Extra- and Intracellular pH in the Brain During Ischaemia,Related to Tissue Lactate Content in Normo- and Hypercapnic rats

The objective of the present study was to assess the relationship between the amount of lactate accumulated during complete ischaemia and the ensuing changes in extra- and intracellular pH (pHe and pHi, respectively). The preischaemic plasma glucose concentration of anaesthetized rats was varied by administration of glucose or insulin, pHe was determined in neocortex with ion-sensitive microelectrodes, and tissue lactate and CO2 contents were measured, tissue CO2 tension being known from separate experiments. The experiments were carried out in both normocapnic [arterial CO2 tension (PaCO2) approximately 40 mm Hg] and hypercapnic (PaCO2 approximately 80 mm Hg) animals. Irrespective of the preischaemic CO2 tension, DeltapHe was linearly related to tissue lactate content. Depending on the preischaemic glucose concentration, DeltapHe varied from <0.4 to >1.4 units. The results thus fail to confirm previous results that the changes in pHe describe two plateau functions (DeltapHe approximately 0.5 and 1.1, respectively), with a transition zone at tissue lactate contents of 17 - 20 mmol kg-1. Changes in pHi given in this study are based on the assumption of a uniform intracellular space. The pHi changed from a normal value of approximately 7.0 to 6.5, 6.1 and 5.8 at tissue lactate contents of 10, 20 and 30 mmol kg-1. The intrinsic (non-bicarbonate) buffer capacity, derived from these figures, was 23 mmol kg-1 pH-1. Some differences in pH and in HCO3- concentration between extra- and intracellular fluids persisted in the ischaemic tissue. These differences were probably caused by a persisting membrane potential in the ischaemic cells.     

Divalent Cations Modulate the Inhibitory Substrate Properties of Murine Glia-derived J1-160 and J1-180 Extracellular Matrix Glycoproteins for Neuronal Adhesion

J1-160 and J1-180 are developmentally late appearing J1 extracellular matrix glycoproteins derived from oligodendrocytes. They prevent adhesion of neurons (but not of astrocytes or fibroblasts) when offered as a substrate in mixture with laminin (Pesheva et al., J. Cell Biol., 109, 1765 - 1778, 1989). In the present study we have examined the influence of divalent cations on the inhibitory substrate properties of J1-160/180 glycoproteins towards adhesion of neurons. By metal chelate affinity chromatography, we show that J1-180, but not J1-160, binds Ca2+, while both J1 components are capable of binding Zn2+ and other divalent metal ions. Divalent cation binding was observed by gel filtration, aggregation assays with coated latex beads and electron microscopic examination to elicit aggregation of the molecules. Divalent cation binding also affects their non-permissive substrate properties towards neurons from early postnatal mouse cerebellum. Without divalent cations, J1-160 and J1-180 are inhibitory for substrate adhesion of neurons independently of the adhesive substrate present (laminin or poly-l-lysine). This effect is neutralized when J1-180 is preincubated with Ca2+ or Zn2+ prior to coating as substrate. In contrast, preincubation with Ca2+ ions does not affect the inhibitory substrate properties of J1-160 under these conditions. These observations show that J1-160/180 molecules may undergo self-aggregation in a divalent cation-dependent mechanism, which correlates with the neutralization of their inhibitory effect on neuronal adhesion. The aggregation state of the molecules may thus influence the process of myelination by a homophilic binding mechanism and determine the effectiveness of neurite extension during central nervous system development and under traumatic conditions in the adult.     

Creatine kinase isoenzyme (CK-BB) in combination to prostatic acid phosphatase measured by RIA in the diagnosis of prostatic cancer

Summary Creatine kinase isoenzyme (CK-BB) measured by mass was used to determine its value in the early diagnosis of prostatic cancer. Sera of patients with prostatic carcinoma of various stages (treated and untreated) were compared to normal male sera and sera of patients with benign hyperplasia of the prostate (BPH) with respect to CK-BB. The sera were simultaneously tested for PAP content. The sensitivity of the CK-BB-RIA was 1.63+/-0.08 g/l and reproducibility in the higher and lower concentration range 7.6% and 10.5%, respectively. CK-BB alone or in combination with PAP is no marker for early detection of prostatic cancer. In individual cases changes occurred similar to those found with a malignant growth of the prostate.     

