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Biomedical subjects

D N Hu

Publications and source records attributed to D N Hu.

At least 37 records · Page 2Linked to original sources
PubMed1998

The identification and differential expression of calcium-binding proteins associated with ocular melanoma.

Calcium-binding proteins may endow tumor cells with properties related to their malignancy and metastatic phenotype. Chromatographic procedures and amino acid sequence analysis were used in this study to identify seven calcium-binding proteins, annexin VI, cap g, annexin V, calmodulin, S100A11, S100B and S100A6, associated with uveal melanoma, the primary ocular tumor of adults. This series of calcium-binding proteins was identified in both primary tumors and cell lines of uveal melanoma. Several of the proteins were shown by immunochemical methods to be differentially expressed between normal uveal melanocytes and malignant melanomas of the uvea. In addition, the expression of S100A6 may correlate with the malignant properties of the tumor.

Adult↗
PubMed1998

TGF-beta2 inhibits growth of uveal melanocytes at physiological concentrations.

The effect of TGF-beta2 on growth of uveal melanocytes in vitro was studied and the dose-dependent inhibitory effect of TGF-beta2 was compared with the known concentration of TGF-beta2 in aqueous humor. Uveal melanocytes were isolated and cultured with medium supplemented with cAMP elevating agents and basic fibroblast growth factor. The uveal melanocytes were plated into multi-well plates. After 24 hr, TGF-beta2 was added to the medium in various concentrations. After 5 days, the cells were detached, counted and compared to the controls. The effect of TGF-beta2 on DNA synthesis (as evaluated by uptake of bromodeoxyuridine) were also tested. TGF-beta2 inhibited growth and DNA synthesis of cultured uveal melanocytes in a dose-dependent manner at concentrations from 0.03-10.0 ng ml-1. The growth-inhibition of TGF-beta2 was present even in serum-free medium. TGF-beta2 had little or no effect on melanogenesis of cultured uveal melanocytes. The serum used for cultivation did not contain active TGF-beta1 or TGF-beta2 as measured by immunoassay. The known amount of active TGF-beta2 in aqueous humor (0.2-0.4 ng ml-1) is sufficient to inhibit the growth of uveal melanocytes. It indicates that TGF-beta2 is a potent growth inhibit factor of uveal melanocytes and may play an important role in maintaining the non-proliferative, relatively quiescence status of uveal melanocytes in vivo.

Adult↗
PubMed1998

Characterization of melanins in human irides and cultured uveal melanocytes from eyes of different colors.

The presence of eumelanin and pheomelanin in irides from eyes of various colors was determined and quantified by a highly specific microanalytical procedure based on chemical degradation. Significant differences in the type of melanin were observed in the stroma and iris pigment epithelium (IPE) fractions obtained by micro-dissection of the iris specimens. Melanin from the IPE is essentially eumelanin, while the pigment in IPE-scraped iris (consisting mainly of stroma plus anterior IPE) proved to be both eumelanic and pheomelanic. A pheomelanic-type pigmentation was associated with green irides, while green-blue mixed-color irides were mostly eumelanic; by contrast, green-brown mixed-color and brown irides could not be placed into either of the two categories and probably feature a mixed pigment content. Blue irides invariably exhibited very low pigment content. Analysis of cultured iridial melanocytes in the growing stage showed a significant shift to pheomelanic pigmentation when compared with those in IPE-scraped tissues, providing evidence that growth of iridial melanocyte induce a marked change of melanin metabolism. After senescence, cultured melanocytes exhibited a marked increase in pigment content, most of the variation was associated with the eumelanin content. These results represent the first direct evidence for the presence of eumelanin and pheomelanin in human irides, and suggest that differences in stromal pigmentation are due not only to the quantity, but also the nature of the melanin pigment.

Cells, Cultured↗
PubMed1998

Pulse radiolysis studies of melatonin and chloromelatonin.

