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T P Dooley

Publications and source records attributed to T P Dooley.

At least 19 recordsLinked to original sources
PubMed1999

Inhibitors of mammalian melanocyte tyrosinase: in vitro comparisons of alkyl esters of gentisic acid with other putative inhibitors.

To discover safe and effective topical skin-lightening agents, we have evaluated alkyl esters of the natural product gentisic acid (GA), which is related to our lead compound methyl gentisate (MG), and four putative tyrosinase inhibitors, utilizing mammalian melanocyte cell cultures and cell-free extracts. Desirable characteristics include the ability to inhibit melanogenesis in cells (IC50 < 100 microg/mL) without cytotoxicity, preferably due to tyrosinase inhibition. Of the six esters synthesized, the smaller esters (e.g. methyl and ethyl) were more effective enzyme inhibitors (IC50 approximately 11 and 20 microg/mL, respectively). For comparison, hydroquinone (HQ), a commercial skin "bleaching" agent, was a less effective enzyme inhibitor (IC50 approximately 72 microg/mL), and was highly cytotoxic to melanocytes in vitro at concentrations substantially lower than the IC50 for enzymatic inhibition. Kojic acid was a potent inhibitor of the mammalian enzyme (IC50 approximately 6 microg/mL), but did not reduce pigmentation in cells. Both arbutin and magnesium ascorbyl phosphate were ineffective in the cell-free and cell-based assays. MG at 100 microg/mL exhibited a minimal inhibitory effect on DHICA oxidase (TRP 1) and no effect on DOPAchrome tautomerase (TRP-2), suggesting that MG inhibits melanogenesis primarily via tyrosinase inhibition. MG and GA were non-mutagenic at the hprt locus in V79 Chinese hamster cells, whereas HQ was highly mutagenic and cytotoxic. The properties of MG in vitro, including (1) pigmentation inhibition in melanocytes, (2) tyrosinase inhibition and selectivity, (3) reduced cytotoxicity relative to HQ, and (4) lack of mutagenic potential in mammalian cells, establish MG as a superior candidate skin-lightening agent.

Animals↗
PubMed1998

Cloning of the human phenol sulfotransferase gene family: three genes implicated in the metabolism of catecholamines, thyroid hormones and drugs.

Phenol sulfotransferases (PST) catalyze the sulfonation of catecholamines, thyroid hormones and phenolic drugs. At least two major forms of human PST enzyme have been characterized biochemically from liver, platelets and other tissues, the phenol-preferring PST (P-PST) and the monoamine neurotransmitter-preferring PST (M-PST). Molecular cloning efforts worldwide over the past 7 years have resulted in the identification of numerous PST cDNA isolates representing alleles of three human PST gene loci termed as STP1, STP2 and STM. All three genes have been mapped precisely to a small region on human chromosome 16p12.1-p11.2 (homologous to mouse chromosome 7), using somatic cell hybrids and cosmid clones. The two most closely related genes, STP1 and STP2, encoding P-PST isozymes have been mapped to a single cosmid clone and are, therefore, in close proximity to one another. STP1 and STP2 are approximately 96% identical at the amino acid sequence level, whereas, the STM gene (encoding M-PST) exhibits a lower level of identity (approximately 93-90.5%) relative to STP1 and STP2. STM is located at a distance of ca. 100 Kb from the STP1 and STP2 doublet. One may speculate that the three genes arose by gene duplication and/or gene conversion in humans. Genomic clones have been sequenced to determine the genomic organization for each of the three highly-related genes. All contain seven coding exons, with conserved intron exon boundaries. Sequencing of individual cDNA isolates of STP1 and STM from various tissues has revealed significant heterogeneity in the 5' nontranslated region, likely due to alternative splicing and/or tissue-specific promoter utilization. DNA polymorphisms have been detected in these genes in the human population and may be useful for molecular genetic studies of the metabolism of endogenous and xenobiotic phenolic molecules. Recent advances in the molecular biology of the human PST gene family are summarized.

