Biomedical subjects
Publications and source records attributed to H Harris.
The abnormal membrane glycoprotein that we previously found to be associated with malignancy in a wide range of different tumours is present in the cell membrane as a dimer and appears to have a specific binding site for glucose.
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Alkaline phosphatase (ALP) components in 8 cell lines of HeLa were examined. Line to line heterogeneity in ALP expression was observed using the criteria of electrophoretic mobility before and after neuraminidase treatment, heat stability, L-phenylanine inhibition, and reactivity against antiplacental ALP antiserum. Six lines contained a placentallike ALP isozyme and varying amounts of a liverlike ALP isozyme. One line contained a liverlike ALP isozyme only. One line contained a new ALP form which was clearly distinguished from the placental, liver, bone, and intestinal ALPs. Thus, derepression of the placental ALP structural locus appeared to have occurred in 6 of the 8 lines. However, where expressed, the placentallike ALP varied electrophoretically from line to line, and in only one case was the mobility identical to that of a common placental ALP phenotype. This phenotypic heterogeneity of the ""derepressed'' placentallike ALP contrasts markedly with the phenotypic stability of other enzymes expressed in HeLa cells.
Alkaline phosphatase (ALP) components in extracts of seven malignant and eleven benign ovarian tumors were characterized using the criteria of electrophoretic mobility before and after neuraminidase treatment, heat stability, L-phenylalanine inhibition and reactivity against antiplacental ALP antiserum. Seven of the eighteen tumors had ALP components which most closely resembled the ALP isoenzyme normally found in placenta and were clearly distinguished from all other tissue ALPs. The proportion of tumors with the placental-like ALP in the malignant group (five out of seven) was significantly greater than the proportion in the benign group (two out of eleven). The fraction (78%) of the malignant tumors with the isozyme represents a larger percentage than has previously been found by examination of cancer patients' sera. The electrophoretic mobilities of the placental-like ALPs in the tumors were in no case identical to the mobilities of any of the six common placental ALP phenotypes. The tumor ALPs may thus be determined by rare variant alleles at the ALP locus, or alternatively, the enzyme molecules may have been subject to structural modification. At least two of these tumors contained an electrophoretically slow. heat-stable, leucine-sensitive ALP, which may correspond to what has been termed the D-variant of placental ALP found in some other tumors.
In an attempt to study mutagenesis in human diploid fibroblasts, clones derived from mass cultures treated with mutagen have been examined by starch-gel electrophoresis for 43 different enzyme loci. A technique of mutagen treatment was devised which facilitated the cloning and which enabled the cells to be exposed to very high doses of EMS and MNNG. Two alterations in phenotype, presumably the result of mutation, were observed, one involving peptidase D (PEPD) and the other phosphoglucomutase (PGM1).
Starch gel electrophoresis and inhibition studies with L-phenylalanine, L-homoarginine, L-leucine, L-leucylglycylglycine, and L-phenylalanylglycylglycine were carried out on a series of human alkaline phosphatases [orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1] derived from fetal and adult liver, kidney, bone, and intestine. No differences between adult and fetal liver, kidney, or bone alkaline phosphatases were observed by either electrophoretic or inhibition studies. However, the fetal intestinal enzyme could be clearly distinguished from the adult intestinal enzyme by its greater anodal electrophoretic mobility and its retardation after treatment with neuraminidase. Even after extensive neuraminidase treatment, its anodal mobility was still slightly greater than that of adult intestinal alkaline phosphatase. Fetal and adult intestinal enzymes showed the same inhibition profiles with the series of inhibitors both before and after treatment with neuraminidase. A survey of intestinal samples from fetuses and premature infants of various gestational ages indicated that the changeover from the synthesis of fetal to adult intestinal enzyme begins at about 28-32 weeks of gestation. The difference between the fetal and adult forms of intestinal alkaline phosphatase may represent the expression of different gene loci or a difference in post-translational modification.
