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Biomedical subjects

H Harris

Publications and source records attributed to H Harris.

At least 19 recordsLinked to original sources
PubMed1992

Induction of tyrosinase in human melanoma cells by L-tyrosine phosphate and cytochalasin D.

Pigmentation of RVH 421 human melanoma cells is induced when cell division is inhibited by cytochalasin D or L-tyrosine phosphate. Increased pigmentation correlates with increased tyrosinase activity when this is monitored over a time-course. Parallel measurements show that the amount of tyrosinase mRNA correlates with enzyme activity in cells growing without these additives. In contrast, in the presence of cytochalasin D or L-tyrosine phosphate, the increase in amount of tyrosinase mRNA is not sufficient to account for the increase in enzyme activity, indicating that these compounds act mainly at a post-transcriptional level.

Blotting, Northern↗
PubMed1992

Total inhibition of involucrin synthesis by a novel two-step antisense procedure. Further examination of the relationship between differentiation and malignancy in hybrid cells.

A novel procedure involving the sequential use of two different antisense constructs has been used to inhibit the synthesis of involucrin in a hybrid cell line formed by the fusion of a human cervical carcinoma cell with a normal human keratinocyte (ESH100P6). In this cell line, and other similar hybrids, malignancy, as measured by progressive growth in vivo, is suppressed; and it has been shown that the keratinocyte imposes its own programme of terminal differentiation on the non-malignant hybrid cell. In particular, involucrin, a precursor of one of the major components of the cornified envelope of mature keratinocytes, continues to be produced. When, however, malignant segregants arise in the hybrid cell population, the terminal differentiation programme of the keratinocyte is not expressed and involucrin ceases to be made. It seemed possible that if the synthesis of involucrin, a critical marker of keratinocyte terminal differentiation, could be completely inhibited, this differentiation programme might be disrupted, and the malignant phenotype might then reappear in the non-malignant hybrids. This question was investigated further in the present paper. Total, and specific, inhibition of involucrin synthesis was indeed achieved by a sequential two-step antisense procedure, which might provide a systematic general method for the complete inactivation of other selected target genes.

Animals↗
PubMed1991

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase.

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase (EC 1.14.18.1) have been developed. The tyrosine hydroxylase assay uses L-[carboxy-14C]tyrosine as the substrate, 14CO2 is released from the products of the hydroxylation and further metabolism of L-[carboxy-14C]tyrosine by incubation with ferricyanide, and measured radiometrically. D-Dopa is a preferable cofactor to L-dopa for the assay. Dopa oxidase activity is measured spectrophotometrically. Dopaquinone, produced on the oxidation of L-dopa, reacts with Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone) to form a pink pigment with an absorbance maximum at 505 nm. Details of the optimisation of conditions for the assays and their specificities for the two enzyme activities are described.

Animals↗
PubMed1991

The stimulation of lymphocyte motility by cultured high endothelial cells and its inhibition by pertussis toxin.

Incubation of rat lymph node lymphocytes with cultured high endothelial cells caused a high proportion of the lymphocytes to lose their round shape and adopt a polar morphology. This shape change was rapid; a substantial increase in the number of polar cells occurred within 15 min. Between 30 and 60% of the lymphocytes became polar. The change was not dependent on protein synthesis. Both lymphocytes which adhered to the cultured high endothelial cells and those which were non-adherent changed shape. Evidence is presented for two pathways for the induction of polarity in non-adherent lymphocytes: (i) transient or weak binding to the high endothelial cells may induce a shape change which persists after lymphocyte detachment and (ii) the high endothelial cells shed or secrete high molecular weight material into the medium which induces the shape change. The rapid change in lymphocyte morphology induced by cultured high endothelial cells was inhibited by pre-incubation of the lymphocytes with low concentrations (4-100 ng/ml) of pertussis toxin. Upon prolonged (2 h) incubation of toxin-treated lymphocytes with cultured endothelial cells a significant proportion of the lymphocytes adhering to the endothelial cells changed shape while the non-adherent lymphocytes remained spherical. This implies that cultured high endothelial cells may stimulate lymphocyte motility by two mechanisms: one which is rapid and pertussis toxin sensitive and one which is slower, pertussis toxin insensitive and dependent on lymphocyte adhesion to the high endothelial cells.

Animals↗
PubMed1990

The human alkaline phosphatases: what we know and what we don't know.