The endogenous indole melatonin and the melatonin receptor agonist 6-chloromelatonin block the proliferation of both dermal and uveal melanoma cells by mechanisms that may involve redox reactions. The interactions of hydrated electrons, the azide radical, hydroxyl radicals and superoxide with melatonin and its 6-chloro analogue have been studied using the technique of pulse radiolysis. The reaction rate constants of eaq- and N3 x with these compounds were found to be dependent on substitution at the sixth position. The rate constants for reaction of 6-chloromelatonin and melatonin with solvated electrons are 4.5 x 10(9) M-1 s-1 and 4.2 x 10(8) M-1 s-1, respectively. The reaction rate constants of N3 x with malatonin and chloromelatonin are 9.8 x 10(9) M-1 s-1 and 3.5 x 10(9) M-1 s-1 and 3.5 x 10(9) M-1 s-1, respectively. Melatonin and 6-chloromelatonin react with hydroxyl radicals at near diffusion controlled rates (1.3 x 10(10) M-1 s-1, 8.2 x 10(9) M-1 s-1). Melatonin and 6-chloromelatonin did not react with superoxide radicals and we calculate an upper limit of 1.0 x 10(4) M-1 s-1 for the rate constant for reaction of melatonin and 6-chloromelatonin with superoxide ion.

Electron Spin Resonance Spectroscopy↗
PubMed1998

Effects of melatonin, its precursors and derivatives on the growth of cultured human uveal melanoma cells.

The effects of melatonin, its precursors and derivatives on the growth of cultured human uveal melanoma cells were studied. The melanoma cells were plated into 24-well plates. Melatonin, its 6-hydroxy or 6-chloro derivative, serotonin, tryptophan or kynurenine was added to the medium in concentrations of 0.001 to 1000 nM. After 5 days the cells were detached, counted, and compared with the controls. Melatonin inhibited the growth of uveal melanoma cell lines in a dose-dependent manner (0.1-10 nM). This growth inhibition occurred at concentrations of melatonin (2 nM) found in human aqueous humour. The melatonin derivatives also inhibited the growth of uveal melanoma cells; 6-chloromelatonin was more potent than melatonin and 6-hydroxymelatonin was the least active (6-chloromelatonin > melatonin > 6-hydroxymelatonin). The precursors of melatonin (tryptophan and serotonin) and the abnormal metabolite of tryptophan (kynurenine) did not inhibit the growth of the melanoma cells, indicating that changes to the metabolic processes of melatonin may play a role in the pathogenesis of uveal melanoma.

Cell Division↗
PubMed1997

Isolation and culture of iris pigment epithelium from iridectomy specimens of eyes with and without exfoliation syndrome.

OBJECTIVE: To culture iris pigment epithelium (IPE) from surgical iridectomy specimens of eyes with and without exfoliation syndrome. METHODS: The IPE was treated to obtain a single cell suspension. Cells were cultured in Ham F12 nutrient mixture, which was supplemented with 30% fetal bovine serum, 50-micrograms/mL [corrected] gentamicin, and 2-mmol/L glutamine. After confluence, the cells were detached using a 0.125% trypsin-0.01% edetic acid solution, resuspended, diluted, and subcultured. The IPE from primary cultures and subcultures was studied by transmission electron microscopy. Immunocytochemical staining was performed. RESULTS: In the primary cultures of IPE from patients with exfoliation syndrome, curved, cross-banded, fine fibrils (diameter, 10-15 nm; periodicity, 10-14 nm) were found on the cell surface. Thicker fibrils (diameter, 24-48 nm; periodicity, 24-36 nm) were found external to the fine fibrils. Subcultures contained mainly fine fibrils. The IPE cells stained positively with anticytokeratin, S100 protein, and vimentin antibodies. CONCLUSION: Iris pigment epithelium can be successfully cultured from eyes with exfoliation syndrome. Studying the production of exfoliation material in vitro should provide information about the pathogenesis of exfoliation syndrome and about the nature of the exfoliation material. The cultivation of normal IPE from surgical specimens provides a source for the study of the growth regulation and pharmacophysiology of IPE in vitro.

Adolescent↗
PubMed1997

Regulation of melanogenesis by human uveal melanocytes in vitro.

The purpose of this study was to investigate factors regulating melanogenesis in cultured human uveal melanocytes. The effects of various substances on the melanin content, tyrosinase activity and growth of cultured uveal melanocytes were tested. 12-O-tetradecanoyl-phorbol-13-acetate (a protein kinase C activator) and various cAMP-elevating agents, including isobutylmethylxanthine, cholera, toxin, and dibutyryl-cAMP increased melanin content per culture, tyrosinase activity and cell numbers of uveal melanocytes in a dose dependent manner. Basic fibroblast growth factor (tyrosine kinase activator) stimulated growth but did not affect melanin content per culture of uveal melanocytes in vitro. These results indicate that cAMP-elevating agents and protein kinase C activator stimulate melanogenesis and growth of cultured uveal melanocytes. Tyrosine kinase activator stimulates growth but not melanogenesis of cultured uveal melanocytes.

