Search PubMed⌕ Search

Biomedical subjects

V Chapman

Publications and source records attributed to V Chapman.

At least 91 records · Page 5Linked to original sources
PubMed1990

A simple theoretical treatment of a competitive enzyme-linked immunosorbent assay (ELISA) and its application to the detection of human blood group antigens.

A theory has been developed to explain the behaviour of a competitive enzyme-linked immunosorbent assay (ELISA) in which an immobilized antigen competes with a liquid-phase antigen for a limiting amount of antibody. The binding of antibody to antigen on a solid surface (microtitre well) is described in terms of Langmuirian adsorption with a binding constant kappa. Two equations are presented to describe the behaviour of the ELISA signal as a function of competing antigen concentration; an exact equation and an approximate equation which can be used when the surface coverage of the immobilized antigen is not known. It is shown how curves of ELISA signal vs. competing antigen concentration depend on K/kappa [antibody]. The theory has been tested using several immobilized blood group A antigens competing with ovarian cyst fluid A substance and found to adequately describe these competitive ELISAs which have a detection limit of approximately 1 ng of blood group antigen.

Binding, Competitive↗
PubMed1990

Use of a HpaII-polymerase chain reaction assay to study DNA methylation in the Pgk-1 CpG island of mouse embryos at the time of X-chromosome inactivation.

A HpaII-PCR assay was used to study DNA methylation in individual mouse embryos. It was found that HpaII site H-7 in the CpG island of the X-chromosome-linked Pgk-1 gene is less than or equal to 10% methylated in oocytes and male embryos but becomes 40% methylated in female embryos at 6.5 days; about the time of X-chromosome inactivation of the inner cell mass.

Animals↗
PubMed1989

Evidence for a net-like organization of lipopolysaccharide particles in the Escherichia coli outer membrane.

Cell wall LPS of Escherichia coli are organized as particles which are visible in the electron microscope, after treatment of the wall with alkali. We now describe alkali treated walls of three E. coli strains with differences in susceptibility to the T4 phage infection. Strain CR63, a usual host for the T4 phage, shows the LPS particles on the murein layer. These particles are absent in alkali treated cell walls of the strain W. Walls of this strain are broken during T4 infection and phages can be seen bearing pieces of membrane attached to their long as well as their short tail fibers. Strain AS19 which is hypersensitive to the lysis from without caused by T4 shows murein layers with no LPS particles on their surface, and networks of LPS particles with bacterial shape. This suggested that LPS are organized in a network of particles which may serve as the skeleton of the cell wall.

Cell Wall↗
PubMed1988

Histocompatibility antigen changes associated with pink-eyed dilute (p) mutations.

The tight linkage between the H-4 histocompatibility locus and the pink-eyed dilute (p) locus raises the possibility that a single gene is responsible for both a histocompatibility antigen and coat color phenotype. To examine this possibility, we have investigated the effects of a spontaneous coat color mutation, pink-eyed unstable (pun), which occurred at the p locus in the C57BL/6J inbred strain, on histocompatibility antigen phenotype. Skin grafts were transplanted from two independently maintained B6-pun substrains to coisogenic, wild-type C57BL/6 recipients; graft rejection uniformly commenced at 6-7 weeks but did not culminate in complete graft destruction as observed in other cases of "crisis" rejection. Neither the onset of rejection time nor the intensity of rejection could be accelerated by introducing new H-2 haplotypes into the wild-type recipients. These results suggested that the pun allele was associated with a histocompatibility antigen not shared with C57BL/6. The pun allele is characterized by a relatively high frequency of reversion to wild-type. Therefore, skin grafts from B6-pun donors were transplanted to homozygous, revertant (+/+) recipients which were subline-matched with the donors; these grafts underwent crisis rejection with the same time of onset of rejection as observed with C57BL/6 recipients. These observations indicate that a new histocompatibility antigen is associated with the pun mutation and is lost upon reversion to wild type; this association is the first demonstration of a link between histocompatibility and coat color phenotypes.

