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Biomedical subjects

D C Bennett

Publications and source records attributed to D C Bennett.

At least 19 recordsLinked to original sources
PubMed1992

Suppression of properties associated with malignancy in murine melanoma-melanocyte hybrid cells.

Murine and human melanoma cells differ relatively reliably from non-tumorigenic melanocytes in certain biological properties. When cultured at low pH, melanocytes tend to be pigmented and melanoma cells unpigmented. The growth of virtually all metastatic melanoma cells is inhibited by phorbol esters such as TPA (12-O-tetradecanoyl phorbol-13-acetate), which stimulate melanocyte growth. Melanocytes fail to grow in suspension culture or produce tumours when implanted in animals, while many melanoma lines can do both. Here we studied which of these properties were dominant in hybrid cells formed by fusion of drug-resistant murine B16-F10RR melanoma cells to melanocytes of the albino and brown lines, melan-c and melan-b. The albino melanocytes are unpigmented but well-differentiated, the brown melanocytes produce pale brown pigment and the melanoma cells are unpigmented under the conditions used. All hybrid colonies observed produced black pigment, except some melan-b/melanoma hybrids when growing sparsely with TPA. Thus pigmentation was generally dominant. 14/15 hybrid lines showed stimulation of proliferation by TPA, as do melanocytes. Most hybrid lines showed no or reduced capacity for growth in suspension, though some grew better in suspension when TPA was present. There was marked suppression of the tumorigenicity of the parental melanoma cells in 4/8 hybrids examined, and tumorigenicity was reduced in the others, despite considerable chromosome loss by the passage level tested. Thus most properties of the non-tumorigenic pigment cells were dominant, as often observed for other cell lineages, and providing further evidence for gene loss in the genesis of malignant melanoma.

Animals↗
PubMed1992

Effects of the developmental colour mutations silver and recessive spotting on proliferation of diploid and immortal mouse melanocytes in culture.

The developmental mouse coat-colour mutations silver (si, chromosome 10) and recessive spotting (rs, chromosome 5, mapping very close to the dominant white spotting or W/c-kit locus), appear to reduce the numbers of functional melanocytes in the skin. They were studied at the cellular level by melanocyte culture. Cellular morphology, differentiation and survival appeared normal. However, both mutations were found to reduce the melanocyte proliferation rate in primary cultures, as measured by [3H]thymidine labelling indices. Two immortal si/si melanocyte lines (designated melan-si1 and melan-si2) and one rs/rs line (melan-rs) were established. Melan-si1 and melan-rs were cloned. All three immortal lines at low passage levels had doubling times significantly greater than those of our other melanocyte lines melan-a, melan-b and melan-c. Thus they retained the phenotype of slow proliferation.

Animals↗
PubMed1991

Dioxin contamination and growth and development in great blue heron embryos.

A great blue heron colony located near a pulp mill in British Columbia failed to fledge young in 1987, with a concurrent sharp increase in polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated dibenzofuran (PCDF) levels in their eggs. In 1988 we tested the hypothesis that the PCDD and PCDF contamination caused reproductive failure by increasing mortality of the heron embryos in ovo. Pairs of great blue heron eggs were collected from three British Columbia colonies with low, intermediate, and high levels of dioxin contamination: Nicomekl, Vancouver, and Crofton, respectively. One egg of each pair was incubated under laboratory conditions at the University of British Columbia (UBC) while the other egg was analyzed for PCDDs and PCDFs. All incubated eggs were fertile. All eggs from the Nicomekl colony hatched, while 13 of 14 eggs from Vancouver and 12 of 13 eggs from Crofton hatched. Subcutaneous edema was observed in 4 of 12 chicks from Crofton and 2 of 13 chicks from Vancouver. No edema was seen in the chicks from Nicomekl. There was a small, but significant, negative regression of plasma calcium concentration, yolk-free body weight, tibia length, wet, dry, and ash weight, beak length, and kidney and stomach weight of the hatched chicks on the tetrachlorodibenzo-p-dioxin (TCDD) level of the paired eggs. Fewer down follicles were present on the heads of TCDD-contaminated chicks. Hence while dioxins did not cause mortality of the heron embryos in ovo, the depression of growth and the presence of edema are suggestive that dioxins at the levels found in the environment have an adverse effect on the development of great blue heron embryos.

