Quantification and preservation of lectin binding by isolated cardiomyocytes

During preparation of cells for experimentation a considerable amount of bound substance is lost. Our aim was to develop a protocol which retained lectin binding to an extent similar to living cells. This procedure would use fixation procedures suited for fluorescent lectin conjugates and gold-conjugates to be visualized by light- and electron microscopy, respectively. We tested glutaraldehyde and paraformaldehyde in different concentrations before and after lectin binding, different buffers and divalent cations, as additives, to determine the effects on preservation of lectin binding. Lectin binding was visualized and semiquantitatively evaluated by image analysis in the light microscope after silver enhancement of lectin-gold conjugates and by using tetramethyl rhodaminyl isothiocyanate (TRITC)-conjugated lectins. Preservation of lectin binding was best visualized with fluorescent lectin conjugates, whereas during silver enhancement procedures of gold-conjugated lectins, a considerable amount of bound lectins was lost. In general, lectin binding to living cells followed by fixation is superior to fixation before lectin binding. Unfavourable combinations of fixatives and buffers can cause a loss of more than 90% bound lectin. In our experiments with freshly isolated guinea pig cardiomyocytes, lectin binding was best when we used Na-cacodylate buffer with glutaraldehyde fixation (0.1%) after binding of lectins to the living cells.     

Ontogeny of J chain expression in pig lymphocytes

The ontogeny of lymphocytes expressing J chain in the cytoplasm (J+) was studied in pig foetuses by the immunofluorescent technique. Peripheral blood lymphocytes were the first J+ cells in prenatal life. The spleen and lymph nodes contained J+ cells in the last days of gestation. J+ cells were found in the lamina propria of the gut and some glands of conventional but not of germ-free piglets. J chain was not detected on or in cell membranes at any developmental stage.     

Prediction of hepatic graft viability before reperfusion: an analysis of effluent from porcine allografts

Rapid and reliable assessment of hepatic graft viability is important for successful orthotopic liver transplantation (OLTx). OLTx was performed in 11 pairs of pigs via a venovenous bypass. Six of these grafts were transplanted immediately (group A), while the other five were preserved in University of Wisconsin (UW) solution for 24 h and then transplanted (group B). All grafts were flushed with 300 ml of chilled (4°C) Ringer's lactate solution before reperfusion of the graft, when 20 ml of effluent from the graft was collected and the concentrations of ammonia, lactic acid, GOT, and LDH were measured. Four of the six pigs in group A survived longer than 3 days, while the other two pigs died of causes other than graft dysfunction. All five pigs in group B died either of hemoperitoneum or hemodynamic instability due to liver failure. The histology of postperfusion biopsies in group A showed minimal pathological changes, while the grafts in group B revealed moderate to severe ischemic injuries. Ammonia and lactic acid in the effluent of group B were significantly higher than those of group A (1511±216 vs 417±333 g/dl and 114.1±12.2 vs 91.4±12.2 mg/dl, respectively; P<0.05 in both cases). Before reperfusion, the rate of total adenine nucleotides in all of the substances in the graft, which were measured using high performance liquid chromatography (HPLC), inversely correlated with the ammonia levels in the effluent. We conclude that an analysis of the effluent, (i.e. the levels of ammonia and lactic acid), flushed from a hepatic graft before reperfusion could serve as a predictor of hepatic graft viability.     

Binding and specificity of major immunoglobulin classes of preformed human anti-pig heart antibodies

Preformed human anti-pig antibodies isolated from perfused pig hearts were used to analyze the binding of various immunoglobulin classes to cultured pig kidney cells. All anti-pig immunoglobulins (i.e., IgG, IgA, and IgM) were localized on the cell surface by the use of an indirect immunofluorescence technique. Anti-pig immunoglobulins also competed for the pig cell surface epitopes with Griffonia simplicifolia lectin (GS-I-B₄), which is specific for -galactosyl residues. This study provides further evidence that preformed human antibodies recognizing -glactosyl-containing epitopes (anti-gal antibodies) could be an important factor in hyperacute rejection of pig organs.     

Reflex peristalsis in the guinea pig isolated ileum is endogenously controlled by kappa opioid receptors

Summary (1) Reflex peristalsis in the circular muscle of the guinea pig ileum was elicited in vitro by sustained luminal distension of the intestinal wall according to 2 cm H₂O and evaluated in terms of the number of peristaltic waves within 15 min intervals. (2) The poorly -selective opioid antagonist naloxone at concentrations of 10^–7 and 10^–6 mol/l increased the frequency of peristaltic contractions within the first 15 min interval, and thereafter in a declining fashion, by 68 and 88%, respectively. The highly -selective opioid antagonist nor-binaltorphimine behaved similarly. It was, by one order of magnitude, more potent but a little less effective than naloxone, i.e., the maximum effect was 57% increase in peristaltic frequency at 10^–8 mol/l. Concentrations of 10^–7 and 10^–6 mol/l had the same effect as 10^–8 mol/l, and 10^–9 mol/l were ineffective. The highly -selective antagonist CTOP-NH₂ and the highly -selective antagonist ICI 174,864 were ineffective up to 10^–6 mol/l. (3) It is concluded that predominantly opioid receptors are used by endogenous opioids under the present conditions to inhibit reflex peristalsis.     

豚鼠肠系膜下神经节细胞的慢自发性去极化电位

目的 :探讨豚鼠肠系膜下神经节 (IMG)细胞慢自发性去极化电位 (SSDP)。方法 :离体细胞内记录技术 ,并观察IMG细胞SSDP发生间隔、持续时间及幅度。结果 :SSDP发生间隔时间、持续时间和幅度较为恒定 ,分别为 44 9.7± 11.0s、198.8±15 .1s和 14.2± 2 .4mV。 5 HT1P受体拮抗剂BRL 2 492 4对SSDP和该细胞的迟慢兴奋性突触后电位 (LS EPSP)无影响。结论 :豚鼠IMG内可能存在SSDP     

氢化可的松对豚鼠离体气管平滑肌收缩活动的影响

目的：研究氢化可的松对豚鼠离体气管平滑肌收缩活动的影响。方法：用组胺和乙酰胆碱引起气管条明显收缩和张力增加。用氢化可的松20-30秒后,观察组胺和乙酰胆碱所引起的气管条收缩活动的改变。结果：用氢化可的松20～30s后,组胺和乙酰胆碱所引起的气管条收缩活动减弱、张力明显降低。结论：氢化可的松可以快速舒张豚鼠气管平滑肌。     

豚鼠螺旋蜗轴动脉SP能神经的结构

目的 观察豚鼠螺旋蜗轴动脉（ＳＭＡ）ＳＰ能神经支配的特点。方法 采用免疫组织化学ＡＢＣ法结合葡萄糖氧化酶－ＤＡＢ－镍染色技术及免疫透射电镜,探讨Ｐ物质（ＳＰ）在ＳＭＡ上的分布特点。结果 ＳＭＡ近端外膜上存在着大量ＳＰ－ＩＲ阳性神经元,在ＳＭＡ远端外膜上可见大量的ＳＰ－ＩＲ阳性神经纤维,相互交错,呈网状结构。结论 ＳＰ的耳蜗血管的调节过程中可能起着重要作用。