Induced resistance in the human non small cell lung carcinoma (NCI-H460) cell line in vitro by anticancer drugs.

Exposure of human non-small cell lung cancer cells (NCI-H460) to gradually increasing concentrations of doxorubicin resulted in the appearance of a new cell line (NCI-H460/R) that was resistant to doxorubicin (96.2-fold) and cross-resistant to etoposide, paclitaxel, vinblastine and epirubicin. Slight cross-resistance to two MDR-unrelated drugs 8-Cl-cAMP and sulfinosine was observed. Flow cytometry analysis showed that the accumulation of doxorubicin in the resistant cells was 88.4% lower than in the parental cells. Also, verapamil significantly decreased the efflux rate in NCI-H460 and NCI-H460/R cells, whereas curcumin inhibited the efflux in NCI-H460 cells only. Gene expression data confirmed the induction of mdr1 (P-gp), as judged by the observed 15-fold increase in its mRNA concentration in doxorubicin-resistant NCI-H460/R cells. In contrast, mrp1 and lrp expression was unaffected by the doxorubicin resistance. Further work should develop a rationale for a novel treatment of NSCLC with appropriate modulators of resistance aimed at improving the outcome of the acquired drug resistance.

Penicillin-induced bursting in motoneurones of the frog spinal cord. Elimination by NMDA antagonist.

The effects of penicillin were investigated in lumbar motoneurones of isolated spinal cord preparation of the frog (Rana ridibunda). Spinal root discharges were recorded and single cell activity was studied with intracellular electrodes. Bath application of 500 IU/ml of penicillin G induced in motoneurones prolonged depolarization shifts, followed by repeated transient depolarizations lasting several hours. In all motoneurones studied, this bursting activity was synchronized with discharges recorded from the ventral root. Blockade of N-methyl-D-aspartate (NMDA) receptors by D,L-2-amino-5-phosphonovaleric acid (50-100 microM) completely eliminated the bursting activity. It is suggested that the spinal cord may be an important locus of the anticonvulsant effect of NMDA antagonists.

The effect of cortical lesion on systemic penicillin epilepsy in rats.

There is a certain recovery of function following brain damage, due to neuronal plasticity. The experiments were performed in order to investigate the effects of cortical lesion on seizural activity in rats induced by systemic application of penicillin. The sensorimotor cortex was unilaterally removed in the lesioned animals, while the control animals were only sham operated or non-operated (before implantation of the electrodes). Seizural activity was recorded by means of electroencephalograms before and after penicillin treatment (1,000,000 I.U./kg, i.p). Testing of penicillin started at least 30 days after cortical lesion. Seizural activity was characterized by spike and wave complexes accompanied by vigilance reduction and sometimes by mild myoclonic jerks in both control and lesioned animals. The early period (about 2 h after penicillin administration) with appearance of the spike-wave discharges with relative increase of the mean total electroencephalogram powers as well as the succeeding period 2.5-5.5 h after penicillin administration) with maximum number of spike-wave discharges did not differ in the electroencephalogram of the control and lesioned animals. The late period of penicillin effect (from 6-11 h after penicillin administration) with frequent spike-wave discharges and still large mean total electroencephalogram powers was observed only in lesioned animals. It is concluded that a cortical lesion destabilizes the brain function in the rat model of epilepsy induced by parenteral administration of penicillin.

A nucleoside analogue, 7-thia-8-oxoguanosine stimulates proliferation of thymocytes in vitro.

7-thia-8-oxoguanosine (immunosine) is a nucleoside analogue with immunoenhancing activity. In this work, its effects on proliferation of thymocytes in vitro were studied. It was found that immunosine stimulated proliferation of thymocytes both of mice and rats. The stimulatory effect depended on antigen presenting cells (APC), since thymocytes depleted of accessory cells did not proliferate to immunosine. In addition, pretreatment of APC with immunosine for 24 h significantly increased proliferation of thymocytes. Immunosine stimulated interleukin 2 (IL-2) production and the expression of activation markers (CD25 and CD71). The upregulation of CD25 (alpha subunit of IL-2R) was detected both on thymocytes and thymic dendritic cells. Proliferation of thymocytes in the presence of immunosine was predominantly mediated by IL-2 since blocking IL-2Ralpha by specific monoclonal antibodies inhibited cell proliferation by 65-85%.

Enhanced serum response element binding activity correlates with down-regulation of c-fos mRNA expression in the rat brain following repeated cortical lesions.

