Search PubMed⌕ Search

Biomedical subjects

S H Kidson

Publications and source records attributed to S H Kidson.

At least 19 recordsLinked to original sources
PubMed2006

Molecular events in early development of the ciliary body: a question of folding.

Ciliary body morphogenesis is a complicated, multi-step process requiring coordinated changes in cell shape, flexure of epithelial sheets and dynamic shifts in mitotic rates. Very little is known of how these cellular events are triggered or regulated. This review summarises current models of ciliary body morphogenesis. The role of intraocular pressure as a driver of morphogenesis is re-evaluated in the light of new information. An update on the role of the lens in ciliary body morphogenesis is presented. In the second part of the review current gene expression data is related to ciliary body morphogenesis. In particular the role of Bmp4 and its downstream target genes are discussed, with novel gene expression patterns of Bmp4 and Tgfbeta1i4 being presented.

Animals↗
PubMed2006

An evaluation of molecular markers for improved detection of breast cancer metastases in sentinel nodes.

BACKGROUND AND OBJECTIVES: In patients with breast cancer (BC), the sentinel node (SN) is the first node in the axillary basin that receives the primary lymphatic flow and can be used to accurately assess the axillary nodal status without removal of the axillary contents. Currently, histology and/or immunohistochemistry are the routine methods of SN analysis. The primary objective of this study was to develop a reproducible reverse transcription (RT) PCR assay, with emphasis on achieving high specificity for accurate detection of BC micrometastases in the SN. To correct for the heterogeneity of BC cells, a multimarker approach was followed, with the further aim of improving the detection rate of the assay. METHODS: In total, 73 markers were evaluated, of which 7 were breast epithelial markers and 66 were either cancer testis or tumour associated antigens. Twelve BC cell lines and 30 SNs (from 30 patients) were analysed using RT-PCR to determine the in vitro and in vivo detection rates for each of the markers. In addition, 20 axillary nodes obtained from a patient with brain death were used as controls to optimise the PCR cycle numbers for all the markers. RESULTS: Of the 30 SNs, 37% (11/30) were positive on haematoxylin and eosin analysis. Extensive immunohistochemical (IHC) analyses of the haematoxylin and eosin negative nodes confirmed the presence of very small numbers of BC cells in an additional 40% (12/30) of SNs. Molecular analysis with the hMAM-A alone identified metastases in 70% (21/30) of SNs. Using MAGE-A3 in combination with hMAM-A identified metastases in 90% (27/30) of patients. Seven SNs (23%) were negative for micrometastases (with haematoxylin and eosin and IHC) but RT-PCR positive for either hMAM-A or MAGE-A3. CONCLUSIONS: As IHC analysis resulted in a 77% detection rate compared with 37% for haematoxylin and eosin analysis, we consider that IHC is essential in order not to miss SN micrometastases. Molecular analysis with hMAM-A and MAGE-A3 allows detection of BC micrometastases with a 90% detection rate. However, the clinical value of histologically negative but RT-PCR positive SNs can only be determined with long term follow up.

Breast Neoplasms↗
PubMed2005

Proliferation and cell shape changes during ciliary body morphogenesis in the mouse.

Very little is known about the structure and development of the ciliary processes in the mouse eye. Our scanning electron microscope (SEM) studies reveal that, unlike other mammals, the ciliary processes form an irregular pattern, crossing over and interweaving rather than lying parallel to one another. Histological and SEM studies from embryonic day (E) 14.5 to postnatal day (P) 7 reveal that the first morphological sign of the ciliary zone is an annular bulge; this is then gradually molded to form discrete ciliary processes. The striking similarity between the developing capillary network and the adult ciliary folds suggests that the patterning template for the ciliary processes could be the underlying capillary network. Cell proliferation measurements and cell height assessments indicated that one of the first events occurring during the morphogenesis of ciliary processes is a proliferative surge around P0 in the outer ciliary epithelium. It is likely that this surge together with increasing cell heights leads to a bulging of this layer. After a slight delay, the inner ciliary epithelium responds by proliferating and extending inward toward the lens. Final shaping of the ciliary processes is achieved through cell height reductions in the inner ciliary epithelium. Thus, in the mouse, the temporal correlation between mitotic and cell height changes during ciliary body morphogenesis suggests that these processes play an integral role in the shaping of ciliary processes.

Animals↗
PubMed2003

Stimulation of melanogenesis by tetradecanoylphorbol 13-acetate (TPA) in mouse melanocytes and neural crest cells.

