounds as [18F]-FDG surrogates primarily due to the low cellular uptake via Glut1.     

TH1/TH2 cytokine secretion of first degree relatives of T1DM patients

Th1/Th2 cytokine imbalance has been demonstrated in Type 1 diabetes (T1DM) patients. We characterized the peak levels, secretory pattern and total cytokine production of the Th1 cytokines (IL-2 and IFN gamma) and Th2 cytokines (IL-4 and IL-10), by stimulated peripheral blood mononuclear cells of twenty six first-degree relatives of T1DM patients, and eleven matched controls. At enrollment, first degree relatives demonstrated a significant increase in peak and overall secretion of IL-2; P<0.01 and P<0.005 respectively and IL-4 cytokine; P<0.05 and P<0.01 respectively, as compared to normal controls. Their mean IFN gamma secretion increased significantly, P<0.05, after one year while their higher IL-2 and IL-4 secretion remained unchanged. Ab-negative and Ab-positive relatives demonstrated a similar cytokine secretion pattern. Four relatives all Ab positive, developed diabetes: Peak IL-4 levels were low in three and markedly decreased within one year in one of these relatives, while peak IL-2 and IFN gamma levels were elevated in all of them. These data demonstrate that secretion of both Th1 and Th2 cytokines is increased in first-degree relatives of T1DM patients independently of their diabetes-associated autoantibodies. The presence of low IL-4 and elevated IL-2 and IFN gamma levels in autoAb positive relatives is associated with progression to overt disease.     

Multivalent carbocyanine molecular probes: synthesis and applications

Synergistic multivalent interactions can amplify desired chemical or biological molecular recognitions. We report a new class of multicarboxylate-containing carbocyanine dye constructs for use as optical scaffolds that not only serve as fluorescent antennas but also participate in structural assembly of the multivalent molecular construct. Three generations of carboxylate-terminating multivalent near-infrared carbocyanine probes from a dicarboxylic acid precursor dye (cypate) were prepared via its imino diacetic acid derivatives. Conjugation of the probes with D-(+)-glucosamine afforded dendritic arrays of the carbohydrates on an inner NIR chromophore core. All the multicarboxylate probes and their glucosamine conjugates have similar NIR spectral properties because conjugation occurred at distal positions to the inner chromophore core, thereby providing consistent and predictable spectral properties for their biological applications. Although light-induced photodamage equally affected the precursor dye, multicarboxylate probes, and their glucosamine derivatives, we observed that octacarboxylcypate (multivalent probe) was remarkably stable in different mediums at physiologically relevant temperatures relative to cypate, especially in basic mediums. Biodistribution studies in tumor-bearing nude mice show that all the glucosamine conjugates localized in the tumor but cypate was almost exclusively retained in the liver at 24 h postinjection. The tumor uptake does not correlate with the number of glucosamine tether on the multicarboxylate probe. Overall, the triglucosamine derivative appears to offer the best balance between high tumor uptake and low retention in nontarget tissues. These results suggest that multivalent molecular beacons are useful for assessing the beneficial effects of multivalency and for optimizing the biological and chemical properties of tissue-specific molecular probes.     

Tissue-print and print-phoresis as platform technologies for the molecular analysis of human surgical specimens: mapping tumor invasion of the prostate capsule

Molecular profiling of human biopsies and surgical specimens is frequently complicated by their inherent biological heterogeneity and by the need to conserve tissue for clinical diagnosis. We have developed a set of novel 'tissue print' and 'print-phoresis' technologies to facilitate tissue and tumor-marker profiling under these circumstances. Tissue printing transfers cells and extracellular matrix components from a tissue surface onto nitrocellulose membranes, generating a two-dimensional anatomical image on which molecular markers can be visualized by specific protein and RNA- and DNA-detection techniques. Print-phoresis is a complementary new electrophoresis method in which thin strips from the print are subjected to polyacrylamide gel electrophoresis, providing a straightforward interface between the tissue-print image and gel-based proteomic techniques. Here we have utilized these technologies to identify and characterize markers of tumor invasion of the prostate capsule, an event generally not apparent to the naked eye that may result in tumor at the surgical margins ('positive margins'). We have also shown that tissue-print technologies can provide a general platform for the generation of marker maps that can be superimposed directly onto histopathological and radiological images, permitting molecular identification and classification of individual malignant lesions.