Bruch''s membrane of the brachymorphic mouse

Bruch's membrane exists between the retinal pigment epithelium and the choriocapillary endothelium. Its structure is very complicated, having five sublayers containing basement membranes of retinal pigment epithelium and choriocapillary endothelium, outer and inner collagenous layers, and a central elastic layer. In the development of Bruch's membrane in normal mice, both basement membranes are created first. Secondarily, collagen fibers are accumulated in the space between these basement membranes and then form a collagenous layer. Finally, the elastic layer elaborated in the collagenous layer separates this into outer and inner collagenous layers. Brachymorphic mice have a disorder in the sulfation pathway, resulting in undersulfation. Consequently, in Bruch's membrane of brachymorphic mice, the expression of decorin, a small proteoglycan containing chondroitin sulfate and an indispensable component in collagen assembly, is at a very low level. It is clear that hypoplasia of the collagenous layer in Bruch's membrane of brachymorphic mice induces a disorder in the following formation of the elastic layer. These findings suggest that the formation of the collagenous layer, regulated with acidic glycoconjugates such as decorin, is important in the development of Bruch's membrane.     

VALIDATION OF A HIGH SENSITIVE IMMUNOENZYMATIC ASSAY TO ESTABLISH THE ORIGIN OF IMMUNOGLOBULINS IN FEMALE GENITAL SECRETIONS

ABSTRACT

Several studies were carried out to characterize the humoral immune response on mucosal genital surfaces. However, the results obtained so far were particularly conflicting due to the absence of validation methods. The aim of this study was to develop and validate a quantitative ELISA method, which is sensitive and reproducible, to measure immunoglobulin and secretory immunoglobulin concentrations in various biological fluids. This quantitative, sensitive (detection limit = 1 µg/L) and reproducible (coefficient of variation <15%) method could be of interest to study the effects of viral infections on mucosal non-specific immune response in genital tract. To explore the humoral response, serum, saliva, vaginal secretions, and cervicovaginal secretions from 18 women, 20–45 years old, were evaluated for total-IgA, secretory IgA, IgM, and IgG. Albumin level was also evaluated by immuno-nephelometry. The secretion rates of immunoglobulins were measured by calculating their relative coefficients of excretion by reference to albumin. Despite large individual variations, median immunoglobulin levels were higher in the endocervical secretions than in the cervicovaginal secretions. When we compared the rates of immunoglobulins in genital fluids, IgG prevalence was higher (80%) in cervicovaginal and endocervical secretions than IgA prevalence (12%). In contrast, digestive mucosal secretions, such as saliva, contained mostly IgA (80%). In cervicovaginal and endocervical secretions, IgG and IgM originated mainly from serum, whereas a local synthesis provided total-IgA and secretory IgA. These results allowed us to raise a possible hypothesis for the origin of immunoglobulins in the genital tract. They illustrated the peculiar feature of the female reproductive tract and the difficulty for this tissue to contribute in the mucosal associated lymphoid tissue. The low secretory-IgA and total-IgA levels could explain the particular sensitivity of the vagina and the cervix to infections.     

Differences in the ultrastructural localization of sulfated glycoconjugates between dentine and bone in the ganoid scales of Polypterus senegalus

ObjectivesSulfated glycoconjugates (S-GCs) are essential components of dentinogenesis and osteogenesis, and are involved in the regulation of the mineralization process. The scales of Polypterus senegalus, which are homologous to teeth and phylogenetically derived from ancestral dermal skeleton, comprise true enamel, dentine, and bone. As part of the phylogenic evolutional studies on mesenchyme-derived hard tissues, we investigated the ultrastructural distribution patterns and histochemical properties of S-GCs in the dentine and bone of scales.MethodsFor detection of S-GCs, a high iron diamine–thiocarbohydrazide–silver proteinate (HID–TCH–SP) staining technique was used.ResultsS-GCs were observed in unmineralized predentine and osteoid, mineralized dentine, osteocyte pericellular matrix, but not in mineralized bone and isopedine. Most S-GCs in the dentine and osteoid were susceptible to testicular hyaluronidase, indicating that they are chondroitin sulfates.ConclusionDistinct differences in the S-GC distribution pattern between the dentine and bone were observed. The S-GC distribution pattern associated with the dentinogenesis of scales is very similar to that of mammalian mantle dentine.     

