Biomedical subjects

V H Latham

Publications and source records attributed to V H Latham.

7 recordsLinked to original sources

PubMed1999

A simple image analysis method for evaluating cell binding to derivatized beads.

We have previously developed a novel method to determine the presence or absence of cell surface lectin receptors using derivatized agarose beads and have used many different derivatized beads in examining other cell surface properties. Thus, we have used beads that were previously utilized mainly for chromatography purposes in a new approach to study cell surface histochemistry. The previous studies analysed cell-bead binding using subjective plus-minus descriptions that suffered from lack of quantification. We needed a quantitative method in this new approach for histochemical cell surface studies. Here we describe a new method using image analysis of photographs that we developed to solve the quantification problem. The histogram feature of Adobe Photoshop was used to analyse cell binding to beads that were isolated by computer. The method is simple and works as more cells bind to the beads, the beads appear darker in color, a feature easily and precisely quantified using Adobe Photoshop.

Animals↗

PubMed1999

A putative role for carbohydrates in sea urchin gastrulation.

Many studies have examined the effects of lectins on embryonic development. Recently, it has been shown that lectins actually enter the blastocoel of sea urchin embryos without microinjection and bind to specific cell types. The present study was performed to examine the effects of lectins on sea urchin gastrulation. Strongylocentrotus purpuratus sea urchin embryos were incubated with several lectins at concentrations from 0.01 microgram/ml to 100 micrograms/ml at 15-28 h in the presence or absence of the preferential binding sugars. The most interesting findings were that the mannose specific lectins Lens culinaris agglutinin (LcH) which binds to secondary mesenchyme cells involved in archenteron anchoring and Pisum sativum (PSA) caused exogastrulation. Wheat germ agglutinin (WGA) which binds to primary mesenchyme cells involved in skeletogenesis caused defective skeletogenesis. Our findings suggest that D-mannose-like residues (LcH and PSA specific sugar) may function in archenteron development and anchoring, while N-acetyl-D-glucosamine-like groups (WGA specific sugar) may contribute to control of primary mesenchyme positioning and function. Specific carbohydrate-containing receptors may, therefore, be of importance in specific gastrulation events.

Animals↗

PubMed1998

Accessing the embryo interior without microinjection.

For decades it has been assumed that in order to insert macromolecules into the embryo blastocoel for numerous experimental purposes, microinjection was required. Microinjection, however, can be only performed on a few embryos at a time, thus precluding many studies that could involve large populations of embryos. Laser scanning confocal microscopy, with its optical sectioning advantage, showed that fluorochrome-labeled macromolecular lectins and bovine albumin enter the blastocoel of living, swimming sea urchin embryos following a period of incubation without microinjection. A procedure is also described that shows macromolecular entry is substantially accelerated in low calcium seawater. The information gained from this study should greatly facilitate experiments on entire populations of millions of embryos at a time that require access of macromolecules to the embryo interior.

Animals↗

PubMed1996

Desktop computer-based image analysis of cell surface fluorescence patterning from a photographic source.

We report the use of standard computer-based image analysis technology to analyze, from a photographic source, individual cell surface receptor site patterns using fluorochrome labeling. The model used in this study was a Strongylocentrotus purpuratus sea urchin embryo labeled with fluorescein isothiocyanate-conjugated wheat germ agglutinin (FITC-WGA) (0.5 mg/ml for 5 min at 15 degrees C incubated with fertilization membrane free embryos). Image capture was performed using desktop-type digital scanning, and the images were imported into Adobe Photoshop for study. All images analyzed were derived from a single photographic negative: 1) the actual micrograph printed from the negative and scanned into a Macintosh IIx computer; 2) the scanned negative itself; and, 3) a high resolution scanning process used with a Kodak Photo CD. Patterns were analyzed using the densitometry feature of Photoshop, and were similar enough from all 3 scanned images to indicate that any of the 3 scanning processes can be used for fluorescence patterning analysis. Adobe Photoshop was also used to highlight, for closer analysis, the fluorescence patterns by producing 3-D effects, border mapping and transition area detailing. The desktop image analysis procedures described here to study fluorescence patterning require no expensive scientific hardware or software.