Animals↗
PubMed1991

Specificity of attenuation control in the ilvGMEDA operon of Escherichia coli K-12.

Three different approaches were used to examine the regulatory effects of the amino acids specified by the peptide-coding region of the leader transcript of the ilvGMEDA operon of Escherichia coli K-12. Gene expression was examined in strains carrying an ilvGMED'-lac operon fusion. In one approach, auxotrophic derivatives were starved of single amino acids for brief periods, and the burst of beta-galactosidase synthesis upon adding the missing amino acid was determined. Auxotrophic derivatives were also grown for brief periods with a limited supply of one amino acid (derepression experiments). Finally, prototrophic strains were grown in minimal medium supplemented with single and multiple supplements of the chosen amino acids. Although codons for arginine, serine, and proline are interspersed among the codons for the three branched-chain (regulatory) amino acids, they appeared to have no effect when added in excess to prototrophs or when supplied in restricted amounts to auxotrophs. Deletions removing the terminator stem from the leader removed all ilv-specific control, indicating that the attenuation mechanism is the sole mechanism for ilv-specific control.

Amino Acid Sequence↗
PubMed1991

Tyrosinase expression and melanogenesis in melanotic and amelanotic B16 mouse melanoma cells.

Tyrosinase activity, abundance, and mRNA transcription were examined in three sublines of the B16 mouse melanoma. Tyrosinase activity and melanin content were highest in the B16-F1 cells, slightly less in the B16-F10, and markedly lower in the B16-F10-DD cells. No differences in the level of tyrosinase mRNA or protein were found in the three different sublines. Thus, the differences in tyrosinase expression arise from the post-translational modification of the enzyme causing its activation or inhibition.

Animals↗
PubMed1990

A primary transcript in spinach chloroplasts that completely lacks a 5' untranslated leader region.

A fifth, previously undetected transcript for the plastid gene encoding the beta subunit of spinach chloroplast ATPase (atpB) has recently been identified in vitro. In this report we show that this transcript is present in vivo and its 5' end is at the translation start codon. When synthesized in vitro using spinach chloroplast RNA polymerase, the 5' end of this novel transcript is located at the beginning of the second codon of the atpB coding sequence. Although this atpB transcript lacks an untranslated leader region, it is an abundant RNA in vivo and is associated with crude polysomal fractions, as are the four larger atpB transcripts. In vitro synthesis of the leaderless transcript does not occur for a plasmid DNA in which the "-35' region of the promoter is deleted.

Adenosine Triphosphatases↗
PubMed1990

An in vitro transcription termination system to analyze chloroplast promoters: identification of multiple promoters for the spinach atpB gene.

Promoters for spinach chloroplast genes were cloned 5' to a strong factor-independent transcription terminator from E. coli. These "minigene" constructions were transcribed in vitro by a transcriptionally active extract of spinach chloroplasts. Transcription of supercoiled DNA templates resulted in synthesis of discretely-sized RNAs that were readily quantifiable. The efficiency of transcription was up to 3.5 RNAs per template. The transcription termination system described in this report was used to identify the primary transcripts for the plastid atpB gene. Four in vivo transcripts for the atpB gene have been previously identified with 5' untranslated leaders of approximately 455, 275, 180 and 100 nucleotides, respectively. In this report we show that the "-455", "-275" and "-180" regions function as chloroplast promoters in vitro. In addition, a fourth promoter was found that yields a primary transcript totally lacking an untranslated leader.

Adenosine Triphosphatases↗
PubMed1990

Identification of the albino mutation of mouse tyrosinase by analysis of an in vitro revertant.