Animals↗
PubMed1997

Identification and cloning of the Mycobacterium avium folA gene, required for dihydrofolate reductase activity.

Dihydrofolate reductase is an essential bacterial enzyme necessary for the maintenance of intracellular folate pools in a biochemically active reduced state. In this report, the Mycobacterium avium folA gene was identified by functional genetic complementation, sequenced, and expressed for the first time. It has an open reading frame of 543 bp with a G + C content of 73%. The translated polypeptide sequence shows 58% identity to the consensus sequence of the conserved regions from eight other bacterial dihydrofolate reductases. Recombinant M. avium dihydrofolate reductase was expressed actively in Escherichia coli, and SDS-PAGE analysis revealed a 20 kDa species, agreeable with that predicted from the polypeptide sequence:

Amino Acid Sequence↗
PubMed1996

Genomic organization and DNA sequences of two human phenol sulfotransferase genes (STP1 and STP2) on the short arm of chromosome 16.

A family of human phenol sulfotransferase genes has been suggested by the cloning of numerous cDNA isolates from different tissues. We have previously cloned and sequenced the STM gene encoding the monoamine neurotransmitter-preferring sulfotransferase, M-PST, and a portion of the STP1 gene encoding the phenol-preferring isozyme, P-PST1 (BBRC 205, 1325-1332; Genomics 18, 440-443). Both genes were mapped to a small region on the short arm of chromosome 16 (BBRC 205, 482-489). Here we report on the sequencing and genomic organization of the STP1 and STP2 genes from a single cosmid clone obtained from chromosome 16p12.1-p11.2. STP1 and STP2 are 95.9% identical at the amino acid sequence level, whereas the STM gene is only 92.9% and 90.5% identical to STP1 and STP2, respectively. Alignment of the genomic sequences indicated that all three genes have 7 coding exons and conserved intron-exon boundaries. These results facilitated the assignment of previously published cDNA isolates as "alleles" of the individual STM, STP1, and STP2 loci on 16p, and provide to us a greater understanding of the complexity and roles of the phenol sulfotransferase gene family in the metabolism of endogenous and xenobiotic agents.

Alleles↗
PubMed1996

Envoplakin, a novel precursor of the cornified envelope that has homology to desmoplakin.

The cornified envelope is a layer of transglutaminase cross-linked protein that is deposited under the plasma membrane of keratinocytes in the outermost layers of the epidermis. We present the sequence of one of the cornified envelope precursors, a protein with an apparent molecular mass of 210 kD. The 210-kD protein is translated from a 6.5-kb mRNA that is transcribed from a single copy gene. The mRNA was upregulated during suspension-induced terminal differentiation of cultured human keratinocytes. Like other envelope precursors, the 210-kD protein became insoluble in SDS and beta-mercaptoethanol on activation of transglutaminases in cultured keratinocytes. The protein was expressed in keratinizing and nonkeratinizing stratified squamous epithelia, but not in simple epithelia or nonepithelial cells. Immunofluorescence staining showed that in epidermal keratinocytes, both in vivo and in culture, the protein was upregulated during terminal differentiation and partially colocalized with desmosomal proteins. Immunogold EM confirmed the colocalization of the 210-kD protein and desmoplakin at desmosomes and on keratin filaments throughout the differentiated layers of the epidermis. Sequence analysis showed that the 210-kD protein is homologous to the keratin-binding proteins desmoplakin, bullous pemphigoid antigen 1, and plectin. These data suggest that the 210-kD protein may link the cornified envelope to desmosomes and keratin filaments. We propose that the 210-kD protein be named "envoplakin."

Amino Acid Sequence↗
PubMed1996

Intersexual phenotypes and sex chromosome complements of five South American opossums (Monodelphis domestica).

