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.     

重组白细胞介素-2纯化过程中影响活性的因素探讨

<正> 白细胞介素-2(Interleukin-2,IL-2)是一种重要的免疫调节剂,具有促进T细胞增殖和分化,调节NK细胞活性,诱导LAK等功能,在肿瘤治疗上有广泛的应用前景。我们的前文就重组菌中IL-2包含体的制备、性质及重组rIL-2的纯化进行了研究,本文就重组rIL-2纯化过程中影响其活性的有关因素进行了探讨。     

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。     

Proton-NMR studies of the effects of ionic strength and pH on the hyperfine-shifted resonances and phenylalanine-82 environment of three species of mitochondrial ferricytochrome c.

Ferricytochromes c from three species (horse, tuna, yeast) display sensitivity to variations in solution ionic strength or pH that is manifested in significant changes in the proton NMR spectra of these proteins. Irradiation of the heme 3-CH3 resonances in the proton NMR spectra of tuna, horse and yeast iso-1 ferricytochromes c is shown to give NOE connectivities to the phenyl ring protons of Phe82 as well as to the beta-CH2 protons of this residue. This method was used to probe selectively the Phe82 spin systems of the three cytochromes c under a variety of solution conditions. This phenylalanine residue has previously been shown to be invariant in all mitochondrial cytochromes c, located near the exposed heme edge in proximity to the heme 3-CH3, and may function as a mediator in electron transfer reactions [Louie, G. V., Pielak, G. J., Smith, M. & Brayer, G. D. (1988) Biochemistry 27, 7870-7876]. Ferricytochromes c from all three species undergo a small but specific structural rearrangement in the environment around the heme 3-CH3 group upon changing the solution conditions from low to high ionic strength. This structural change involves a decrease in the distance between the Phe82 beta-CH2 group and the heme 3-CH3 substituent. In addition, studies of the effect of pH on the 1H-NMR spectrum of yeast iso-1 ferricytochrome c show that the heme 3-CH3 proton resonance exhibits a pH-dependent shift with an apparent pK in the range of 6.0-7.0. The chemical shift change of the yeast iso-1 ferricytochrome c heme 3-CH3 resonance is not accompanied by an increase in the linewidth as previously described for horse ferricytochrome c [Burns, P. D. & La Mar, G. N. (1981) J. Biol. Chem. 256, 4934-4939]. These spectral changes are interpreted as arising from an ionization of His33 near the C-terminus. In general, the larger spectral changes observed for the resonances in the vicinity of the heme 3-CH3 group in yeast iso-1 ferricytochrome c with changes in solution conditions, relative to the tuna and horse proteins, suggest that the region around Phe82 is more open and that movement of the Phe82 residue is less constrained in yeast ferricytochrome c. Finally, it is demonstrated here that both the heme 8-CH3 and the 7 alpha-CH resonances of yeast ferricytochrome c titrate with p2H and exhibit apparent pK values of approximately 7.0. The titrating group responsible for these spectral changes is proposed to be His39.     

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.     

Plasmin and the regulation of tissue-type plasminogen activator biosynthesis in human endothelial cells.

Plasmin inhibited the biosynthesis of tissue-type plasminogen activator (tPA) antigen by human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner. The amount of tPA antigen found in the 24-h conditioned medium of cells treated with 100 nM plasmin for 1 h was 20-30% of that in the control group. However, in contrast to tPA, such treatment led to a 3-fold increase in plasminogen activator inhibitor (PAI) activity, whereas the amount of PAI type 1 antigen was unchanged. The effects of plasmin on HUVEC were binding- and catalytic activity-dependent and were specifically blocked by epsilon-aminocaproic acid. Microplasmin, which has no kringle domains, was less effective in reducing tPA antigen biosynthesis or enhancing PAI activity in HUVEC. Kringle domains of plasmin affected neither tPA antigen nor PAI activity of the cells. Other proteases including chymotrypsin, trypsin, and collagenase at comparable concentrations did not have a significant effect on the biosynthesis of tPA antigen or PAI activity of HUVEC. Thrombin stimulated the biosynthesis of tPA and PAI-1 antigens by HUVEC. Thrombin also stimulated an increase in the protein kinase activity in HUVEC, whereas plasmin inhibited the protein kinase activity of the cells. It is possible that plasmin regulates the biosynthesis of tPA in HUVEC through the signal transduction pathway involving protein kinase.     

Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat

Summary Genomic in situ hybridization was used to identify alien chromatin in chromosome spreads of wheat, Triticum aestivum L., lines incorporating chromosomes from Leymus multicaulis (Kar. and Kir.) Tzvelev and Thinopyrum bessarabicum (Savul. and Rayss) Löve, and chromosome arms from Hordeum chilense Roem. and Schult, H. vulgare L. and Secale cereale L. Total genomic DNA from the introgressed alien species was used as a probe, together with excess amounts of unlabelled blocking DNA from wheat, for DNA:DNA in-situ hybridization. The method labelled the alien chromatin yellow-green, while the wheat chromosomes showed only the orange-red fluorescence of the DNA counterstain. Nuclei were screened from seedling root-tips (including those from half-grains) and anther wall tissue. The genomic probing method identified alien chromosomes and chromosome arms and allowed counting in nuclei at all stages of the cell cycle, so complete metaphases were not needed. At prophase or interphase, two labelled domains were visible in most nuclei from disomic lines, while only one labelled domain was visible in monosomic lines. At metaphase, direct visualization of the morphology of the alien chromosome or chromosome segment was possible and allowed identification of the relationship of the alien chromatin to the wheat chromosomes. The genomic in-situ hybridization method is fast, sensitive, accurate and informative. Hence it is likely to be of great value for both cytogenetic analysis and in plant breeding programmes.     

Binding of anti-human-interleukin-6 monoclonal antibodies to synthetic peptides of human interleukin-6 studied using surface plasmon resonance.

Biosensor technology employing surface plasmon resonance (SPR) detection provides a highly-sensitive (sub ng), non-extrinsic labelling approach for monitoring protein interactions in real-time. We have used this approach to map the binding sites on human interleukin-6 (hIL-6) for a series of anti-hIL-6 monoclonal antibodies (mAbs). Epitopes were localised by monitoring the ability of ten synthetic peptides, spanning the sequence of hIL-6, to inhibit the binding of anti-hIL-6 mAbs to immobilised hIL-6. Peptide P8 (Pro139-Gln153) inhibited binding of anti-IL-6-mAbs 1, 2 and 7. To increase the sensitivity of detection of antibody-synthetic peptide interactions, a procedure was developed for immobilising the synthetic peptides directly to the sensor surface of the SPR instrument. From this study, association equilibrium constants of 2.1 x 10(6)M-1 and 3.6 x 10(4)M-1 were calculated for the mAb7-immobilised P8 and mAb7-free P8 interactions, respectively.