A novel, sensitive detection system for high-density microarrays using dendrimer technology.

To improve signal detection on cDNA microarrays, we adapted a fluorescent oligonucleotide dendrimeric signal amplification system to microarray technology. This signal detection method requires 16-fold less RNA for probe synthesis, does not depend on the incorporation of fluorescent dNTPs into a reverse transcription reaction, generates a high signal-to-background ratio, and can be used to allow for multichannel detection on a single chip. Furthermore, since the dendrimers can be detected individually, it may be possible, by employing dendrimer-binding standards, to calculate the numbers of bound cDNAs can be estimated. These features make the dendrimer signal detection reagent ideal for high-throughput functional genomics research.

Prospective use of DNA microarrays for evaluating renal function and disease.

At the forefront of the revolution in human genomics is DNA microarray technology, which evaluates expression levels or genotypes of thousands of genes simultaneously, by means of miniaturization and parallel processing. Furthermore, advances in bioinformatics will result in the creation of large databases, which will require complex software programming for structural analysis. Over the next decade, DNA microarrays, combined with sophisticated informatics and genomic databases, will provide molecular fingerprints of disease processes and prognoses. This review provides an update on DNA microarray technology and its application to renal diseases.

Mapping sperm binding domains on the sea urchin egg receptor for sperm.

The receptor on the surface of the egg of the sea urchin Strongylocentrotus purpuratus that mediates species-specific binding of sperm is a 350-kDa cell surface glycoprotein. Earlier studies established that a recombinant protein encompassing a major portion of the N-terminal half of the receptor inhibited fertilization when tested in a competitive fertilization bioassay. To identify in more detail the sites in this domain of the receptor that are involved in binding sperm, a series of deletion constructs were expressed as glutathione S-transferase fusion proteins and tested for inhibitory activity in a fertilization bioassay. In addition, a novel assay for directly testing the sperm binding activity of these proteins was developed. In this assay we quantitated sperm binding to recombinant proteins representing various domains of the receptor immobilized on glutathione agarose beads. Using this new assay, two domains in the N-terminal half of the receptor were found to be involved in sperm binding. One of the peptide domains, composed of 247 amino acids, binds both the sperm of S. purpuratus and the sperm of another genus of sea urchin, Lytechinus pictus. In contrast, binding to the second domain consisting of a 32-amino-acid residue peptide was found to be genus specific; no binding of L. pictus sperm was observed. A working model is proposed incorporating these findings with earlier studies on the function of the oligosaccharide chains of the receptor. In this model it is postulated that the sperm initially interact with the nonspecific binding domain on the polypeptide and the sulfated O-linked oligosaccharide chains of the receptor. This interaction is followed by binding to the more specific polypeptide binding site on the receptor. It is proposed that only subsequent to binding at this second site can gamete fusion occur.

Sea urchin egg receptor for sperm: the oligosaccharide chains stabilize sperm binding.

Sulfated O-linked oligosaccharides from the sea urchin egg receptor have been shown to bind to acrosome-reacted sperm and to inhibit fertilization in a competitive bioassay. However, the inhibitory activity of these isolated chains was much lower than that of a recombinant protein representing a portion of the extracellular domain of the receptor. Because the isolated oligosaccharides lacked the potential polyvalency that they might have when linked to the polypeptide backbone, in the current study we asked if their inhibitory activity could be increased by chemically coupling them to a protein to form a neoglycoprotein. Using a recombinant fragment of the receptor we could not detect an oligosaccharide dependent increase in inhibitory activity with this neoglycoprotein, probably because of the much higher inhibitory activity of the polypeptide backbone. Therefore, we examined the activity of the oligosaccharides coupled to a protein lacking the ability to inhibit fertilization, namely, bovine serum albumin. A marked increase in the inhibitory activity of the oligosaccharides was observed with this neoglycoprotein. Finally, because inhibition by the oligosaccharides and the polypeptide was measured in an end point assay, namely, inhibition of fertilization, we sought a more direct, kinetically sensitive way to measure their properties. Accordingly, an assay was devised (R.L. Stears and W.J. Lennarz, unpublished observations) involving measurement of sperm binding to beads that was dependent on the presence of the receptor or its components. This assay revealed that sperm binding to beads via the recombinant protein peaked at 10 sec and then declined. In contrast, binding mediated by neoglycosylated recombinant protein reached a plateau. Thus, binding of sperm to the oligosaccharides resulted in a more stable interaction than that observed in binding to the polypeptide backbone.

Trigeminal nerve and ganglion in the Meckel cave: appearance at CT and MR imaging.

