Fluorescein-conjugated Lectins Research Articles

Use of two conventional staining methods to assess the acrosomal status of stallion spermatozoa

The acrosome is a highly specialised region of the spermatozoon that is essential for fertilisation. Defects or dysfunction of this structure have been associated with fertility problems in man and various domestic species including stallions. Current methods of evaluating the acrosome of stallion spermatozoa are time consuming and require specialised equipment, which is cost prohibitive to the average practitioner. To evaluate 2 conventional stains (Dip Quick and Spermac) and determine their usefulness in assessing acrosome integrity in stallions as compared with specific acrosomal labelling with a fluorescein-conjugated lectin - a method that has been validated for acrosome status evaluation in stallions. In vivo experimental design. Semen from 6 mature Miniature horse stallions of known fertility was collected on 5 separate occasions. To increase the number of reacted acrosomes, portions of each ejaculate were incubated with the calcium ionophore, A23187. Ejaculates were divided and semen samples were processed according to recommendations for fluorescein-conjugated peanut lectin, Pisum sativum agglutin, Dip Quick, and Spermac staining methods. Slides were evaluated independently by 2 separate investigators. Spermatozoa were classified as having intact, reacting, reacted or defective acrosomes. All parameters obtained by both investigators, using all 3 staining methods were highly correlated (P<0.001). There was no statistical difference (P>0.05) between investigators or staining method for the percentages of intact or reacted acrosomes. However, there was a significant difference between investigators and staining methods for determining reacting acrosome percentages (P<0.05). Dip Quick and Spermac stains are useful for determining intact vs. reacted acrosomes for stallion spermatozoa.

A Novel Ovarian Xenografting Model to Characterize the Impact of Chemotherapy Agents on Human Primordial Follicle Reserve

Many chemotherapeutic agents, especially of the alkylating family, alter fertility in premenopausal females. However, it is not practically possible to quantify and characterize the impact of cancer drugs on ovarian reserve in a clinical setting. Thus, our specific aim was to develop a xenograft model to characterize the in vivo impact of chemotherapy agents on human ovary. Ovarian pieces from 24 weeks old abortuses were xenografted s.c. to severe combined immunodeficient mice (n = 52). Animals received either a single dose of 200 mg/kg of cyclophosphamide or the vehicle. Grafts were recovered from the control and treated mice 12 to 72 h after the cyclophosphamide injection and serially sectioned for primordial follicle counts. Apoptosis was assessed with terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Platelet/endothelial cell adhesion molecule 1 (PECAM-1) staining, as well as intra-vital fluorescein-conjugated lectin and Evans blue labeling were done to assess microvasculature by confocal microscopy. Although there was 12% reduction in primordial follicle density by 12 h following treatment (P > 0.05), the follicle loss increased significantly at 24 h (53%, P < 0.01) and peaked at 48 h (93%, P < 0.0001). TUNEL staining peaked at 12 h, earlier than the diminishment in follicle numbers, and decreased thereafter. Xenograft vascularization pattern was similar to non-xenografted tissue, indicating appropriate in vivo drug delivery. The impact of cyclophosphamide on primordial follicle reserve in our human ovarian xenograft model is consistent with the clinical gonadotoxicity of this drug. Human ovarian xenografting is a promising model to characterize the gonadotoxic effects of current and emerging cancer drugs without a need for lengthy clinical studies.

Differentiation of mitral cell dendrites in the developing main olfactory bulbs of normal and naris-occluded rats

