Enzyme-amplified rate conductimetric immunoassay

A new immunoassay technique based on measurement of conductance changes in solutions is described. The assay employs an immobilized monoclonal antibody to capture a protein analyte along with a second antibody to the same analyte, conjugated to an enzyme capable of producing ions which are measured conductimetrically. Urease was selected as the enzyme, because it produces, from urea, four ions for each catalytic event. The analyte studied was human chorionic gonadotropin in serum. Higher concentrations of analyte during incubation with immobilized antibody and antibody-urease conjugate led to increased binding of the latter. After removal of unbound conjugate, urea solution was added and the rate of conductance change measured in the bulk substrate solution. Experiments, performed in polystyrene microtiter wells using a specially designed electrode, demonstrated the ability to measure 30 picomolar concentrations of human chorionic gonadotropin with a 30-s rate measurement. Urease proved to be an excellent labeling enzyme, retaining its activity under the nonionic conditions necessary to maintain low background conductance. Good agreement was obtained between observed rates and those expected from conductimetric theory and known physical parameters. The potential utility of the conductimetric immunoassay lies in the fabrication of biosensor devices for simplification and cost reduction of immunochemical-based instrumentation. Further improvements to the technique are proposed to achieve lower detection limits.     

The "linker" region (amino acids 38-47) of the disintegrin elegantin is a novel inhibitory domain of integrin alpha5beta1-dependent cell adhesion on fibronectin: evidence for the negative regulation of fibronectin synergy site biological activity

Disintegrins are a family of potent inhibitors of cell-cell and cell-matrix adhesion. In this study we have identified a region of the disintegrin elegantin, termed the "linker domain" (amino acids 38-47), with inhibitory activity toward alpha(5)beta(1)-mediated cell adhesion on fibronectin (Fn). Using a chimeric structure-function approach in which sequences of the functionally distinct disintegrin kistrin were introduced into the elegantin template at targeted sites, a loss of inhibitory function toward alpha(5)beta(1)-mediated adhesion on Fn was observed when the elegantin linker domain was substituted. Subsequent analysis comparing the inhibitory efficacies of the panel of elegantin-kistrin chimeras toward CHO alpha(5) cell adhesion on recombinant Fn III(6-10) fragments showed that the loss of inhibitory activity associated with the disruption of the elegantin linker domain was dependent upon the presence of a functional Fn III(9) synergy site within the Fn III(6-10) substrate. This suggested that the elegantin linker domain inhibits primarily the activity of the Fn synergy domain in promoting alpha(5)beta(1) integrin-mediated cell adhesion. Construction of a cyclic peptide corresponding to the entire region of the elegantin linker domain showed that this domain has intrinsic alpha(5)beta(1) inhibitory activity comparable with the activity of the RGDS peptide. These data demonstrate a novel biological function for a disintegrin domain that antagonizes integrin-mediated cell adhesion.     

Structure, function, and motility of vacuoles in filamentous fungi

Current information on the structure and function of motile tubular vacuoles in Pisolithus tinctorius and other fungi is reviewed. The use of fluorochromes to label the vacuole lumen is evaluated and observations on the structure and motility of vacuoles in P. tinctorius are differentiated from possible artifacts. The styryl dyes FM4-64 and MDY-64, used in yeast to demonstrate endocytosis, show little or no labeling of internal membranes in undamaged P. tinctorius cells. This agrees with our data showing that other probes for endocytosis such as Lucifer yellow CH are not taken up by hyphal tip cells. Overall, the observations do not support endocytosis in hyphal tips. It has been suggested that tubular vacuole systems carry out longitudinal transport, and evidence in favor of this hypothesis is evaluated. New data are presented to show that many of the large vacuoles in subapical cells are attached to the plasma membrane and are relatively immobile, while video sequences show movement of fluorochrome in pulses along a series of several large vacuoles, all interconnected via tubules. Tubular vacuoles from thick sections of hyphae processed under anhydrous conditions are shown by X-ray microanalysis to contain relatively high levels of P and K, as seen previously in the larger vacuoles. These results provide further evidence for a role of the tubular vacuoles in longitudinal transport of P. Copyright 1998 Academic Press.     