Animals↗
PubMed1988

Androgen regulation of murine beta-glucuronidase expression: identification and characterization of a nonresponse variant.

One of the major features of beta-glucuronidase (GUS) expression in inbred strains of the house mouse, Mus musculus, is the responsiveness of this enzyme to androgen stimulation in tubule cells of the kidney. Both GUS-specific and nonspecific mutations have been described which define genes that serve to control this response. During examination of the expression of GUS in the interbreeding subspecies, Mus hortulanus, a new GUS haplotype was uncovered that is characterized, in part, by a lack of GUS response to androgen stimulation in an apparently responsive kidney. Blot hybridization analyses of kidney RNA with a radiolabeled murine GUS cDNA shows this lack of response to be reflected in GUS mRNA levels. The difference in heat stability of GUS activity between M. hortulanus and a responsive inbred strain, ICR/Ha, was utilized to assess the contribution of each parent to kidney levels of GUS in androgen-treated and -untreated F1 progeny of these strains. The results, together with preliminary genetic studies, suggest that the element controlling this responsiveness (or the lack thereof) is cis-active and tightly linked to the GUS structural gene on chromosome 5. It is not known whether this element is identical to another GUS-specific, cis-active element, Gus-r, which also controls the androgen response of GUS in mouse kidney.

Androgens↗
PubMed1987

Differential methylation of the ornithine carbamoyl transferase gene on active and inactive mouse X chromosomes.

Ornithine carbamoyl transferase (Oct) is an X-linked gene which exhibits tissue-specific expression. To determine whether methylation of specific CpG sequences plays a role in dosage compensation or tissue-specific expression of the gene, 13 potentially methylatable sites were identified over a 30-kilobase (kb) region spanning from approximately 15 kb upstream to beyond exon II. Fragments of the Mus hortulanus Oct gene were used as probes to establish the degree of methylation at each site. By considering the methylation status in liver (expressing tissue) versus kidney (nonexpressing tissue) from male and female mice, the active and inactive genes could be investigated on active and inactive X-chromosome backgrounds. One MspI site, 12 kb 5' of the Oct-coding region, was cleaved by HpaII in liver DNA from males but not in kidney DNA from males and thus exhibited complete correlation with tissue-specific expression of the gene. Six other sites showed partial methylation, reflecting incomplete correlation with tissue-specific expression.

Animals↗
PubMed1986

The egasyn gene affects the processing of oligosaccharides of lysosomal beta-glucuronidase in liver.

The accumulation of the relatively large amounts of beta-glucuronidase in microsomal fractions of normal mice depends on formation of complexes with the protein egasyn. Unexpectedly, it was found that the egasyn gene also affects the processing of beta-glucuronidase, which is segregated to lysosomes. In egasyn-positive mice lysosomal beta-glucuronidase from liver has a mean pI of 5.9 with a minor proportion at pI 5.4, whereas in egasyn-negative mice the proportion of the two lysosomal forms is reversed. Combined experiments measuring susceptibility to neuraminidase and to endoglycosidase H and specific binding to Ricinus communis lectin-agarose columns showed that the alterations in isoelectric point were associated with a decrease in complex oligosaccharides of lysosomal beta-glucuronidase in egasyn-positive mice. Since this alteration occurs not only in a congenic strain carrying the Eg0 gene but also in several other inbred strains that are homozygous for this gene, it is considered to be a genuine effect of the Eg gene rather than other genes that might regulate oligosaccharide processing. Also, the alteration is likely to be a result of direct physical interaction of the egasyn protein and lysosomal beta-glucuronidase, since a second lysosomal enzyme, beta-galactosidase, which does not form complexes with egasyn, is unaffected. The results suggest a model in which egasyn not only causes accumulation of beta-glucuronidase in the microsomal compartment but also acts upon the precursor to lysosomal beta-glucuronidase to alter its interaction with trans-Golgi-apparatus processing enzymes.