From within an albino melanocyte line grown in vitro we identified and cloned cells that apparently had reverted to wild type. We sequenced a part of the tyrosinase gene, encompassing a candidate mutation, from wild-type, albino, and revertant cell DNAs. The revertant cells contain, on one chromosome, a perfect base reversion to the wild-type sequence of this candidate mutation, proving that this is the sole defect in the tyrosinase gene of albino mutant mice. The revertant cells readily regain the albino phenotype after freezing and thawing. Taking advantage of a Dde I restriction site created by the albino mutation, we demonstrated that the regained phenotype is due to allele loss involving the wild-type chromosome. The Dde I site also allowed us to show that all inbred albino mice carry the same mutation and so must be derived from the same progenitor.

Albinism↗
PubMed1990

Characterization of TRP-1 mRNA levels in dominant and recessive mutations at the mouse brown (b) locus.

The mouse brown locus encodes a putative membrane-bound metalloenzyme, tyrosinase-related protein-1 (TRP-1). We have examined the effect on mRNA expression of the locus of a number of mutant alleles. The common null mutant allele, brown, produces wild-type levels of TRP-1 mRNA, which is nonfunctional. Another recessive allele, cordovan-Harwell, has an intermediate, dark-brown phenotype and produces only very low levels of presumably normal TRP-1 mRNA. Two dominant alleles appear to act by killing the melanocyte in which they are expressed. One of them, Light, has normal size and amounts of TRP-1 mRNA. The other, White-based brown, produces no detectable TRP-1 mRNA. It has a gross DNA rearrangement at the 5' end, and we speculate that this results in activation of transcription of sequences not usually seen in melanocytes, and that this is toxic to the cell. The relationship between phenotype and molecular structure at the locus is discussed, and we draw some general principles applicable to other developmental genes.

Animals↗
PubMed1990

Phenotypic rescue of mutant brown melanocytes by a retrovirus carrying a wild-type tyrosinase-related protein gene.

A mouse cDNA for the developmentally controlled, melanocyte-specific protein, tyrosinase-related protein 1 (TRP-1), was previously cloned and reported to show genetic linkage with the coat-colour locus brown (b) on mouse chromosome 4. The cDNA has been inserted into a retroviral vector derived from Moloney murine leukaemia virus, under the control of the human histone H4 promoter. This vector was used to infect melanocytes of the immortal line melan-b, which are homozygous for the b mutation and which display light brown pigmentation in culture. Infected cultures containing between 0.2 and 2 copies of provirus per cell displayed an altered phenotype: 20-50% of cells now had the black to dark brown colour characteristic of cultured wild-type (Black, B/B) mouse melanocytes. Thus the TRP-1 gene complements the brown mutation. We conclude that TRP-1 is the product of the wild-type b-locus.

Animals↗
PubMed1989

Cloned mouse melanocyte lines carrying the germline mutations albino and brown: complementation in culture.

We have established two new immortal lines of mouse melanocytes, melan-b and melan-c, from mice homozygous for the brown (b) and albino (c) mutations respectively. Both lines were derived through differentiation in vitro of embryonic epidermal melanoblasts. The brown melanocytes are visibly brown by light microscopy, and centrifuged cell suspensions form brown pellets. The albino melanocytes form white pellets and contain abundant unpigmented premelanosomes as shown by transmission electron microscopy. Like normal, non-immortal melanocytes and like the immortal black melanocyte line melan-a, both lines show little or no growth in a standard, serum-supplemented medium, but proliferate well in the presence of 12-o-tetradecanoyl phorbol-13-acetate (TPA). Sustained growth of the albino cells also requires either keratinocyte feeder cells or 2-mercaptoethanol (2-ME). The modal chromosome numbers are 39 for melan-b and 40 (diploid) for melan-c. Neither line is tumorigenic in nude mice. Heterokaryons between the two lines can be constructed and form wild-type, black pigment. Melanocyte lines can now be reproducibly generated from mice of different strains, and provide tools for molecular studies of germline coat-colour mutations. These two lines provide elegant means to study the developmentally controlled expression of the two complementary genes, B and C, with black melanin pigment as a readily detectable natural marker.

Animals↗
PubMed1989

Mechanisms of differentiation in melanoma cells and melanocytes.