The morphological differentiation of mitral cell dendrites during embryonic and early postnatal development was examined in the main olfactory bulb of rats to determine a possible role of afferent activity in the development of the dendrites. Mitral cells and olfactory nerve fibers were labeled with 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate (DiI) and fluorescein-conjugated lectin (Ulex europeus agglutinin-I), respectively. Morphogenesis of mitral cell dendrites proceeded as previously described (Malun and Brunjes [1996] J. Comp. Neurol. 368:1–16); that is, undifferentiated dendrites with radial orientation were transformed into a single primary dendrite having a glomerular tuft and secondary dendrites extending tangentially into the external plexiform layer. Quantitative examinations in both pre- and postnatal rats revealed that the differentiation of primary dendrites, including tuft formation, increases in diameter and decreases in branching, started before birth, whereas differentiated secondary dendrites were only observed in postnatal animals. Mitral cells with more than two primary dendrites were found after embryonic day 21. The proportion of the mitral cells with differentiated dendrites increased postnatally. At postnatal day 10, almost all mitral cells had fully differentiated dendrites, and mitral cells with multiple primary dendrites were no longer seen. No significant change was found during development in the number of stem dendrites that arose directly from the cell body. Unilateral naris occlusion started on postnatal day 1 retarded differentiation of primary and secondary dendrites, and increased the proportion of mitral cells with multiple primary dendrites. These finding revealed that differentiation of mitral cell primary dendrites precedes that of secondary dendrites, and suggested that the differentiation of secondary dendrites proceeds in an activity-dependent manner. J. Comp. Neurol. 418:402–410, 2000. © 2000 Wiley-Liss, Inc.

Differentiation of mitral cell dendrites in the developing main olfactory bulbs of normal and naris-occluded rats

The morphological differentiation of mitral cell dendrites during embryonic and early postnatal development was examined in the main olfactory bulb of rats to determine a possible role of afferent activity in the development of the dendrites. Mitral cells and olfactory nerve fibers were labeled with 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI) and fluorescein-conjugated lectin (Ulex europeus agglutinin-I), respectively. Morphogenesis of mitral cell dendrites proceeded as previously described (Malun and Brunjes [1996] J. Comp. Neurol. 368:1-16); that is, undifferentiated dendrites with radial orientation were transformed into a single primary dendrite having a glomerular tuft and secondary dendrites extending tangentially into the external plexiform layer. Quantitative examinations in both pre- and postnatal rats revealed that the differentiation of primary dendrites, including tuft formation, increases in diameter and decreases in branching, started before birth, whereas differentiated secondary dendrites were only observed in postnatal animals. Mitral cells with more than two primary dendrites were found after embryonic day 21. The proportion of the mitral cells with differentiated dendrites increased postnatally. At postnatal day 10, almost all mitral cells had fully differentiated dendrites, and mitral cells with multiple primary dendrites were no longer seen. No significant change was found during development in the number of stem dendrites that arose directly from the cell body. Unilateral naris occlusion started on postnatal day 1 retarded differentiation of primary and secondary dendrites, and increased the proportion of mitral cells with multiple primary dendrites. These finding revealed that differentiation of mitral cell primary dendrites precedes that of secondary dendrites, and suggested that the differentiation of secondary dendrites proceeds in an activity-dependent manner.

The expulsion of Echinostoma trivolvis from C3H mice: differences in glycoconjugates in mouse versus hamster small intestinal mucosa during infection.

Mucosal glycoconjugates were examined in C3H mice and in hamster small intestines infected with Echinostoma trivolvis and in uninfected rodents, using periodic-acid Schiff (PAS) and high-iron diamine-alcian blue (HID-AB) staining and three different fluorescein-conjugated lectins: Triticum vulgaris agglutinin (WGA), Helix pomatia agglutinin (HPA) and Griffonia simplicifolia agglutinin (GSA-II). Lectin-labelling by electron microscopy was also undertaken with WGA and HPA lectin-gold probes. HID-AB stain demonstrated that the most mature goblet cells of the mouse villi contain sulfomucins, whereas those of hamsters contain sialomucins. The expression of lectin-binding sites and the intensity of the lectin binding in the small intestines were changed by echinostome infection. Specific differences in the reaction to mucin glycoproteins were clearly observed between the mouse and hamster intestines infected with E. trivolvis; lectin-binding to hyperplastic goblet cells and crypts in the infected mice increased, while no marked increase in the number of goblet cells and reaction to the glycoconjugates were observed in the infected hamsters. These findings indicate that the expression of terminal N-acetyl-D-galactosamine, sialic acid and N-acetyl-D-glucosamine increased in mucins secreted from hyperplastic goblet cells associated with E. trivolvis infection in mice. No marked increase in these glycoconjugates occurred in hamster infections. These findings reflect clear differences in infectivity of E. trivolvis in C3H mice versus hamsters.

