Age-related loss of the GABA synthetic enzyme glutamic acid decarboxylase in rat primary auditory cortex.

Age-related changes within the auditory brainstem typically include alterations in inhibitory neurotransmission and coding mediated by GABA and glycinergic circuits. As part of an effort to evaluate the impact of aging on neurotransmission in the higher auditory centers, the present study examined age-related changes in the GABA synthetic enzyme, glutamic acid decarboxylase (GAD), in rat primary auditory cortex (AI), which contains a vast network of intrinsic and extrinsic GABAergic circuits throughout its layers. Message levels of the two GAD isoforms found in brain, GAD(65) and GAD(67), and GAD(67) protein levels were compared in young adult, middle-aged and aged rats using in situ hybridization and quantitative immunocytochemistry, respectively. For comparison, age-related GAD changes were also assessed in the parietal cortex and hippocampus. Significant age-related decreases in GAD(65&67) messages were observed in AI layers II-VI of aged rats relative to their young adult cohorts. The largest changes were identified in layer II (GAD(65): -26.6% and GAD(67): -40.1%). GAD(67) protein expression decreased significantly in parallel with mRNA decreases in all layers of AI. Adjacent regions of parietal cortex showed no significant GAD(67) protein changes among the age groups, except in layer IV. As previously described, GAD(67) message and protein levels in selected hippocampal regions were significantly reduced in aged rats. Age-related GAD reductions likely reflect decreases in both metabolic and pre-synaptic GABA levels suggesting a plastic down-regulation of normal adult inhibitory GABA neurotransmission. Consistent with the present findings, functional studies in primate visual cortex and preliminary studies in AI find coding changes suggestive of altered inhibitory processing in aged animals. An age-related loss of normal adult GABA neurotransmission in AI would likely alter temporal coding properties and could contribute to the loss in speech understanding observed in the elderly.

Mechanisms of radiosensitization in bromodeoxyuridine-substituted cells.

The radiosensitization of exponentially-growing V79-171 cells whose DNA has been substituted by bromodeoxyuridine (BrdU) in place of thymidine is decreased if acetone is present during irradiation. Acetone, at a concentration of 1 mol dm-3, removes the majority of the increase in double-strand breaks (dsbs) caused by BrdU substitution, but only removes approximately half of the increase in cell killing. The decrease in cell radiosensitization coincides with the removal of the additional dsbs. The protection afforded by acetone against dsbs is assumed to be due to its ability to scavenge hydrated electrons, thought to be the active species causing the increased DNA damage in the presence of BrdU. The residual component of BrdU radiosensitization which could not be removed by treatment with acetone may be due to either a subset of nonscavengable, lethal dsbs or the influence of BrdU on the fixation of potentially-lethal damage (Iliakis et al. 1992). Cells substituted with BrdU are not sensitized to hydroxyl radicals (from hydrogen peroxide). Also, the enhanced levels of single-strand break (ssb) and dsb production in cells substituted with BrdU arise from analogous events (i.e. increases in the yield of ssbs). These studies support the locally multiply damaged site theory of lesion (dsb) production (Ward 1981) and, in the case of BrdU-substituted cells, the increase in dsbs appears to be due to the production of additional ssbs by hydrated electrons at sites of multiple damage.

Optimization of the polymerase chain reaction with regard to fidelity: modified T7, Taq, and vent DNA polymerases.

The fidelity of DNA polymerases used in the polymerase chain reaction (PCR) can be influenced by many factors in the reaction mixture. To maximize the fidelity of DNA polymerases in the PCR, pH, concentrations of deoxynucleoside triphosphates, and magnesium ion were varied. Denaturing gradient gel electrophoresis was used to separate the polymerase-induced mutants from wild-type DNA sequences. Thermolabile modified T7 DNA polymerase, thermostable Taq, and Vent DNA polymerases were studied. Fidelity of all three DNA polymerases was sensitive to concentrations of deoxynucleoside triphosphates, magnesium ion, and pH. Within conditions that permitted efficient amplification, optimization with regard to these three factors yielded an average error rate in error/base pair incorporated of 7.2 x 10(-5) for Taq, 4.5 x 10(-5) for Vent, and 4.4 x 10(-5) for modified T7 (Sequenase) DNA polymerases.

Radiosensitization of Chinese hamster V79 cells by bromodeoxyuridine substitution of thymidine: enhancement of radiation-induced toxicity and DNA strand break production by monofilar and bifilar substitution.

