Functional complementation by electroporation of human BACs into mammalian fibroblast cells

Bacterial Artificial Chromosomes (BACs) have been used to complement a metabolic defect and to transfer a drug resistance marker into mammalian cells by electroporation. The selectable markers are stable and the recipient cells have BAC DNA integrated into the chromosomes as shown by fluorescent in situ hybridization, PCR and Southern hybridization.     

Gene therapy for malignant gliomas

Malignant gliomas are attractive targets for gene therapy because of their relatively well-localized distribution. Several new strategies have been devised that target different aspects of glioma biology. Gene transfer can be used to synthesize chemotherapy drugs that block DNA synthesis within these highly mitotic tumors. New genes can be introduced that restore the functions of mutated tumor suppressor genes or block the molecular pathways needed for tumor angiogenesis. Alternatively, the immune response to these tumors can be augmented by the local production of cytokines. Finally, viruses themselves can be used as tumoricidal agents by designing viruses that selectively replicate and destroy tumor cells. The advantages and limitations of these approaches are discussed in the context of their possible application to the treatment of these highly lethal malignancies.     

Maternal depressive disorder and contextual risk: contributions to the development of attachment insecurity and behavior problems in toddlerhood

Research has shown that offspring of depressed caregivers are at increased risk for maladaptive development and emotional difficulties. Specifically, infants and toddlers of depressed mothers have been shown to evidence higher percentages of insecure attachments and more behavioral difficulties than offspring of nondisordered mothers. However, even in studies that reveal significant differences between children of depressed and nondepressed caregivers, a substantial number of children with depressed caregivers do not evidence dysfunction. Such findings have resulted in increased attention to the broader social context in which children of depressed mothers develop. This investigation examined the direct influences of maternal depression on child development, as well as the role of contextual risks that may be particularly heightened in families with depressed parents. Toddlers with depressed mothers evidenced significantly more insecure attachments than did toddlers with nondisordered mothers, and this difference was not accounted for by contextual risk. In predicting child behavior problems, contextual risk was found to mediate the relation between maternal depression and child behavior problems. Father-report data on child behavior corroborated the mother report data. Results are discussed in terms of the diversity of functioning in offspring of depressed caregivers that can be attributed to varied levels of contextual risk accompanying depression.     

Effects of ethanol administration on components of the ubiquitin proteolytic pathway in rat liver

Hepatic protein accumulation during ethanol administration may result partly from an ethanol-elicited decline in hepatic protein degradation, which we have previously shown. We conducted the current studies to examine the effects of ethanol administration on the levels of hepatic ubiquitin, an 8.5-kd protein which is an important mediator of extralysosomal protein catabolism. Rats were pair-fed liquid diets containing either ethanol (36% of calories) or isocaloric maltose-dextrin for 1 to 5 weeks. Ubiquitin was immunochemically quantified by competitive enzyme-linked immunosorbent assay (ELISA) in crude cytosol fractions from whole liver and in 12,000g supernatants of hepatocyte lysates. Ubiquitin levels in hepatic cytosol fractions of ethanol-fed rats exceeded those of controls by about 30%. Isolated hepatocytes from ethanol-fed animals also showed a 40% to 75% elevation of ubiquitin above that in cells of pair-fed controls and this difference exceeded the relative rise in hepatocellular protein. In hepatocyte lysates subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, we detected monomeric ubiquitin and higher molecular mass ubiquitin-protein conjugates. However, the immunoblot analyses revealed no quantitative changes in the level of either free or conjugated ubiquitin. The ubiquitin conjugating activity of crude and diethyl aminoethyl-fractionated liver cytosols of ethanol-fed rats had equal capacities to those from controls in catalyzing the formation of ubiquitin-protein conjugates. Our findings indicate that chronic ethanol consumption increased the level of immunoreactive ubiquitin in rat liver. This may have resulted from enhanced ubiquitin production because of an ethanol-elicited stress response and/or decreased catabolism of ubiquitin and its conjugates. Our findings also provide no indication that the ethanol-elicited reduction in hepatic proteolysis is because of a ubiquitin-mediated mechanisms.     

Regulation of insulin-like growth factors I and II and their binding proteins in human bone marrow stromal cells by dexamethasone

Müller cells are highly permeable to potassium ions and play a major role in maintaining potassium homeostasis in the vertebrate retina during light-evoked neuronal activity. Potassium fluxes across the Müller cell's membrane are believed to underlie the light-evoked responses of these cells. We studied the potassium currents of turtle Müller cells in the retinal slice and in dissociated cell preparations and their role in the genesis of the light-evoked responses of these cells. In either preparation, the I-V curve, measured under voltage-clamp conditions, consisted of inward and outward currents. A mixture of cesium ions, TEA, and 4-AP blocked the inward current but had no effect on the outward current. Extracellular cesium ions alone blocked the inward current but exerted no effect on the photoresponses. Extracellular barium ions blocked both inward and outward currents, induced substantial depolarization, and augmented the light-evoked responses, especially the OFF component. Exposing isolated Müller cells to a high potassium concentration did not cause any current or voltage responses when barium ions were present. In contrast, application of glutamate in the presence of barium ions induced a small inward current that was associated with a substantially augmented depolarizing wave relative to that observed under control conditions. This observation suggests a role for an electrogenic glutamate transporter in generating the OFF component of the turtle Müller cell photoresponse.     

Water filtration of the forearm in short- and long-term diabetes mellitus

Blood flow and capillary filtration coefficient (CFC) were measured by strain-gauge plethysmography on the upper and lower third of the forearm in 9 normal subjects and 29 well regulated patients with diabetes mellitus of varying duration (less than 10 years, 10 to 20 years, and more than 20 years). There was no difference in blood flow in the four groups, but CFC was significantly increased in long-term diabetes (duration above 20 years) when measured at the distal part of the forearm near the wrist. Calculations showed that this was probably due to the relatively high contribution of connective tissue in this part of the forearm. Increased water filtration in connective tissue in long-term diabetics is in accordance with earlier findings of a lowered subcutaneous interstitial fluid albumin concentration in long-term diabetics, this being explained by an increase in net water outflux from the microcirculation.