Repeated lesions of rat cerebral cortex result in transient peaks in the level of the c-fos transcript, but after the second lesion, this peak is substantially diminished. Using this lesion paradigm, we have analyzed the participation of the c-fos promoter elements SRE and DSE in the regulation of c-fos transcription. Following a single lesion, SRE/DSE binding activity peaked at 2 h, subsequent to the maximal levels of c-fos mRNA and parallel to the peak of c-Fos protein. After a second lesion (reinduction), 4 h following the initial lesion, SRE/DSE binding activity peaked after only 30 min and was significantly higher than following the first lesion. Once again, this peak occurred after the peak of c-fos mRNA expression and parallel with the second peak of c-Fos protein expression. These results suggested that the SRE and DSE promoter elements participated in the induction and down-regulation of c-fos transcription in vivo and suggested the possible involvement of Fos protein in its own regulation. The ability of Fos/Fra proteins to participate in a transcriptional complex was confirmed in gel-shift experiments with an AP-1 element, and the biphasic trend of binding activity was observed. Supershift experiments were performed to directly determine whether Fos protein was participating in SRE and/or DSE transcriptional complexes. No alterations in the position or intensity of the shifted band were observed using Fos/Fra antiserum suggesting that Fos/Fra proteins could be involved in c-fos down-regulation through mechanisms other than direct participation in the SRE/DSE transcription complex.

GAP-43 mRNA expression in early development of human nervous system.

The temporal and spatial distribution of GAP-43 mRNA in early human development, from 6 to 23 gestational weeks (g.w.), was examined by in situ hybridization histochemistry. GAP-43 mRNA was expressed as early as 6 g.w. in all regions of developing nervous system, the spinal cord, brainstem, cerebellum, diencephalic and telencephalic regions. Although the pronounced level of expression persisted during the entire examined period, the intensity of expression varied along the spatial axis over time. Analysis at the cellular level revealed that early on in development (6 g.w.) GAP-43 mRNA was expressed in the entire neuroblast population. With the onset of differentiation, at 13-23 g.w., GAP-43 mRNA expression had switched to the neurons that are in the process outgrowth. The highest level of GAP-43 mRNA expression was localized in the regions consisting of differentiating neurons, such as the cortical plate and intermediate zone of the telencephalic wall, and several delineated subcortical and thalamic nuclei. The spatial and temporal pattern of GAP-43 mRNA expression obtained suggests a possible dual role of GAP-43 in the development of the human nervous system: in the embryonic brain it could be involved in fundamental processes underlying cell proliferation; in the fetal brain its expression is specifically correlated with differentiation and the outgrowth of axons.

Differential effects of amphetamine and phencyclidine on the expression of growth-associated protein GAP-43.

The purpose of the present study was to test changes in the expression of growth-associated protein (GAP-43) after chronic treatment with two different psychotomimetic drugs: amphetamine and phencyclidine. Rats were treated chronically for 7 days (twice daily) with 5 mg/kg of amphetamine and phencyclidine and sacrificed after 2, 5 or 7 days of treatment, and following 7, 14 or 21 days of recovery after full treatment (7 days). Separate groups of rats were treated on the same regiment with haloperidol, and control group was treated with vehicle. To determine the effects of different psychotomimetic drugs on the expression of GAP-43 we have used Northern blotting and quantitative in situ hybridization. Treatment with amphetamine induced decrease of GAP-43 mRNA expression, that was detected also during recovery period, up to 14 days after the last day of 7 days treatments. On the contrary, PCP induced increase of GAP-43 mRNA expression, that was detectable from the first days of treatment until 21 days after the last day of treatment. Treatment with haloperidol did not produce significant changes in GAP-43 mRNA expression. It can be suggested that GAP-43 upregulation upon phencyclidine treatment occurs as a result of functional activation of pathways able to participate in remodeling, while amphetamine showed neurotoxic effect, decreasing expression of GAP-43 mRNA.

Changes in neuropeptide levels after brain damage in rats.

The physiological and pathophysiological roles of neuropeptides are still not clear. The aim of our study was to detect long lasting changes of vasoactive-intestinal peptide (VIP), somatostatin (SOM) and substance P (SP) contents in the rat cerebral cortex and hippocampus after brain lesion. The experiments were performed on groups of adult male Wistar rats. The first group consisted of animals with unilateral ablation of the sensorimotor cortex performed at the age of 60 days. The second group was a control one (rats of the same age but with an intact brain). Both groups of animals were sacrificed at the age of 90-105 days and radioimmunoassay was used to determine amounts of VIP, SOM and SP. The mean values of VIP levels were decreased significantly only in contralateral cortical areas, while there was an increase of SP in lesioned animals. Our results suggest that descrete changes in neuropeptide levels occur during restorative processes after brain lesion.