Acetylglucosaminidase↗
PubMed1985

Genetic variation for enzyme structure and systemic regulation in two new haplotypes of the beta-glucuronidase gene of Mus musculus castaneus.

Two new haplotypes of the [Gus] gene complex have been characterized following their transfer from Mus musculus castaneus, where they were found, to a C57BL/6J genetic background. The [GUS]CS haplotype carries a new structural allele, Gus-scs, coding for enzyme with decreased thermolability and lacking an antigenic site present in other beta-glucuronidase allozymes. The [Gus]CL haplotype carries another new structural allele, Gus-scl, that codes for enzyme with increased thermolability and possessing the antigenic site. Both CS and CL beta-glucuronidase have the same catalytic activity/molecule as the standard B allozyme from C57BL/6J mice. Mice carrying either the [Gus]CS or [Gus]CL haplotype have reduced enzyme activity in all tissues examined at all stages of development. The reduced enzyme activity is partially accounted for by reduced rates of enzyme synthesis, and the remainder probably results from increased rates of enzyme turnover. beta-Glucuronidase mRNA levels in these mice were not reduced suggesting that the observed reduction in enzyme synthesis is due to a decreased efficiency of translation for CS and CL mRNA.

Alleles↗
PubMed1984

Kinetics of beta-glucuronidase induction by androgen. Genetic variation in the first order rate constant.

Measurements of enzyme activity, rates of protein synthesis, and mRNA activity suggest that the induction of beta-glucuronidase in mouse kidney in response to androgen is regulated at a pretranslational level. Following an initial lag period, the rate and extent of induction follow the rules of simple turnover kinetics and can be described in terms of a zero order rate constant for acquisition of mRNA activity (ka) and a first order rate constant for loss of activity (kb). Genetic variation in kb, described here for the first time, alters the half-time and extent of induction. Variation in kb is independent of previously described variation in ka and, unlike changes in ka, is not associated with change in the lag time. The DNA sequences determining kb, like those determining ka, are genetically linked to the structural gene for beta-glucuronidase. Following the removal of androgen, beta-glucuronidase activity, rate of synthesis, and mRNA activity all decline rapidly with half-lives of 1-2 days. Even in the most rapidly inducing strains, this is significantly faster than the half-time for induction determined by kb. Furthermore, genetic variation in kb does not affect the rate of de-induction. These facts suggest that kb may not describe the turnover of beta-glucuronidase mRNA, but rather the turnover of another step in the induction process.

Animals↗
PubMed1984

Methylation patterns of repetitive DNA sequences in germ cells of Mus musculus.

The major and the minor satellite sequences of Mus musculus were undermethylated in both sperm and oocyte DNAs relative to the amount of undermethylation observed in adult somatic tissue DNA. This hypomethylation was specific for satellite sequences in sperm DNA. Dispersed repetitive and low copy sequences show a high degree of methylation in sperm DNA; however, a dispersed repetitive sequence was undermethylated in oocyte DNA. This finding suggests a difference in the amount of total genomic DNA methylation between sperm and oocyte DNA. The methylation levels of the minor satellite sequences did not change during spermiogenesis, and were not associated with the onset of meiosis or a specific stage in sperm development.

Animals↗
PubMed1983

Distribution of the T4D bacteriophage binding sites on the Escherichia coli B surface.

A study of the distribution of the T4D bacteriophage binding sites on the Escherichia coli B bacterial surface has shown that: (1) the number of binding sites per unit surface area is larger during growth period than during the division period, (2) the density of the binding sites on one-half of the bacterial cell is larger than the density of binding sites on the other half; (3) in newly-divided bacteria, the maximal binding site density is situated at one pole; (4) as bacteria grow, this maximum shifts to the middle of the cell; (5) when the septum is established, the middle of the cell becomes very poor in phage binding sites activity, and (6) phage adsorbs in clusters or in groups following curved lines around the bacterial cell.