In vitro studies have shown that the phorbol ester, 12-tetradecanoylphorbol 13-acetate (TPA) induces neural crest cell differentiation into melanocytes, and stimulates proliferation and differentiation of normal melanocytes. As TPA is not a physiological agent, its action is clearly mimicking some in vivo pathway involved in these processes. An understanding of the effect of TPA on the expression of melanogenic genes will therefore provide valuable insight into the molecular mechanisms regulating melanocyte differentiation. In this study, we utilized primary cultures of neural crest cells and an immortalized melanocyte cell line (DMEL-2) which proliferates in the absence of TPA, to explore the effects of TPA on key melanogenic effectors. In neural crest cells, TPA was found to be necessary for both microphthalmia associated transcription factor (Mitf) up-regulation and for melanin synthesis. Using northern blots, we show that in DMEL-2 cells, TPA significantly increases the messenger ribonucleic acid (mRNA) levels of the tyrosinase gene family (tyrosinase, Tyrp1 and Dct) and the expression of Mitf. Western blots demonstrate that in these TPA-treated cells there is a concomitant increase in Tyr, Tyrp1 and glycosylated Dct protein levels. Pax3, a known Mitf regulator, is unaltered by TPA treatment. This study demonstrates the utility of a novel cell line for investigating the long-term effects of TPA on melanogenesis and provides an understanding of how TPA enhances mouse melanocyte differentiation.

Animals↗
PubMed2003

KUVA (khellin plus ultraviolet A) stimulates proliferation and melanogenesis in normal human melanocytes and melanoma cells in vitro.

BACKGROUND: Khellin is a naturally occurring furochromone which, when combined with artificial ultraviolet (UV) A or solar irradiation (KUVA), is reported to repigment vitiligo skin as effectively as PUVA photochemotherapy. The exact mechanism of KUVA-induced repigmentation is unknown. OBJECTIVES: The aim of this study was to test the effect of khellin and KUVA on proliferation and melanogenesis of normal human melanocytes and Mel-1 melanoma cells in vitro. METHODS: Cultured normal human melanocytes, Mel-1 melanoma cells and fibroblasts were treated with khellin, UVA and KUVA and the effect on proliferation determined by cell counting. The effect on melanogenesis was determined by a radiometric melanin formation assay. Changes in gene expression and protein synthesis were determined by Northern blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analyses. RESULTS: Khellin stimulated proliferation of Mel-1 melanoma cells and melanocytes at concentrations between 1 nmol L-1 and 0.5 mmol L-1 with a peak effect at 0.01 mmol L-1 khellin. In contrast, khellin inhibited proliferation of fibroblasts over the entire concentration range tested. At concentrations above 0.5 mmol L-1, khellin was cytotoxic to both melanocytic cells and fibroblasts. Exposure of khellin-treated cells to single doses of UVA between 150 and 280 mJ cm-2 resulted in an enhanced proliferative effect. Khellin and KUVA also stimulated the melanogenic enzyme activity of pigmented cells, with the most effective treatment being 0.01 mmol L-1 khellin with 250 mJ cm-2 UVA. Western blot, Northern blot and RT-PCR analysis revealed that these increases in melanogenic enzyme activity were not due to increases in gene expression or protein synthesis. UVA treatment resulted in an increase in enzyme glycosylation and this correlated with the increase in melanogenesis. CONCLUSIONS: We conclude that khellin activated by UVA stimulates melanocyte proliferation and melanogenesis. Our results point to the possibility that current treatment regimens might be improved if reduced khellin doses are applied and suggest that improved delivery vehicles be tested.

Blotting, Northern↗
PubMed2003

Accurate molecular detection of melanoma nodal metastases: an assessment of multimarker assay specificity, sensitivity, and detection rate.