Literature is reviewed on the mechanisms of differentiation in mammalian melanoma cells and normal melanocytes. Pigment cells are particularly useful for studies requiring the observation of differentiation in living cells, for example, studies of commitment. Topics discussed include melanin synthesis and other markers of pigment cell differentiation; stochastic models of differentiation and commitment; the lability of early stages of differentiation; extracellular factors affecting pigment cell differentiation, with implications for intracellular controls; the role of proliferation and the cell cycle in differentiation, and the relative roles of changes in transcription, translation, and posttranslational processes.

Animals↗
PubMed1987

A line of non-tumorigenic mouse melanocytes, syngeneic with the B16 melanoma and requiring a tumour promoter for growth.

An immortal line of pigmented melanocytes, "melan-a", has been derived from normal epidermal melanoblasts from embryos of inbred C57BL mice. The conditions favouring proliferation of these cells largely resemble those for normal, non-established mouse melanoblasts and melanocytes, and include a low extracellular pH and the presence of a tumour promoter, tetradecanoyl phorbol acetate (TPA) or teleocidin. Melan-a cells have the diploid chromosome number and do not form tumours in syngeneic or nude mice. They are therefore the first known line of non-tumorigenic mouse melanocytes, although an aneuploid melanocyte line of untested tumorigenicity has been reported (Sato et al., 1985). Melan-a cells are syngeneic with the B16 melanoma and its sublines, and provide an excellent parallel non-tumorigenic line for studies of the cellular and molecular basis of melanoma malignancy.

Animals↗
PubMed1987

Differentiation apparently repressed by the nucleus. Rapidly-induced pigmentation of enucleated melanoma cells.

There is evidence for cytoplasmic control over gene expression in cell differentiation, but still very little is known of the intracellular mechanism, nuclear, cytoplasmic, or both, which actively initiates the differentiation of one cell type into another. Here the role of the cytoplasm was examined in the induction of differentiation of cultured mouse melanoma cells by melanocyte-stimulating hormone and alkaline medium. Intact cells were compared with cytoplasts, cells enucleated by centrifugation in the presence of cytochalasin D (CD). Surprisingly, early inductions of pigment (melanin) synthesis and of the principal melanin-synthesizing enzyme activity, tyrosinase, could be achieved in cytoplasts. Indeed these early changes were slower in nucleated cells and were accelerated by the inhibitor of protein synthesis, cycloheximide. Thus the initial activation of tyrosinase and melanin synthesis--although not necessarily any other or later aspects of melanoma cell differentiation--is apparently controlled through a labile, transcription- and translation-dependent repression. To our knowledge this is a novel mechanism for the initiation of differentiation; its generality remains to be tested.

Animals↗
PubMed1986

The Shrunken gene on chromosome 9 of Zea mays L is expressed in various plant tissues and encodes an anaerobic protein.

The Shrunken gene, located on the short arm of chromosome 9 of Zea mays, encodes the enzyme sucrose synthase (EC 2.4.1.13). The gene is known to be expressed in the endosperm of the developing maize kernel and seems to be involved in sucrose breakdown prior to starch synthesis. We have analyzed different tissues of the maize plant for transcripts of the Shrunken gene and have found rather high transcription rates in the etiolated shoot and the primary root of the germinating kernel. If the etiolated seedlings are illuminated, the transcript level drops by about 95% in the greening plant parts (1st and 2nd leaves) which are active in photosynthesis. A very low transcript level is found in mature green leaves where sucrose is formed from products of photosynthesis via a separate pathway. Upon anaerobic stress of the young seedling, the level of Shrunken transcripts increases 10 and 20 times in shoot and root tissue respectively. Apparently anaerobic induction supersedes the negative control that is observed after illumination in the 1st and 2nd leaves. From the experiments outlined here we conclude that the anaerobic protein 87 (ANP87, Hake et al. 1985) is encoded by the Shrunken locus. While the expression of the Shrunken gene varies in different tissues and in response to external stimuli, transcription of the second sucrose synthase (B) gene seems to be irresponsive to anaerobic stress and to be expressed at a similar low level in all of the tissues examined.

Alcohol Dehydrogenase↗