Binding Sites↗
PubMed1982

Structural characteristics of the short-tail fibers of T4 bacteriophage.

The characteristics of pure preparations of short-tail fibers of bacteriophage T4 have been studied in the optical and electron microscope. Three main structures were observed: 1) spheres of 8.1 nm diameter; 2) fibers 43 nm long and 3.8 nm thick; and 3) fibers 54 nm long and 3.2 nm thick. Both types of fibers exhibited a regular beaded appearance. The 43-nm fibers were the most abundant structure. During the process of purification of the short-tail fibers, the formation of aggregates was observed each time the material containing the short-tail fibers was dialyzed against saline solutions. These aggregates became increasingly fibrous (as observed in the optical microscope) as the material used was increasingly enriched in short-tail fibers. Finally, most of the aggregates were of the fibrous type when they were formed from a purified preparation of short-tail fibers. In the electron microscope, it was found that the filamentous aggregates were organized in well-defined bundles. The amino acid composition of the highly purified short-tail fibers was also determined. Among the known fibrous proteins, the ones that most resemble the amino acid composition of the short-tail fibers are actin and fibrinogen. These observations are discussed in relation to the T4 short-tail fiber structure and their localization on the hexagonal baseplate of the T4 tail structure.

Amino Acids↗
PubMed1982

Expression of beta-glucuronidase haplotypes in prototype and congenic mouse strains.

A gene complex consists of a structural gene with its associated regulatory information; together they behave as the functional and evolutionary unit of mammalian chromosomes. The use of congenic lines, in which alternate forms, or haplotypes, of a gene complex are transferred into a common genetic background by repeated backcrossing, provides a means of comparing the regulatory properties of different haplotypes of a gene complex without the complications introduced by extraneous genetic differences. We have now carried out such a study of the A, B, and H haplotypes of the beta-glucuronidase gene complex, [Gus], in mice. These haplotypes were derived from strains A/J, C57BL/6J, and C3H/HeJ and were compared against the C57BL/6J genetic background. Enzyme structure was compared in terms of charge (isoelectric point), stability (rate of thermal denaturation), substrate affinity (for 4 MU glucuronide), and antigenicity (reactivity with a standard antibody). Compared to the B form, the enzyme coded by the A haplotype has a lower isoelectric point, and that coded by the H haplotype is less stable. The decreased stability is the result of a lower activation energy for the thermal denaturation reaction. These differences were maintained in the congenic strains. All three enzyme forms showed identical substrate affinities. Antigenicity per enzyme unit was also identical for all three, indicating that none lacks an antigenic site possessed by the others and that they all possess the same catalytic activity per molecule. The expression of alleles of the Gus-t temporal locus within the gene complex was not affected by transfer into the C57BL/6 genetic background. The same developmental switches in enzyme activity were seen in each case. Transfer into the C57Bl/6 background also did not affect expression of the Gus-r regulator determining androgen inducibility of beta-glucuronidase synthesis in kidney epithelial cells. However, enzyme accumulation in induced cells was altered when the haplotypes were transferred into the C57BL/6 genetic background. Since the rate of synthesis was not affected, it suggests that the genetic differences between strains that are not linked to the [Gus] complex affect the rate of enzyme loss by degradation or secretion. Beta-Glucuronidase in liver is present in both lysosomes and endoplasmic reticulum (microsomes). The relative amount of enzyme at each site depended on both the indentity of the structural allele and the function of unlinked genetic modifiers. Within the C57BL/6 background the percentage of total enzyme present in the microsome fraction was the order A greater than B greater than H. For the H form of the enzyme the percentage was appreciably greater in the C3H genetic background compared to C57BL/6. As expected, then, the [Gus] complex contains all of the genetic determinants of enzyme structure detected by thermal stability and isoelectric point measurements...

Animals↗
PubMed1981

Chromosome and cellular origin of choriocarcinoma.

