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W H Lewis

Publications and source records attributed to W H Lewis.

At least 55 records · Page 3Linked to original sources
PubMed1989

Enzyme-linked immunosorbent assay for alpha-fetoprotein in human serum.

A simple and inexpensive enzyme-linked immunosorbent assay for determination of serum alpha-fetoprotein concentration, with results available within 4 h, is described. The assay has a working range of 2-100 ng/ml, with within- and between-batch coefficients of variation (n = 24) at different concentrations between 8.9 and 12.7%. Results on 80 patients' sera (AFP concentrations 0-175 ng/ml) correlate well with those by radioimmunoassay. Turn around time (4 h) is shorter than with similar immunoassays currently available.

Carcinoma, Hepatocellular↗
PubMed1989

Enzyme-linked immunosorbent assay for free thyroxin in human serum.

We describe a simple enzyme-linked immunosorbent assay (ELISA) in which microtiter plates are used for determining the free thyroxin concentration (FT4) in serum. Only commercially available chemicals and reagents are needed, including the antiserum. The working range is 1 to 60 ng/L. Turnaround time is about 4 h. Within-run coefficients of variation (CV) for FT4 concentrations of 9.0, 19.0, and 35.0 ng/L were less than 5%; between-run CVs were 10% to 11%. Results by a radioimmunoassay method for 150 sera correlated well (r = 0.922).

Enzyme-Linked Immunosorbent Assay↗
PubMed1989

Amplification of N-myc and ornithine decarboxylase genes in human neuroblastoma and hydroxyurea-resistant hamster cell lines.

The genes for the M2 subunit of ribonucleotide reductase (RRM2), ornithine decarboxylase (ODC1), and 55,000-Daltons protein (P5), are amplified in hydroxyurea-resistant hamster and human cell lines. These genomic sequences have been mapped to hamster chromosome 7 and to human chromosome 2p24-25 near the cytogenetic location of the N-myc gene. We now report that genomic sequences homologous to N-myc are amplified in hydroxyurea-resistant hamster lung cell line, 600H, and the N-myc gene segregates with hamster chromosome 7 in mouse-hamster somatic cell hybrids. The conserved linkage group consisting of the RRM2, ODC1, P5, and N-myc in the hamster and human genomes prompted our investigation of human neuroblastomas. We report here that genomic DNA from 1 of 6 primary neuroblastoma tumors containing amplified N-myc also contains amplified sequences homologous to a hamster ODC cDNA.

Adolescent↗
PubMed1988

Structure and rearrangement of the T cell receptor J alpha locus in T cells and leukemic T cell lines.

The vast majority of T cells express an antigen receptor (TcR) composed of an alpha/beta heterodimer. The alpha and beta chains are encoded for by a set of variable (V), joining (J) and constant (C) region genes. Unlike the J genes of the beta chain which are limited in number and are clustered close to the constant region, the J alpha genes are spread over an 85-kilobase DNA region, upstream of the C alpha gene. We have isolated the complete J alpha locus, bounded on the 5' side by the C sequence of the delta gene and on the 3' side by the C sequence of the alpha gene. The experiments described here demonstrate that the J gene segments extend 75 kb 5' of C alpha and participate equally in generating the diversity of the alpha chain in peripheral T cells. Similarly, in leukemic T cell lines, rearrangements occurred over the entire locus and involved both alleles. Densitometry data suggest that in most peripheral T cells both alleles also are rearranged; thus, allelic exclusion in the alpha locus does not occur at the level of rearrangement. In three cell lines, an identical rearrangement has occurred on one allele in a region located 10 kb from the 5' end of the locus.

Genes↗
PubMed1988

Specific antigen detection by immunoelectroendosmosis.

A novel approach to the immunological stage of immunoblotting is described. The method employs the phenomenon of electroendosmosis to bring the antibodies sequentially into contact with the antigen. Using a three-stage peroxidase antiperoxidase system for human group specific protein (Gc) phenotyping, it has been shown that the method has comparable sensitivity, is more rapid and is more economical of antibodies than a similar multi-stage immerse-and-wash system.

Antigens↗
PubMed1988

Homozygous deletion of a DNA marker from chromosome 11p13 in sporadic Wilms tumor.

A random DNA fragment, probe p2.3 (locus D11S87), was cloned from the 11p13 region between a translocation breakpoint associated with familial aniridia and another translocation breakpoint associated with childhood T-cell leukemia. The D11S87 locus maps between the catalase (CAT) locus and the beta subunit of follicle stimulating hormone (FSHB). The D11S87 locus is deleted in a Wilms tumor patient with a constitutional deletion of 11p and in a case of sporadic Wilms tumor (WiT-13) apparently with normal karyotype. In the WiT-13 tumor both maternal and paternal chromosomes 11 are retained; D11S87 is deleted homozygously and FSHB hemizygously. These results suggest two mutational events resulting in homozygous deletion in this patient. The D11S87 homozygous deletion was also demonstrated in WiT-13 nude mouse heterotransplants and in fibroblast-like cell line derived from the primary tumor. The minimum size of the deletion was estimated to be 30 kb as determined by cosmid screening and hybridization. As homozygous deletions in the 11p13 region have not been previously reported for sporadic Wilms tumors, these findings place the D11S87 locus within or approximate to the Wilms tumor gene.