Centrifugation on Percoll gradient enhances fluorescent lectin binding on human sperm: a flow cytometric analysis.

Centrifugation on a Percoll gradient is commonly used to enrich sperm preparations in mobile forms prior to in vitro fertilization attempts. It has been suggested that this method also induces the capacitation of sperm, a step preceding the acrosomal reaction allowing egg fertilization. The modifications of the acrosomal membrane involved in these physiological events likely include alterations of glycosylation patterns. This hypothesis was investigated by comparing the membrane binding of 15 fluorescein-conjugated lectins on 60 samples of sperm, before and after Percoll centrifugation. The numbers of labeled sperm and their mean fluorescence intensity, recorded in flow cytometry, significantly increased for 10 and 14 of the 15 lectins tested. Microscopic examination of the labeled sperm showed that the acrosome and equatorial plates were more often labeled after Percoll centrifugation, confirming the hypothesis that this method modifies the glycosylation pattern of structures important for egg fertilization.

The occurrence of calcofluor and lectin binding polysaccharides in the outer wall of arbuscular mycorrhizal fungal spores

The hyaline outer wall of some spores of arbuscular mycorrhizal fungi is of important diagnostic value in identifying species but little is known about the chemical characteristics of this wall. With the use of the fluorochrome calcofluor and of the fluorescein-conjugated lectin wheat germ agglutinin we observed that chitin is one of the main components of the hyaline outer wall of four species of Glomus , and that neither chemical nor enzymic treatments of the spores were capable of changing the binding properties of this polysaccharide.

Developmentally-regulated lectin binding in the embryonic mouse telencephalon

Cell-surface carbohydrate epitopes are important determinants in cell-cell and cell-matrix interactions, and oligosaccharide groups are structural components of many growth factor receptors and cell adhesion molecules. These epitopes may participate in the regulation of stem cell proliferation and differentiation during central nervous system development. To further understand these cellular phenomena, it is important to define the changes in neuroepithelial cell-surface carbohydrate expression during development. We used a panel of fluorescein-conjugated lectins to label live, freshly dissociated cells from the embryonic day 11 to 18 (E11 to E18) mouse telencephalon. The intensity and heterogeneity of lectin labeling was assessed by flow cytometry. The lectins that we examined exhibited widely varying levels of labeling intensity. Lectins with the highest degree of binding included cholera toxin B subunit (CTB), which binds primarily to the gangliosides GM1 and GD1b, phaseolus vulgaris erythroagglutinating lectin (PHA-E), which binds to a variety of cell adhesion molecules, and wheat germ agglutinin (WGA). Many lectins showed increasing labeling intensity and cellular heterogeneity as development progressed. To determine if the observed cellular heterogeneity in lectin binding reflected biological differences in neuroepithelial cell subpopulations, cells from the E14 telencephalon were separated into two populations based on their intensity of CTB labeling using a fluorescence activated cell sorter. The population of weakly CTB labeled cells contained more than four times as many cells in S-phase of the cell cycle than the population of intensely CTB labeled cells. These observations suggest that lectin cytochemistry and flow cytometry can be useful in identifying specific cell subpopulations of neuroepithelial precursor cells during development, allowing their isolation and characterization in vitro.