Pyruvate kinase in human brain tumours. Its significance in the treatment of gliomas

Summary Pyruvate kinase isozyme distribution was studied in 101 intracranial tumours of various nature and origin, and in normal human brain (both foetal and adult). In foetal brain, five different forms could be detected by electrophoresis (K₄, K₃M, K₂M₂, KM₃, and M₄). In adult brain, the M₄ type, K₃M hybrid, and K₄ are present; the M type is largely predominant. Alanine inhibition of pyruvate kinase can be used to discriminate between M and K-type pyruvate kinase. The results obtained in an alanine inhibition test are in agreement with the electrophoretic pattern. Pyruvate kinase from foetal brain and brain of a newborn is more inhibited compared with pyruvate kinase from adult brain. In adult brain a high residual activity of pyruvate kinase is found in the presence of alanine. Well differentiated neuroepithelial tumours,i.e., astrocytomas, oligodendrogliomas, and ependymomas showed also relatively high residual activities, though less than in normal adult brain. On the contrary, in poorly differentiated gliomas low residual activity was found. Alanine inhibition of pyruvate kinase correlates well with degree of differentiation of these tumours. There is also a strong correlationship between alanine inhibition of pyruvate kinase and one year survival after total or subtotal resection of gliomas in adults.When in gliomas the residual activity is determined not in the centre of the tumour but more towards the periphery, much higher residual activity is found. It is suggested that brain biopsies in which a residual activity higher than 70% is found probably contain no tumour in the paraffin slides.Poorly differentiated gliomas were characterized by the presence of type K, and the hybrids K₃M. In well differentiated gliomas, besides K₄ and K₃M, M₄ was also present. Alanine inhibition was in agreement with the electrophoretic pattern in all tumours. In children (age 1–11 years) gliomas showed no correlation between the distribution of pyruvate kinase isozymes and the histological classification and grading. Of the non-neuro-epithelial tumours studied relatively high residual activities were found for pyruvate kinase in haemangioblastomas, chromophobe adenomas, and craniopharyngiomas. This was also found in an arteriovenous malformation. Other non-neuroepithelial tumours showed much less residual activity. These included benign tumours, meningiomas, neurilemmomas, malignant metastatic tumours, and fibrosarcomas. It was also found in cavernomas. The determination of pyruvate kinase activity in the presence of alanine may be useful for the diagnosis and treatment of intracerebral tumours, in particular gliomas of adults.The alanine inhibition test is a reliable quantitative procedure. It can be performed in 10 minutes, and may well fit in the scope of a surgical procedure.     

Subthalamic neuron activity related to tremor and movement in Parkinson's disease

Single cell activity recorded in the subthalamic nucleus (STN) of Parkinson's patients and the effect of tremor, passive and voluntary movement upon the same cells are described. Three types of cells were distinguished by the pattern of discharge: tonic, phasic and rhythmic. They all demonstrated high mean firing rates (65, 59 and 69 Hz, respectively). Simultaneous recordings of muscle activity and tremor helped in defining cell activity. The implantation of the definitive stimulating electrode in the patients was based on the number of STN cells related to tremor, active and passive movements (mean = 68%) along the track chosen. Cells were related to tremor (n = 21; 11%), modified the discharge with differences in the amplitude of tremor (n = 4), and changed the rate and pattern when tremor stopped spontaneously or artificially (n = 6). Movement-related cells (n = 97; 51%) showed a cyclic activity correlated with phases of the movement, or modified the firing rate along the performance of the movement. Tremor and movement-related cells (n = 11; 6%) revealed an interesting sensory-motor integrative function.     

