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Geometric morphometrics is routinely used in ecology and evolution and morphometric datasets are increasingly shared among researchers, allowing for more comprehensive studies and higher statistical power (as a consequence of increased sample size). However, sharing of morphometric data opens up the question of how much nonbiologically relevant variation (i.e., measurement error) is introduced in the resulting datasets and how this variation affects analyses. We perform a set of analyses based on an empirical 3D geometric morphometric dataset. In particular, we quantify the amount of error associated with combining data from multiple devices and digitized by multiple operators and test for the presence of bias. We also extend these analyses to a dataset obtained with a recently developed automated method, which does not require human-digitized landmarks. Further, we analyze how measurement error affects estimates of phylogenetic signal and how its effect compares with the effect of phylogenetic uncertainty. We show that measurement error can be substantial when combining surface models produced by different devices and even more among landmarks digitized by different operators. We also document the presence of small, but significant, amounts of nonrandom error (i.e., bias). Measurement error is heavily reduced by excluding landmarks that are difficult to digitize. The automated method we tested had low levels of error, if used in combination with a procedure for dimensionality reduction. Estimates of phylogenetic signal can be more affected by measurement error than by phylogenetic uncertainty. Our results generally highlight the importance of landmark choice and the usefulness of estimating measurement error. Further, measurement error may limit comparisons of estimates of phylogenetic signal across studies if these have been performed using different devices or by different operators. Finally, we also show how widely held assumptions do not always hold true, particularly that measurement error affects inference more at a shallower phylogenetic scale and that automated methods perform worse than human digitization.

The wild-type agouti-banding pattern for hair is well characterized in lower mammals such as mice. The switch between eumelanin and pheomelanin in bands in the hair results from the interaction of alpha-melanocyte stimulating hormone and agouti signal protein through the melanocortin 1 receptor on melanocytes. However, such banding patterns have not been described to date in higher mammals. We now report such 'agouti'-banding patterns that occur in several subspecies of baboons, and characterize those hairs using chemical and immunohistochemical methods. Hair and skin samples were obtained from the dorsa of adult male baboons of different subspecies (Papio cynocephalus hamadryas (PCH) and Papio cynocephalus anubis (PCA)). The hairs were excised with scissors into the gray and the white bands of the PCH subspecies and into the black and the yellow bands of the PCA subspecies, and were analyzed for total melanin, eumelanin, and pheomelanin by spectrophotometric and chemical methods. Hairs in the PCA subspecies oscillate between a eumelanic band (with high melanin content) and a pheomelanic band, while hairs in the PCH subspecies oscillate between a eumelanic band (with low melanin content) and a non-pigmented band. Those chemical data are consistent with the histological appearance of the hair bulbs stained by the Fontana-Masson technique. The difference in the melanin content between PCH and PCA subspecies is most likely related to tyrosinase levels, as suggested by the presence of unpigmented muzzle in the PCH subspecies compared with the black muzzle in the PCA subspecies.

The bioavailability of drugs administered topically or orally depends on their metabolism by epithelial enzymes such as the cytosolic sulfotransferases (SULT). Reverse transcriptase-polymerase chain reaction (RT-PCR) methods were established to detect expression of 8 SULT genes and 4 arylsulfatase (ARS) genes in human tissues of epithelial origin and in cultures of normal and transformed (cancer) cells. The results indicate: (i) SULT 1A1, 1A3, ARSC, and ARSD genes are ubiquitously expressed; (ii) expression is frequently similar between cell lines and corresponding tissues; (iii) SULT gene expression in normal cultured cells is generally comparable to the expression in associated transformed (cancer) cell lines; (iv) SULT 1A1 promoter usage is mainly tissue specific; however, both promoters are frequently used in SULT 1A3 expression; and (v) the expression profile of SULT 1A1, 1A3, 1E1, and 2B1a/b suggests that one or more of these isoforms may be involved in the cutaneous sulfoconjugation of minoxidil and cholesterol., (Copyright 2000 Academic Press.)

