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Biomedical subjects

R White

Publications and source records attributed to R White.

At least 181 records · Page 10Linked to original sources
PubMed1995

Identification of neurofibromatosis 1 (NF1) homologous loci by direct sequencing, fluorescence in situ hybridization, and PCR amplification of somatic cell hybrids.

Using fluorescence in situ hybridization (FISH), we have identified seven NF1-related loci, two separate loci on chromosome 2, at bands 2q21 and 2q33-q34, and one locus each on five other chromosomes at bands 14q11.2, 15q11.2, 18p11.2, 21q11.2-q21, and 22q11.2. Application of PCR using NF1 primer pairs and genomic DNA from somatic cell hybrids confirmed the above loci, identified additional loci on chromosomes 12 and 15, and showed that the various loci do not share homology beyond NF1 exon 27b. Sequenced PCR products representing segments corresponding to NF1 exons from these loci demonstrated greater than 95% sequence identity with the NF1 locus. We used sequence differences between bona fide NF1 and NF1-homologous loci to strategically design primer sets to specifically amplify 30 of 36 exons within the 5' end of the NF1 gene. These developments have facilitated mutation analysis at the NF1 locus using genomic DNA as template.

Animals↗
PubMed1995

Microsatellite instability in colorectal adenocarcinoma cell lines that have full-length adenomatous polyposis coli protein.

Almost 20% of colon cancers are characterized by genomic instability at simple repeated sequences. This instability is the result of a deficient DNA mismatch repair system. Sporadic, as well as hereditary carcinomas of the proximal colon display this effect. In this study, we examined colorectal adenocarcinoma cell lines, with or without wild-type adenomatous polyposis coli (APC) protein, for the presence of microsatellite instability. The three cell lines that maintained full-length APC protein also displayed the highest level of instability, suggesting a negative correlation between APC mutations and microsatellite instability. This data, in addition to other studies that show a negative correlation between microsatellite instability and mutations in p53 and K-ras, support the idea of a second pathway for colorectal cancer development.

Adenocarcinoma↗
PubMed1995

Precise mapping of t(12;14) leiomyoma breakpoint on chromosome 14 between D14S298 and D14S540.

Uterine leiomyoma is a common tumor of smooth muscle cell origin often characterized by the presence of a balanced t(12;14)(q13-15;q24.1) chromosomal translocation. This breakpoint on chromosome 14 had previously been placed between the markers SPTB and D14S77, a region estimated to span 7 cM. In this study we have used a meiotic breakpoint mapping panel to construct a high resolution genetic map of this interval. Markers that mapped within this interval were used to analyze DNA from a somatic cell hybrid containing the t(12;14) translocated chromosome. The results of this analysis localize the t(12;14) breakpoint on chromosome 14 between D14S298 and D14S540, between which no meiotic recombination was detected. This sets the stage for identifying the gene(s) disrupted by the chromosomal translocation by defining the markers that flank the translocation breakpoint.

Base Sequence↗
PubMed1995

Alternatively spliced adenomatous polyposis coli (APC) gene transcripts that delete exons mutated in attenuated APC.

Reverse transcription-PCR combined with either (a) restriction enzyme digestion and repeat PCR or (b) ligase chain reaction has identified two new alternatively spliced transcripts of the adenomatous polyposis coli (APC) gene. In one of these transcripts exons 1-4 and the first 16 bases of exon 5 are deleted; in the other exons 2-4 and the first 16 bases of 5 are deleted. Both transcripts use an intraexonic splice acceptor in exon 5. These transcripts delete exons mutated in attenuated APC (3 and 4) and could account for the reduction in severity of this variant.

Alternative Splicing↗
PubMed1995

A YAC contig spanning the dominant retinitis pigmentosa locus (RP9) on chromosome 7p.

The dominant retinitis pigmentosa locus RP9 has previously been localized to 7p13-p15, in the interval D7S526-D7S484. We now report refinement of the locus to the interval D7S795-D7S484 and a YAC contig of approximately 4.8 Mb spanning this region and extending both distally and proximally from it. The contig was constructed by STS content mapping and physically orders 29 STSs in 28 YAC clones. The order of polymorphic markers in the contig is consistent with a genetic map that has been assembled using haplotype data from the CEPH pedigrees. This contig will provide a primary resource for the construction of a transcriptional map of this region and for the identification of the defective gene causing this form of adRP.

Base Sequence↗
PubMed1995

A gene (DLG2) located at 17q12-q21 encodes a new homologue of the Drosophila tumor suppressor dIg-A.