Animals↗

PubMed1995

Rapid identification of lectin receptors and their possible function in sea urchin cell systems.

An assay using lectin derivatized agarose beads to rapidly and inexpensively identify cell surface lectin receptors was recently described by Latham et al. (1995). In this earlier study, the assay was tested on large, early stage sea urchin embryo cells. In this study this assay was used to examine lectin receptors on small, later stage sea urchin embryo cells that are more typical of cells that most investigators deal with, to ascertain if cell size is a determining factor in the assay's validity. The results indicated that the assay is a valid method to identify lectin receptors on small as well as large cells. Twenty-three hour Strongylocentrotus purpuratus embryo cells strongly bound Triticum vulgaris, concanavalin A, Artocarpus integrifolia and Vicia villosa using both the agarose bead and fluorescence assays, while three other lectins, Ulex europaeus I, Lotus tetragonolobus and Lens culinaris did not strongly bind to the cells using these two assays. As in earlier studies agglutinability results did not correlate well with results using the two other assays. In all cases where lectin bead binding, fluorescent lectin binding or lectin-mediated agglutination occurred, specific sugars reduced the observed binding. The second part of this study examined the putative role of concanavilin A receptors in a specific cellular interaction: sperm-egg binding. Concanavalin A inhibited fertilization of dejellied sea urchin eggs when their vitelline layers were intact and to a lesser extent when their vitelline layers were removed. This effect was counteracted by alpha methyl glucose. The major differences between these studies and previous work is that here concanavalin A was washed out after incubation with eggs, making it more likely that results reflect binding to cell surface lectin receptors rather than toxicity. In addition, performing the experiments on eggs with or without vitelline layers provided information on the location of concanavalin A receptors that may function in sperm-egg interaction.

Agglutination Tests↗

PubMed1995

A rapid lectin receptor binding assay: comparative evaluation of sea urchin embryo cell surface lectin receptors.

Lectin receptor binding assays, such as those that utilize fluorescence, radioactivity or electron microscopy are not designed for rapidly screening hundreds of cell types for the presence or absence of specific lectin receptors. An assay is described here that is designated for this purpose. It utilizes lectins derivatized to agarose beads and can be used to screen many cell types in min. This assay was used to examine lectin receptors on the surfaces of 1-8 cell stage Strongylocentrotus purpuratus sea urchin embryos. The same cells were also assayed using standard fluorescence and agglutinability procedures to ascertain the type of information obtained by the new assay and how it correlates with results from the standard methods. The bead results correlated well with results using fluorescent lectin. Only wheat germ agglutinin bound very strongly in both bead and fluorescence assays, while concanavalin A, Dolichos biflorus, Lens culinaris and Tetragonolobus purpureas did not bind or bound weakly using both methods. Results using a third method, lectin mediated cell agglutination, did not correlate with the bead or fluorescence assays. Lectin receptors were also examined on embryos prepared by two different methods of preventing formation of fertilization membranes, so that coat-free cell surfaces could be studied, the standard dithiothreitol method and a new method using alpha-amylase. Lectin receptors on the cell surfaces of embryos prepared by both methods were nearly identical. The possible functions of WGA receptors, the most prevalent lectin receptors of those studied, that were uniformly present throughout early development of this sea urchin species, are considered.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Producing exposed coat-free embryos.

Production of embryos that are free of tough outer coats facilitates studies that are not possible with embryos surrounded by impenetrable envelopes. This report describes a new procedure for preventing formation of fertilisation membranes in the sea urchin (Lytechinus pictus) model. This procedure involves treating unfertilised eggs with the enzyme alpha-amylase, which cleaves alpha-1,4 glucosidic bonds in the vitelline layer. A major advantage of this method is that it is very well defined and completely controllable with alpha-amylase inhibitor. The results suggest that intact alpha-1,4 glucosidic bonds are essential for vitelline layer integrity required for formation of the fertilisation membrane. Eggs treated with alpha-amylase possessed the same surface lectin receptors as untreated eggs and, as shown by light and transmission electron microscopy, produced healthy, cleaving embryos that were free of fertilisation envelopes.

Animals↗