Cannabinoid withdrawal is dependent upon PKA activation in the cerebellum

Region-specific up-regulation of the cyclic AMP pathway is considered an important molecular mechanism in the origin of the somatic manifestations of the withdrawal syndrome to known drugs of abuse. Nevertheless, the existence of a withdrawal syndrome after prolonged cannabinoid administration has long been a controversial issue. Recent studies, in different species, have shown that withdrawal to prolonged cannabinoid exposure precipitated by the cannabinoid antagonist SR141716A is characterized by physical signs underlying impairment of motor coordination. Interestingly, cannabinoid withdrawal is accompanied by an increase of adenylyl cyclase activity in the cerebellum. Here, we investigate the functional role of the cyclic AMP pathway in the cerebellum in the establishment of cannabinoid withdrawal. We show that after SR141716A precipitation of cannabinoid withdrawal, basal and calcium-calmodulin-stimulated adenylyl cyclase activities as well as active PKA in the cerebellum increase in a transient manner with a temporal profile which matches that of the somatic expression of abstinence. Selectively blocking the up-regulation of the cyclic AMP pathway in the cerebellum, by microinfusing the cyclic AMP blocker Rp-8Br-cAMPS in this region, markedly reduced both PKA activation and the somatic expression of cannabinoid withdrawal. Our results (i) directly link the behavioural manifestations of cannabinoid withdrawal with the up-regulation of the cyclic AMP pathway in the cerebellum, pointing towards common molecular adaptive mechanisms for dependence and withdrawal to most drugs of abuse; (ii) suggest a particular role for the cerebellum as a major neurobiological substrate for cannabinoid withdrawal.     

Norepinephrine regulation of growth hormone release from goldfish pituitary cells. II. Intracellular sites of action

Previous results suggest that norepinephrine decreases growth hormone (GH) release in goldfish by means of alpha-2 adrenoceptor activation. The intracellular mechanisms by which norepinephrine inhibits GH release were examined in the present study using dispersed goldfish pituitary cells. In 2-h static incubation experiments, norepinephrine and the alpha-2 agonist clonidine decreased basal GH release and the GH responses to stimulation by the dopamine D1 agonist SKF38393 and two native gonadotropin-releasing hormones (GnRH). Norepinephrine also reduced GH responses to the adenylate cyclase activator forskolin, two protein kinase C (PKC) activators (phorbol ester and synthetic diacylglycerol), and two Ca2+ ionophores (ionomycin and A23187). Similarly, norepinephrine applied as a 1-h pulse in cell column perifusion experiments reduced basal GH release and abolished the GH response to a 5-min pulse of arachidonic acid. In goldfish, D1-stimulated GH release is mediated by AC-, arachidonic acid-and Ca2+-dependent pathways, whereas GnRH action is coupled to PKC-and Ca2+-dependent mechanisms. These results suggest that norepinephrine activation of alpha-2 receptors inhibits ligand-induced GH secretion by actions subsequent to activation of these second messenger cascades. To further characterize norepinephrine mechanisms of action on unstimulated hormone release, the ability of norepinephrine and an alpha-2 agonist to affect activation of two second messenger cascades under basal conditions was also investigated. Static incubation with clonidine reduced cAMP production in a time-and dose-dependent manner, suggesting that norepinephrine inhibitory action can also be expressed at the level of cAMP production. Resting intracellular free calcium levels in single, identified goldfish somatotropes was unaffected by norepinephrine. However, the inhibitory effects of norepinephrine on basal GH secretion was not observed in the presence of a voltage-sensitive Ca2+ channel agonist. Whether these channels are targets for norepinephrine action on unstimulated GH release requires further investigation.     