Intersexual opossums (Monodelphis domestica) from a large captive colony are described. These are the first naturally existing New World (didelphoid) intersexual marsupials for which reproductive phenotype and sex chromosome constitution are reported. One animal was XX, two were XY, and two were XO; all had lower body weight than normal males or females and the overall appearance of females. They were first recognized as abnormal by the presence of a small flaccid, nonstalked scrotum, markedly smaller than the scrotum of a normal male but in an equivalent position cranial to the cloacal aperture. Each scrotum contained a core of fatty connective tissue, but none contained testicular tissue. Teat patterns, seen only after close shaving of the hair over the area of the teat field, varied within and between the various sex genotypes, with one XY and one XO having the paired rudiments typical of normal males. All individuals had gonads, with no transabdominal migration. In the XX intersex there were mature ovaries with Graffian follicles, but in the XY and XO intersexes there was gonadal dysgenesis. The urogenital tract of all was female in appearance but was immature except in the XX intersex. Development of the scrotum and of the teat primordia can be explained on the basis of regulatory gene influences on the X chromosome. Intersex incidence in the colony is probably much higher than that observed because of ascertainment bias.

Animals↗
PubMed1995

Human dehydroepiandrosterone sulfotransferase. Purification, molecular cloning, and characterization.

Human tissues possess at least four distinct forms of cytosolic ST, three of which are involved in the sulfation of steroids. DHEA-ST is responsible for the majority of hydroxysteroid and bile acid sulfation in human tissues and abundant levels of the enzyme are present in human liver and adrenal tissues. In the adult human adrenal, DHEA-ST has been localized immunologically to the zona reticularis of the adrenal cortex. No age- or gender-related differences in the expression of DHEA-ST activity in adult human liver cytosols have been reported. The cDNA encoding DHEA-ST has been isolated from a human liver cDNA library and expressed in both mammalian COS cells and E. coli. Purification and molecular characterization studies suggest a single form of DHEA-ST in human tissues. The properties of DHEA-ST expressed in either mammalian or bacterial cells are very similar to those of the native enzyme. DHEA-ST can also bioactivate a number of procarcinogens to reactive electrophilic forms. Hydroxymethyl PAHs are sulfated and bioactivated at a relatively rapid rate by DHEA-ST, whereas 1'-hydroxysafrole and N-hydroxy-2-acetylaminofluorene are bioactivated to a lesser extent.

Adrenal Glands↗
PubMed1995

Mouse rhodanese gene (Tst): cDNA cloning, sequencing, and recombinant protein expression.

Rhodanese (thiosulfate sulfurtransferase) is expressed at high levels in liver and is involved in the detoxification of cyanide. The full-length cDNA corresponding to the mouse rhodanese gene (Tst), which is located on chromosome 15, was cloned by PCR amplification of a liver cDNA library and subjected to DNA sequencing. Alignment of the rhodanese cDNA sequences from mouse and rat, which we previously cloned (Biochem. J. 275:227-231), revealed 97.3 percent identity at the protein level and 94.6 percent identity at the DNA level. When the mouse and rat cDNAs were expressed under the control of IPTG-inducible promoters in E. coli, the cell extracts exhibited cyanide-metabolizing activity, indicating that both genes encode functional rhodanese molecules.

Amino Acid Sequence↗
PubMed1995

Analysis of Batten disease candidate genes STP and STM.

We have sequenced a large proportion of the open reading frames (ORFs) of two phenol sulphotransferase gene transcripts (STP and STM) from three patients with Batten disease. This was done using reverse transcription and PCR amplification of total RNA followed by direct sequencing of the PCR products. No mutations or changes have been observed in either gene after sequencing 93% of the STP ORF and 72% of the STM ORF. Work is in progress to finish sequencing both genes which will allow the confirmation or exclusion of these phenol sulphotransferases having a role in the development of Batten disease.

Arylsulfotransferase↗
PubMed1995

Phenol sulfotransferases: candidate genes for Batten disease.