Cytosolic sulfotransferases (ST) catalyze the sulfation of various phenolic agents, catecholamines, thyroid hormones, steroids, drugs, and procarcinogens, usually resulting in the inactivation and subsequent excretion of the compound. My laboratory's efforts have focused on the cloning of the human phenol-sulfating (PST) members of this gene superfamily, implicated in the bioactivation of the hair growth stimulant, minoxidil. At least two major forms of human PST enzymes have been characterized biochemically, the phenol-preferring PST (P-PST), and the catecholamine-preferring PST (M-PST). Various cDNAs have been cloned representing alleles of 3 gene loci termed as STP1, STP2, and STM, which were all mapped precisely to a small region on human chromosome 16p and to the homologous region of mouse chromosome 7. Human cosmid genomic clones have been sequenced to determine the genomic organization for each of the 3 highly-related genes. All contain 7 coding exons, with conserved intron-exon boundaries, and presumptive alternative tissue-specific promoters. At least one of the 3 PST-encoding genes is responsible for forming minoxidil sulfate in the lower outer root sheath of anagen hair follicles. The steroid sulfating genes, STD and STE, have been cloned by other laboratories. The isozyme products of these genes sulfate DHEA and estrogens, respectively. I hypothesize that either STE or STD is involved in the formation of cholesterol sulfate (CS) in epidermal keratinocytes. CS has been demonstrated by other groups to be an activator of keratinocyte Protein Kinase Ceta, which subsequently results in the activation of epidermal transglutaminase and formation of the cornified envelop. STE or STD might also be involved in bioinactivation of estrogens and androgens within skin. Our recent unpublished results have focused on elucidating the patterns of ST gene expression in cultured keratinocytes and fibroblasts derived from human skin using RT-PCR, to understand which of the 5 different ST genes in involved in the regulation of keratinocyte differentiation and minoxidil-induced hair growth.

We have established cell lines from benign cutaneous melanocytic lesions and from melanoma-affected lymph nodes of monodelphis domestica, the laboratory opossum (a South American marsupial now widely maintained in captive colonies for experimental purposes). Unlike melanoma cell lines currently available from humans and other mammals, the opossum lines are derived from cells transformed in vivo by experimentally controlled exposure to ultraviolet B (UVB) radiation of known spectral composition. Differences in the patterns of protein expression among cell lines at different stages of the UVB-induced melanoma cascade can be identified by proteome analysis and will provide a useful basis for comparisons with human and mouse melanoma cell lines. Powerful new two-dimensional (2D) gel electrophoresis technologies and sophisticated bioinformatics programs make it possible to carry out qualitative and quantitative analyses of the entire protein complement expressed by the genome (proteome) of a specific cell type. One area of biology particularly well suited to proteome analysis is carcinogenesis. It is now feasible, for example, to attempt to characterize the full repertoire of proteins, including all the antigenic determinants at the cell surface and in the cytosol, during the carcinogenic cascade from normal progenitor cells, to benign tumor cells, and finally, to highly invasive metastatic cells. Proteome analyses have been initiated with the cell lines from M. domestica.

Cytosolic phenol sulfotransferases (PST) catalyze the sulfation/sulfonation of various phenolic agents, including catecholamines, thyroid hormones, and drugs (e.g., minoxidil and acetaminophen), which usually results in the inactivation and subsequent excretion of the compound. Our recent efforts have focused on the cloning and sequencing of the human gene family encoding the PST isozymes, and the results are summarized in this article. Multiple PST cDNA isolates have been cloned in various laboratories representing alleles of three phenol sulfotransferase 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). The two most closely related genes, STP1 and STP2, encode isozymes of phenol-preferring PST (P-PST) and have been mapped to a single genomic cosmid clone, thus in proximity to one another. The STM gene encoding the monoamine neurotransmitter-preferring PST (M-PST) exhibits a lower level of similarity relative to STP1 and STP2. 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 from various tissues has revealed heterogeneity in the 5' nontranslated regions, likely due to tissue-specific promoter utilization (or perhaps alternative splicing). DNA and protein polymorphisms have been identified in the population and may be useful for molecular genetic studies of the variability in the metabolism of catecholamines, thyroid hormones, and phenolic drugs, and possibly neuropsychiatric or other metabolic disorders.

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:

Hand preference for signing and for nonsign actions was examined longitudinally in 24 young children (3 deaf, 21 hearing) with deaf parents. Most of these children showed a strong preference for their right hands in their sign production. This preference emerged early in their development, was relatively consistent over time, and predicted mature hand preference. Although most of the children also preferred to use their right hands in nonsign actions, their right-hand preference for signing was much stronger. Hand preference scores for two types of nonsign actions, communicative gestures and object actions, were significantly correlated with those for signing. Hand preference also was linked to rate of motor development but not to sign language acquisition. These findings are discussed with regard to current conceptualizations about the interrelationships among language, motor processes, and laterality.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Litters of suckling young of the laboratory opossum (Monodelphis domestica) were irradiated with UV light from sunlamps with a spectral emission peak at 302 nm (UVB) to induce melanocytic nevi. Total doses of 0.87-5.0 kJ/m2 were divided equally among up to 14 exposures during the 19 days from birth. Of 358 sucklings exposed, 217 survived to weaning, and 22 (10%) possessed a nevus when shaved and examined at or after weaning. Affected animals were then exposed 3 times/week to 125 J/m2 of UVB for up to 45 weeks to promote progression to malignancy. Nevi of 8 of the 20 chronically-exposed animals progressed to malignant melanoma with metastases to lymph node(s). Cell cultures were prepared from affected nodes to confirm that pigmented nodal cells were metastatic melanomas. One established cell line (TD15L) contained highly pigmented, dendritic, malignant melanoma cells. These cells, injected s.c. as xenogeneic grafts into athymic nude mice, remained viable in the subcutis and were moderately tumorigenic in the dermis. UVR exposure of Monodelphis sucklings is a novel, effective, and proficient way of initiating melanocytic lesions for studies on susceptibility and progression to melanoma, and the cell lines derived from these melanomas will provide promising new reagents for chemotherapy and immunotherapy investigations.

An in vitro cell culture assay was developed to identify inhibitors of melanogenesis and agents which produce cytostatic or cytotoxic effects specifically in melanocytes. A total of 50 compounds related to tyrosine, dihydroxyphenylalanine, and hydroquinone (HQ) were tested in vitro in order to determine their effects upon a murine melanocyte cell line, Mel-Ab, that produces copious amounts of melanin in culture. The agents that demonstrated an inhibition of growth or pigment production by 50% (IC50) at < 100 micrograms/ml were considered active. The cytotoxicity of melanocyte-active compounds were also tested in vitro on a control nonmelanocyte cell line (HT 1080), using a simple crystal violet staining method to quantitate adherent cell number after treatment. The cell culture assay was validated with known potent melanocyte cytotoxic agents, including HQ and 4-S-cysteaminylphenol (4-S-CAP). Although most cytotoxic chemicals were nonspecific in this primary screen (i.e. killing both Mel-Ab and HT-1080 cells), several of the compounds tested exhibited high melanocyte-specific cytotoxicity, similar to HQ and 4-S-CAP. Potentially these compounds may be useful as either antimelanoma or skin depigmentation agents. All of the compounds identified as active in this primary screen were cytotoxic or cytostatic to melanocytes, except for the methyl ester of gentisic acid, which uniquely inhibited the de novo synthesis of melanin without cytotoxicity.

The oncogenic transformation of epidermal melanocytes produces primary cutaneous melanoma. In this article, previously published cytogenetic, biochemical, molecular biology, and cell biology studies of cutaneous melanoma oncogenesis are reviewed. A variety of laboratory animal models have been developed for studies of the induction of melanoma, including mice, the laboratory opossum Monodelphis domestica, Sinclair swine, and Xiphophorus fish. Some of the advantages and disadvantages of these animal models are presented for comparison to human melanoma. Cytogenetic and loss of heterozygosity studies over the past decade have demonstrated that human metastatic melanomas contain numerous chromosomal abnormalities, and that normal melanocytes have putative tumor suppressor genes that are presumably deleted or inactivated in transformed melanocytes to yield malignant melanoma cells. The status of research efforts to identify the putative tumor suppressor genes of human chromosomes 1p, 6q, and 9p, implicated in sporadic and familial melanoma, is presented. Furthermore, the roles of ultraviolet radiation, genetic susceptibility, dominant oncogenes, growth factors, the p53 gene, antioxidant enzymes, and DNA tumor viruses in the formation of cutaneous primary melanoma are discussed.

We have recently cloned a cDNA encoding the human phenol-preferring phenol sulfotransferase (P-PST) enzyme. An oligonucleotide primer pair based on the human STP (representing sulfotransferase, phenol-preferring) cDNA sequence was synthesized and was employed in polymerase chain reaction (PCR) amplification of human genomic DNA to identify a 525-bp DNA fragment. The DNA sequence of this portion of the STP gene, near the 5' end of the coding region, was determined. The amplified genomic fragment contained two small introns of 104 and 89 bp. When DNA samples from a human-hamster somatic cell hybrid panel were screened by PCR using these primers, only those hybrids that contained human chromosome 16 were positive for the 525-bp genomic fragment. To identify the specific region on chromosome 16 that contained the STP gene, PCR amplification reactions were performed on a human-mouse somatic cell hybrid panel containing defined portions of human chromosome 16. The results indicated that STP is localized proximal to the gene for protein kinase C, beta 1 polypeptide (PRKCB1), in the region from the distal portion of 16p11.2 to p12.1. The human STP gene maps near the locus for Batten disease (CLN3). Furthermore, we have determined by genotyping of murine interspecific backcross progeny that the homologous gene in mouse (Stp) localizes to the syntenic region of mouse chromosome 7 near the D7Mit8 (at 54 cM) and D7Bir1 markers.

