Cytochemistry and immunolocalisation of polysaccharides and proteoglycans in the endosperm of green Arabica coffee beans.

The major noncellulosic polysaccharides and proteoglycans in the coffee bean (Coffea arabica) cell wall are (galacto)mannans and arabinogalactan proteins. Immunological and chemical probes demonstrated that the mannans and arabinogalactan proteins were located continuously across the width of the cell wall, but that the concentration of different structural epitopes within these polysaccharide types showed considerable spatial variation. For the mannans this was implied by the striated pattern demonstrated by fluctuation of the affinity between the mannan monoclonal antibody BGM C6 and (galacto)mannan. The arabinogalactan proteins labelled by the Yariv reagent and the arabinogalactan protein-specific antibody LM2 appeared to be located in all regions of the wall except the middle lamella, but showed some differences in intensity of labelling. However, the LM6 antibody, specific for (1-->5)-alpha-arabinan epitopes, was located only as a compact region adjacent to the cell lumen in the body of the endosperm; though, it did label throughout the wall of epidermal cells. This implied that either some of the more highly arabinosylated arabinogalactan proteins contained contiguous 5-arabinosyl residues or that a rhamnogalacturonan which contained 5-arabinosyl residues as side chains existed in the cell wall. In either case the polymers were very restricted in their distribution. A second category of pectin, a homogalacturonan detected by JIM7, was located only in the middle lamella region. The architecture of the wall, as revealed by resin etching, appeared to reflect the chemical heterogeneity, with three distinct physical zones identifiable in a cross section across a single wall.

Localization of Cell Wall Polysaccharides during Kiwifruit (Actinidia deliciosa) Ripening.

The localization of pectin, cellulose, xyloglucan, and callose was compared in kiwifruit (Actinidia deliciosa [A. Chev.] C. F. Liang and A. R. Ferguson var. deliciosa "Hayward") at harvest, at the end of the first phase of softening, and when ripe. Pectin was visualized using three different methods: labeling of galacturonic acid residues, labeling of negatively charged groups, and labeling with JIM 5 (nonesterified residues) and JIM 7 (methyl-esterified) monoclonal antibodies. Labeling of pectin gave different results depending on the detection system used. Differences related to patterns of change during ripening and to spatial distribution of label intensity. Cell wall pectin was available for labeling at all stages of fruit softening, but no clear differentiation of the middle lamella region was seen, although JIM 5 binding predominated where the middle lamellae joined the intercellular spaces in unripe fruit. Negatively charged groups (cationic gold labeling) and, to a lesser extent, galacturonic acid residues (Aplysia depilans gonad lectin labeling) were preferentially located near the cell wall/plasma membrane boundary. The lack of strong binding of the JIM antibodies indicated that the reactive groups were inaccessible. Cellulose remained intact and labeled densely across the wall at all stages of fruit ripening. Distribution of xyloglucan was patchy at harvest but was scattered throughout the wall later in ripening. Alterations to labeling of xyloglucan indicated that some epitopes were differentially exposed. Plasmodesmatal regions were clearly different in composition to other wall areas, showing an absence of cellulose labeling, specific pectin labeling, and callose presence. A similar predominance of pectin labeling compared with cellulose also occurred at the middle lamella wedge near intercellular spaces.

The enantiomers of zacopride: an intra-species comparison of their potencies in functional and anxiolytic models.

1. The 5-HT3 receptor antagonist, zacopride, and its enantiomers, R(+)-zacopride and S(-)-zacopride, were examined in three pharmacological models: (i) 5-HT-induced depolarization of the mouse isolated vagus nerve preparation, (ii) the 5-HT-evoked von Bezold-Jarisch reflex in the mouse, and (iii) the mouse light:dark box model of anxiety. Other standard 5-HT3 receptor antagonists were also included for comparison in these studies. 2. Racemic zacopride, and both of the enantiomers, displayed potent 5-HT3 receptor antagonist activity in the isolated vagus nerve and in the von Bezold-Jarisch model. No 5-HT3 receptor agonist or partial agonist effects of these compounds were detected. 3. In the isolated vagus nerve, R(+)-zacopride and ondansetron were surmountable 5-HT3 receptor antagonists (pA2 values of 9.3 and 8.3, respectively), whereas racemic zacopride, S(-)-zacopride and tropisetron were insurmountable antagonists, markedly suppressing the maximum response to 5-HT. 4. In vivo, racemic zacopride, R(+)-zacopride, S(-)-zacopride and WAY100289 were potent antagonists of the 5-HT-evoked von Bezold-Jarisch reflex, with minimum effective doses (lowest dose required to reduce the reflex by > or = 85%; MED85) of 1.0, 3.0, 0.3 and 3.0 micrograms kg-1, s.c., respectively. 5. Racemic zacopride, R(+)-zacopride and S(-)-zacopride were active in the mouse light:dark box model of anxiety, with similar potencies (minimum effective dose 1 microgram kg-1, s.c.) and similar active dose-ranges (1-1000 micrograms kg-1, s.c.). 6. The doses of racemic zacopride, R( + )-zacopride and S(-)-zacopride required to block 5-HT3receptors in vivo correlated reasonably well with their potencies in an anxiety model within the same species. In these studies, there was no evidence of a marked difference between the anxiolytic potencies ofR( + )-zacopride and S(-)-zacopride.

Coagulation studies and fistula blood flow during erythropoietin therapy in haemodialysis patients.

An increased incidence of fistula thrombosis has been reported in haemodialysis patients treated with recombinant human erythropoietin (rHuEpo). The present study sought to investigate this problem by measuring fistula blood flow, blood viscosity, and a variety of tests of coagulation and haemostasis in a group of ten haemodialysis patients treated with rHuEpo. Fistula blood flow did not alter during the first 12 months of rHuEpo despite a significant increase in whole-blood viscosity. Bleeding time improved in all ten patients after 4 months of therapy, and this improvement was maintained at 12 months. There were no significant changes in one-stage prothrombin time, kaolin cephalin clotting time, whole-blood clotting time, prothrombin consumption index, plasma fibrinogen factor VII, factor VIII, antithrombin III, or platelet aggregability to ADP over the first 4 months of rHuEpo. In contrast, protein C decreased from 84.3 to 66.4% (P less than 0.01) and protein S from 124.1 to 68.3% (P less than 0.001) over the first 4 months. By 8 and 12 months, the concentrations of these substances had returned to pretreatment values. The levels of protein C and S attained at 4 months are known to predispose to thrombosis, and it is possible that this effect may contribute to the increased incidence of fistula thrombosis observed in haemodialysis patients treated with rHuEpo.

Human red blood cells inhibit endothelium-derived relaxing factor (EDRF) activity.

We have used a bioassay-cascade system to investigate the inhibitory effects of human red blood cells on EDRF activity. The vascular smooth muscle relaxant effect of EDRF released from an endothelium-intact pig coronary artery by the calcium ionophore A23187 was inhibited by washed red cells. This inhibition of EDRF activity by red cells was similar to that expected from their haemoglobin content. This study provides further evidence that in vivo EDRF acts as a local autocoid.