Batten disease (juvenile-onset neuronal ceroid lipofuscinosis; JNCL) is an autosomal recessive neurodegenerative disorder, characterized by the cytosomal accumulation of autofluorescent proteolipopigments in neurons and other cell types. The Batten disease gene (CLN3) has not yet been identified, but has been mapped to a small region of human chromosome area 16p12.1-p11.2. We recently reported the fortuitous discovery that the cytosolic phenol sulfotransferase gene (STP) is located within this same interval of chromosome 16p. Since phenol sulfotransferase is expressed in neurons, can sulfate lipophilic phenolic compounds, and is mapped near CLN3, STP is considered as a candidate gene for Batten disease. YAC and cosmid cloning results have further substantiated the close proximity of STP and a highly related sulfotransferase (STM), encoding the catecholamine-preferring enzyme, to the CLN3 region of chromosome 16p. In this report, we summarize some of the recent progress in the identification of two phenol sulfotransferase genes (STP and STM) as positional candidate genes for Batten disease.

Arylsulfotransferase↗
PubMed1995

UV-induced melanoma. A karyotype with a single translocation is stable after allografting and metastasis.

Metastatic melanoma cell lines were derived from a lymph node of a laboratory opossum, Monodelphis domestica, which had been exposed to mid-wavelength ultraviolet radiation (UVB) initially as a suckling young, and subsequently as a shaved juvenile and adult. The melanoma cell lines were dendritic and pigmented in vitro and contained a t(6;8)(p13;q13) as the only cytogenetic abnormality. The translocation was detected in 15% of primary cultures (passage 2) from the affected lymph node and in 100% of two ring-clone-derived lines, L1 and L2. The breakpoint or resulting partial trisomy of chromosomes 8 may have played a functional role in the tumorigenesis or metastasis of the tumor. The t(6;8) served as a convenient cytogenetic marker for allogeneic grafting studies in Monodelphis. The L2 cells were allografted subcutaneously (s.c.) into genetically diverse suckling young at 3 weeks of age and resulted in the growth of invasive, pigmented, primary and metastatic lesions affecting lymph nodes, lung, and other tissues. Metastatic variant cell lines, M1 and M3, were derived from the affected lungs of two animals and both lines demonstrated the same t(6;8), without additional numerical or structural chromosomal abnormalities. The maintenance of karyotypic stability with a single translocation during in vivo tumor growth and dissemination in this new allografting model is quiet remarkable, as most human metastatic melanomas exhibit multiple structural and numerical cytogenetic abnormalities.

Animals↗
PubMed1995

A new allogeneic model for metastatic melanoma.

Metastatic melanoma cells, clonally derived from an affected lymph node of an ultraviolet-irradiated laboratory opossum, were allografted subcutaneously into suckling young, juveniles and adults to determine their tumorigenicity and metastatic potential. All injected 1- and 3-week-old suckling young survived well beyond weaning at 8 weeks. One died 12 weeks after injection from the effects of rampant metastatic involvement, while the rest were killed 13 to 26 weeks after injection. At necropsy, most animals showed extensive primary tumour growth, many showed metastasis to nodes and/or lungs, and in some there was dissemination to distant sites including liver and spleen. Animals injected as juveniles or adults rejected the allografts. Injection of allogeneic malignant melanoma cells during early postnatal development facilitates successful, long-term allografting and metastasis without concomitant immunosuppressive agents. Developmental lack of self-recognition (immunological immaturity) or induced tolerance may be responsible. This unique model system will be useful for further metastasis studies and may be valuable for investigations of novel antineoplastic therapies.

Animals↗
PubMed1995

Immortalized rat whisker dermal papilla cells cooperate with mouse immature hair follicle buds to activate type IV procollagenases in collagen matrix coculture: correlation with ability to promote hair follicle development in nude mouse grafts.