Blotting, Southern↗
PubMed1987

The gene for ornithine decarboxylase is co-amplified in hydroxyurea-resistant hamster cells.

We have constructed a cDNA library from the highly hydroxyurea-resistant hamster cell line 600H in which the activity of ribonucleotide reductase is elevated more than 80-fold. Using the technique of differential hybridization, we have isolated a number of cDNA clones from this library which are homologous to genomic DNA sequences amplified in the 600H cell line compared to the V79 parental line. One of these cDNA clones by sequence analysis was found to code for ornithine decarboxylase. This was confirmed by in vitro translation of poly(A+) RNA isolated by hybridization-selection followed by immunoprecipitation with antiserum specific for mouse ornithine decarboxylase. Genomic sequences homologous to the cDNA clone were shown to be sequentially amplified 6-20-fold in hamster cell lines selected stepwise for resistance to increasing concentrations of hydroxyurea. Genomic sequences homologous to a cDNA for the M2 subunit of ribonucleotide reductase were also amplified in these cell lines, and the degree of M2 sequence amplification corresponded to the degree of amplification of ornithine decarboxylase sequences, suggesting that the two genes had been co-amplified during the selection of the hydroxyurea-resistant phenotype.

Amino Acid Sequence↗
PubMed1987

Gene for M1 subunit of ribonucleotide reductase is amplified in hydroxyurea-resistant hamster cells.

The hydroxyurea-resistant Chinese hamster cell line 600H has been shown to have greatly elevated quantities of ribonucleotide reductase. This increase in enzyme activity is due to an increased level of both the M1 and M2 subunit activities. The M1 subunit has been purified from the 600H cell line and shown to consist of a series of six protein spots with apparent molecular weights of 88,000 daltons, but with varying isoelectric points in the range of pH 6.5-7.0. Western blot analyses with antisera against the M1 and M2 proteins indicated that both subunit proteins are present in elevated quantities in the 600H cell line when compared to the wild-type V79 cell line. Southern blot analyses with genomic DNA from the series of stepwise-selected hydroxyurea-resistant cell lines leading to 600H showed that, in latter steps of selection, genomic sequences homologous to a mouse M1 cDNA have undergone a fivefold amplification. This was accompanied by a four- to eightfold increase in the single M1 homologous mRNA.

Animals↗
PubMed1987

Ribonucleotide reductase M2 subunit sequences mapped to four different chromosomal sites in humans and mice: functional locus identified by its amplification in hydroxyurea-resistant cell lines.

The sites of sequences homologous to a murine cDNA for ribonucleotide reductase (RR) subunit M2 were determined on human and murine chromosomes by Southern blot analysis of interspecies somatic cell hybrid lines and by in situ hybridization. In the human genome, four chromosomal sites carrying RRM2-related sequences were identified at 1p31----p33, 1q21----q23, 2p24----p25, and Xp11----p21. In the mouse, M2 sequences were found on chromosomes 4, 7, 12, and 13 by somatic cell hybrid studies. By Southern analysis of human hydroxyurea-resistant cells that overproduce M2 because of gene amplification, we have identified the amplified restriction fragments as those that map to chromosome 2. To further confirm the site of the functional RRM2 locus, two other cDNA clones, p5-8 and S7 (coding for ornithine decarboxylase; ODC), which are coamplified with RRM2 sequences in human and rodent hydroxyurea-resistant cell lines, were mapped by Southern and in situ hybridization. Their chromosomal map positions coincided with the region of human chromosome 2 (p24----p25) that also contains one of the four RRM2-like sequences. Since this RRM2 sequence and p5-8 and ODC are most likely part of the same amplification unit, the RRM2 structural gene can be assigned to human chromosome 2p24----p25. This region is homologous to a region of mouse chromosome 12 that also carries one of numerous ODC-like sequences. In an RRM2-overproducing mouse cell line, we found amplification of the chromosome 12-specific restriction fragments. Thus, we conclude that mouse chromosome 12 carries the functional locus for RRM2.

Animals↗
PubMed1987

Chromosomal assignment of amplified genes in hydroxyurea-resistant hamster cells.