Dysfunctional regulation of alphaCaMKII and syntaxin 1B transcription after induction of LTP in the aged rat

Syntaxin 1B and alphaCaMKII are two genes that are upregulated after the induction of LTP and appear to underlie different mechanisms of synaptic plasticity. alphaCaMKII is directly implicated in strengthening the synapses that have been modified, whereas syntaxin 1B has been implicated in a mechanism for the propagation of synaptic plasticity within neural circuits. In these experiments we have investigated whether the regulation of these genes is altered after the induction of LTP in aged rats. We found, three hours after the induction of LTP in the dentate gyrus, that aged rats could be subgrouped into those in which LTP was maintained and those in which LTP had decayed back to basal levels. Both genes were upregulated in young adult rats, whereas there was a differential pattern of LTP-induced expression in the aged rats. Dendritic alphaCaMKII was upregulated in aged rats only when LTP was maintained. In contrast, regulation of syntaxin 1B and alphaCaMKII was absent in the granule cell bodies of the aged rats regardless of whether LTP was maintained or not. These results suggest that molecular mechanisms implicated in two aspects of hippocampal synaptic plasticity malfunction during normal ageing and therefore may have some contributory role in the decline in memory function routinely observed in ageing.     

Effect of two complex platinum salts on human sperm motility and acrosome reaction

Hydrogen hexachloroplatinate, H2PtCl6, has been shown to induce the human sperm acrosome reaction in vitro. However, the molecular mechanism underlying this exocytic process has not been studied. Therefore, two structurally and chemically different platinum (Pt) compounds, the potent sensitizer sodium-hexachloro-platinate-(IV), Na2[PtCl6], and the nonimmunogenic tetraamineplatinum-(II)-chloride, [Pt(NH3)4]Cl2, were selected for the experiments. Their effects on human sperm function and second messenger pathways were investigated. Washed human spermatozoa were treated with different concentrations of both Pt salts (0.5-1000 microM) during or after capacitation for 3 h at 37 degrees C. In addition, spermatozoa were incubated with Pt salts in calcium-free medium or in the presence of the protein kinase A+C inhibitor H7. Sperm motility was evaluated by computer-assisted sperm analysis; acrosomal loss was detected by triple staining. Compared with the controls (6.6+/-2.4%), the percentages of living acrosome-reacted spermatozoa showed a significant dose-dependent increase (P<0.001) after 3 h of incubation with Na2[PtCl6] (7.9+/-4.2% for 0.5 microM 25.0+/-2.9% for 1 mM) and [Pt(NH3)4]Cl2 (7.9+/-3.9% to 21.0+/-5.8%). Sperm motility was markedly reduced in samples containing the highest concentrations of the Pt salts. The acrosome reaction was also significantly increased when spermatozoa had first been capacitated and then treated with both Pt salts. Calcium-free medium had no effect on the ability of both Pt salts to induce the acrosome reaction. However, incubation of Na2[PtCl6] in the presence of H7 tendentiously decreased the percentage of acrosome-reacted spermatozoa. In conclusion, complex Pt salts such as Na2[PtCl6] or [Pt(NH3)4]Cl2 influence human sperm functions by inducing the acrosome reaction during or after capacitation. This stimulatory effect is independent of calcium and seems to be dependent on protein kinase A or C.     

LHRH拮抗剂Cetrorelix对子宫内膜癌细胞生长周期的影响及其机理

目的 :研究LHRH拮抗剂Cetrorelix对子宫内膜癌细胞生长周期及周期相关蛋白的影响 ,探讨其抑制内膜癌细胞生长的机理。方法 :用流式细胞仪细胞周期分析及Westernblotting蛋白分析法 ,研究在Cetrorelix的作用下子宫内膜癌细胞系HEC 1A细胞生长周期及相关周期蛋白的改变。结果 :1 0 -5mol/LCetrorelix可导致HEC 1A细胞生长停滞于G2 /M期 ,而与G2 /M期停滞相关的p5 3 ,磷酸化p5 3 (phospho p5 3 ) (丝氨酸 3 92 )及磷酸化cdc2 (phospho cdc2 ) (酪氨酸 1 5 )蛋白水平均显著增高。结论 :Cetrorelix抑制内膜癌细胞增殖作用的机理是结合细胞表面受体后引起一系列抑制性信号传递 ,导致细胞周期停滞于G2 /M期 ,主要表现为G2 期停滞。其中p5 3激活及cdc2磷酸化失活是引起细胞周期停滞的重要因素