During embryonic development presumptive hair follicle cells of epithelial and mesenchymal origin are determined in defined body locations. This is followed by rapid proliferation of epithelial cells and associated penetration into the dermis in response to as yet undetermined signals. A collagen matrix culture system, which maintains the three-dimensional relationships of hair follicle cells to each other, was developed to study the regulation of the enlargement of immature hair follicles and the accompanying remodeling of the dermis. In studies with a heterogeneous dermis-derived preparation of murine hair follicles, ranging in size from the earliest down-growing budding cell mass to hair-forming follicles, we had previously shown that cell proliferation was stimulated by cholera toxin and epidermal growth factor, but only the epidermal growth factor-stimulated proliferation was accompanied by digestion of the collagen matrix due to release of collagenolytic enzymes. Further studies revealed that transforming growth factor-alpha also stimulated hair follicle cell proliferation and collagenase release. However, although transforming growth factor-beta inhibited the transforming growth factor-alpha-stimulated proliferation, it enhanced the release and activation of collagenases and other gelatin-degrading enzymes detectable by gelatin zymography. Stimulation of collagenolytic activity depended on the three-dimensional hair follicle structure and did not occur in monolayer cultures of hair follicle cells. Comparison of hair follicle buds with more developed dermis-derived hair follicles, plated at the same cell density (based on DNA content), suggested that a greater fraction of cells in the bud-stage follicle responded to the growth factors by release of collagenases. Possibly only the cells in the advancing portion of growing hair follicles that are closest to the dermal papilla cell cluster produce the collagenases in response to growth factors. To examine the participation of dermal papilla cells in collagenase release and activation, several immortalized rat whisker dermal papilla cell lines were co-cultured with mouse hair follicle buds. Co-culture resulted in a marked enlargement of follicles as well as activation of the 92-kDa type IV collagenase, produced by hair follicle buds, that correlated with ability of the dermal papilla cells to stimulate hair formation in grafts of hair follicle buds on nude mice. Dermal papilla cells cultured alone produced the 72-kDa type IV collagenase, which was also activated during co-culture with hair follicle buds. Thus, two activities, both relevant for hair follicle development, namely, cell proliferation and release and activation of collagenases, have been stimulated in immature hair follicle buds by either growth-factor supplementation or interaction with dermal papilla cells.(ABSTRACT TRUNCATED AT 400 WORDS)

High selectivity for covalent reaction at adenine N-3 within duplex DNA is a distinguishing feature of the CC-1065 and duocarmycin classes of natural products. Studies of the base and sequence selectivity exhibited by duocarmycins and CC-1065-based alkylating agents have focused on characterization of the predominant covalent adenine adducts that are formed. While information about minor DNA reaction products could provide valuable insights to our understanding the DNA recognition and reactivity properties of these agents, little characterization of such adducts by these agents has appeared in the literature. To broaden our structure-reactivity understanding of these DNA alkylating compounds, comparative investigations of the covalent sequence selectivity exhibited by compounds containing altered cyclopropapyrroloindole (CPI) alkylating subunits such as duocarmycin A were undertaken using the DNA polymerase inhibition assay. We were surprised to identify with this assay a DNA sequence with an unusual propensity for covalent reaction with duocarmycin A at a guanine nucleotide. Using the heat strand breakage assay with a duplex oligonucleotide containing this interesting sequence, we confirmed the site of alkylation to be the indicated guanine in the sequence 5'-CGCGTTG*GGAG-3'. The trimethoxyindole-CPI analog of duocarmycin A does not alkylate this guanine, suggesting that there are interesting features to the duplex recognition/reactivity exhibited by duocarmycin A. Herein we describe our identification of the first DNA sequence which covalently reacts with duocarmycin A at a guanine nucleotide in the absence of additional minor groove binding agents.