A review of the human alkaline phosphatases dealing specifically with (1) the gene loci, (2) characterization and discrimination of the various enzymes, (3) polymorphism at the enzyme level, (4) cDNA and gene structures, (5) membrane binding, (6) the carbohydrate moieties, (7) hypophosphatasia, (8) alkaline phosphatases in malignancies, (9) function.

Alkaline Phosphatase↗
PubMed1990

Congenital rubella syndrome associated with calcific epiphyseal stippling and peroxisomal dysfunction.

An infant girl had the clinical and immunologic findings of congenital rubella syndrome but also had arthrogryposis multiplex and calcific epiphyseal stippling. Spastic quadriparesis developed, and both physical and behavioral development were slow. Increased spasticity of the legs at 5 1/2 years was related not to progressive rubella encephalomyelopathy but to spinal cord compression by abnormal cartilaginous tissue. The presence of a peroxisomal disorder was demonstrated by a greatly increased level of phytanic acid and slightly increased levels of hexacosanoate in serum and by reduced activity of peroxisomal dihydroxyacetone phosphate acyltransferase and a slightly increased ratio of cytosolic to peroxisomal catalase activity in cultured fibroblasts. A reduction in the number and size of peroxisomes was demonstrated in cultured fibroblasts, and a needle biopsy specimen of the liver also showed the peroxisomes to have a smaller diameter than usual. We recommend that any child with epiphyseal stippling be assessed for peroxisomal disease and that the potential for spinal cord compression by dysplastic bone or cartilage be recognized. The association of peroxisomal dysfunction with congenital rubella has not been described previously. The interaction between rubella virus infection and peroxisomal function may need further investigation.

Calcinosis↗
PubMed1989

Localization of two alkaline phosphatase genes to the proximal region of mouse chromosome 1.

Using a human cDNA clone encoding the intestinal form of alkaline phosphatase, we have identified and mapped by RFLP analysis in a Mus spretus x C57BL/6J interspecies backcross two alkaline phosphatase genes which segregate independently on the proximal part of mouse Chromosome 1. The gene order and intergene distances were determined by standard backcross analysis as: centromere- Len-2 - 19.0 cM - Akp-3 - 20.0 cM - Akp-4 - 2.0 cM - Ren-1.

Alkaline Phosphatase↗
PubMed1989

The effect of antisense RNA to fibronectin on the malignancy of hybrids between melanoma cells and normal fibroblasts.

An expression vector containing a cDNA complementary to 1.3 kb of the 5' coding sequences of the fibronectin gene in the antisense orientation with respect to its promoter was introduced by electroporation into hybrids between melanoma cells and normal fibroblasts in which malignancy was suppressed. Immunofluorescence analysis of clones transfected with the antisense cDNA showed a dramatic decrease in the amount of fibronectin on the cell surface compared to that seen on the surface of the untransfected hybrid cells or of cells transfected with fibronectin cDNA in the sense orientation or with the expression vector alone. Four out of five clones transfected with the antisense cDNA were highly tumorigenic, whereas transfectants containing either the sense fibronectin construct or the expression vector alone remained non-tumorigenic. These results suggest that antisense RNA to fibronectin may be able to abrogate the suppression of malignancy imposed on the hybrid cells by the fibroblast parent.

Animals↗
PubMed1989

First identification of a gene defect for hypophosphatasia: evidence that alkaline phosphatase acts in skeletal mineralization.

Hypophosphatasia is a heritable disorder characterized by defective osteogenesis and deficient liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. Severe forms of the disease are inherited in an autosomal recessive fashion. We examined cultured skin fibroblasts from twelve patients with severe hypophosphatasia. All were deficient in L/B/K ALP activity, yet produced normal levels of the corresponding mRNA. Sequence analysis of L/B/K ALP cDNA isolated from one of the patient-derived fibroblast lines revealed a point mutation that converted amino acid 162 of mature L/B/K ALP from alanine to threonine. The patient was homozygous and the parents, who are second cousins, heterozygous for this mutation. Introduction of the mutation into an otherwise normal cDNA disrupted the expression of active enzyme, demonstrating that a defect in the L/B/K ALP gene resulted in hypophosphatasia and that the enzyme is, therefore, essential for normal skeletal mineralization.

Alkaline Phosphatase↗
PubMed1989

Analysis of liver/bone/kidney alkaline phosphatase mRNA, DNA, and enzymatic activity in cultured skin fibroblasts from 14 unrelated patients with severe hypophosphatasia.