Adult↗
PubMed1997

Melatonin inhibits growth of cultured human uveal melanoma cells.

The effects of melatonin on the growth of human uveal melanoma cells were studied in vitro. Three continuous uveal melanoma cell lines were tested. Cells were plated into multi-well plates. After 24 h, melatonin was added to the medium at concentrations from 0.001 to 1000 nM. Cells were collected and counted after 5 days and compared with the controls. Melatonin inhibited the growth of melanoma cells in a dose-dependent manner (0.1-10 nM) with a mean inhibition rate of 50%. The uptake of bromodeoxyuridine (BrdU) by the melanoma cells was also measured. Melatonin inhibited the uptake of BrdU of melanoma cells at concentrations of 0.1-10 nM with a mean inhibition rate of 40%. These results indicate that melatonin may offer a new treatment for metastatic uveal melanoma.

Bromodeoxyuridine↗
PubMed1997

Tissue culture of adult human retinal ganglion cells.

PURPOSE: We wished to isolate and cultivate adult human retinal ganglion cells (RGC) from donor eyes. METHODS: Small pieces of retina from donor eyes were plated in dishes and cultured with Ham's F12 medium with 10% serum for organ culture. For cell culture, cells were isolated by mechanical or enzymatic dissociation methods and cultured with F12 medium with 10% serum, with or without nerve growth factor (NGF) and/or basic fibroblast growth factor (bFGF). RESULTS: In organ cultures, no neurite outgrowth from the retinal explants was observed. In cell cultures for which mechanical dissociation methods were used, the few cells that could be isolated showed poor viability. Better results were obtained with enzymatic dissociation methods. When cultured with medium supplemented with bFGF, some cells attached, spread, and sent out numerous dendrites, morphologically similar to RGC. These cells stained positively for neurofilaments and Thy-1 and negatively for glial fibrillary acidic protein (GFAP), indicating they were RGC. CONCLUSIONS: Cell cultures of human RGC can be established. This is a potential model system for studying effects of damaging and protective factors on RGC in vitro.

Adult↗
PubMed1995

Melanogenesis by human uveal melanocytes in vitro.

PURPOSE: To study melanogenesis by cultured human uveal melanocytes, and the relationship between melanin production by uveal melanocytes in vitro with the degree of iris pigmentation in vivo. METHODS: Melanin content, melanin production, and tyrosinase activity of cultured uveal melanocytes derived from eyes of various iris color were measured at different stages of cultivation. RESULTS: Cultured uveal melanocytes maintained a constant level of melanin content, expressed tyrosinase activity, and produced measurable amounts of melanin in vitro. Melanosomes in different stages were seen ultrastructurally. Melanin production correlated directly with the degree of iris pigmentation of the eyes from which the uveal melanocytes were isolated. Tyrosinase activity of cultured uveal melanocytes from black versus white donors was significantly different, but, among white donors, there was no correlation with iris pigmentation or with melanin production in vitro. CONCLUSION: Cultured uveal melanocytes can produce melanin in vitro. Cultured uveal melanocytes isolated from eyes of different iris color maintained their inherent capacity for melanogenesis. Therefore, cultured uveal melanocytes are an excellent model system for studying melanogenesis in uveal melanocytes in vitro.

Black People↗
PubMed1993

Studies of human uveal melanocytes in vitro: growth regulation of cultured human uveal melanocytes.

PURPOSE: The authors studied the growth requirements and growth regulation of cultured human adult uveal melanocytes (UM). METHODS: The effect of various mitogens and growth factors on the growth of UM were tested separately or combined on cultured UM in multiwell plates. RESULTS: Basic fibroblast growth factor (bFGF) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulate the growth of UM. Without these agents, the UM did not grow or survive. A cyclic adenosine monophosphate (cAMP) stimulator, such as isobutylmethylxanthine or cholera toxin, stimulated growth in the presence of bFGF. Fetal bovine serum (FBS) is also required for growth. In its absence, UM did not grow, even in the presence of bFGF and cAMP stimulators. Other substances, such as epidermal growth factor, acidic FGF, nerve growth factor, and platelet-derived growth factor had no stimulating effects on the growth of UM. CONCLUSIONS: Three classes of agents are required for the growth of UM in vitro: bFGF or TPA, a cAMP stimulator, and FBS. Adult human UM cultured in medium containing all these agents grew well and could be passaged for many generations.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Studies of human uveal melanocytes in vitro: isolation, purification and cultivation of human uveal melanocytes.