We have isolated a novel cDNA that maps distal to BRCA1 at 17q12-q21. The total sequence predicts a protein of 576 amino acids with three conserved regions: a 90-amino-acid repeat domain, a SH3 (src homology region 3) motif, and a guanylate kinase domain. These conserved regions are shared among members of the discs-large family of proteins that include human p55, a membrane protein expressed in erythrocytes, rat PSD-95/SAP90, a synapse protein expressed in brain, Drosophila dIg-A, a septate junction protein expressed in various epithelia, and human and mouse ZO-1 and canine ZO-2, two tight junction proteins. dIg-A has been shown to act as a tumor suppressor, and the other members may all be involved in signal transduction through specialized membrane domains with highly organized cytoskeletons and thus are potential tumor suppressors. Since allelic loss has been reported in the 17q12-q21 region in breast and ovarian cancer and it appears that BRCA1 is not the target of the losses, we looked for somatic alterations in DLG2 in sporadic breast tumors. No evidence for mutation was found, making it unlikely that DLG2 is involved in sporadic breast cancer.

Amino Acid Sequence↗
PubMed1995

Isolation and mapping of a gene encoding a novel human ADP-ribosylation factor on chromosome 17q12-q21.

A gene encoding a low-molecular-weight GTP-binding protein was isolated from a retinal cDNA library and mapped to human chromosome 17q12-q21. Comparison of the predicted protein with the protein databases revealed striking homology to the family of ADP-ribosylation factors (ARFs), which are thought to be involved in membrane trafficking and protein secretion. The greatest homology observed was with the rat ARF-like 4 protein (ARL4), with which it shared 58% identity, while the more highly conserved human ARF1 and ARF3 proteins each shared 46% identity. Inspection of the predicted new protein showed that it contained each of the six conserved motifs that are required for guanine nucleotide binding and hydrolysis, and thus it is probably a novel ARF isoform. We have designated the new protein and its corresponding gene ARF4L.

ADP-Ribosylation Factor 1↗
PubMed1995

Template-switching during DNA synthesis by Thermus aquaticus DNA polymerase I.

Recombinant DNA molecules are often generated during the polymerase chain reaction (PCR) when partially homologous templates are available [e.g., see Pääbo et al. (1990) J. Biol. Chem. 265, 4718-4721]. It has been suggested that these recombinant molecules are a consequence of truncated extension products annealing to partially homologous templates on subsequent PCR cycles. However, we demonstrate here that recombinants can be generated during a single round of primer extension in the absence of subsequent heat denaturation, indicating that template-switching produces some of these recombinant molecules. Two types of template-switches were observed: (i) switches to pre-existing templates and (ii) switches to the complementary nascent strand. Recombination is reduced several fold when the complementary template strands are physically separated by attachment to streptavidin magnetic beads. This result supports the hypothesis that either the polymerase or at least one of the two extending strands switches templates during DNA synthesis and that interaction between the complementary template strands is necessary for efficient template-switching.

Base Sequence↗
PubMed1995

The CEPH consortium linkage map of human chromosome 11.

The CEPH consortium framework map of chromosome 11 is presented. The map was generated from CEPH family DNAs with 181 probe/enzyme combinations contributed by 20 laboratories. Seventy-seven of the loci are defined by microsatellite polymorphisms that can be typed by the PCR. A total of 42 loci have been placed on the map with likelihood support of at least 1000:1. The female, male, and sex-average maps extend for 179.6, 110.8, and 145.3 cM, respectively. The largest interval on the sex-average map is less than 11 cM, and the average distance between uniquely placed loci is 4 cM. The genotypic data obtained for map construction have been used to identify the positions of crossovers on the chromosomes of CEPH family children, allowing the localization of new markers without computationally intensive likelihood models and providing a basis for efficient extension of the linkage map to higher resolution.

Adult↗
PubMed1995

Genomic organization of the neurofibromatosis 1 gene (NF1).