Mycotic keratitis — an underestimated mycosis

Mycotic keratitis, an important ophthalmologic problem, especially in outdoor workers in the tropics, is frequently caused by filamentous fungi such as species of Fusarium, Aspergillus and Curvularia, and by yeast-like fungi such as Candida. A rapid, presumptive diagnosis can be made by recognition of certain typical clinical features and by direct microscopic detection of fungi in corneal scrapings stained by various methods. The diagnosis is confirmed by culture. In difficult cases, microbiological studies on corneal biopsies or histopathological studies on tissue sections may need to be performed. The use of fluorescein-conjugated lectins and similar diagnostic tools is aimed at providing rapid, species-specific detection of fungi in corneal tissue. Antifungal therapy must be instituted as soon as the diagnosis is made. While keratitis due to Aspergillus, Candida and dematiaceous fungi can be successfully treated by many of the currently available polyenes and azoles, the treatment of Fusarium keratitis still frequently requires the use of pimaricin or econazole. Treatment by the oral and parenteral routes may prove useful in severe mycotic keratitis. Surgery may need to be performed on cases unresponsive to medical therapy or where serious complications are likely to occur. The pathogenesis of mycotic keratitis appears to involve agent factors, such as invasiveness and toxigenicity, and host factors, such as trauma and intrinsic defects in resistance. Areas for future research include the development of rapid, species-specific diagnostic aids, of broad-spectrum antifungal compounds active by various routes, and of therapeutic modalities which act on the fungus and on molecules involved in the pathogenesis of the condition.

LECTIN BINDING BY CRUSTACEAN CUTICLE: THE CUTICLE OF CALLINECTES SAPIDUS THROUGHOUT THE MOLT CYCLE, AND THE INTERMOLT CUTICLE OF PROCAMBARUS CLARKII AND OCYPODE QUADRATA

ABSTRACT Pieces of dorsal carapace from the crayfish Procambarus clarkii, the ghost crab Ocypode quadrata, and the blue crab Callinectes sapidus were fixed in Rossman's fluid or alcoholic Formalin, decalcified, paraffin-embedded, and probed with up to 21 fluorescein-conjugated lectins in order to determine the composition and distribution of their carbohydrate moieties. Unfixed, frozen sections were also employed. Tissue was examined from the crayfish and the ghost crab at intermolt (C4), and from the blue crab at premolt (all D stages), ecdysis, postmolt (all A and B stages, and stage C,) and intermolt. Different lectins showed different binding patterns at intermolt, but patterns of binding for individual lectins were consistent among the different species. A number of lectins showed different binding patterns at different stages of the molt cycle. Lectin binding was influenced by the method of fixation, with Rossman's-fixed tissue binding more lectins and more intensely. The overall pattern of lectin binding indicated that different carbohydrate moieties exist in the different layers of the integument, and that the availability of these lectin-binding sites may change over the molt cycle.

Echinostoma caproni and E. trivolvis alter the binding of glycoconjugates in the intestinal mucosa of C3H mice as determined by lectin histochemistry.

Mouse (C3H) mucosal glycoconjugates were examined in normal small intestines and intestines infected with Echinostoma caproni or E. trivolvis using six different fluorescein-conjugated lectins: Triticum vulgaris agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), Ricinus communis agglutinin I (RCA-I), Glycine max soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), and Arachis hypogaea peanut agglutinin (PNA). The expression of lectin-binding sites and the intensity of the binding of lectins in the mouse small intestines were changed by infection with the echinostomes. Specific differences in the reaction to glycoproteins were clearly observed between the mouse intestines infected with E. caproni and those infected with E. trivolvis. In E. caproni infection, binding of most of the lectins to the villi was remarkably reduced in accord with the villous atrophy and loss of goblet cells. In contrast, in E. trivolvis infection, the binding of WGA, RCA-I and DBA was reduced in the microvillar surfaces, but binding of UEA-I and SBA were unchanged compared to the control intestines. The lectin binding to goblet cells in E. trivolvis-infected mice mostly increased. These observations may reflect the marked increase in goblet cells and the less severe damage in the villi of E. trivolvis infection compared to E. caproni infection. Most of the glycoconjugates were slightly reduced in the hyperplastic crypts except for N-acetyl glucosamine. It is possible that glucose metabolism in the host intestines infected with E. trivolvis was activated, resulting in an increase in the rate of mucin synthesis as well as qualitative changes in mucus, thereby mediating the expulsion of the worms.

Analysis of the responses of human spermatozoa to A23187 employing a novel technique for assessing the acrosome reaction.

