Nonlinear sequence-dependent structure of nigral dopamine neuron interspike interval firing patterns.

Firing patterns of 15 dopamine neurons in the rat substantia nigra were studied. These cells alternated between two firing modes, single-spike and bursting, which interwove to produce irregular, aperiodic interspike interval (ISI) patterns. When examined by linear autocorrelation analysis, these patterns appeared to reflect a primarily stochastic or random process. However, dynamical analysis revealed that the sequential behavior of a majority of these cells expressed "higher-dimensional" nonlinear deterministic structure. Dimensionality refers to the number of degrees of freedom or complexity of a time series. Bursting was statistically associated with some aspects of nonlinear ISI sequence dependence. Controlling for the effects of nonstationarity substantially increased overall predictability of ISI sequences. We hypothesize that the nonlinear deterministic structure of ISI firing patterns reflects the neuron's response to coordinated synaptic inputs emerging from neural circuit interactions.     

Antitumor agents-CLXXV. Anti-tubulin action of (+)-thiocolchicine prepared by partial synthesis

(+)-Thiocolchicine (2b) was prepared from (±)-colchicine (1) in a five-step reaction sequence that included chromatographic separation of appropriate camphanylated diastereomers. Acid hydrolysis of the (+)-diastereomer, followed by acetylation, yielded the desired product 2b. (+)-Thiocolchicine has 15-fold lower inhibitory activity against tubulin polymerization than (−)-thiocolchicine, and is 29-fold less potent for inhibiting growth of human Burkitt lymphoma cells. The enantiomer 2a, prepared from the (−)-camphanylated diastereomer, had potent activity in all assays comparable to that of (−)-thiocolchicine prepared by other methods. These results support the hypothesis that the proper configuration of colchicine-related compounds is an important requirement for their anti-tubulin action.     

Characterization of a plasma membrane-associated phosphoinositide-specific phospholipase C from soybean

Phosphoinositide-specific phospholipase C (PI-PLC) is a key signal transducing enzyme which generates the second messengers inositol trisphosphate and diacylglycerol in mammalian cells. A cDNA clone (PI-PLC1) encoding a phosphoinositide-specific phospholipase C was isolated from soybean by screening a cDNA expression library using an anti-(plasma membrane) serum. Genomic DNA gel blot analysis suggested that the corresponding gene is a member of a multigene family. The deduced amino acid sequence of the soybean PI-PLC1 isozyme contains the conserved X and Y regions, found in other PI-PLCs. It is closely related to mammalian δ-type PI-PLCs, Dictyostelium discoideum PI-PLC and yeast PI-PLC1 in terms of the arrangement of the conserved region. Unlike mammalian δ-type PI-PLCs and yeast PI-PLC1, the putative Ca²⁺-binding site of the soybean PI-PLC1 is located in the region spanning the X and Y domains, and the N-terminal region is truncated. FLAG epitope-tagged PI-PLC1 fusion protein purified from transgenic tobacco plants showed phosphoinositide-specific phospholipase C activity. Heterologous expression of the soybean PI-PLC1 cDNA in a yeast PI-PLC1 deletion mutant complemented the lethality phenotype of haploid PI-PLC1 disruptants. Immunoblot analysis of the cell fractions prepared from transgenic tobacco plants over-expressing the FLAG epitope-tagged PI-PLC1 fusion protein indicated that the protein encoded by the PI-PLC1 cDNA was localized in the cytosol and plasma membrane.     

Effect of temperature and baroreceptor stimulation on reflex venomotor responses

To investigate the interaction of thermal reflexes and baroreflexes in the control of the peripheral veins, we studied in supine humans the effects of lower body negative pressure (LBNP) and neck suction (NS) on forearm veins at ambient temperatures (Ta) of 18, 28, and 37 degrees C. Forearm venous volume (FVV)-venous pressure (FVP) relations (forearm venous capacitance) on six subjects showed an increase from 18 through 28 to 37 degrees C (P less than 0.001). Heart rate increased (P less than 0.001) and forearm venous capacitance decreased (P less than 0.001) in proportion to the level of LBNP applied from 20 to 50 Torr at all Ta. At 50 Torr LBNP, FVV at 30 cmH2O, FVP decreased from control values of 2.5, 3.8, and 4.4 to 1.6, 2.7, and 3.4 ml/100 ml at 18, 28, and 37 degrees C, respectively. We also studied venomotor responses using the occluded limb technique. Although LBNP caused venoconstriction, NS applied either alone or during LBNP produced no change in venomotor tone. Therefore we concluded that carotid baroreceptors play little role in reflex venomotor adjustments. Since changes in mean arterial and pulse pressures during LBNP did not account for the observed venomotor responses, we concluded that low-pressure baroreceptors initiate significant venoconstrictor reflexes over a wide range of Ta.     