Bromodeoxyuridine (BrdU) competes with thymidine (TdR) for incorporation into DNA of exponentially growing V79-171 cells. Such cells show an enhancement of the radiation response as determined by clonogenic survival and DNA damage measured by filter elution techniques after doses up to 15 Gy. The degree of radiosensitization for both survival and rates of alkaline and neutral elution are dependent on percentage BrdU substitution and independent of whether BrdU is in one strand only (monofilar) or both strands (bifilar) of the DNA duplex: e.g., for 16% BrdU substitution distributed either monofilarly or partially bifilarly, there is an enhancement factor for Do of 1.55. At this percentage substitution, the enhancement factor for the rate of alkaline elution is 1.75 and that for the rate of neutral elution is 1.54. The greater the percentage BrdU substitution, the larger was the enhancement ratio for survival and radiation-induced strand breaks in both monofilarly and bifilarly substituted cells. The increase in cell radiosensitivity caused by BrdU substitution shows a better correlation with the increase in radiation-induced double-strand breaks than with the increase in radiation-induced single-strand breaks.

Hypoxic toxicity of misonidazole in a glucose-6-phosphate dehydrogenase deficient mutant Chinese hamster ovary cell line.

The metabolic activation of misonidazole (MISO) and its effects on the hexose monophosphate pathway (HMP) and on cell viability were studied in hypoxic mutant Chinese hamster ovary (CHO) cells deficient in glucose-6-phosphate dehydrogenase and their parent wildtype cells. The metabolic activation of MISO was similar in both cell lines as indicated by the binding of 14C-MISO to the acid-insoluble fraction of these cells; it was decreased by the absence of glucose. In the wildtype CHO cells, MISO caused a significant stimulation of the activity of the HMP while in the mutant CHO cells no HMP activity was measurable, even in the presence of MISO. In both cell lines clonogenicity began to decline after 2 hr and trypan blue exclusion after 4 hr of hypoxic incubation. The effect of MISO on both parameters of cell viability was somewhat more pronounced in the wildtype CHO cells. This difference became especially significant at the longer incubation times. The results indicate that reducing equivalents for the metabolic activation of MISO are provided not only by the HMP but that pathways other than the HMP, such as glycolysis or pathways starting from mitochondrial tricarboxylates, are of similar or even greater importance in this respect.

Dependence of misonidazole binding on factors associated with hypoxic metabolism.

The binding of misonidazole (MISO) to macromolecules in hypoxic cells is believed to require metabolic reduction. Several factors in the cells' environment, such as pH, glucose, lactate and MISO concentration could affect the capacity of metabolic reduction. Modulation of the binding of MISO by these factors was studied by exposing exponential EMT6/Ro cells to MISO under extremely hypoxic conditions. No binding was observed under aerobic conditions. There was no difference in the binding of 0.02 mM MISO at varying concentrations of glucose from 0.015 mM to 5 mM. Thus, for diagnostic purposes with concentrations of MISO lower than 0.02 mM, little effect of glucose concentration is expected. However, with 5 mM MISO, the binding of MISO increased with increasing glucose concentration (3-fold increase after 2 hours incubation in 5 mM glucose relative to 0.015 mM glucose). At intermediate MISO concentrations (0.1 mM to 5 mM); the higher the MISO concentration, the greater was the increase in binding due to 5 mM glucose. There was no detectable effect of lactate (0, 3 and 10 mM) at pH 7.2 on the binding of MISO either in 0.015 mM or 5 mM glucose. However, a decrease of pH (from 7.2 to 6.5) decreased the binding of MISO in 5 mM glucose but not in 0.015 mM glucose. These data indicated that the binding of MISO is a multi-step process, which involves the concentrations of both glucose (probably via reducing equivalents) and MISO.

The role of glycolysis and hexose monophosphate pathway in the hypoxic toxicity of misonidazole.

The metabolic activation of misonidazole (MISO) and its effects on the hexose monophosphate pathway (HMP) and clonogenicity were studied in hypoxic EMT6/Ro, wildtype Chinese hamster ovary (CHO) and mutant CHO cells deficient in glucose-6-phosphate dehydrogenase. In all three cell lines metabolic activation of MISO, as indicated by the binding of 14C-MISO to the acid-insoluble fraction of these cells, was increased by the presence of glucose. In EMT6/Ro cells and wildtype CHO cells, MISO caused a significant stimulation of the activity of the HMP while in the mutant CHO cells no HMP activity was measurable, even in the presence of MISO. Loss of clonogenicity induced by MISO occurred markedly earlier in EMT6/Ro cells than in the CHO cells. In the latter cells, however, only a small difference was observed between the wildtype and mutant cell line. From these results it is concluded that not only the HMP but also glycolysis and other, glucose-independent, metabolic pathways are able to provide electrons for the reductive activation of MISO and hence contribute to the hypoxic toxicity of this compound.