BACKGROUND: The application of lymphoscintigraphy followed by sentinel lymph node (SN) biopsy to patients with primary melanoma has revolutionised the ability to identify accurately, yet conservatively, those patients who harbour occult nodal metastases. The molecular detection of SN micrometastases facilitates the cost effective analysis of the entire SN using multiple markers. Currently, a lack of marker specificity is the main barrier preventing the molecular evaluation of SN tissue from becoming clinically applicable. AIMS: To develop a reproducible multimarker reverse transcription-polymerase chain reaction (RT-PCR) assay, with the emphasis on achieving high specificity for the accurate detection of melanoma metastases in nodal tissue. METHODS: Three pigment cell specific (PCS) markers-tyrosinase, Pmel-17, and MART-1-and one cancer testis antigen (CTA)-MAGE-3-were selected for use in a multimarker RT-PCR assay. The conditions for this assay were optimised. RESULTS: High specificity was achievable for each marker by optimising the PCR cycle number such that unwanted transcripts (that is, illegitimate transcripts and/or specific transcripts from other low abundance nodal cell types) remained undetectable in appropriate controls (normal visceral nodes). Tyrosinase was 100% specific at 40 PCR cycles, MAGE-3 and MART-1 at 35 PCR cycles, and Pmel-17 at 30 PCR cycles. Tyrosinase proved to be the most sensitive marker, detecting 10 melanoma cells in 0.1 g of nodal tissue. CONCLUSIONS: Excellent reproducibility of the entire nodal processing and RT-PCR protocol for the detection of very low numbers of melanoma cells in nodal tissue was shown, although there is a risk of false positives using the PCS markers alone, because of an approximate 4-8.5% incidence rate of nodal nevi in melanoma draining SNs (these nevi being absent in all other normal nodes). MAGE-3 was shown to be the only marker that is not expressed by melanocytes. However, because not all melanomas express MAGE-3, it is recommended that more emphasis should be placed on the development of a panel of CTA markers to ensure a zero false positive rate and to provide optimum detection.

Antigens, Neoplasm↗
PubMed2001

SV-40 large T antigen reversibly inhibits expression of tyrosinase, TRP-1, TRP-2 and Mitf, but not Pax-3, in conditionally immortalized mouse melanocytes.

Transformation of mouse melanocytes with a variety of exogenous oncogenes or chemical carcinogens frequently results in irreversible loss of pigmentation. We have infected mouse melanocytes with a temperature-sensitive mutant of the simian virus 40 (SV40) large tumour antigen to study the molecular mechanisms underlying depigmentation during melanocyte transformation. The results show that, out of six cell lines analyzed at the permissive temperature of the oncoprotein, three epidermal and two dermal melanocyte clones remained pigmented and retained the ability to synthesize melanin and to express the melanocyte-specific genes, including tyrosinase, tyrosinase related protein-1, tyrosinase related protein-2 and Mitf. In contrast, one dermal melanocyte clone (DMEL-3) gradually depigmented. This depigmentation was characterized by enhanced growth and down-regulation of melanocyte-specific gene expression. When the oncogene was inactivated by culture at the non-permissive temperature, the pigmented phenotype in DMEL-3 cells could be rescued, and there was a corresponding time-dependent increase in melanocyte-specific gene expression. After extended passage, this rescue could not be achieved. Our results provide direct evidence for the role of the SV40 large T antigen in melanocyte de-differentiation. Expression of Pax-3, a transcription factor implicated in melanocyte differentiation, was unaltered during the SV40-initiated de-differentiation, and de-differentiated melanocytes expressed normal levels of Pax-3 message. We speculate on the mechanism by which the oncoprotein might be regulating Mitf gene expression and of the role of Pax-3 in this process.

3T3 Cells↗
PubMed2000

Haploinsufficiency of the transcription factors FOXC1 and FOXC2 results in aberrant ocular development.

Anterior segment developmental disorders, including Axenfeld-Rieger anomaly (ARA), variably associate with harmfully elevated intraocular pressure (IOP), which causes glaucoma. Clinically observed dysgenesis does not correlate with IOP, however, and the etiology of glaucoma development is not understood. The forkhead transcription factor genes Foxc1 (formerly Mf1 ) and Foxc2 (formerly Mfh1 ) are expressed in the mesenchyme from which the ocular drainage structures derive. Mutations in the human homolog of Foxc1, FKHL7, cause dominant anterior segment defects and glaucoma in various families. We show that Foxc1 (+/-)mice have anterior segment abnormalities similar to those reported in human patients. These abnormalities include small or absent Schlemm's canal, aberrantly developed trabecular meshwork, iris hypoplasia, severely eccentric pupils and displaced Schwalbe's line. The penetrance of clinically obvious abnormalities varies with genetic background. In some affected eyes, collagen bundles were half normal diameter, or collagen and elastic tissue were very sparse. Thus, abnormalities in extracellular matrix synthesis or organization may contribute to development of the ocular phenotypes. Despite the abnormalities in ocular drainage structures in Foxc1 (+/-)mice, IOP was normal in almost all mice analyzed, on all genetic backgrounds and at all ages. Similar abnormalities were found in Foxc2 (+/-)mice, but no disease-associated mutations were identified in the human homolog FKHL14 in 32 ARA patients. Foxc1 (+/-)and Foxc2 (+/-)mice are useful models for studying anterior segment development and its anomalies, and may allow identification of genes that interact with Foxc1 and Foxc2 (or FKHL7 and FKHL14 ) to produce a phenotype with elevated IOP and glaucoma.