The nude mouse graft model for testing the hair-forming ability of selected cell populations has considerable potential for providing insights into factors that are important for hair follicle development and proper hair formation. We have developed a minimal component system consisting of immature hair follicle buds from newborn pigmented C57BL/6 mice and adenovirus E1A-immortalized rat vibrissa dermal papilla cells. Hair follicle buds contribute to formation of hairless skin when grafted alone or with Swiss 3T3 cells, but produce densely haired skin when grafted with a fresh dermal cell preparation. The fresh dermal cell preparation represents the single cell fraction after hair follicles have been removed from a collagenase digest of newborn mouse dermis. It provides dermal papilla cells, fibroblasts, and possibly other important growth factor-producing cell types. Rat vibrissa dermal papilla cells supported dense hair growth at early passage in culture but progressively lost this potential during repeated passage in culture. Of 19 E1A-immortalized, clonally derived rat vibrissa dermal papilla cell lines, the four most positive clones supported hair growth to the extent of approximately 200 to 300 hairs per 1-2 cm2 graft area. The remaining clones were moderately positive (five clones), weakly positive (three clones), or negative (seven clones). Swiss 3T3 cells prevented contraction of the graft area but did not appear to affect the number of hairs in the graft site produced by dermal papilla cells plus hair follicle buds alone. The relatively low hair density (estimated 1-5% of normal) resulting from grafts of hair follicle buds with the most positive of the immortalized dermal papilla cell clones compared to fresh dermal cells suggests that optimal reconstitution of hair growth requires some function of dermal papilla cells partially lost during the immortalization process and possibly the contribution of other cell types present in the fresh dermal cell preparation, which is not supplied by the Swiss 3T3 cells. The current graft system, comprising hair follicle buds and immortalized dermal papilla cell clones, provides an assay for positive or negative influences on hair growth exerted by added selected cell types, growth factors, or other substances. Characterization of the phenotype of the dermal papilla cell lines, which differ in their ability to support hair growth when grafted with hair follicle buds, may provide insight into specific dermal papilla cell properties important for their function in this system.

We developed and evaluated an in vivo athymic nude mouse model for tumor growth, angiogenesis, metastasis, and antineoplastic drug development. Melanoma cell lines expressing beta-galactosidase encoded by the Escherichia coli lac Z gene have been created by infecting an immortal murine melanocyte cell line with a recombinant retrovirus expressing the v-Ha-ras oncogene and lac Z to generate the MRB (melanoma, ras, beta-galactosidase) cell lines. The amelanotic, phorbol ester-independent, transformed melanoma cell lines developed tumors rapidly when injected subcutaneously into nude mice, as well as experimental lung metastases when injected i.v. into the tail vein. beta-galactosidase-expressing subcutaneous tumors and lung metastases stained blue with X-gal. The melanomas produced in nude mice have been characterized by using various histochemical and immunohistochemical staining methods to detect melanoma- and endothelial-cell-specific markers to determine the extent of neovascularization in MRB nude mouse tumors. Optimal staining of endothelial cells involved in tumor angiogenesis was observed by using ADPase activity and antiangiotensin-converting enzyme antibody staining. Attempts at indirect quantification of metastatic tumor cell number within the lung by either beta-galactosidase enzymatic activity or ELISA immunoreactivity were unsuccessful. However, the MRB cell lines should be useful in screening for and studying the mechanisms of action of antineoplastic, antimetastatic, and angiostatic drugs in vivo in athymic nude mice.

The cDNA encoding full-length human p11 calpactin I light chain has been cloned and subjected to DNA sequencing. The open reading frame specifies a 97-amino-acid residue protein that surprisingly is identical to the p11 sequences of two mammalian ungulate species, cow and pig. However, the previously reported p11 polypeptide sequences of mouse and rat exhibited 8-9% nonidentity to human p11. These mammalian sequence comparison results are unexpected in view of current molecular cladistic theories that suggest a closer relationship between primates and rodents, rather than primates and ungulates. The mouse p11 gene has been previously mapped to chromosome 3 at a position syntenic with a centromeric-proximal region on human chromosome 1, and the human p11 cDNA clone is likely to be useful in physical mapping on chromosome 1.

Involucrin is a precursor of the insoluble protein envelope that is assembled in the outermost layers of the epidermis. The coding sequence of the protein contains a number of short tandem repeats that have been greatly altered during mammalian evolution. We have characterised eight mouse monoclonal antibodies raised against human involucrin, all of which bind to the protein in immunoprecipitation, immunoblot and immunohistochemical preparations. Each antibody was screened for cross-reactivity with gorilla, owl monkey, dog and pig involucrin and with a fragment of the human protein, expressed in lambda gt 11, that includes the entire early region of the modern segment of repeats. Three antibodies recognised involucrin in all of these assays. Four antibodies recognised primate involucrins and the lambda gt 11 fragment. One antibody, which showed cross-reactivity with lower molecular weight proteins, only recognised primate involucrins and therefore bound outside the early region of the modern segment. Since the antibodies can be used to detect involucrin both biochemically and histologically, in a range of species, they will have applications in further studies of the expression, function and evolution of the protein.

