Solid-state (207)Pb NMR studies of lead-group 16 and mixed transition-metal/lead-group 16 element-containing materials.

(207)Pb solid-state NMR studies have been conducted on binary lead-group 16 and mixed transition-metal/lead group 16 materials, correlating the NMR chemical shifts of the materials with their structures. The experimental results show that the (207)Pb chemical shifts are strongly influenced by the local electronic structure. Data are reported for lead selenide, lead selenate, calcium plumbate, strontium plumbite, barium plumbite, lead borate, lead zirconate, lead tungstate, lead meta-tantalate, lead niobate, lead molybdate, lead meta-vanadate, lead sulfite, and lead sulfate.

Profiling human gene expression with cDNA microarrays.

As a result of genomics initiatives worldwide, it has become increasingly easy to obtain cDNA clones representing the 3 ends of many human genes. This unit describes methods that allow these clones to be used as hybridization detectors in a highly parallel assay of gene expression. Protocols are provided for preparing cDNA microarrays, extracting RNA from cells of interest and preparing fluorescently labeled cDNA representations of the message pools, and hybridizing the labeled cDNAs to the microarrays.

Profiling human gene expression with cDNA microarrays.

As a result of genomics initiatives worldwide, it has become increasingly easy to obtain cDNA clones representing the 3' ends of many human genes. This unit describes methods that allow these clones to be used as hybridization detectors in a highly parallel assay of gene expression. Protocols are provided for preparing cDNA microarrays, extracting RNA from cells of interest and preparing fluorescently labeled cDNA representations of the message pools, and hybridizing the labeled cDNAs to the microarrays.

Molecular classification of cutaneous malignant melanoma by gene expression profiling.

The most common human cancers are malignant neoplasms of the skin. Incidence of cutaneous melanoma is rising especially steeply, with minimal progress in non-surgical treatment of advanced disease. Despite significant effort to identify independent predictors of melanoma outcome, no accepted histopathological, molecular or immunohistochemical marker defines subsets of this neoplasm. Accordingly, though melanoma is thought to present with different 'taxonomic' forms, these are considered part of a continuous spectrum rather than discrete entities. Here we report the discovery of a subset of melanomas identified by mathematical analysis of gene expression in a series of samples. Remarkably, many genes underlying the classification of this subset are differentially regulated in invasive melanomas that form primitive tubular networks in vitro, a feature of some highly aggressive metastatic melanomas. Global transcript analysis can identify unrecognized subtypes of cutaneous melanoma and predict experimentally verifiable phenotypic characteristics that may be of importance to disease progression.

Frequent alterations of evolutionarily conserved regions of chromosome 1 in human malignant melanoma.

Recurring alterations of chromosome 1 represent the most frequent site of structural chromosome abnormalities across all human solid tumors, including human cutaneous malignant melanoma. In melanoma, breakpoints involving chromosome 1 appear to accumulate most frequently at the paracentromeric regions, and secondly, to cluster within 1p36. Of interest, these three band regions (1p11-12, 1q21, and 1p36) were simultaneously recognized by a single YAC clone which was isolated from sequences mapping to 1q21. This observation indicates the common and highly conserved nature of sequences residing within these three bands. Because of this finding, we have examined the possible association of these recurring sites of rearrangements of chromosome 1 in malignant melanoma. To elucidate genomic alterations in these regions, we have analyzed melanoma samples simultaneously by fluorescence in situ hybridization (FISH) using both the YAC clone encoding 1p11, 1q21, and 1p36 homologous sequences, and an alpha-satellite probe for the chromosome 1 centromere. Twelve of 20 (60%) randomly selected melanoma cell lines showed detectable rearrangements in one or more of the chromosome 1 band regions. These results provide support for the notion that the homology between these regions is associated with chromosomal instability, and possibly, is of biologic relevance in malignant melanoma.

Microdissection based cloning of a translocation breakpoint in a human malignant melanoma.