The connection between absence-like seizures and hypothermia induced by penicillin: possible implication on other animal models of petit mal epilepsy.

In this study we investigated the relationship between penicillin-induced hypothermia and petit mal epilepsy induced by this proconvulsant antibiotic. In order to find a possible dose-dependent relationship, we used two doses: 1500.000 and 1000.000 U/kg b.wt., both known as being sufficient to induce absence-like attacks with subsequent spike and wave discharges (SWD) in electrocorticogram (ECoG). Because of experimental data suggesting penicillin binding to benzodiazepine receptor recognition site, we also studied penicillin-induced changes in body temperature after diazepam pretreatment. Results of this study clearly show that penicillin in doses known to induce petit mal-like epilepsy concomitantly induces statistically significant dose-dependent decrease in body temperature. Pretreatment with diazepam completely prevents both penicillin-induced hypothermia and SWDs. On the other hand, both the diazepam and mixed diazepam + penicillin treatments did not significantly alter body temperature. These results suggest, however, that at least some of the penicillin effects described could be assigned to its binding to the benzodiazepine receptor recognition site at GABA(A) ionophore. This may have an important clinical implication because the inhibitory action of penicillin at the benzodiazepine receptor recognition site could account for the mechanism of penicillin-induced unspecific encephalopathies in humans. The relationship between petit mal epilepsy and hypothermia sheds new light on the action mechanisms of penicillin-induced absence seizures.

The kinetics of hypoxanthine efflux from the rat brain.

The brain efflux of radiolabelled hypoxanthine in the rat was rapid in the first minute after injection [K(eff)(i)=0.21+/-0.06 min(-1)], which was saturable with a V(max)=13.08+/-0.81 nM min(-1) g(-1), and a high K(m,app) (67.2+/-13.4 microM); the K(i,app) for inosine was 31.5+/-7.6 microM. Capillary depletion analysis indicated that hypoxanthine accumulates in neurons and glia with the time. From cross-inhibition studies with different purines and pyrimidines, it suggests that these molecules could also be important substrates for this carrier.

Permeability of the blood-cerebrospinal fluid and blood-brain barriers to thyrotropin-releasing hormone.

The permeability of the blood-cerebrospinal fluid (CSF) barrier to 3H-labelled thyrotropin-releasing hormone (TRH), was studied at the blood-tissue interface of the isolated perfused choroid plexus of the sheep, using a rapid (less than 30 s), single circulation paired-tracer dilution technique, in which D-[14C]mannitol serves as an extracellular marker. Arterio-venous loss of 14C radioactivity reflects the percentage of the D-mannitol dose that crosses the blood-CSF barrier using a non-specific pathway. This loss suggests that the choroidal epithelium is moderately leaky. Cellular uptake of TRH, estimated by directly comparing venous dilution profiles of [3H]TRH and D-[14C]mannitol was independent of this leakiness. The unidirectional transport of TRH could not be saturated with unlabelled TRH at a concentration as high as 10 mM, but was markedly reduced by 10 mM proline and by the inhibitor of amidase and aminopeptidase activity, bacitracin (2 mM). Permeability of the blood-brain barrier to [3H]TRH was studied in the adult rat, employing the intracarotid injection technique of Oldendorf in which [14C]butanol served as an 'internal standard'. Brain-uptake of 3H radioactivity corrected for residual vascular space indicated a low extraction from the blood of TRH during a 15 s period of exposure to the peptide. Self-inhibition of [3H]TRH uptake by unlabelled TRH (10 mM) could not be demonstrated, but L-proline (10 mM) and bacitracin (2 mM) strongly inhibited this uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoamine-containing neurons in the Aplysia brain.

The localization of monoamine-containing neurons in the CNS of Aplysia depilans has been studied by fluorescent histochemistry (the glyoxylic acid condensation method) and microspectrofluorimetry. Yellow fluorescent nerve cells and fibers show the emission maximum at 515-520 nm which corresponds to that of serotonin fluorophore in a model system. Green fluorescent nerve cells have the emission maximum at 485 nm which corresponds to that of catecholamine. Central catecholamine-containing neurons were found in cerebral, buccal, pedal and unpaired abdominal ganglia. The majority of them were revealed in cerebral ganglia (about 40). Serotonin-containing neurons are abundant in cerebral and pedal ganglia. More than 30 serotonin-containing nerve cells were localized in cerebral ganglia. In the right pedal ganglion approximately 100 neurons were revealed; in the left one about 150. In the abdominal ganglion all nerve cells of this chemical type (except one) are located in the right hemiganglion. The results are summarized in corresponding schemes.