CC-1065, a cyclopropylpyrroloindole (CPI), is a highly potent antitumor DNA-alkylating agent. We have devised a simple method to detect CPI bonding sites on double-stranded DNA (dsDNA). The technique utilizes a modified form of bacteriophage T7 polymerase, Sequenase, to synthesize a radiolabeled nascent strand from dsDNA that has been reacted in vitro with the CC-1065 analogue U-73975 (adozelesin). The reaction products were electrophoresed on sequencing gels containing 8 M urea and visualized by autoradiography. The transit of this DNA polymerase is inhibited at the sites where CPIs are bound to the template strand. Thus, the enzyme stalls or stops at the nucleotide immediately adjacent to the modified base, resulting in the accumulation of DNA strands at these sites and in diminished read-through beyond these sites in a set of CPI-treated DNA molecules. The precise positions of polymerase inhibition can be determined by comparison of CPI-treated and unreacted DNA reactions. This modified dideoxynucleotide sequencing technique has been used to establish the sequence selectivity of U-73975. Approximately 1 kilobase of dsDNA has been analyzed to derive a consensus canonical bonding sequence, 5'(T/A)-T/A-T-A*-(C/G)-(G), where A* is the site of U-73975 alkylation and parentheses denote deoxynucleotide preferences. Noncanonical sites were also found at poly(A) sites. This technique yielded a consensus sequence for U-73975 bonding that is similar to, but not identical with, the published consensus obtained for CC-1065 by a modified Maxam and Gilbert sequencing technique. We have also examined the bonding of [3H]U-73975 to the DNA of viable cultured mammalian cells, using gel electrophoresis and autoradiographic techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

The nontumorigenic, immortal line of murine melanocytes, Mel-ab, requires the continual presence of biologically active phorbol esters for growth (R.E. Wilson et al., Cancer Res., 49:711-716, 1989). Comparable treatments of B16 murine melanoma cells result in partial inhibition of cell proliferation. The role of protein kinase C (PKC) in the modulation of growth of cells from these two melanocytic cell lines has been investigated. Significant levels of PKC were present in quiescent Mel-ab cells as determined by Western blotting, whereas no immunoreactive protein was detected in cell extracts from either proliferating Mel-ab or B16.F1 cells. Phosphorylation of a Mr 80,000 protein, which by one- and two-dimensional gel analysis comigrated with the known Mr 80,000 protein substrate of PKC in fibroblasts, was induced in 12-O-tetradecanoylphorbol-13-acetate-stimulated quiescent Mel-ab cells but not in proliferating Mel-ab cells or B16.F1 melanoma cells. Direct measurement of PKC activity in these cells demonstrated a 10-fold greater level of activity in quiescent Mel-ab cells (262 +/- 50 pmol/min/mg SD) compared with growing cells (22.8 +/- 11.8 pmol/min/mg SD). An intermediate level of activity was detected in proliferating B16.F1 melanoma cells (148.5 +/- 20.4 pmol/min/mg SD). The subcellular distribution of PKC was dependent upon the growth state of the cells such that quiescent Mel-ab cells displayed a higher level of activity in the cytosol, whereas growing Mel-ab cells displayed greater activity in the particulate fraction. Like many other transformed lines, B16.F1 melanoma cells constitutively expressed the majority of enzyme activity in the particulate fraction. Measurement of [3H]phorbol ester binding in intact cells paralleled the PKC activation data such that quiescent Mel-ab cells displayed binding of 1612 +/- 147 cpm/10(6) cells, whereas proliferating Mel-ab and B16.F1 melanoma cells displayed binding of 652 +/- 28 and 947 +/- 81 cpm/10(6) cells, respectively. Membrane-permeant diacylglycerol analogues, which activated but did not down-regulate PKC, were devoid of growth-stimulating effects on melanocytes, even in the presence of the specific diacylglycerol kinase inhibitor, R59022. Together, these data show that PKC down-regulation, and not activation, correlates with the growth of melanocytes in culture.

The precise biochemical mechanism and site(s) of action by which minoxidil stimulates hair growth are not yet clear. Minoxidil sulfate is the active metabolite of minoxidil, with regard to smooth muscle vasodilation and hair growth. Formation of minoxidil sulfate is catalyzed by specific PAPS-dependent sulfotransferase(s) and minoxidil-sulfating activities have been previously reported to be present in liver and hair follicles. One of these minoxidil-sulfating enzymes has been purified from rat liver (rat minoxidil sulfotransferase, MST) and a rabbit anti-MST antibody has been prepared. Using this anti-MST antibody, we have immunohistochemically localized minoxidil sulfotransferase in the liver and anagen hair follicles from rat. In rat pelage and vibrissa follicles, this enzyme is localized within the cytoplasm of epithelial cells in the lower outer root sheath. Although the immunolocalization of MST might not necessarily correlate with the MST activity known to be present in anagen follicles, the results of this study strongly suggest that the lower outer root sheath of the hair follicle may serve as a site for the sulfation of topically applied minoxidil.

