DNA mismatch repair and O6-alkylguanine-DNA alkyltransferase analysis and response to Temodal in newly diagnosed malignant glioma

PURPOSE: We evaluated the response to Temodal (Schering-Plough Research Institute, Kenilworth, NJ) of patients with newly diagnosed malignant glioma, as well as the predictive value of quantifying tumor DNA mismatch repair activity and O6-alkylguanine-DNA alkyltransferase (AGT). PATIENTS AND METHODS: Thirty-three patients with newly diagnosed glioblastoma multiforme (GBM) and five patients with newly diagnosed anaplastic astrocytoma (AA) were treated with Temodal at a starting dose of 200 mg/m2 daily for 5 consecutive days with repeat dosing every 28 days after the first daily dose. Immunochemistry for the detection of the human DNA mismatch repair proteins MSH2 and MLH1 and the DNA repair protein AGT was performed with monoclonal antibodies and characterized with respect to percent positive staining. RESULTS: Of the 33 patients with GBM, complete responses (CRs) occurred in three patients, partial responses (PRs) occurred in 14 patients, stable disease (SD) was seen in four patients, and 12 patients developed progressive disease (PD). Toxicity included infrequent grades 3 and 4 myelosuppression, constipation, nausea, and headache. Thirty tumors showed greater than 60% cells that stained for MSH2 and MLH1, with three CRs, 12 PRs, three SDs, and 12 PDs. Eight tumors showed 60% or less cells that stained with antibodies to MSH2 and/or MLH1, with 3 PRs, 3 SDs, and 2 PDs. Eleven tumors showed 20% or greater cells that stained with an antibody to AGT, with 1 PR, 2 SDs, and 8 PDs. Twenty-five tumors showed less than 20% cells that stained for AGT, with 3 CRs, 12 PRs, 4 SDs, and 6 PDs. CONCLUSION: These results suggest that Temodal has activity against newly diagnosed GBM and AA and warrants continued evaluation of this agent. Furthermore, pretherapy analysis of tumor DNA mismatch repair and, particularly, AGT protein expression may identify patients in whom tumors are resistant to Temodal.     

Differential respiratory muscle recruitment induced by clonidine in awake goats

The purpose of this study was to test the hypothesis that dysrhythmic breathing induced by the alpha2-agonist clonidine is accompanied by differential recruitment of respiratory muscles. In adult goats (n = 14) electromyographic (EMG) measurements were made from inspiratory muscles (diaphragm and parasternal intercostal) and expiratory muscles [triangularis sterni (TS) and transversus abdominis (Abd)]. EMG of the thyroarytenoid (TA) muscle was used as an index of upper airway (glottal) patency. Peak EMG activities of all spinal inspiratory and expiratory muscles were augmented by central and peripheral chemoreceptor stimuli. Phasic TA was apparent in the postinspiratory phase of the breathing cycle under normoxic conditions. During dysrhythmic breathing episodes induced by clonidine, TS and Abd activities were attenuated or abolished, whereas diaphragm and parasternal intercostal activities were unchanged. There was no tonic activation of TS or Abd EMG during apneas; however, TA activity became tonic throughout the apnea. We conclude that 1) alpha2-adrenoceptor stimulation results in differential recruitment of respiratory muscles during respiratory dysrhythmias and 2) apneas are accompanied by active glottic closure in the awake goat.     

Sequence of the gene encoding a highly thermostable neutral proteinase from Bacillus sp. strain EA1: expression in Escherichia coli and characterisation

Ammonium assimilation was studied by feeding [15N]ammonium to actively growing mycelium of Agaricus bisporus. Products of ammonium assimilation were analysed using 15N-NMR. Participation of glutamine synthetase, glutamate synthase and NADP-dependent glutamate dehydrogenase was determined by inhibiting glutamine synthetase with phosphinothricin and glutamate synthase with azaserine. Our results clearly indicate that, under the conditions used, ammonium assimilation is mainly catalysed by the enzymes of the glutamine synthetase/glutamate synthase pathway. No indications were found for participation of NADP-dependent glutamate dehydrogenase. Furthermore, 15N-labelling shows that transamination of glutamate with pyruvate to yield alanine is a major route in nitrogen metabolism. Another major route is the formation of N-acetylglucosamine. Compared to the formation of N-acetylglucosamine there was only a limited formation of arginine.     

Coupling potentials in CA1 neurons during calcium-free-induced field burst activity

Small amplitude depolarizations (fast prepotentials, spikelets) recorded in mammalian neurons are thought to represent either dendritic action potentials or presynaptic action potentials attenuated by gap junctions. We have used whole-cell recordings in an in vitro calcium-free model of epilepsy to record spikelets from CA1 neurons of the rat hippocampus. It was found that spikelet appearance was closely correlated with the occurrence of dye coupling between pyramidal neurons, indicating that both phenomena share a common substrate. Spikelets were characterized according to waveform (amplitude and shape) and temporal occurrence. Spikelet amplitudes were found to be invariant with neuronal membrane potential, and their pattern of occurrence was indistinguishable from patterns of action potential firing in these cells. Voltage and current recordings revealed a spikelet waveform that was usually biphasic, comprised of a rapid depolarization followed by a slower hyperpolarization. Numerical differentiation of spike bursts resulted in waveforms similar to recorded spikelet sequences, while numerical integration of spikelets yielded waveforms that were indistinguishable from action potentials. Modification of spikelet waveforms by the potassium channel blocker tetraethylammonium chloride suggests that spikelets may arise from both resistive and capacitive transmission of presynaptic action potentials. Intracellular alkalinization and acidification brought about by perfusion with NH4Cl caused changes in spikelet frequency, consistent with reported alterations of field burst activity in this model of epilepsy. These results suggest that spikelets result from gap junctional communication, and may be important determinants of neuronal activity during seizure-like activity.     

Bilateral symmetry of biomechanical properties in rat femora

In many studies, bone healing and remodeling have been examined in various animal models using one femur as a control for the contralateral femur based on the assumption that they are bilaterally symmetrical. Symmetry studies have been limited mainly to geometrical properties. The purpose of this study was to determine whether or not there is symmetry in the mechanical properties of rat femora. Two strain gauges were attached to the anterior surface parallel to the long axis of explanted femora of retired female breeder and 120-day-old male Sprague Dawley rats. Femora were mechanically tested in cantilever bending and the strain values were recorded. Moments of inertia, cortical areas, and moduli of elasticity were determined from strains and cross-sectional properties. Female femora showed a bilateral strain difference of less than 2.2% and an elastic modulus difference of less than 8.7%. Males had less than 2.0% and 7.9% differences for strain and elastic moduli, respectively. Statistical analysis showed no significant difference between left and right femoral strain values for the females, but modulus differences were significant different at the p = 0.05 level. There was no significant difference in strain and modulus values for the males, indicating mechanical and geometrical symmetry of their femora.