PURPOSE: To demonstrate the anatomy of the Meckel cave, which is normally seen by means of dissection, with high-resolution computed tomography (CT) and magnetic resonance (MR) imaging. MATERIALS AND METHODS: Twenty cadaver specimens were scanned with 1-mm contiguous axial and coronal CT sections. Seven specimens were also scanned with 1-mm contiguous parasagittal sections and were dissected for correlation with CT images. Two volunteers also underwent high-resolution, fast spin-echo MR imaging. MR images were compared with the CT images and the results of dissection. RESULTS: Dissection and CT and MR imaging demonstrated that the trigeminal nerve within the Meckel cave consists of numerous small fibers and that the trigeminal ganglion consists of a small amount of solid tissue. CONCLUSION: In vivo, high-resolution, fast spin-echo imaging can demonstrate the anatomy of the trigeminal nerve in the Meckel cave because of improved spatial and contrast resolution.

Developmental expression of the sea urchin egg receptor for sperm.

Little is known about the biochemical changes underlying the morphological differentiation of the sea urchin egg during oogenesis. Because of this and the essential role of gamete recognition in fertilization, we studied the developmental expression of the recently identified egg surface receptor for sperm during oogenesis. Consecutive stages of ovaries undergoing oogenesis over a 4-month time course were examined morphologically and assessed with respect to content of sperm receptor mRNA, as well as the content and subcellular distribution of the sperm receptor glycoprotein. Although in early oocyte stages neither mRNA encoding for the receptor nor receptor glycoprotein was detectable, at the last two stages of development the level of receptor mRNA accumulation increased dramatically. This finding correlated well with immunoblot analyses which established that sperm receptor protein was only detectable at the last two stages of egg maturation. Interestingly, immunocytochemistry showed that the formation of the receptor correlated temporally and spatially with the formation of cortical granules. In the earlier of these two stages of maturation, the receptor population identified by immunoblotting was found by immunocytochemistry to be restricted to the cortical granules and small vesicles in the cytoplasm. In contrast, at the last stage of egg maturation, sperm receptor was also detected at the surface of the oocyte, localized predominantly to the microvilli. Two receptor populations appear to exist, one in cortical granules and a second at the cell surface that may be formed via secretory vesicles. The late appearance of the receptor on the plasma membrane during oogenesis is consistent with its biological role in binding sperm to the mature egg cell surface.

Diagnosis of bowel obstruction on plain abdominal radiographs: significance of air-fluid levels at different heights in the same loop of bowel.

OBJECTIVE: Differential air-fluid levels are two distinct air-fluid interfaces on horizontal-beam abdominal radiographs that are at different heights but within the same loop of bowel. Differential air-fluid levels have been considered by many to be strong evidence of mechanical bowel obstruction, but others have found this sign unreliable for differentiating mechanical from adynamic obstructions. Neither opinion is supported by evidence from large series of patients. Accordingly, we determined the efficacy of differential air-fluid levels for distinguishing mechanical from adynamic bowel obstruction. MATERIALS AND METHODS: We identified patients who had a total of 62 episodes of proved mechanical bowel obstruction and 38 episodes of adynamic obstruction through a computer search of medical records and radiographic files. On horizontal-beam abdominal radiographs of these patients, the presence and height of intestinal differential air-fluid levels were determined by the consensus of two experienced radiologists. These data were then statistically analyzed to determine the usefulness of differential air-fluid levels for distinguishing between mechanical and adynamic bowel obstructions. RESULTS: Plain films showed differential air-fluid levels in 32 (52%) of the 62 episodes of mechanical obstructions compared with 11 (29%) of the 38 adynamic obstructions, giving a sensitivity for mechanical obstruction of 0.52 and a specificity of 0.71. As the minimum significant height of differential air-fluid levels increased, specificity increased and sensitivity decreased. The positive predictive value also increased as differential air-fluid level heights increased, reaching a level of 0.86 or greater at 20 mm. CONCLUSION: The presence of differential air-fluid levels is an insensitive method of determining if a bowel obstruction is mechanical, because only a small proportion of mechanical obstructions have differential air-fluid levels. In our population of patients, however, a differential air-fluid level of 20 mm or greater was moderately suggestive that a bowel obstruction was mechanical in nature.

Biological calcium absorption edge imaging using monochromatic synchrotron radiation.

Soft X-ray contact absorption edge images of unfixed, unstained biological specimens were made using monochromatic synchrotron radiation. X-ray contact replicas of unfixed, hydrated biological specimens at the nitrogen absorption edge and above and below the CaLIII absorption edge were compared to comparative conventional morphological and elemental high-resolution imaging methods (scanning and transmission electron microscopy, TEM-histochemistry and TEM-X-ray microanalysis). Soft X-ray absorption edge images made above the calcium absorption edge clearly revealed morphological detail and identified regions ladened with calcium as verified by TEM histochemistry of identical spores. Similarly, nitrogen absorption edge images identified residual nitrogenous material in the spore resuspension medium, and non-viable spores with nitrogen loss due to protoplast disaggregation.