Neurofibromatosis 1 maps to chromosome band 17q11.2, and the NF1 locus has been partially characterized. Even though the full-length NF1 cDNA has been sequenced, the complete genomic structure of the NF1 gene has not been elucidated. The 5' end of NF1 is embedded in a CpG island containing a NotI restriction site, and the remainder of the gene lies in the adjacent 350-kb NotI fragment. In our efforts to develop a comprehensive screen for NF1 mutations, we have isolated genomic DNA clones that together harbor the entire NF1 cDNA sequence. We have identified all intron-exon boundaries of the coding region and established that it is composed of 59 exons. Furthermore, we have defined the 3'-untranslated region (3'-UTR) of the NF1 gene; it spans approximately 3.5 kb of genomic DNA sequence and is continuous with the stop codon. Oligonucleotide primer pairs synthesized from exon-flanking DNA sequences were used in the polymerase chain reaction with cloned, chromosome 17-specific genomic DNA as template to amplify NF1 exons 1 through 27b and the exon containing the 3'-UTR separately. This information should be useful for implementing a comprehensive NF1 mutation screen using genomic DNA as template.

Amino Acid Sequence↗
PubMed1995

Generation of cytotoxic effector cells against human melanoma.

Metastatic or tumor-draining lymph nodes from six of nine melanoma patients undergoing lymph node dissection for metastatic melanoma generated cytotoxic T cells against autologous melanoma when these lymph node cells were treated by in vitro sensitization and recombinant interleukin-2 (IL-2). During the initial lymphocyte culture (2-6 weeks), cross-reactivity with autologous tumor cells, K562 and Daudi cells was usually noted. Cold-target inhibition assay with K562 and Daudi showed K562/Daudi-associated antigens on melanoma cells. During the later phase of lymphocyte culture with repeated in vitro sensitization (over 6-10 weeks), cytotoxicity was noted against autologous and allogeneic melanoma cells but not against K562. Daudi cells or autologous fibroblasts. Repeated in vitro sensitization resulted in the selection of specific cytotoxic lymphocytes against melanoma. Cold-target inhibition assay with autologous and allogeneic melanoma cells revealed shared and individual antigens. Using blocking monoclonal antibodies, MHC-restricted killing was noted in the autologous system. Further, both the autologous and allogeneic systems could be mediated through adhesion molecules such as ICAM-1 and LFA-3 on melanoma cells and LFA-1 on T cells. This study suggests that a constellation of cytotoxic effector cells and melanoma-associated antigens may be pivotal in tumor killing. Thus, future adoptive immunotherapy should modulate and enhance this complex interaction.

Adult↗
PubMed1995

Microbial transformation of N-heptyl physostigmine, a semisynthetic alkaloid inhibitor of cholinesterase.

The microbiological transformation of N-heptyl physostigmine (L-693,487) (1), a semisynthetic physostigmine cholinesterase inhibitor, was investigated using Verticillium lecanii MF 5713 (ATCC 74148), Acremonium sp MF 5723 (ATCC 74164) and Actinoplanes sp MA 6559 (ATCC 53771). Nine microbial metabolites (2-10) of 1 were isolated and purified using reversed-phase HPLC. The structures of the metabolites were established using spectroscopic techniques including MS and NMR. Some of the microbial metabolites were identical to metabolites present in urine of a dog treated with 1.

Acremonium↗
PubMed1995

Bacterial adhesion measurements on soft contact lenses using a Modified Vortex Device and a Modified Robbins Device.

S. marcescens 8100 and P. aeruginosa 15442 were used to study bacterial adhesion to hydrogel contact lenses which had not been worn. Bacterial removal from unworn lens materials was assessed with a calibrated vortex device modified with a digital rpm readout and fitted with a test tube attachment (MVD). The MVD, which relies on a whirlpool-like force to remove the bacteria, showed that bacteria adhered to the same degree to etafilcon A, vifilcon A and polymacon lenses under standardized conditions. Tracking the isoenzyme patterns of these bacterial species over time showed instability of S. marcescens upon repeated passage. This instability was not evident with P. aeruginosa. Bacterial adhesion of P. aeruginosa 15442, to human worn and unworn etafilcon A materials was determined with a Modified Robbins Device. The MRD was closed off at both ends stopping medium and bacterial movement after 1 h of fluid flow over the lens surface. The results show that immediately following this 1-h period more bacteria adhere to unworn contact lenses than to worn lenses. However, bacterial counts were equivalent on worn and unworn lenses following 5 h of static incubation.

Bacterial Adhesion↗
PubMed1995

Identification of phosphorylation sites in the mouse oestrogen receptor.