Protocols for the use of A23187 in assessing the ability of human spermatozoa to acrosome-react and exhibit sperm-oocyte fusion have been developed and the results compared in two independent cohorts of infertile patients. Both bioassays were found to depend upon such factors as the dose and formulation of A23187, the duration of exposure, and the amount and type of protein used to supplement the medium. An optimal protocol for the hamster oocyte penetration test comprised a 3-hour exposure to 1.25-2.5 microM ionophore and gave penetration rates of 93.2 +/- 3.2% (11.26 +/- 1.27 sperm/egg) for a group of 33 fertile donors compared with 63.0 +/- 5.4% (4.73 +/- 0.81 sperm/egg) for a cohort of 56 patients consulting for infertility (P < 0.001). Higher doses (5.0-10.0 microM) of A23187 caused an inhibition of sperm-oocyte fusion in association with a loss of motility, although the integrity of sperm plasma membrane did not appear to be compromised and high rates (approximately 80%) of acrosome reaction were observed. A protocol for assessing the ability of viable human spermatozoa to acrosome-react in response to A23187 was developed, employing a fluorescein-conjugated lectin in concert with the hypoosmotic swelling test, which gave values of 20.1 +/- 2.6% and 13.6 +/- 1.6% for groups of fertile donors (n = 29) and infertile patients (n = 32) respectively (P < 0.05). Although only acrosome-reacted spermatozoa were capable of fusing with zona-free hamster oocytes, there was no significant correlation between the proportion of acrosome-reacted cells and the levels of sperm-oocyte fusion observed in two independent groups of patients, indicating that these bioassays are measuring different aspects of human sperm function. These results have implications for the way in which the responses of human spermatozoa to ionophore treatment are quantified and interpreted.

Toxin resistance and reduced secretion in a mouse L-cell mutant defective in herpes virus propagation.

The mouse L-cell mutant gro29 was selected originally for its inability to propagate herpes simplex virus; it shows severe defects in virus egress and the transport and processing of viral glycoproteins after infection. In this report, we show that uninfected gro29 cells display pleiotropic changes in protein secretion, oligosaccharide processing, and sensitivity to the toxins ricin and modeccin. Specifically, the rate of secretion of a nonglycosylated protein, human growth hormone, was reduced 70% in gro29 cells compared with the parental L cells. A direct measurement of the transport capacity of Golgi membranes in a cell-free assay suggests that gro29 cells contain less functional Golgi than parental cells. Despite this deficiency, N-linked oligosaccharides were processed efficiently in mutant cells, although there were differences in the structure of the mature forms. Lectin intoxication assays revealed that gro29 cells were cross-resistant to killing by the cytotoxic lectins ricin and modeccin, but not to wheat germ agglutinin, Ricinus communis agglutinin RCA120, or leucoagglutinin. Fluorescence labeling using fluorescein-conjugated lectins showed that uninfected gro29 cells expressed relatively few ricin-binding molecules, suggesting a possible mechanism for toxin resistance. These studies provide evidence that the processes of protein secretion, lectin intoxication, and herpes virus maturation and egress may share a common cellular component.

Lectin-binding profile of plasmacytoid monocytes

The lectin-binding profile of plasmacytoid monocytes, also known as plasmacytoid T cells, was studied in 18 axillary lymph nodes draining invasive breast carcinomas. The plasmacytoid monocytes bound fluorescein-conjugated lectins from Canavalia ensiformis (Con A), Phaseolus vulgaris (PHA-L), Arachis hypogaea (PNA), Ricinus communis (RCA I and II), and Triticum vulgaris (WGA), but no reaction was found with those from Dolichos biflorus (DBA) and Ulex europaeus (UEA I). Fluorescence was bright and the reactivity was distributed in a granular pattern in the cytoplasm of plasmacytoid monocytes, a staining pattern similar to that of monocytes and macrophages. B and T lymphocytes usually exhibited weak staining with the same lectins, and this was limited to the cell membrane. Plasmacytoid monocytes were originally thought to be closely related to the T-cell system. The results of recent immunocytologic studies and the lectin-binding profile of plasmacytoid monocytes observed in this study, however, suggest that these cells are related to the mononuclear phagocytic system.