1. Inhibition studies have been carried out on a series of ALPs derived from liver, bone, kidney, placenta and intestine, using L-phenylalanine, L-homoarginine, L-leucine, L-leucyl-glycyl-glycine and L-phenylalanyl-glycyl-glycine as inhibitors. 2. No differences between liver, bone and kidney ALPs with any of the inhibitors were observed. 3. L-phenylalanine and L-homoarginine give a major degree of discrimination between liver/bone/kidney ALP on the one hand, and placental and intestinal ALPs on the other. L-leucyl-glycyl-glycine and L-phenylalanyl-glycyl-glycine give a major degree of discrimination between placental ALP on the one hand, and intestinal ALP and liver/bone/kidney ALP on the other. L-leucine discriminates between the three classes, but to a lesser degree. Minor degrees of discrimination between placental and intestinal ALPs occur with L-phenylalanine and L-homoarginine and between intestinal and liver/bone/kidney ALPs with L-leucyl-glycyl-glycine and L-phenylalanyl-glycyl-glycine. By using an appropriate combination of inhibitors the ALPs can be separated into three clearly distinct categories: placental, intestinal and liver/bone/kidney. 4. The six common placental ALP phenotypes as defined by electrophoresis show identical inhibition profiles with the series of inhibitors. The same profile was found for several rare electrophoretic variants. However, two rare electrophoretic variants (P-187 and P-92) each encountered once in a sample of 225 plancentae, showed significantly deviant inhibitions with the various inhibitors and also differed from each other. From the electrophoretic patterns, both of these rare phenotypes appear to be heterozygotes. P-187 probably corresponds to the so-called D-variant previously described. P-92 represents a new type of placental ALP variant with an aberrant inhibition profile. In both cases the particular rare allele concerned evidently alters the primary structure of the enzyme so that it has an altered electrophoretic mobility and also an altered sensitivity to inhibition for each of the different inhibitors. 5. Treatment of the various ALPs with neuraminidase to remove sialic acid residues does not affect their inhibition characteristics or activities.
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Tumours were induced by chemical carcinogens in chimaeric mice made by the amalgamation of two embryos each producing a different electrophoretic variant of the enzyme glucose phosphate isomerase. Although, in these chimaeras, the smallest samples of normal tissue that could be analysed almost invariably contained both isoenzymes, almost all the tumours contained only one. An analysis of the size of the clones that formed the chimaeric epidermis permitted the conclusion that the epidermal tumours produced could not have risen from more than 8 cells; and the most probable interpretation of the data is that these tumours were clonal growths.
Cell fusion provides a genetic test for linkage between cellular phenotypic markers and malignancy. We have examined hybrid cells in which malignancy is suppressed and malignant segregants derived from them, to test whether the reappearance of malignancy is regularly associated with loss or abnormality of actin cables or microtubules. Immunofluorescence studies did not reveal any difference between the malignant and non-malignant hybrids in the number and distribution of microtubules that they contained. All the hybrids, whether tumorigenic or not, contained elaborate patterns of cytoplasmic microtubules. Furthermore, no consistent relationship was observed between the absence of actin cables and malignancy.
A significant difference between blacks and whites in the distribution of red cell galactokinase (GALK) has been found by Tedesco et al. [2]. From the shapes of the distributions, it was inferred that whites are essentially all homozygous for one allele (GALKA), but blacks are polymorphic. A second allele (GALKP), for lower GALK activity, is presented at high frequency in blacks but rare or absent in whites. This paper presents a method which, assuming the genetic model presented, estimates the genotype composition of the black sample. We make some reasonable biochemical assumptions and fit a mixture of three normal distributions to the black data to obtain an estimate of p, the frequency of GALKA in blacks. The fit of the model to the data is excellent and the best estimate of p is .217 +/- .025. Since admixture of white genes in blacks from the United States is known to be about 20%, the value of p implies that virtually all GALKA alleles were introduced by admixture, and that the ancestral black population was monomorphic for GALKP. If whites are indeed monomorphic for GALKA, they differ from unmixed blacks by a full gene substitution at the locus for GALK.
This report has considered three approaches to the prenatal diagnosis of the severe, early onset form of hypophosphatasia. Two of these approaches, ultrasonography and the determination of the bone/liver isozymes of alkaline phosphatase (ALP) in cultured amniotic fluid cells, have proven useful diagnostically. The third method, assay of the bone/liver isozyme activity or total activity in supernatant amniotic fluid, was not informative for the affected fetus we studies. Failure to visualize a well-defined fetal skull after 16 weeks of pregnancy when the level of alpha-fetoprotein in the amniotic fluid is normal should arouse the suspicion of hypophosphatasia. Because the disease is known to manifest clinical variabiltiy, studies to detect both the biochemical defect as well as the structural manifestations should be considered. The combined use of ultrasonography, analysis of amniotic fluid alpha-fetoprotein, and the measurement of the bone/liver ALP in cultured amniotic fluid cells would appear to be the best approach to the prenatal diagnosis.
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The use of L-glutamate dehydrogenase (GLUD) as a reagent in staining mixtures to detect the isozymes of enzymes which catalyze the production of ammonia has been investigated. Methods have been devised for the electrophoresis and detection, using GLUD, of seven enzymes: cytidine deaminase, adenosine deaminase, adenosine monophosphate deaminase, arginase, argininosuccinase, D-amino acid oxidase, and D-aspartate oxidase. GLUD-linked staining methods appear to be sensitive, specific, and of general application.
A method for the starch gel electrophoresis of human L-glutamate dehydrogenase (GLUD) is described, as is the tissue distribution of GLUD detected by this method. Extracts of livers from 200 Whites were analyzed without demonstration of an electrophoretic variant. The molecular size was estimated to be 330,000 and the isoelectric point pH 4.83.