Distribution of glycoconjugates in normal rat ventral prostate and their use as markers of androgen-controlled secretory function in culture

The distribution of glycoconjugates in uncultured and cultured rat ventral prostate was studied by using eight fluorescent lectins. The prostate pieces were cultured in defined medium with or without testosterone for 1-14 days. Each lectin revealed a characteristic binding pattern. Con A, LCA, WGA, and RCA I stained both epithelial and interstitial components. SBA and PNA were specific for the epithelium: SBA stained the region of the Golgi complex; PNA showed the brightest fluorescence in the apical part of the cells representing the region of secretory granules. In culture without testosterone the epithelial cells gradually lost their fluorescence, whereas the stromal fluorescence increased. The basement membrane was disorganized. With testosterone the integrity of the epithelium and stroma was maintained, and the staining pattern of the lectins was in the main similar as in vivo. However, at 14 days a change in the staining pattern of apical cytoplasm with PNA was noted, indicating that in long-term cultures, in addition to testosterone, other hormones and growth factors are necessary to complete especially the last stages of the secretory process in the epithelial cells.     

Preliminary Study of the Lectin Receptor Expression in Adenocarcinomas of the Gastrointestinal Tract

Ten cases of gastrointestinal adenocarcinomas were histochemically investigated fortheir lectin receptors by avidin—biotin-peroxidase complex(ABC)method.Results showed thatgastric adenocarcinomas expressed more types of lectin receptors than large boweladenocarcinomas.Positive rates of Glc/Man-and GlcNAc-specific lectin receptors in these tu-mors were kigher than those of Gal/GalNAc-and Fuc-specific lectin receptors.From the stom-ach to rectum,the spectrum of the lectin receptors in pericarcinomatous tissues graduallychanged from the gastric type to the rectal type.The expression of lectin receptors ingustrointestinal adenocarcinomas was obviously different Jrom that in their correspondingpericarcinomatous tissues.The increase and decrease of lectin receptor expression,theappearance and disappearance of some types of lectin receptors as well as the change of thereceptor distribution are indirectly regulated by cellular genome.     

Neoplastic cells as targets of spontaneously cytotoxic lymphocytes: studies with natural killer-like cell lines

Summary Native natural killer (NK) cells comprise a heterogeneous family of lymphocytes distributed among several organs, which display spontaneous cytotoxic reactions directed against a broad range of tumor targets. In these studies, murine cell lines have been established in vitro following the selective expansion of bone marrow-and spleen-derived killer progenitors in culture medium supplemented with interleukin-2. Several clones of independent origin have been characterized in order to determine the extent of their phenotypic and functional diversity. With few exceptions most of them were found to be highly effective in lysing a variety of tumor cell lines, to share common cell surface alloantigens, lectin-binding receptors, and cytochemical markers. The presence of prominent azurophilic cytoplasmic granules is the most characteristic ultrastructural feature of these cells. In attempting to elucidate the nature of membrane components specifically recognized by NK cells we compared several isogenic tumor cell variants selected on the basis of their differential NK susceptibility, immunogenicity, metastatic potential or resistance to cytotoxic plant lectins. Sialylated glycoconjugates exposed on the external face of the tumor cell membrane appear to be essential determinants in the interaction between NK cells and their targets. Permanent cell lines retaining most of the functional attributes of endogenous NK cells may prove instrumental in understanding their role during tumor progression.     