Immunofluorescent lectin binding patterns and glycoprotein co-localization in the developing murine molar tooth

Fluorescein-conjugated lectins were used in conjunction with antibodies to laminin, tenascin and amelogenin to investigate saccharide expression in the developing tooth germ. At the bud stage, peanut agglutinin (PNA) binding demonstrated residues that may be d-galactose-(β1 → 3) dGalNAc, and this staining occurred after the expression of tenascin. Only the cap-stage enamel organ suprabasal cells and the enamel knot stained intensely with Ulex europeus agglutinin-I, but not Lotus tetragonolobus agglutinin, implying the transient presence of blood group H type I oligosaccharides. At the late stages of amelogenesis, enamel synthesis is preceded by en bloc loss of inner enamel basement membrane components. Before this, Bandeiraea (Griffonia) simplicifolia — I (BSL-I) staining was lost from post-mitotic ameloblasts, suggesting that a glycosylated species is initially removed. Additionally, PNA was co-localized with amelogenin protein, suggesting that it may express β- d-galactosyl sequences. These results indicate that the glycosylation patterns of matrix components during odontogenesis may be important as they vary in a manner similar to that of the well-known glycoproteins.

Metacyclogenesis of Leishmania spp: Species-Specific In vitro Transformation, Complement Resistance, and Cell Surface Carbohydrate and Protein Profiles

Metacyclic (stationary) and logarithmic (log) forms of promastigotes of Leishmania donovani and Leishmania major were characterized in several ways. The highly active metacyclic forms were larger with more protein and less carbohydrate. The flagellum increased in length 2.4 times in L. major as compared to 1.8 times in L. donovani. Resistance to complement-mediated lysis by normal human serum of in vitro grown Leishmania promastigotes was related to the species, the growth phase in culture, and also the temperature. Metacyclic forms of both species had a much increased resistance to killing by normal serum at different temperatures. Differences in membrane-exposed carbohydrates were detected by fluorescein-conjugated lectins. Peanut agglutinin and Ulex agglutinin I differentiated log and stationary phase promastigotes of L. major. Higher amounts of acid phosphatase were demonstrated in the metacyclic phase. Differences in polypeptides were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides of approximately 51 and 114 kDa were found exclusively in metacyclic promastigotes of both species, whereas 38- and 23-kDa polypeptides were lost or reduced during transformation from log to metacyclic phase promastigotes of L. donovani. In addition, a 75-kDa polypeptide was expressed only in metacyclic promastigotes of L. major.

Abnormalities of lectin histochemistry in familial polyposis coli and hereditary nonpolyposis colorectal cancer

A transformation in the composition of colonic glycoconjugates has been described in adenomas, carcinomas, and certain premalignant conditions. These changes have been detected histochemically by the labeling patterns of fluorescein-conjugated lectins, which bind specific carbohydrate structures on fixed tissue sections. This study was performed to determine whether abnormal lectin binding patterns are present in tissues from patients genetically predisposed to colonic neoplasms and whether these patterns could be used as phenotypic markers for inheritance of the genotype. Lectin staining patterns of 22 colectomy specimens from patients with familial polyposis coli (FPC) and rectal biopsy specimens from 47 patients at risk for hereditary nonpolyposis colorectal cancer (HNPCC) (also known as Lynch syndromes I and II) were compared with rectal biopsy specimens from 27 sex-matched controls. The fluorescein-conjugated lectins included the agglutinins derived from peanut, Dolichos biflorus, Ulex europeus, and wheat germ (including the succinylated derivative). Using a technique for quantitating lectin binding on the tissue sections that provided a score from 0 to 400, labeling with certain lectins was found to vary slightly as a function of age and sex. Histologically normal mucosa from patients with FPC bound significantly less wheat germ agglutinin but significantly more U. europeus and succinylated wheat germ agglutinins than controls. Adenomas and dysplastic flat mucosa from the colectomy specimens of patients with FPC showed significantly less binding with D. biflorus, succinylated wheat germ, and wheat germ agglutinins than controls. Rectal tissues from patients at risk for HNPCC were found to bind significantly less peanut agglutinin and D. biflorus agglutinin than controls. Of interest, staining of the tissues by peanut and wheat germ lectins increased as a function of patient age; the control subjects were older than the patients with familial colon cancer, which could possibly account for the observations made with these two lectins. These results provide evidence that the premalignant colonic epithelium in familial polyposis and the hereditary nonpolyposis colon cancer syndromes may be biologically different and indicate that glycoconjugate modifications are early events in the evolution of the neoplastic phenotype.