Hypophosphatasia is a heritable disorder characterized by defective bone mineralization and a deficiency of liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity in serum and tissues. Severe forms of the disease, which are generally lethal in infancy, are inherited in an autosomal recessive fashion. The gene defects that produce hypophosphatasia are poorly understood, but many are likely to occur at the L/B/K ALP locus. To investigate these gene defects, we analyzed L/B/K ALP DNA, RNA, and enzyme activity in cultured dermal fibroblasts from 14 patients with perinatal or infantile hypophosphatasia and from 12 normal individuals. Southern blot analyses of the L/B/K ALP genes from patients and controls revealed identical restriction patterns. Control fibroblast ALP activity correlated with the corresponding L/B/K ALP mRNA levels estimated by blot hybridization analysis and densitometry (r = .94, P less than .0001). In contrast, fibroblasts from the hypophosphatasia patients were deficient in ALP enzyme activity but expressed apparently full-sized L/B/K ALP mRNA at normal levels. Bone specimens from one of the patients were examined and found to be deficient in histochemical ALP but contained immunologic cross-reactive material detected by anti-human liver ALP antiserum. Our results demonstrate that the deficiency of ALP activity in fibroblasts from 14 patients with severe hypophosphatasia is not due to decreased steady-state levels of the corresponding mRNA. The presence of enzymatically inactive L/B/K ALP protein in one of these patients is consistent with a point mutation or small in-frame deletion in the coding region of L/B/K ALP gene.

Alkaline Phosphatase↗
PubMed1989

The biology of tumour suppression.

Evidence is presented for the view that suppression of malignancy in hybrids between malignant and normal cells is achieved in one of two ways. The normal cell either imposes its own pattern of terminal differentiation on the hybrid or it complements a genetic block in the differentiation programme of the malignant cell. The progressive multiplication of malignant cells in the animal is seen as a secondary consequence of blocked differentiation. Karyological examination of hybrids in which malignancy is suppressed is entirely consistent with the view that programmes of differentiation may be governed in a pleiotropic fashion by single genes.

Animals↗
PubMed1988

Sequence and characterization of the human intestinal alkaline phosphatase gene.

At least four genes encode the human alkaline phosphatases (ALPs). The genes encoding three of these proteins (intestinal, placental, and placental-like ALPs), are linked on the long arm of chromosome 2, while the fourth gene (encoding liver/bone/kidney ALP) is located on chromosome 1. One of the linked genes, intestinal alkaline phosphatase, has been isolated on two overlapping phage clones and sequenced in its entirety. The gene is composed of 11 exons interrupted by 10 introns. Introns in intestinal, placental, and liver/bone/kidney ALPs occur at analogous positions (see accompanying articles), confirming that these genes arose from a single ancestral ALP gene. Multiple intestinal ALP mRNA species can be detected in RNA isolated from adult and fetal intestine and from cell line RNAs. In cell line RNA, the various species are the result of differential use of at least three of the four polyadenylation signals present in the intestinal ALP gene. A 125-base pair fragment located 5' to the first exon can function as a promoter in mammalian cells. This region contains two putative transcription signals, a TATA-like sequence and a consensus binding site for the transcription factor Sp1.

Alkaline Phosphatase↗
PubMed1988

Structure of the human liver/bone/kidney alkaline phosphatase gene.

In man, there are multiple forms of alkaline phosphatase encoded by at least three homologous genes: placental, intestinal, and liver/bone/kidney. This report describes the characterization of the human liver/bone/kidney alkaline phosphatase locus. The gene appears to exist as a single copy in the haploid genome and is comprised of 12 exons distributed over more than 50 kilobases. In liver, kidney, SAOS-2 human osteosarcoma cells, and cultured fibroblasts, there is a single major start for transcription situated about 25 nucleotides downstream of an A/T-rich motif. The promoter region is extremely G/C-rich, is relatively abundant in the dinucleotide CpG, and contains four copies of the consensus sequence for SP1 binding (GGGCGG). The liver/bone/kidney alkaline phosphatase gene is at least five times larger than the intestinal and placental alkaline phosphatase genes, mainly due to intron size differences. Intron-exon junctions occur at analogous positions in all three genes, but there is an extra non-coding exon at the 5' end of the liver/bone/kidney alkaline phosphatase gene. The relevance of our findings with respect to the evolution of the human alkaline phosphatase multigene family is discussed.

Alkaline Phosphatase↗