Temporal and spatial preferences of c-fos mRNA expression in the rat brain following cortical lesion.

The expression of the proto-oncogene c-fos is increased in neuronal cells by a number of stimuli and the usefulness of this gene as a marker of neuronal activity has been demonstrated. The temporal and spatial expression of c-fos mRNA following the induction of a unilateral cortical lesion have been investigated in the rat brain by Northern blot analysis and in situ hybridization histochemistry. It was observed that the lesion evoked a rapid increase (20-fold) in the content of c-fos mRNA in the ipsilateral cortex, whereas in the contralateral cortex c-fos mRNA expression was more modest (7-fold). In the whole hippocampus a large and very rapid increase (17-fold) of c-fos mRNA expression was detected. The effect of a cortical lesion on Ca2+ uptake and membrane potential was also investigated. Using synaptosomes as a model system, we have provided evidence that Ca2+ entry via membrane depolarization increases in coordination with c-fos gene expression in neuronal cells. The principal conclusions from this study are that cortical lesions induce transient expression of the c-fos gene in specific neuronal cells of the rat brain.

The characteristics of nucleobase transport and metabolism by the perfused sheep choroid plexus.

The uptake of nucleobases was investigated across the basolateral membrane of the sheep choroid plexus perfused in situ. The maximal uptake (U(max)) for hypoxanthine and adenine, was 35.51+/-1.50% and 30.71+/-0.49% and for guanine, thymine and uracil was 12.00+/-0.53%, 13.07+/-0.48% and 12.30+/-0.55%, respectively with a negligible backflux, except for that of thymine (35.11+/-5.37% of the U(max)). HPLC analysis revealed that the purine nucleobase hypoxanthine and the pyrimidine nucleobase thymine can pass intact through the choroid plexus and enter the cerebrospinal fluid CSF so the lack of backflux for hypoxanthine was not a result of metabolic trapping in the cell. Competition studies revealed that hypoxanthine, adenine and thymine shared the same transport system, while guanine and uracil were transported by a separate mechanism and that nucleosides can partially share the same transporter. HPLC analysis of sheep CSF collected in vivo revealed only two nucleobases were present adenine and hypoxanthine; with an R(CSF/Plasma) 0.19+/-0.02 and 3.43+/-0.20, respectively. Xanthine and urate, the final products of purine catabolism, could not be detected in the CSF even in trace amounts. These results suggest that the activity of xanthine oxidase in the brain of the sheep is very low so the metabolic degradation of purines is carried out only as far as hypoxanthine which then accumulates in the CSF. In conclusion, the presence of saturable transport systems for nucleobases at the basolateral membrane of the choroidal epithelium was demonstrated, which could be important for the distribution of the salvageable nucleobases, adenine and hypoxanthine in the central nervous system.

The characteristics of basolateral nucleoside transport in the perfused sheep choroid plexus and the effect of nitric oxide inhibition on these processes.

The single pass paired dilution technique was used to measure the uptake of nucleosides across the basolateral face of the isolated in situ perfused sheep choroid plexus (CP). The uptake of labelled adenosine and guanosine into the CP was large (approximately 35%) whereas that of thymidine was less (approximately 15%). The addition of 0.5 mM unlabelled adenosine to the perfusate inhibited the uptake of labelled adenosine by 66%, guanosine by 100% and that of thymidine by 50%, whereas the addition of 0.5 mM unlabelled thymidine caused complete self-inhibition. The backflux of adenosine was very small which may indicate a high rate of cellular metabolism or a flux into cerebrospinal fluid (CSF). The addition of 0.5 mM unlabelled adenosine did not alter the backflux of adenosine, but increased that of guanosine and thymidine. The entry of radioactivity derived from adenosine across the apical side of the CP cells into the newly formed CSF was determined as a 'CSF uptake index' relative to [14C]butanol and found to be about 25%; however, HPLC analysis revealed that the majority of this activity was hypoxanthine, and not adenosine. The complete inhibition of nitric oxide synthase caused a significant reduction in adenosine uptake into the CP and an increase in backflux for this molecule. It would appear that the uptake for adenosine by the CP is governed by the rate of cellular metabolism and not by the rate of transport into the cells of the choroid plexus whereas for guanosine and thymidine, transport is of greater importance.

Effect of early cortical lesion on the acute model of epilepsy.