Kinetoplastid Flagellates: Surface-Reactive Carbohydrates Detected by Fluorescein-Conjugated Lectins

Membrane-associated carbohydrate residues of 3 isolates of Leishmania derived from etiological agents of visceral leishmaniasis (VL), postkala-azar dermal leishmaniasis (PKDL), and cutaneous leishmaniasis (CL), as well as 2 other nonpathogenic insect gut kinetoplastid flagellates, Bodo sp. and Herpetomonas sp., were characterized with the aid of 8 fluorescein-conjugated lectins. Four lectins, concanavalin A, Dolichos biflorus, phytohemagglutinin P, Ricinus communis agglutinin, bound to all kinetoplastid flagellates at different concentrations. All Leishmania promastigotes showed reactions with Ulex agglutinin. Although these lectins were bound to all kinetoplastids, the site and intensity of binding was different. All skin-dwelling Leishmania parasites, viz., Leishmania donovani of PKDL and Leishmania tropica of CL showed unique selectivity toward peanut agglutinin (PNA), soybean agglutinin, and wheatgerm agglutinin (WGA). More interestingly, Herpetomonas showed positive fluorescence with PNA and WGA, whereas Bodo was negative. The results demonstrated that no lectin could distinguish between the pathogenic and nonpathogenic status of kinetoplastid flagellates. Moreover, the antigenic (carbohydrate) profiles of Herpetomonas corresponded more closely to those of L. tropica, whereas Bodo shared some common lectin receptors with L. donovani of VL.

Rapid Detection of Acanthamoeba Cysts in Corneal Scrapings by Lactophenol Cotton Blue Staining

To the Editor. — Acanthamoeba keratitis is increasingly being reported from developing countries where contact lens wear is not necessarily a predisposing factor. 1 This is mainly due to the increased awareness and heightened suspicion of the occurrence of this form of keratitis. Currently, direct microscopic examination of smears of ulcer scrapings stained by the Giemsa, Gram's, or Wright's staining techniques affords the simplest and least expensive means of arriving at a rapid presumptive diagnosis of this condition. In such smears, however, the organisms may not always be readily distinguished from the surrounding tissue cells. Other methods, involving the use of calcofluor white, 2 fluorescent antibodies, 3 and fluorescein-conjugated lectins, 4 are very specific, but they are elaborate, expensive, and require the use of special microscopes. We have treated three patients with Acanthamoeba keratitis where the diagnosis was first suspected by the detection of double-walled, cystlike structures in corneal scrapings

Detection of hemicelluloses specific to the cell wall of tracheary elements and phloem cells by fluorescein-conjugated lectins

Binding of fluorescein-conjugated wheat-germ agglutinin (F-WGA) and some other lectins to tissues from various plants were examined by epifluorescence microscopy. F-WGA bound specifically to the walls of tracheary elements (TEs) and phloem cells of pea roots. The binding sites in TEs were localized only in the secondary thickening and became evident at very early stages of differentiation. Fluorescein-conjugated derivatives ofSolanum tuberosum lectin,Lycopersicon esculentum lectin, andDatura stramonium lectin, which bind N-acetylglucosamine residues as WGA, also bound to the secondary thickening of TEs of pea roots. The binding sites for F-WGA were not removed by extraction with hot EDTA and proteinase K, but removed by extraction with an alkali solution. The alkali-extracted binding sites from the roots were precipitated together with hemicelluloses by 80% ethanol. These results indicate that the binding sites are not present on pectins, proteins, or cellulose, but hemicelluloses. Localized distribution of the binding sites for F-WGA in TEs was found also in a variety of angiosperm plants.