Animals↗
PubMed1999

The forkhead/winged-helix gene, Mf1, is necessary for the normal development of the cornea and formation of the anterior chamber in the mouse eye.

Mf1, which encodes a winged-helix/forkhead transcription factor, is the murine homolog of human FKHL7, mutated in individuals with autosomal dominant inherited dysgenesis of the anterior segment of the eye (Axenfeld-Reiger anomaly). Mouse embryos homozygous for null mutations in Mf1 (Mf1(lacZ) and Mf1(ch)) show severely abnormal development of the anterior segment. The cornea fails to separate from the lens, resulting in the complete absence of an anterior chamber. There is no differentiation of the inner corneal endothelial layer, as judged by electron microscopy and by absence of labeling with monoclonal antibody to zonula occludens protein 1, a normal component of occluding junctions in wild-type endothelial cells. In addition, the mutant corneal stroma is disorganized and the epithelium thicker than normal. The Mf1 gene is normally expressed in the periocular mesenchyme at E11.5 but is downregulated as the corneal endothelium differentiates. In contrast, Mf1(lacZ) expression persists longer in mutant corneal mesenchyme, and abnormal expression is also seen in the mutant corneal epithelium. Based on classical studies with the chick embryonic eye, a model is proposed for the differentiation of the mammalian corneal endothelium from mesenchyme in response to putative signals from the lens. Possible roles for Mf1 in this process are discussed.

Animals↗
PubMed1999

Erythropoietin production in anemia associated with experimental cancer.

Serum erythropoietin (EPO) concentrations reportedly are depressed in patients with chronic disorders such as cancer, rheumatoid arthritis, and acquired immunodeficiency syndrome. We evaluated serum EPO levels in mice with tumors and found that the EPO response was appropriate for the associated anemia during the major part of the disease process. The levels of the hormone increased as the anemia worsened in association with progression of the disease. The increased EPO levels were comparable to those of controls with a similar degree of experimentally induced anemia. Only during the terminal stages of cancer, when the animals were severely cachectic, were serum EPO concentrations lower than in controls with a similar degree of anemia. These findings suggest that a blunted EPO response in experimental cancer occurs only in association with advanced disease.

Anemia, Hemolytic↗
PubMed1999

The detection of circulating melanoma cells correlates with tumour thickness and ulceration but is not predictive of metastasis for patients with primary melanoma.

We analysed peripheral blood samples from 143 patients with primary melanoma (PM) for the presence of tyrosinase mRNA by reverse transcription-polymerase chain reaction (PCR) to determine whether the early detection of circulating melanoma cells (CMCs) is of clinical value in monitoring melanoma progression. Ten of the patients (7%) with PM had detectable CMCs. The percentage of PCR-positive patients was higher for stage II patients (9.0%) than for stage I (5.3%), but the difference was not significant. A significantly higher percentage (P<0.05) of PCR-positive patients were found to have tumours greater than 1.5 mm in thickness or had ulcerated tumours. This suggests that tumour thickness and ulceration are the two most significant prognostic factors. The detection rate of 9% for patients with stage II disease is much lower than would be expected, since 23.9% (16 out of 67) of the stage II patients subsequently developed metastases. Of these 16 patients, only one was PCR-positive, 1 week before the metastases became clinically evident. Thus, the current technique fails to predict the likelihood of developing metastatic disease (P=0.3485). The other nine PCR-positive patients had not developed metastases after a median follow-up period of 4 years. It is concluded that this technique for the detection of CMCs is of limited clinical value in predicting the likelihood of metastasis in patients with PM. It is suggested that the detection of micrometastases in other anatomical compartments, such as sentinel lymph nodes, should be explored for the identification of patients at risk for developing metastases.

Disease Progression↗
PubMed1998

Molecular cloning and sequence analysis of a chicken cDNA encoding tyrosinase-related protein-2/DOPAchrome tautomerase.

We have cloned and sequenced a chicken cDNA encoding an l-DOPAchrome tautomerase (DCT) from an embryonic melanocyte cDNA library. The chicken DCT gene encodes a deduced protein of 516 amino acids (aas) and shares 69.2% and 69.9% aa sequence identity with the deduced mouse and human DCT proteins, respectively. Northern blot hybridisation analysis reveals a DCT transcript of 3.5kb in RNA from the retinal pigment epithelium (RPE) of chick embryos. Genomic Southern blot hybridisation analysis suggests that the chicken DCT gene consists of several introns and spans between 15 and 30kb of the chicken genome. This study completes the sequencing of all the members of the chicken tyrosinase-related protein gene family and provides evidence that this gene family is conserved between avians and mammals.