The experiments were performed in order to investigate the sparing of function following early postnatal cortical lesion in the acute rat model of epilepsy. Sensorimotor cortex was unilaterally removed at 9 and 10 days of postnatal age in lesioned animals, while control animals were only sham operated (at the same early stage of life) or non-operated (before implantation of the electrodes). Seizure activity was recorded by means of electroencephalograms at adult stage of life induced by parenteral administration of penicillin (1,000,000 I.U./kg, i.p.). Our results showed that when the cortical lesion was performed in infancy (on the contrary to the lesion performed in adulthood) there was no prolongation of seizure activity in an acute model of epilepsy.

Histochemical study of biogenic monoamines in early ("Prenervous") and late embryos of sea urchins.

Treatment of the embryos of sea urchins with glyoxylic acid results in the appearance of luminescence which is indicative of the presence of biogenic monoamines. At the early stages of development (cleavage divisions, blastula, gastrula) the histochemical method reveals a tryptamine-like substance which is first found in all embryonic cells and later is concentrated mainly in the cells of the primary gut and ciliary bands. At the stages of prism and pluteus there appear neuron-like cells containing dopamine. The inhibitors of monoamine oxidase and neurotoxins reliably increase the histochemical reaction to monoamines only in late embryos which suggests a change in the properties of monoaminergic systems in the course of embryogenesis.

Ganglioside GM1 and GM3 in early human brain development: an immunocytochemical study.

The distribution of GM1 and GM3 gangliosides in human brain development between gestational week (g.w.) 6 and 15 was demonstrated by an immunocytochemical approach using polyclonal anti-GM1 and anti-GM3 antibodies. The first appearance of GM1- and GM3-positive cells was recorded as early as in g.w.6. Both antibodies labeled the cells in the ventricular zone of the telencephalic wall, with radially oriented fibers toward the pial surface, which represent radial glia cells with glia fibers. The intensive GM3 immunoreactivity was also exhibited in proliferating cells in the ventricular zone between g.w.6 and 12. During the period from g.w. 12 to 15, characterized by a rapid multiplication of neurons and glia cells, an increased number of GM1- and GM3-positive cells was observed. Prominent GM1 ganglioside staining was observed at the surface of the cell bodies in the ventricular zone. Besides surface labeling in migrating cells, GM1 immunoreactivity was identified inside the soma in the regions of cortical plate and subplate. GM1 immunoreactivity was more pronounced on the membrane of neuronal cells migrating along radial glia fibers, especially at the contact site between neuronal and glial cells. The GM3 ganglioside was localized mostly inside the soma, showing a granular immunoreactivity pattern. Our observations confirm the presence of GM1 and GM3 gangliosides in neuronal and glial cells in early human brain development. The involvement, especially of GM1 ganglioside in glia-neuronal contacts during migration of neuroblasts to their final destination, as well as the presence of GM3 ganglioside in proliferative cells in the ventricular zone of the telencephalic wall was also recorded.

Correlation between triplet repeat expansion and computed tomography measures of caudate nuclei atrophy in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant, progressive disorder characterized by choreic movements, cognitive decline, and psychiatric manifestations. Eleven patients with HD were retrospectively selected from a larger group of 42 patients based on the similar, early onset of the disease (between 21 and 30 years) and the same duration of HD at the moment of computed tomography (CT) examination (5 years). A significant correlation between the number of CAG trinucleotides and the bicaudate index or the frontal horn index, two indices of caudate atrophy, was found in this group of patients. Our results, although in a small number of patients, suggest that the striatal degeneration, assessed by CT measures, is primarily regulated by the size of expanded CAG repeats.

Amphetamine and haloperidol modulatory effects on Purkinje cell activity and on EEG power spectra in the acute rat model of epilepsy.

The modulation of cerebellar Purkinje cell activity and EEG from parietal cortex was studied in the rat model of epilepsy induced by penicillin under acute haloperidol and amphetamine treatment. The discharge pattern of Purkinje cells showed tendency towards inhibition and EEG power spectra increased after parenteral administration of penicillin (1000000 IU/kg, i.p.). Acute haloperidol treatment (1 mg/kg, i.p.), performed after the development of penicillin induced epileptic episodes, elicited a prominent excitation of Purkinje cell discharges associated with parallel increase in mean EEG power spectra. However, acute DL-amphetamine treatment induced marked suppression of Purkinje cell discharges as well as outstanding decrease of the mean EEG power spectra. These results indicate that cerebellar Purkinje cells may be important in the control of seizure activity and that noradrenergic influences are relevant.