The effectiveness of superoxide dismutase and allopurinol was evaluated in preventing reperfusion injury in Sprague-Dawley rats utilizing a limb replantation model after 6 hours of warm ischemia. Immediately prior to reperfusion of the replanted limb the animals received (intravenously) either a single bolus of superoxide dismutase (16,000 units/kg), a single bolus of allopurinol (45 mg/kg), both agents, or only control solutions. In the rats that received only control solutions, 30% of the limbs survived the ischemic insult. The group that received both superoxide dismutase and allopurinol had a 75% limb survival (P = 0.005). The animals that had only superoxide dismutase or allopurinol had 58% and 60% of the replanted limbs survive, respectively. This study suggests that the administration of superoxide dismutase and allopurinol may be helpful in the prevention of reperfusion injury in skeletal muscle in an acute injury setting (without pretreatment) and may thereby improve limb salvage after a significant ischemic period.

A nontumorigenic line of murine melanocytes, Mel-ab, has been transfected with the v-Ha-ras gene under transcriptional control of the Moloney murine leukemia virus long terminal repeat. Transfectants produced rapidly growing undifferentiated melanomas in recipient mice. The inhibition of melanin production in transformed cells, observable both in vitro and in vivo, suggests that ras may affect melanocyte cytodifferentiation. Mel-ab cells require the continual presence of 12-O-tetradecanoylphorbol-13-acetate, or other activators of protein kinase C, for in vitro growth. Transfectants expressing v-Ha-ras no longer manifested this requirement and were actually growth inhibited by the addition of protein kinase C activators. These results are consistent with the notion that ras acts via the protein kinase C pathway in conferring autonomous growth on Mel-ab cells.

We have transfected the polyoma middle T oncogene into an immortal murine melanocyte cell line, Mel-ab. This highly pigmented line is phorbol ester (TPA) dependent for in vitro growth, suggesting activation and/or down regulation of Protein Kinase C (PKC) is essential for mitogenesis. Moreover, cells of this line do not form tumours when injected subcutaneously into immunocompetent or immunoincompetent mice. Here we show that PyMT alone is sufficient to produce TPA-independence and transformation to the tumourigenic state in transfected Mel-ab cells. Western blot analysis shows that middle T overcomes the TPA requirement by a mechanism independent of PKC down regulation though this does appear to occur when Mel-ab cells are grown continuously in TPA. These results suggest that PyMT is not exerting its transforming effect by PKC down regulation, but conceivably at some later stage of second messenger signalling, possibly through PyMT-c-src protein kinase activity.

Forty-four youngsters with precordial murmurs and carotid bruits were evaluated clinically and independently, using pulsed Doppler ultrasound. The precordial murmur was evaluated with M-mode echocardiography combined with Doppler flow evaluation, and the carotid bruit was evaluated with peripheral vascular sector scan with Doppler flow evaluation. These ultrasonic techniques can identify abnormal blood flow at anatomic sites such as the aortic valve and in the carotid arteries. The patients had no symptoms and their condition, except for six, was mild enough that catheterization was not indicated. The clinical diagnosis of aortic stenosis was made in 30 children, and nine were thought to have no heart disease. On the basis of the ultrasonic examinations, 28 patients were diagnosed as having aortic stenosis and seven subjects had no intracardiac turbulence. However, there was disagreement in 14 instances; four of the six clinical "normals" were found to have aortic stenosis by pulsed Doppler echocardiography; six patients diagnosed as having mild aortic stenosis on a clinical basis were found to have no aortic abnormality. The results confirm that aortic stenosis usually presents as a murmur maximal in the aortic area, which is associated with a carotid bruit. Unfortunately, in at least one-fourth of the cases the murmur was not maximal at the aortic area, and a carotid bruit was found in several normal subjects. Since the consequences of over- or under-diagnosis of aortic stenosis are substantial, careful thought should be given to the differential diagnosis, and if possible, pulsed Doppler echocardiography should be utilized for a definitive statement of aortic valve-induced turbulence.