Chromosome translocations in human malignancies have identified the genomic location of several important growth-regulatory sequences (e.g., cellular oncogenes and suppressor genes). Melanomas are characterized by recurring chromosome alterations, including deletion or translocations of the long arm of chromosome 6 (6q). This report details our efforts to clone the t(1;6)(q21;q14) breakpoint in a malignant melanoma to further our understanding of the biology of these tumors. The strategy utilized combined microdissection of the translocation chromosome, development and characterization of a DNA microclone library, isolation of cosmids and YACs from the breakpoint region, ordering of clones by two-color metaphase/interphase fluorescence in situ hybridization, and finally, identification of a YAC spanning the translocation breakpoint. By analogy to other tumor systems, molecular examination of the chromosome 6 breakpoint may provide insight into the pathobiology of this important neoplasm.

A disease locus for hereditary haemorrhagic telangiectasia maps to chromosome 9q33-34.

Hereditary haemorrhagic telangiectasia (HHT), or Osler-Weber-Rendu disease, is an autosomal dominant vascular dysplasia of unknown pathogenesis leading to 'widespread' dermal, mucosal and visceral telangiectases and recurrent haemorrhage. We have mapped the HHT gene, by linkage analysis, to markers on 9q33-34 in two large multi-generation families. Haplotype analysis and mapping of recombination breakpoints gives a 4 cM interval between D9S61 and D9S63 as the most likely location of the gene. The closest marker, D9S65, is estimated to be within 1 cM of the gene and shows a combined lod score of 11.41. Two potential candidate genes, COL5A1 and ZNF79, are also located within 9q33-34. These results provide a starting point for the eventual cloning of the HHT gene.

A comparison of 131I-labeled monoclonal antibody 17-1A treatment to external beam irradiation on the growth of LS174T human colon carcinoma xenografts.

Inhibition of growth of LS174T human colon cancer xenografts in athymic nude mice due to 131I-labeled MoAb 17-1A treatment was compared to inhibition due to different single doses of 60Co external radiation. From those data, conditions which produced equivalent radiobiological end points could be identified and compared to dose estimates calculated using a technique analogous to the Medical Internal Radiation Dose (MIRD) Committee formalism. The tumor growth rate in mice injected with a single intraperitoneal administration of 300 microCi of 131I-labeled MoAb was reduced relative to tumor growth in untreated control animals and in mice administered unlabeled MoAb and was found to be similar to the growth rate of tumors given a single 6 Gy dose of 60Co radiation. Furthermore, the growth rate of tumors in mice that received three injections of 300 microCi of 131I-labeled MoAb on days 9, 16 and 28 after tumor cell injection was similar to the growth rate of tumors given a single 60Co dose of 8 or 10 Gy. The biodistribution data for 125I-labeled 17-1A MoAb were used to calculate total doses for the tumor and various normal tissues in animals given a single administration of 131I-labeled 17-1A MoAb. The absorbed radiation dose in tumor was approximately five times higher than in normal tissues. The results of the present study indicate that the tumor growth inhibition produced by the administration of radiolabeled antibody can equal that produced by up to 10 Gy of external beam radiation. In addition, the MIRD calculations allow comparison of this form of low dose radiation to external photon irradiation.

Comparative binding and preclinical localization and therapy studies with radiolabeled human chimeric and murine 17-1A monoclonal antibodies.