Forskolin as a novel lipolytic agent

Forskolin is a novel lipolytic agent which elevates cAMP and FFA release in rat adipocytes in a manner different from existing lipolytic factors. This effect of Forskolin is potentiated by all lipolytic hormones tested, i.e. epinephrine, ACTH, and glucagon and is also reversible. The same batch of adipocytes can be repeatedly stimulated after washing. The effective concentration of Forskolin is in the micromolar range. Its action is due to an activation of cAMP synthesis by adenylate cyclase. There is no effect on cAMP hydrolysis. In contrast to stimulation by lipolytic hormones, Forskolin-activated membrane adenylate cyclase was not further stimulated by GPP(NH)P. These results suggest that Forskolin may be a useful analytical agent in the study of adenylate cyclase mediated function in intact adipocytes.     

1,25-双羟胆钙化醇及其受体含量检测的实验研究

 建立了一种改良的血清1,25-双羟胆钙化醇(1,25-Dihydroxycholecalciferol,DHCC)超微量放射受体检测(RRA)技术。完成了灵敏度、精密度、准确度、稳定性及特异性等技术指标。报告了我国健康青年血清DHCC正常值;检测了先天性佝偻病、青春期佝偻病病人及患肾性骨病奶牛等血清DHCC水平。 根据配体与受体相互结合的定量关系,建立了DHCCR(DHCC受体)检测技术。在游离与结合配基分离方面,除建立与比较了DCC(葡聚糖包埋的活性炭)及HAP(羟基磷灰石)方法外,还首次将IEF(等电聚焦电泳)应用于DHCCR分离技术。对佝偻病鸡小肠粘膜上皮细胞受体含量进行了检测并比较了鸡小肠、输卵管壳腺及肝组织DHCCR含量。     

Proteinases inhibit H(+)-ATPase and Na+/H+ exchange but not water transport in apical and endosomal membranes from rat proximal tubule.

A marked increase in water permeability can be induced in Xenopus oocytes by injection of mRNA from tissues that express water channels, suggesting that the water channel is a protein. In view of this and previous reports which showed that proteinases may interfere with mercurial inhibition of water transport in red blood cells (RBC), we examined the influence of trypsin, chymotrypsin, papain, pronase, subtilisin and thermolysin on water permeability as well as on ATPase activity, H(+)-pump, passive H+ conductance, and Na+/H+ exchange in apical brush-border vesicles (BBMV) and endosomal (EV) vesicles from rat renal cortex. H+ transport was measured by Acridine orange fluorescence quenching and water transport by stopped-flow light scattering. As measured by potential-driven H+ accumulation in BBMV and EV, proteinase treatment had little effect on vesicle integrity. In BBMV, ecto-ATPase activity was inhibited by 15-30%, Na+/H+ exchange by 20-55%, and H+ conductance was unchanged. Osmotic water permeability (Pf) was 570 microns/s and was inhibited 85-90% by 0.6 mM HgCl2; proteinase treatment did not affect Pf or the HgCl2 inhibition. In EV, NEM-sensitive H+ accumulation and ATPase activity were inhibited by greater than 95%. Pf (140 microns/s) and HgCl2 inhibition (75-85%) were not influenced by proteinase treatment. SDS-PAGE showed selective digestion of multiple polypeptides by proteinases. These results confirm the presence of water channels in BBMV and EV and demonstrate selective inhibition of ATPase function and Na+/H+ exchange by proteinase digestion. The lack of effect of proteinases on water transport by mercurials. We conclude that the water channel may be a small integral membrane protein which, unlike the H(+)-ATPase and Na+/H+ exchanger, has no functionally important membrane domains that are sensitive to proteolysis.     

In vivo mechanical properties of leukocytes during adhesion to venular endothelium

Transient deformations of leukocytes (WBCs) were studied during their saltation along post-capillary venous endothelium (EC) in mesentery of the rat. During intermittent adhesion of WBCs to EC, prevailing fluid shear stresses, tau wall, resulted in a stepwise loading of the WBC upon attachment with a transient increase in length, L(t), and reduction in height, H(t). Measurements of L(t) and H(t) from frame-by-frame analysis of video recordings were modelled as the simple shear of a standard linear viscoelastic solid to facilitate calculation of the elastic (k1, k2) and viscous (mu) elements with k1 in parallel with serial elements k2 and mu. The magnitude of tau wall was determined from measurements of red cell velocity within the venule. During the spontaneous adhesion of WBCs, a value of cell viscosity (mu) of 45 Poise was determined. Stimulating adhesion by topical application of the chemoattractant FMLP resulted in a 15-fold increase of mu to 668 Poise. Transient deformations during topical application of cytochalesin B to disrupt actin fibers within the WBC, yielded a 40% reduction in k1, compared to an 80% reduction with colchicine which disrupts the microtubule structure. Thus, colchicine treated cells appear to be twice as deformable as cells treated with cytochalesin. During adhesion stimulated by the cytokine Interleukin-1, mu increased 50% without changes in k1 and k2, possibly due to slight activation of the WBC.