Galectins as markers of aggressiveness of mouse mammary carcinoma: towards a lectin target therapy of human breast cancer

Summary Galectins, β-galactoside binding proteins, expressed selectively in human breast carcinoma are attractive targets to employ lectin-aimed therapeutics. We examined β-galactoside binding potency of neoplastic cells using fluorescein-labelled synthetic glycoconjugates as probes for flow cytometry. As a result, surface β-galactoside binding proteins/galectins were discovered on mouse mammary carcinoma cells in vitro and in vivo unlike non-malignant cells from the several tissues; and asialo-GM1 ganglioside carbohydrate part - containing probe was the most specific one. However, in liver and lung metastatic cells galectins seem to be expressed within cytoplasm and/or nuclei. Galectin expression correlated directly with aggressive tumour potential in the A/Sn transplantable model similar to findings in several human breast carcinoma cell lines. However, galectin expression was reduced during tumour progression in more aggressive forms of spontaneous BLRB mammary carcinomas like it was shown for human breast carcinoma specimens. Analysis of the histopathological data led, however, to the conclusion that galectin expression hardly might be a suitable marker of aggressiveness of heterogeneous mammary carcinomas as the observed level of galectin expression is influenced by the amount of the stroma in a tumour sample and/or probably, galectin expression inversely correlates with tumour aggressiveness during the initial and advanced steps of mammary tumour progression. We conclude that surface β-galactoside binding proteins/galectins that are selectively expressed during mouse mammary carcinoma progression, similarly to human breast carcinomas, seem to be proper targets for asialo-GM1-vectored cytotoxics and our mouse model system might be a relevant instrument to further test novel modes of anti-breast cancer therapy. An erratum to this artcile can be found at     

Regulated expression of the Leishmania major surface virulence factor lipophosphoglycan using conditionally destabilized fusion proteins

Surface glycoconjugates play important roles in the infectious cycle of Leishmania major, including the abundant lipophosphoglycan (LPG) implicated in parasite survival in the sand fly vector and the initial stages of establishment in the mammalian host macrophage. We describe a system for inducible expression of LPG, applying a novel protein-based system that allows controlled degradation of a key LPG biosynthetic enzyme, UDP-galactopyranose mutase (UGM). This methodology relies on a mutated FK506-binding protein (FKBP) destabilizing domain (dd) fused to the protein of interest; in the absence of rapamycin analogs, such as Shld1, the dd domain is destabilized, leading to proteasomal degradation, whereas drug treatment confers stabilization. Tests in L. major using dd fusions to a panel of reporters and cellular proteins confirmed its functionality, with a high degree of regulation and low background, and we established the kinetics of protein activation and/or loss. Two inexpensive and widely available ligands, FK506 and rapamycin, functioned similarly to Shld1, without effect on Leishmania growth or differentiation. We generated parasites lacking UGM through deletion of the GLF gene and substitution with a ddGLF fusion construct, either as chromosomal knockins or through episomal complementation; these showed little or no LPG expression in the absence of inducer, whereas in its presence, high levels of LPG were attained rapidly. Complement lysis tests confirmed the correct integrity of the Leishmania LPG coat. These data suggest that the dd approach has great promise in the study of LPG and other pathways relevant to parasite survival and virulence.     

Glycobiology of the Leishmania parasite and emerging targets for antileishmanial drug discovery

Importance of the field: Parasitic diseases that pose a threat to human life include leishmaniasis – caused by protozoa of Leishmania species. Existing drugs have limitations due to deleterious side effects like teratogenicity and factors like cost and drug resistance, thus furthering the need to develop this area of research.

Areas covered in this review: We came across drug targets, very recently characterised, cloned and validated by genomics and bioinformatics. We bring these promising drug targets into focus so that they can be explored to their fullest.

What the reader will gain: In an effort to bridge the gaps between existing knowledge and future prospects of drug discovery, we found interesting studies validating drug targets and paving the way for better experiments to be designed. In a few cases, novel pathways have been characterized, while in others, well established pathways when probed further, led to the discovery of new drug targets.

Take home message: The review constitutes a comprehensive report on upcoming drug targets, with emphasis on glycosylphosphatidylinositol (GPI)-anchored glycoconjugates along with related biochemistry of enolase, glycosome and purine salvage pathways, as we strive to bring ourselves a step closer to being able to combat this deadly disease.