This study was undertaken to determine if a duplex scanner equipped with a new 10 MHz probe could accurately evaluate microvascular anastomotic patency. The overall predictive accuracy of the duplex scanner was 90% (p less than 0.0001) with no difference noted among the three main anastomotic groups examined--acute artery, acute vein, and long-term artery. There was, however, a statistically significant difference (p less than 0.05) in the ability to interpret vessels that were patent (100%) versus those that were partially occluded (73%) or occluded (88%). It is believed that the duplex scanner has potential applications to preoperatively and intraoperatively study microvessels and postoperatively to supplement other techniques in monitoring acute and long-term anastomotic patency.

In the bacterial plasmid ColE1 the control of initiation of DNA replication is mediated by the interaction of two complementary RNA molecules, the replication primer and RNA1. The rate of interaction between RNA1 and the primer RNA in vitro can be increased by the product of the ColE1 rop gene, a 63-amino-acid polypeptide. We have investigated the role of the Rop protein in suppressing the incompatibility defects of 13 RNA1-mutant alleles. These RNA1 mutants are defective due to single nucleotide mismatches with their target, the primer RNA. The rop gene suppresses the defective behavior of most of the RNA1 point mismatch mutants in vivo. However, certain mutations that map in stems I and III of RNA1 are not suppressed by rop. The interaction of wild-type and mutant species of RNA1 with ColE1 replication primer transcripts was studied in vitro in the presence or absence of purified Rop protein. The Rop protein is known to increase the rate of wild-type RNA1-primer interaction about twofold in vitro. This enhancement was also observed for mutant RNA1 species having point alterations or a deletion of the 5' terminus of RNA1, which is consistent with the in vivo suppression results. The implications of these results on the mechanism of Rop activity are considered.

Undifferentiated mouse embryonal carcinoma (EC) cells are capable of transactivating the adenovirus EIIa promoter in the absence of its normal transactivator, E1A protein, suggesting that EC cells contain an E1A-like activity. In an effort to identify where this activity appears during normal mouse development, mouse oocytes and preimplantation embryos were injected with plasmids containing the EIIa promoter coupled to various reporter genes. These expression vectors were fully active in human 293 cells where E1A is present, but were inactive in differentiated fibroblast cell lines unless cotransfected with the E1A gene. In mouse oocytes and preimplantation embryos, EIIa promoter activity in the absence of adenovirus E1A protein was equivalent to or greater than activity of the HSV thymidine kinase promoter coupled to a strong enhancer. Coinjection of the E1A gene failed to stimulate EIIa activity further, perhaps because c-myc protein, which has been reported to transactivate this promoter, was already present at high levels in mouse oocytes. Activation of the EIIa promoter in the absence of E1A was unique to mouse oocytes and preimplantation embryos because gene expression from an EIIa promoter introduced into transgenic mice was observed only in the adult ovary, and particularly in the oocytes. In addition, post-implantation transgenic embryos failed to express the E1A-activatable reporter gene, thereby indicating that expression from the EIIa promoter is restricted to the relatively undifferentiated stages of oogenesis and preimplantation development. These data suggest that cellular promoters of the class that can be transactivated by E1A may serve uniquely to initiate transcription of genes that are needed for preimplantation development.

The control of DNA replication initiation in the plasmid ColE1 is mediated by RNAI, a 108 nucleotide plasmid-encoded RNA that is entirely complementary to the 5'-terminal region of the replication primer RNA. RNAI acts in trans to inhibit primer maturation. Previously, we constructed a plasmid in which the ColE1 RNAI was separated from the primer and placed under transcriptional control of the Serratia marcesens tryptophan promoter. This plasmid provides RNAI in trans in vivo and mediates ColE1-type incompatibility. To determine the critical structural and functional domains of RNAI, we have undertaken a mutational analysis of the RNAI gene carried by this plasmid. We have selected mutants that no longer mediate ColE1-type incompatibility in trans. From the DNA sequences of 18 mutants we have identified mutations at nine new sites in RNAI. In addition, we have determined the secondary structural features of several mutant RNAI species and compared them to wild-type RNAI. Analysis of these mutations has revealed several key features of RNAI secondary structure and function. The domains of RNAI identified in this work which are essential for its function are: the single-stranded loop regions; the integrity of the double-stranded stems; and the single-stranded 5' terminus.

A flow-fluorimetric high-performance liquid chromatographic assay for 6-methylthioguanine in urine has been developed. This compound is a major catabolite of 6-thioguanine, an important drug in cancer chemotherapy. The metabolite was extracted from alkaline urine with ethyl acetate which was injected onto a reversed-phase high-performance liquid chromatographic system for separation and detection. The method is simple, rapid and sensitive to below 500 ng ml-1 which is below the levels encountered following a therapeutic dose of 6-thioguanine. Another metabolite was chromatographically separated from 6-methylthioguanine and partially characterised.