Murine MoAb 17-1A is an IgG2a antibody reactive with a gastrointestinal cancer-associated cell surface antigen. Human-mouse chimeric 17-1A MoAbs were constructed in which the murine variable region of 17-1A was joined with human IgG1, IgG2, IgG3, and IgG4 constant regions. Human-mouse IgG1, IgG2, and IgG4 chimeric antibodies were compared with the parental murine antibody and its F(ab')2 fragments for their ability to bind to colon carcinoma cells in vitro, for their blood clearance in normal nude mice, and for their localization and tumor growth inhibition of colon carcinoma xenografts in nude mice. Indirect immunofluorescence experiments with fluorescein-conjugated goat anti-mouse or goat anti-human antibody verified that the substitution of human constant regions in the chimeric MoAbs did not significantly alter the ability of the murine variable region to bind to colon adenocarcinoma cell lines (LS174T, SW948, and C0112). The immunoreactivities of 125I-labeled murine and chimeric 17-1A MoAbs measured in a live cell-binding assay with LS174T, SW948, and C0112 cells revealed that chimeric IgG1, IgG4, and 17-1A F(ab')2 were comparable to murine 17-1A while chimeric IgG2 showed lower binding. The blood half-lives of 125I-labeled murine 17-1A, its F(ab')2 fragments, and chimeric IgG1, IgG2, and IgG4 in normal nude mice determined by serial eye bleeding were 7.5, 0.5, 5.2, 6.9, and 1.9 days, respectively. In biodistribution studies at 4 days after injection of 125I-labeled MoAbs in nude mice bearing LS174T tumors, chimeric IgG1 had the highest tumor concentration of 20.5% injected dose/g with a tumor/blood ratio of 3.2. 131I-labeled murine 17-1A administered in a single injection of 300 microCi or 3 injections of about 300 microCi each to nude mice bearing established LS174T tumors inhibited tumor growth, whereas a comparable amount of unlabeled murine 17-1A did not inhibit tumor growth. 131I-labeled chimeric IgG1 MoAb showed a similar level of tumor growth inhibition. The results of the present study indicate that 17-1A chimeric IgG1 antibody may be the best choice for clinical radioimmunodetection and radioimmunotherapy studies.

Lipid microvesicles and their association with procoagulant activity in urine and glomeruli of rabbits with nephrotoxic nephritis.

The procoagulant activity (PCA) in urine of rabbits with nephrotoxic nephritis was characterized. Most of the PCA in urine was pelleted by centrifugation at 50,000 X g but was not pelleted together with cells and casts at 1,000 X g. PCA appeared in the void volume of a Sepharose 4B column but would not pass through a 0.2-micron filter. Ultrastructural studies using both transmission and scanning electron microscopy showed that urine PCA was associated with lipid vesicles 0.1 to 1-micron in diameter. These vesicular structures were shown to promote fibrin formation from recalcified plasma on a 0.2-micron filter surface. This microvesicular PCA was mostly Factor VII-like as judged by clotting assay using human factor-deficient plasmas. Aggregates of vesicles were present in urine as granular casts. Ultrastructural studies of rabbit kidney showed similar vesicular structures in the proximal tubular lumen and budding from glomerular epithelial cells. Fibrin was seen adjacent to both glomerular endothelial cells and epithelial cells in association with vesicular structures. We conclude that microvesicles in urine carry a procoagulant signal which is tissue factor/Factor VII-like. We speculate that these vesicles may come from the glomerulus by budding off from glomerular epithelial cells, or macrophages.

Amount of antibody is critical for immune complex displacement by charge competition from both rabbit glomeruli and anionic beads.

The purpose of this study was to determine the feasibility of displacing cationized bovine serum albumin (CBSA) immune complexes from glomeruli by charge competition. An in vitro model identified protamine as an effective agent for displacing 125I-CBSA from anionic beads (dextran sulfate-coated Sepharose 4B). Anti-CBSA serum prevented displacement of 125I-CBSA from anionic beads in a dose-dependent fashion. When 125I-CBSA was injected intravenously into rabbits 98% of 125I-CBSA disappeared from blood within 5 min, at which time CBSA was visualized by immunofluorescence in glomerular capillary walls but not in liver, muscle, skin, spleen or lung. By 24 h 90% of 125I-CBSA had disappeared from glomeruli. In contrast, injection of anti-CBSA antibody caused persistence of 125I-CBSA in kidney (particularly along glomerular capillary walls) for more than 7 days (detected by counting 125I in kidney, by radionuclide imaging and by immunofluorescence). Protamine administration (50 mg intravenously daily for 6 days) caused significant reduction of 125I-CBSA trapped in kidney only if the amount of anti-CBSA injected was small. Protamine did not significantly displace 125I-CBSA from glomeruli if the anti-CBSA dose was larger. Therefore both in vivo and in vitro displacement of 125I-CBSA by protamine depended upon the amount of antibody. We conclude that although charge dependent displacement of immune complexes from glomeruli is probably feasible using protamine this approach would only work in the presence of small amounts of antibody.