Phosphorylation sites in the mouse oestrogen receptor, expressed in COS-1 cells in the presence of 17 beta-oestradiol, have been mapped by solid phase microsequencing. The receptor was first radio-labelled with [32P]orthophosphate and a number of 3H- or 14C-labelled amino acids, immunopurified and then tryptic peptides were separated by thin layer chromatography or high performance liquid chromatography. Amino acid sequence analysis indicated that Ser-122, Ser-156, Ser-158 and Ser-298 were phosphorylated. The substitution of Ser-122 and Ser-298 with alanine had a negligible effect on the transcriptional activity of the receptor in transfected cells. However, a reduction of transcriptional activity was observed when Ser-122 was mutated in the context of mutations in a putative amphipathic alpha-helix involved in AF-2 activity. Thus a region of AF-1 that encompasses Ser-122 appears to interact with AF-2 in the full-length receptor.

3T3 Cells↗
PubMed1995

Peritoneal dialysis solutions reverse the hemodynamic effects of nitric oxide synthesis inhibitors.

Nitric oxide (NO) synthesis is inhibited by a variety of L-arginine analogs including NG-nitro-L-arginine methyl ester (L-NAME) and NG NG-dimethylarginine (ADMA). ADMA is present in elevated concentrations in renal failure and potentially could alter microcirculatory hemodynamics during peritoneal dialysis (PD). This investigation utilized the techniques of intravital microscopy to quantitate the mesenteric arteriolar hemodynamic effects of PD solutions during NO synthesis inhibition. L-NAME (100 microns) produced maximum arteriolar vasoconstriction to 74% of baseline diameter (19.9 +/- 2.2 vs. 26.9 +/- 1.4 microns, P < 0.001, N = 10) and ADMA (100 microns) to 68% (20.5 +/- 2.5 vs. 30.1 +/- 2.0 microns, P < 0.01, N = 6). L-NAME decreased red blood cell velocity to 44% of baseline velocity (3.8 +/- 0.8 vs. 8.5 +/- 1.1 mm/second, P < 0.001) and ADMA to 52% (5.1 +/- 1.1 vs. 9.8 +/- 0.9 mm/second, P < 0.01, N = 6). Despite NO synthesis inhibition, standard PD solutions reversed these hemodynamic effects with both 1.5% and 4.25% Dianeal (Baxter) rapidly reversing the vasoconstriction and restoring blood flow back to baseline values. When Dianeal and L-NAME were simultaneously superfused, no L-NAME induced vasoconstriction occurred and Dianeal maintained vasodilatory properties despite L-NAME (P < 0.01, N = 5). This investigation reaffirms that basal levels of NO are important in maintaining normal hemodynamics in the mesenteric microcirculation. Reversal of the L-NAME induced arteriolar hemodynamic effects by Dianeal suggests that the endogenous NO synthesis inhibitor ADMA has no significant effects in the regulation of the mesenteric microvascular arteriolar hemodynamics during PD. Since these PD solutions remain vasoactive despite NO synthesis inhibition, this suggests that these PD solutions possess vasoactive properties primarily through a NO independent mechanism.

Animals↗
PubMed1995

The tetranucleotide repeat polymorphism D21S1245 demonstrates hypermutability in germline and somatic cells.

Six novel polymorphic short sequence repeats were identified and localized on the linkage map of human chromosome 21 by genotyping the CEPH reference pedigrees. One of these markers, the tetrameric (AAAG)n repeat D21S1245, was found to be hypermutable. In the DNAs from lymphoblastoid cell lines of members of the 40 CEPH families a total of 18 new alleles were detected. These new alleles, sometimes appearing in mosaic forms, arose equally in paternal and maternal DNAs, and could be equally larger or smaller than the alleles from which they were derived. The larger alleles of D21S1245 are more prone to be converted to new alleles. None of the new alleles with mosaicism were present in the corresponding genomic blood DNA, and therefore originated during or after the establishment of the lymphoblastoid cell lines; half of the new alleles without mosaicism were also found in genomic blood DNA of the appropriate CEPH individuals. The range of germline mutation rate observed in the 716 meioses examined was 0.56-1.4 x 10(-2); the range of somatic mutations observed in the 405 cell lines examined was 1.96-3.46 x 10(-2). This is one of the most hypermutable microsatellite repeat polymorphism in the human genome detected to date. D21S1245, is highly polymorphic (heterozygosity of 0.96) and maps between D21S231 and D21S198.

Alleles↗
PubMed1995

Formation and resolution of bilateral traumatic pulmonary pseudocysts.

A case of bilateral traumatic pulmonary pseudocysts, an uncommon complication of blunt chest trauma, is presented. The radiologic changes were followed over 6 months until spontaneous resolution had occurred. The mechanism of formation of traumatic pseudocysts and the diagnostic implications are discussed.

Adult↗