We have shown previously that cDNAs for the M1 and M2 subunits of ribonucleotide reductase, ornithine decarboxylase (ODC), and p5-8, a 55,000-Dalton protein, hybridize to amplified genomic sequences in a highly hydroxyurea-resistant hamster cell line. We have extended these observations to include two additional, independently isolated, hydroxyurea-resistant cell lines: SC8, a single-step hamster ovary cell line, and KH450, a multistep human myeloid leukemic cell line, have also undergone genomic amplification for sequences homologous to ODC and p5-8 cDNAs. However, neither SC8 nor KH450 contains amplified genomic sequences homologous to an M1 cDNA probe. A panel of mouse-hamster somatic cell hybrids was used to map sequences homologous to M1, M2, ODC, and 5-8 cDNAs in the hamster genome. The M2, ODC, and p5-8 cDNAs hybridized to DNA fragments that segregated with hamster chromosome 7. In contrast, M1 cDNA hybridized to DNA fragments that segregated with hamster chromosome 3. These data suggest that the genes RRM2, (M2), ODC, and p5-8, but not RRMI (M1), are linked and may have been co-amplified in the selection of the hydroxyurea-resistant hamster and human cell lines.

Animals↗
PubMed1987

Allergic reactivity of Parthenium hysterophorus (Santa Maria feverfew) pollen: an unrecognized allergen.

Parthenium hysterophorus (PH), a member of the Asteraceae, is a ubiquitous weed found in great profusion in the United States Gulf coast area. A plant survey carried out in Texas Gulf coast demonstrated that around Corpus Christi, PH is the most prevalent weed. We have studied known ragweed-sensitive patients living in Austin and Corpus Christi, by radioallergosorbent test (RAST) analysis for PH and Ambrosia psilostachya (AP; western ragweed). Nine of ten patients from Austin were RAST-positive to PH and AP; 8 of the 10 patients had positive scores to both pollen extracts. All of the 8 patients from Corpus Christi were positive to both PH and AP. The Austin group showed a significant correlation between AP and PH RAST scores while no correlation between RAST values was seen in the Corpus Christi group. RAST inhibition analysis in this highly selected population did reveal patients that had true sensitivity to PH pollen alone or who were sensitive to both PH and AP pollen. This suggests that in areas where it is highly prevalent PH pollen is an overlooked cause of allergic disease.

Allergens↗
PubMed1987

Nonrandom loss of maternal chromosome 11 alleles in Wilms tumors.

A series of gene probes for chromosome 11 has been used to study the genetic events associated with the development of Wilms tumor. Examination of DNA samples from five patients with Wilms tumor in whom the tumors showed loss of chromosome 11 alleles and their parents indicate that alleles lost in the tumors are of maternal origin. These data suggest that the parental derivation of chromosome 11 alleles lost in these Wilms tumors is not random.

Alleles↗
PubMed1987

Genetic mechanisms of tumor-specific loss of 11p DNA sequences in Wilms tumor.

Wilms tumor, a common childhood renal tumor, occurs in both a heritable and a nonheritable form. The heritable form may occasionally be attributed to a chromosome deletion at 11p13, and tumors from patients with normal constitutional chromosomes often show deletion or rearrangement of 11p13. It has been suggested that a germinal or somatic mutation may occur on one chromosome 11 and predispose to Wilms tumor and that a subsequent somatic genetic event on the normal homologue at 11p13 may permit tumor development. To study the frequency and mechanism of such tumor-specific genetic events, we have examined the karyotype and chromosome 11 genotype of normal and tumor tissues from 13 childhood renal tumor patients with different histologic tumor types and associated clinical conditions. Tumors of eight of the 12 Wilms tumor patients, including all viable tumors examined directly, show molecular evidence of loss of 11p DNA sequences by somatic recombination (four cases), chromosome loss (two cases), and recombination (two cases) or chromosome loss and duplication. One malignant rhabdoid tumor in a patient heterozygous for multiple 11p markers did not show any tumor-specific 11p alteration. These findings confirm the critical role of 11p sequences in Wilms tumor development and reveal that mitotic recombination may be the most frequent mechanism by which tumors develop.

Child↗
PubMed1985

Molecular analysis of gene deletion in aniridia--Wilms tumor association.

Hybrid clones were produced from the fusion of Chinese hamster cells and human fibroblasts from a patient with the aniridia-Wilms tumor association (AWTA). The DNA from the parental cells and the hybrid clones was screened by Southern blot and DNA hybridization with probes for the human insulin and Ha-ras-1 genes. Two alleles for the Ha-ras-1 gene were shown to exist in the AWTA cells by restriction fragment length polymorphism. One hybrid clone, containing a single allele for Ha-ras-1 was shown to contain a single chromosome 11 with a cytogenetically visible deletion at 11p13. The DNA from this hybrid contained the human genes for insulin, A gamma-globin, G gamma-globin, Ha-ras-1, and calcitonin, but lacked any human sequences homologous to a human catalase cDNA. This clone was also shown to express human lactate dehydrogenase A (LDH A) activity. These data indicate that the deletion of the affected chromosome in this AWTA patient begins distal to LDH A and includes band 11p13, but does not extend to calcitonin or other genes thought to be located in the distal half of chromosome 11p.

Animals↗