Modulation of the hypoxic toxicity and binding of misonidazole by glucose.

The hypoxic toxicity and binding of misonidazole (MISO) requires metabolic reduction. The influence of glucose on the toxicity and binding of MISO was studied because glucose is a major substrate for the supply of NADPH through the hexose monophosphate pathway (HMP). Hypoxic EMT6/Ro cells (10(6) cells ml-1) were incubated with varying concentrations of glucose (0.015 mM to 5 mM). The initial rate of glucose transport was found to increase linearly with the extracellular glucose concentration up to 5 mM (0.038 nmol glucose 10(-6) cells sec-1). About 1.5 percent of the total glucose consumed went through the HMP for hypoxic cells in 5 mM glucose. The rate of HMP progressively decreased as the glucose concentration was lowered. When exposed to 5 mM MISO, the HMP was stimulated. This stimulation declined from 3.2 times in 5 mM glucose to barely detectable below 1 mM glucose. Both the hypoxic toxicity and binding of 5 mM MISO to the acid-insoluble fraction were decreased as the concentration of glucose was lowered. Below 0.5 mM glucose, no significant toxicity due to MISO was observed. There was an initial burden of 2.5 nmol MISO 10(-6) cells bound with little toxicity. After this initial burden, the terminal slope was 1.8 mol MISO bound 10(-6) cells (63 percent decrease in the surviving fraction). These results indicate that glucose concentrations lower than 5 mM can decrease the HMP rate and the toxicity and binding of MISO to hypoxic cells, and imply that calibration curves with normal and low glucose concentrations should be used to estimate the possible hypoxic fraction when MISO is used as a hypoxic probe in vivo.

Decreased hypoxic toxicity and binding of misonidazole by low glucose concentration.

The modulation of the hypoxic toxicity and binding of Misonidazole (MISO) by glucose and lactate was studied by exposing exponential EMT6/Ro cells to 5 mM MISO under hypoxic conditions. The concentrations of glucose used were 0.015 mM and 5 mM, and the concentrations of lactate were 0, 3 and 10 mM. There was no significant hypoxic toxicity due to MISO in the absence of glucose. However, with 5 mM glucose, after a latent period of 0.5 hours, there was a rapid decrease in cell survival to less than 0.1% at 2.5 hours incubation in 5 mM MISO. The binding of MISO was also increased by glucose. The amount of MISO bound to cells in 0.015 mM glucose leveled off at 2 nmoles MISO/7 X 10(5) cells at 1 hour, whereas the binding in 5 mM glucose continued to increase to more than 5 nmoles MISO/7 X 10(5) cells after 3 hours incubation. There was no detectable effect of lactate on the binding of MISO to the cells either in 0.015 mM or 5 mM glucose. The high affinity of this binding was indicated by the lack of exchange of radioactive MISO with non-radioactive MISO even after 2 hours of incubation. These data showed that glucose concentrations could modify the toxicity and binding of MISO to hypoxic cells.

Primary multipotential malignant neoplasm of bone: chondrosarcoma associated with squamous cell carcinoma.

A primary neoplasm of the proximal humerus in a 68-year-old woman was unique histologically in that it contained both malignant cartilaginous and squamous cell components. The epithelial differentiation was confirmed by the demonstration of keratin by immunohistochemical techniques and of basement membrane, tonofilaments, and well-formed desmosomes by electron microscopy. The patient died 3 1/2 years after the onset of symptoms, without clinical evidence of either a primary tumor elsewhere or metastasis. The differential diagnosis from other bone tumors with epithelial differentiation, such as adamantinoma and "primitive multipotential primary sarcoma," is discussed. This is a rare primary neoplasm of bone of unknown histogenesis. Intermutability or metaplasia between mesenchymal and epithelial tissues is a possibility. The tumor probably originated from multipotential stem cells with the ability to undergo biphasic or dual differentiation toward mesenchymal and epithelial elements.