The preferential association of choriocarcinoma with complete moles has long been recognized. In fact, of all forms of pregnancy that lead to choriocarcinoma, the risk associated with hydatidiform moles is 2000 to 4000 times greater than that of normal pregnancy or abortion. In order to corroborate the cell lineage between hydatidiform moles and choriocarcinoma, we investigated the chromosomal constitution and polymorphisms in the latter cells. Three choriocarcinomas from three women with a previous history of hydatidiform moles were examined chromosomally. Two hundred thirteen cells were analyzed for their chromosome number by conventional Giemsa staining; karyotyping of 33 cells with Q- and C-banding revealed 13 kinds of structural abnormalities unequivocally identified as of clonal origin. Two cases exhibited involvement of the long arm of chromosome 1 in rearrangements. Complete homozygosity and exclusive inheritance of a paternal genome would be expected if the tumor arose from a preceding molar pregnancy. However, the heterozygosity observed in choriocarcinoma cells suggests no common cellular lineage with complete moles; the latter originate through fertilization of an "empty egg" by a haploid sperm, followed by duplication of the chromosomes. Several possibilities have to be considered for the origin of choriocarcinomas, since it seems unlikely that alterations in the paracentric areas of the marker chromosomes occurred during tumor development. One possibility is that moles which are characterized by heterozygosity may transform into choriocarcinomas. Another possibility is that such tumors may originate from trophoblasts of the conceptuses rather than from moles.

Adult↗
PubMed1981

Defective packing of an unusual DNA in a virulent Erwinia phage, Erh 1.

A newly isolated bacteriophage, Erh 1, for Erwinia herbicola, has been characterized. This virulent phage has been found to have an elongated rod-like head and a short complex tail structure. One major protein and 5 minor proteins have been identified as phage components. The head structure was found to be transparent, flexible and could be twisted or flattened by various treatments. The DNA, isolated from highly purified phage particles, was linear, double-stranded, had a G-C content of 46-47% and displayed two unique features. (1) The isolated phage DNA molecules were highly heterogeneous in contour lengths; the most prevalent molecules had a length of 6-12 mu, but molecules have been measured with lengths ranging from 2.3 mu to 37 mu. (2) One or two long single-stranded regions, "gaps," ranging in length from 0.3 to 3.1 mu with an average length of 1.4 +/- 0.7 mu, were found in about 25% of the phage DNA molecules. Upon density gradient centrifugation of p32 labeled phage, it was found that most of the DNA was contained in apparently noninfectious, defective particles, with densities ranging from 1.34 to 1.41 while most of the infectious particles were found in fractions of density of 1.44 +/- 0.02. When used to multiply infect cells, the lower density particles were able to complement each other and form infectious centers. Further, it was found that infectious particles themselves were heterogeneous and had sedimentation constants varying from 600 S to 1400 S. From the distribution of DNA sizes in these particles, the variations in sedimentation behavior, and the flexibility of the head structure of most particles, it appears that the head structure is formed first and then the DNA is packed inefficiently into this head structure. Apparently, most phage particles are only partly filled and do not contain a complete genome while a few others may contain a large amount of redundant viral DNA.

Bacteriophages↗
PubMed1981

Polymerization of Escherichia coli bacteriophage T4D short tail fibers into filaments and filament bundles.

Crude preparations of short tail fibers of E. coli T4D bacteriophage were found to be readily soluble in 0.01 M EDTA, but after dialysis against H2O, and a 10-fold concentration, the fibers aggregated into long filaments and filament bundles. However, since the filaments from these crude preparations were associated with nonfilamentous material, the fibers were purified in the presence of 0.01 M EDTA. After dialysis of the purified short tail fiber preparation against water, and 10-fold concentration, a precipitate was again formed that could be readily separated by low speed centrifugation. In micrographs, this precipitated material showed large aggregates of short tail fibers but did not show well-defined filaments. However, when these aggregates were incubated with 0.02 M MgSO4 and 0.06 M KC1, well-defined filaments were again formed.

Electrophoresis, Polyacrylamide Gel↗