PURPOSE: To develop the methods for isolation and cultivation of human uveal melanocytes (UM) from adult donor eyes. METHODS: After removal of the pigment epithelium, the uvea was pretreated in trypsin solution at 4 degrees C overnight, incubated at 37 degrees C with trypsin for 1 hr, then incubated with collagenase for 3 hr. Released cells were collected each hour during the incubation and cultured with F12 medium supplemented with fetal bovine serum, basic fibroblast growth factor, isobutylmethylxanthine and cholera toxin. Contaminant cells were eliminated by adding a selective cytotoxic agent, geneticin, when necessary. RESULTS: These methods provide pure melanocyte cultures with high cell yields, good viability, and rapid growth rates. UM isolated and maintained using these methods can be passaged 23 times for a period of 7 mo for more than 35 population doublings. This is comparable to results obtained with cultured neonatal dermal melanocytes and exceeds results obtained with adult dermal melanocytes cultured in media supplemented with phorbol ester, isobutylmethylxanthine, and cholera toxin. CONCLUSION: A method for isolation and cultivation of UM has been developed that yields satisfactory results. Cultured UM may be useful in in vitro studies of UM physiology and may allow development of in vitro models of the pathogenesis of uveal malignant melanoma.

Adult↗
PubMed1992

Cleft lip with or without cleft palate in Shanghai, China: evidence for an autosomal major locus.

Orientals are at higher risk for cleft lip with or without cleft palate (CL +/- P) than Caucasians or blacks. We collected demographic and family data to study factors contributing to the etiology of CL +/- P in Shanghai. The birth incidence of nonsyndromic CL +/- P (Shanghai 1980-87) was 1.11/1,000, with a male/female ratio of 1.42. Almost 2,000 nonsyndromic CL +/- P probands were ascertained from individuals operated on during the years 1956-83 at surgical hospitals in Shanghai. Detailed family histories and medical examinations were obtained for the probands and all available family members. Genetic analyses of the probands' families were performed under the mixed model with major locus (ML) and multifactorial (MFT) components. The hypotheses of no familial transmission and of MFT alone could be rejected. Of the ML models, the autosomal recessive was significantly most likely and was assumed for testing three complex hypotheses: (1) ML and sporadics; (2) ML and MFT; (3) ML, MFT, and sporadics. None of the complex models were more likely than the ML alone model. In conclusion, the best-fitting, most parsimonious model for CL +/- P in Shanghai was that of an autosomal recessive major locus.

Asian People↗
PubMed1992

Isolation and cultivation of human iris pigment epithelium.

There have been very few attempts to isolate and culture human iris pigment epithelium (IPE). Earlier efforts that used whole iris explant methods did not achieve pure cultures of IPE. We have developed methods for separating the IPE from the iris stroma of post-mortem eyes that avoid contamination by other cell types. Three different isolation methods were studied: direct dissection, enzyme digestion, and enzyme-assisted microdissection. The latter method yielded the best results. After treatment with enzyme solution, the IPE was easily separated from the stroma under the stereomicroscope and subsequently cultured with supplemented F12 medium. With this method, approximately 2.3 x 10(5) cells were isolated from each iris with an average viability of 90.2%. IPE cells isolated from 19 of 24 eyes grew to confluence in primary culture. The IPE could be maintained in pure culture for many generations over several months with up to 20 population doublings. Cultured IPE demonstrated cytokeratin and S-100 protein by immunocytochemistry. Some of these cells also displayed desmin, indicating origin from the anterior IPE. Cultured IPE cells retained most of the characteristics of IPE in vivo, such as apical/basal polarization, microvilli, and many cell junctions. Gradual dilution of pigment occurred in the dividing IPE cells, suggesting an inability to produce melanin in vitro. A subpopulation of the IPE cells contained myofilaments by electron microscopy, also indicating a anterior IPE origin. This method provides a source for large numbers of human IPE cells and could be useful in studies of the biology of IPE and the role of IPE in pathogenesis of several eye diseases, most notably exfoliation syndrome and its associated glaucomas.

Adult↗
PubMed1991

Genetic aspects of antibiotic induced deafness: mitochondrial inheritance.

Analysis of 36 pedigrees with a positive family history of aminoglycoside antibiotic induced deafness, ascertained in a population of 483,611 in Zhabei District in Shanghai, showed that the susceptibility to antibiotic ototoxicity was transmitted by females exclusively, indicating mitochondrial inheritance. Reanalysis of 18 other published pedigrees confirmed this conclusion.

Aminoglycosides↗