Procoagulant activity in normal human urine associated with subcellular particles.

Procoagulant activity (PCA) in normal human urine was found to be sedimented by centrifugation at X 100,000g. Therefore, studies were done to identify the structures associated with the procoagulant activity. Transmission electron microscopy of the X 100,000g pellet revealed numerous membrane-bound vesicles as well as fibrous material. Filtration of normal urine through a 0.2-micron filter removed more than 90% of the procoagulant activity. Scanning electron microscopy of the filter surface revealed 0.1 to 1.1 micron particles and fibrous material. By centrifugation at pH 3 and 5 the fibrous material and particles were separated. The procoagulant activity remained with the particles in each case. The fibrous material was shown to be Tamm-Horsfall protein by SDS-PAGE and Western blotting using anti-Tamm-Horsfall protein serum. Purified Tamm-Horsfall protein itself was not procoagulant. Therefore, PCA in normal human urine is associated with particles 0.1 to 1.1 micron in diameter which appear to be lipid membranes in various arrangements.

Procoagulant activity in glomeruli and urine of rabbits with nephrotoxic nephritis.

A telescoped model of nephrotoxic nephritis in the rabbit, using guinea pig antiglomerular basement membrane IgG in rabbits preimmunized with guinea pig IgG, reproducibly induced crescentic nephritis. Procoagulant activity (PCA) was measured in sieve-isolated glomeruli that had been either sonicated or cultured for 48 hours. In both sonicated and cultured glomeruli PCA peaked on days 5 and 6. The time course for appearance of PCA corresponded precisely with the appearance of proteinaceous material containing fibrin in Bowman's space as measured by a light microscopic histologic scoring system and confirmed by immunofluorescence and electron microscopy. Glomerular PCA returned to baseline by days 9 and 10 in spite of progression of glomerular injury. PCA also appeared in urine. Urine PCA peaked on day 8 and persisted through day 12 when glomerular PCA had returned to baseline. Glomerular and urine PCA were characterized using human coagulation factor-deficient plasmas and antithromboplastin IgG. Both glomerular PCA and urine PCA were inhibited by antithromboplastin IgG, showing that thromboplastin (tissue factor) contributed to PCA. The PCA in glomerular sonicates was dependent on factor X, but independent of factor VII or Hageman factor, suggesting that factor VII was present. Following glomerular culture for 48 hours the PCA had changed and in some cases was dependent on Hageman factor, factor IX, and factor VII for full PCA expression. Urine PCA was uniformly Hageman factor dependent and sometimes independent of factors VII and X. No active thrombin was present. The forms of glomerular and urine PCA were, therefore, complex. They seemed to be primarily driven by thromboplastin but also appeared to require the presence of the intrinsic coagulation pathway for full expression of PCA.

Acute hypotension due to platelet serotonin-induced chemoreflexes after intravenous injection of dextran sulfate in the rabbit.

The hypotension and bradycardia observed after intravenous injection of dextran sulfate in rabbits was prevented by prior depletion of circulating platelets, but was not prevented by depletion of the third component of complement or Hageman factor. Dextran sulfate injection caused immediate thrombocytopenia with temporary localization of platelets within lungs. Morphological analysis revealed platelet aggregates in lung capillaries. The platelets had changed shape and were in the process of degranulating. Serotonin and histamine levels in blood increased approximately 5-fold and 7-fold, respectively, after dextran sulfate injection. The cardiovascular events following dextran sulfate injection were mimicked by intravenous serotonin but not by intravenous histamine injection, although a combination of serotonin and histamine reproduced the pattern of blood pressure changes better than did either agent alone. Quantification of platelets trapped in lung revealed that the potential release of serotonin from trapped platelets could account for the rise in plasma serotonin concentration and the hemodynamic changes observed. Both the dextran sulfate-induced cardiovascular effects and serotonin-induced hypotension were markedly diminished by cutting vagus and depressor nerves, and were virtually abolished by carotid ligation in addition to nerve section. These results support the concept that platelet activation within rabbit lungs may cause hypotension via serotonin-induced chemoreflexes.