An in vivo nude mouse graft model and an in vitro collagen matrix culture system were used to study interactions of immature hair follicle buds from newborn mice with clonally derived AdE1A-12S-immortalized rat whisker dermal papilla cell lines. Of the 19 available dermal papilla cell lines, four consistently supported good hair follicle development and hair growth in grafts. Seven cell lines were clearly negative in this assay, and the remaining eight cell lines yielded poor to moderate hair growth. As a correlate to in vivo extracellular matrix remodeling accompanying hair follicle development, type IV collagenase activity in the medium from cocultures of dermal papilla cells and hair follicle buds was analyzed by gelatin zymography. Hair follicle buds cultured alone secrete primarily the 92-kDa type IV procollagenase. Cocultivation of hair follicle buds with eight of the dermal papilla cell lines resulted in activation of this proenzyme and activation of the 72-kDa and 92-kDa type IV procollagenases produced by the dermal papilla cells. Seven of these eight dermal papilla cell lines support hair growth in the graft system. In the absence of dermal papilla cells, several growth factors induced activation of the 92-kDa procollagenase secreted by hair follicle buds cultured in serum-free medium: epidermal growth factor, transforming growth factor alpha, acidic fibroblast growth factor, and keratinocyte growth factor. The current working hypothesis is that a) hair follicle epithelial cells interact with dermal papilla cells in coculture by mutual induction of growth factors and cytokines that stimulate the release and activation of matrix remodeling proteases; and b) the ability of dermal papilla cells to interact with hair follicle epithelial cells in this way may be crucial for controlled dermal matrix remodelling during HF development.

Animals↗
PubMed1995

Chromosome 16 microdeletion in a patient with juvenile neuronal ceroid lipofuscinosis (Batten disease).

The gene that is involved in juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten disease--CLN3--has been localized to 16p12, and the mutation shows a strong association with alleles of microsatellite markers D16S298, D16S299, and D16S288. Recently, haplotype analysis of a Batten patient from a consanguineous relationship indicated homozygosity for a D16S298 null allele. PCR analysis with different primers on DNA from the patient and his family suggests the presence of a cytogenetically undetectable deletion, which was confirmed by Southern blot analysis. The microdeletion is embedded in a region containing chromosome 16-specific repeated sequences. However, putative candidates for CLN3, members of the highly homologous sulfotransferase gene family, which are also present in this region in several copies, were not deleted in the patient. If the microdeletion in this patient is responsible for Batten disease, then we conclude that the sulfotransferase genes are probably not involved in JNCL. By use of markers and probes flanking D16S298, the maximum size of the microdeletion was determined to be approximately 29 kb. The microdeletion may affect the CLN3 gene, which is expected to be in close proximity to D16S298.

Alleles↗
PubMed1994

Genomic organization and DNA sequence of the human catecholamine-sulfating phenol sulfotransferase gene (STM).

The human monoamine neurotransmitter-preferring phenol sulfotransferase (M-PST) plays an essential role in the sulfation of catecholamines, such as dopamine. The cDNA encoding M-PST has been reported, and we have recently identified cosmid clones from human chromosome 16p11.2 for this gene, STM. Plasmid subclones derived from the STM cosmid clones were subjected to dideoxynucleotide chain termination sequencing to determine the genomic organization and DNA sequence of STM. The gene encoding full-length STM is approximately 6.4 kb and contains 8 exons and 7 introns.

Animals↗
PubMed1994

Mapping of two phenol sulphotransferase genes, STP and STM, to 16p: candidate genes for Batten disease.

The cytosolic phenol sulphotransferase gene (STP) was mapped to a region of chromosome 16, within the interval defined by human-rodent somatic cell hybrid breakpoints CY160(D) and CY12, which contains FRA16E. YAC and cosmid clones from this 16p interval were screened for the presence of STP. Two non-overlapping cosmid contigs were identified which contain STP-like sequences. Sequencing of these STP-like sequences confirmed that STP is contained within contig 343.1 and maps proximal to FRA16E, and that a related sulphotransferase STM, encoding the catecholamine-sulphating enzyme, is contained within contig 55.4 and maps to the adjacent hybrid interval CY12-CY180A. Thus two phenol sulphotransferase genes (STP and STM) have been finely localised to chromosome 16p12.1-p11.2, to the same region as CLN3, the gene for Batten disease. Both genes are therefore candidate genes for Batten disease.

Animals↗