Choromański L, Beat D A, Nordin J H, Pan A A, Honigberg B M
Z Parasitenkd. 1985;71(4):443-58. doi: 10.1007/BF00928347.
Fluorescence emitted by individual cells of several Trichomonas vaginalis strains, nearly all of which were cloned, incubated with fluorescein-conjugated lectins in the absence (experimental) or presence (control) of inhibitory sugars, or else in phosphate-buffered saline alone (autofluorescence) was measured with a Leitz MPV Compact microfluorometer. Irrespective of whether the organisms were postfixed in formalin or glutaraldehyde, the relative fluorescence emitted by the cells was closely comparable, provided that appropriate neutral density filters were employed. However, autofluorescence was much higher for glutaraldehyde-fixed trichomonads. Therefore, although better preserved and more amenable to subsequent manipulations, such organisms were found unsuitable for use in "qualitative" titration of the fluorescence emitted by various strains. Provided that the necessary precautions were taken, comparable fluorescence readings were obtained with trichomonads affixed to glass slides by heat (41 degrees C, on a section spreader) or by a cytologic centrifuge (Cytospin 2). Large numbers of concanavalin A (Con A)-binding sites were present on organisms of all strains, irrespective of their virulence for human patients and as estimated by the subcutaneous mouse assay; these sites were shown with the aid of D-mannose to be mannose or mannose-related residues. More binding sites for soybean agglutinin (SBA) were found on the virulent than on avirulent strains. On the basis of inhibition experiments, the sugar residues mainly responsible for these differences appeared to be D-lactose residues. Similar differences were observed with Ricinus communis agglutinin Type I (RCA I), for which D-galactose was employed as the competing sugar. However, with two cloned strains the situation with regard to RCA I binding was reversed - more of the lectin bound to a mild than to a virulent strain. The results obtained with Ricinus communis Type II agglutinin (RCA II) were often similar to those noted for RCA I; however, in most instances the inhibition with N-acetyl-D-galactosamine (GalNAc) was lower. Furthermore, the results noted with the GalNAc-specific agglutinins from Dolichus biflorus and Helix pomatia suggested that only very few GalNAc residues were available for binding on the surfaces of all T. vaginalis strains examined in the course of this study. Statistical analyses of fluorescence emitted by four clones of each, Balt 42 (virulent) and JH31A (avirulent) T. vaginalis strain upon incubation with Con A and SBA revealed homogeneity of these strains with regard to the number of the specific surface saccharide residues, D-mannose and D-lactose.(ABSTRACT TRUNCATED AT 400 WORDS)
用Leitz MPV Compact微型荧光计测量了几种阴道毛滴虫菌株（几乎所有菌株都是克隆株）的单个细胞发出的荧光。这些菌株在不存在（实验组）或存在（对照组）抑制性糖的情况下，或仅在磷酸盐缓冲盐水中（自发荧光）与荧光素偶联的凝集素一起孵育。无论生物体是用福尔马林还是戊二醛进行后固定，只要使用合适的中性密度滤光片，细胞发出的相对荧光就具有高度可比性。然而，戊二醛固定的滴虫的自发荧光要高得多。因此，尽管这种生物体保存得更好，更便于后续操作，但发现它们不适用于对各种菌株发出的荧光进行“定性”滴定。只要采取必要的预防措施，通过加热（41摄氏度，在切片涂抹器上）或通过细胞离心机（Cytospin 2）固定在载玻片上的滴虫，可获得可比的荧光读数。所有菌株的生物体上都存在大量的伴刀豆球蛋白A（Con A）结合位点，无论它们对人类患者的毒力如何，通过皮下小鼠试验估计；借助D-甘露糖显示这些位点是甘露糖或与甘露糖相关的残基。在有毒力的菌株上发现的大豆凝集素（SBA）结合位点比无毒力的菌株更多。根据抑制实验，造成这些差异的主要糖残基似乎是D-乳糖残基。用I型蓖麻凝集素（RCA I）观察到类似的差异，使用D-半乳糖作为竞争糖。然而，对于两个克隆株，RCA I结合的情况则相反——凝集素与温和菌株结合的比与有毒力的菌株结合的更多。用II型蓖麻凝集素（RCA II）获得的结果通常与RCA I的结果相似；然而，在大多数情况下，用N-乙酰-D-半乳糖胺（GalNAc）的抑制作用较低。此外，用来自双花扁豆和苹果螺的GalNAc特异性凝集素得到的结果表明，在本研究过程中检测的所有阴道毛滴虫菌株的表面上，只有极少数GalNAc残基可用于结合。对Balt 42（有毒力）和JH31A（无毒力）阴道毛滴虫菌株的四个克隆株在与Con A和SBA孵育后发出的荧光进行统计分析，结果显示这些菌株在特异性表面糖残基、D-甘露糖和D-乳糖的数量方面具有同质性。（摘要截短至400字）