Amino Acid Sequence↗
PubMed1997

An improved and combined reverse transcription-polymerase chain reaction assay for reliable detection of metastatic melanoma cells in peripheral blood.

The application of reverse transcription-polymerase chain reaction (RT-PCR) for detection of metastatic melanoma cells in peripheral blood has been used by a number of different scientific groups with both encouraging and discouraging results. We describe an improved, combined RT-PCR assay for tyrosinase mRNA, which can detect a single, viable melanoma cell in 10-15 ml of peripheral blood. The assay has been found to be highly sensitive, reproducible, cost-effective and clinically applicable. Furthermore, we developed a simple but effective procedure which prevents carry-over contamination. We present evidence indicating that the chance of obtaining normal melanocyte contamination with the needle prick during blood sampling is only 2% among all skin categories. It was found that the phenomenon of illegitimate transcription does not contribute to sporadic false positives. We conclude that the assay, as described here, is of clinical relevance and is a potentially powerful tool for studying various aspects of melanoma biology.

DNA Primers↗
PubMed1997

The regulation of tyrosinase gene transcription.

Tyrosinase is one of the key enzymes essential for melanogenesis. The control of its activity rests in part at the level of transcriptional regulation. The 5' promoter regions of the human, mouse, chicken, quail, snapping turtle, and frog tyrosinase sequences have been isolated and the mechanisms regulating the activity of these sequences are beginning to be elucidated. This review provides an update on the following aspects of tyrosinase gene regulation: basal promoter elements that determine the site of transcription initiation for RNA polymerase II; the cis-acting elements and DNA-binding factors that mediate melanocyte-specific expression of the tyrosinase gene; promoter elements involved in the temporal control of tyrosinase gene expression; additional elements that may be required to achieve wild-type levels of gene expression; and specific elements that may be required for modulation of tyrosinase gene expression in response to humoral factors or external stimuli that are known to influence the amounts of melanin synthesized by fully differentiated melanocytes. The wild type expression of tyrosinase is the result of the interaction of many different factors and it is becoming evident that certain elements and factors play more than one role in this process.

Animals↗
PubMed1996

The cloning and characterization of chick tyrosinase from a novel embryonic cDNA library.

Very little is known about the genes involved in the regulation of avian skin and feather pigmentation. In mammals, two gene families have been identified as being important for the regulation of melanin biosynthesis. To isolate the avian equivalents of these families, we have generated an embryonic chick melanocyte cDNA library. Neural crest cells from 500 black chick embryos were cultured under conditions supportive of melanocyte differentiation and proliferation. A cDNA library was constructed and screened with a mouse tyrosinase cDNA probe. Nineteen clones were obtained, seven of which cross-hybridized to a mouse tyrosinase cDNA on Southern blots. The longest of these clones, B8.3 (1.9 kb), was sequenced and found to share 99.7% nucleotide and 99.8% amino acid sequence homology to a reported chick tyrosinase cDNA. Both Northern blot analysis and in situ hybridization demonstrated that clone B8.3 was expressed in the retinal pigment epithelium of chick embryos. Our results suggest therefore that the cDNA library described here may allow the cloning of novel melanogenic genes.

Amino Acid Sequence↗
PubMed1996

Characteristic sequences in the promoter region of the chicken tyrosinase-encoding gene.

We have isolated and sequenced a genomic DNA sequence encoding chicken tyrosinase (TYR) that includes 2125 nt of 5' flanking sequence, the first exon and a part of the first intron. The 5' flanking sequence was able to drive transcription of a reporter gene in immortalised quail neural crest cells. The sequence, which is the most extensive to be reported for a lower vertebrate TYR gene to date, was further analyzed using primer extension and computer-aided homology searches. Transcription initiation appears to occur at heterogeneous start points and in the absence of a TATA box, but may be mediated via a potential initiator (Inr) element and Sp1-binding motif. We have identified two evolutionarily conserved regions within the 5' flanking sequence that may be functionally significant, as they contain regulatory elements previously reported to play a role in melanocyte-specific expression of TYR in mammals. This study contributes towards an understanding of the requirements for melanocyte-specific TYR expression in lower vertebrates.

Amino Acid Sequence↗