Rapid and sensitive GC/MS characterization of glycolipid released Galalpha1,3Gal-terminated oligosaccharides from small organ specimens of a single pig

Pig to human xenotransplantation is considered a possible solution to the prevailing chronic lack of human donor organs for allotransplantation. The Galalpha1,3Gal determinant is the major porcine xenogeneic epitope causing hyperacute rejection following human antibody binding and complement activation. In order to characterize the tissue distribution of Galalpha1,3Gal-containing and blood group- type glycosphingolipids in pig, acid and nonacid glycosphingolipids were isolated from the kidney, small intestine, spleen, salivary gland, liver, and heart of a single pig obtained from a semi-inbred strain homozygous at the SLA locus. Glycolipids were analyzed by thin-layer immunostaining using monoclonal antibodies, and following ceramide glycanase cleavage as permethylated oligosaccharides by gas chromatography, gas chromatography-mass spectrometry, and matrix- assisted laser desorption/ionization mass spectrometry. The kidney contained large amounts of Galalpha1,3Gal-containing penta- and hexasaccharides having carbohydrate sequences consistent with the Galalpha1,3nLc4and Galalpha1,3Lexstructures, respectively. The former structure was tentatively identified in all organs by GC/MS. The presence of extended Galalpha1,3Gal-terminated structures in the kidney and heart was suggested by antibody binding, and GC/MS indicated the presence of a Galalpha1,3nLc6structure in the heart. The kidney, spleen, and heart contained blood group H pentaglycosylceramides based on type 1 (H-5-1) and type 2 (H-5-2) chains, and H hexaglycosylceramides based on the type 4 chain (H-6-4). In the intestine H-5-1 and H-6-4 were expressed, in the salivary gland H-5-1 and H-5-2, whereas only the H-5-1 structure was identified in the liver. Blood group A structures were identified in the salivary gland and the heart by antibody binding and GC/MS, indicating an organ- specific expression of blood group AH antigens in the pig.      

Detection and quantification of Lyme spirochetes using sensitive and specific molecular beacon probes

Background  

Lyme disease, caused by Borrelia burgdorferi, affects a large number of people in both the USA and Europe. The mouse is a natural host for this spirochete and is widely used as a model system to study Lyme pathogenesis mechanisms. Since disease manifestations often depend upon the spirochete burden in a particular tissue, it is critical to accurately measure the bacterial number in infected tissues. The current methods either lack sensitivity and specificity (SYBR Green), or require independent analysis of samples in parallel to quantitate host and bacterial DNA (TaqMan). We have developed a novel molecular beacon-based convenient multiplex real-time quantitative PCR assay to identify and detect small numbers of B. burgdorferi in infected mouse tissues.     

CORTICAL ONTOGENY IN ROOTS. I. ZEA MAYS

Serial growth stages of young Zea mays primary roots were analyzed for patterns of ground meristem ontogeny. The number of cell layers in the cortex decreases from approximately 15 to 11 during early root growth. The cortex arises mostly by periclinal divisions in the outer portions of the ground meristem at levels 50–150 μm from the meristem tip, although some layers of outer cortex arise beyond 150 μm. The proendodermis contributes 3–5 cell layers to the cortex, but this contribution diminishes during early seedling growth as anticlinal divisions occur in the proendodermis. The relationship between the ground meristem and protoderm changes at the tip of the meristem during root elongation.     

APICAL ORGANIZATION IN ROOTS OF THE CONVOLVULACEAE

A sampling of radicles and germinated primary roots was obtained for several species of the Convolvulaceae in order to study ontogenetically the organization of the protomeristem. The findings differ from earlier studies in that only a few radicles and primary roots exhibited proto-meristems with layered initials. Most radicles exhibited a pattern of layered initials in which the outer cell layers of cortex were not aligned with a layer of cortical initials but with cells at the base of the columella. In long primary roots the protomeristem, consisting of central cylinder initials, lateral rootcap-epidermal initials, and common initials for the cortex and columella, was the general pattern for the members of the family. The reorganization of the protomeristem from a layered condition to common initials generally occurred upon germination. Different expressions of intermediate patterns indicated that periclinal divisions in the outer layers of cortex near the initials or in the immediate derivatives of the cortical initials were responsible for cellular displacements and new cell alignments. After reorganization, periclinal divisions in the peripheral portions of the common initials maintained the cellular continuity from the cortex into the columella. In addition, a few primary roots with a degenerated columella and a transversal meristem were observed.