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Biomedical subjects

K H Sit

Publications and source records attributed to K H Sit.

At least 55 records · Page 3Linked to original sources
PubMed1992

Coarse to fine hair conversion induced by zinc in C57/6J mice.

The cytotoxic effects of excessive zinc is well documented but its effect on cytodifferentiation appears relatively unknown. We show here that chronic administration (25 days) of zinc chloride in C57/6J mice (11 to 56 micrograms/g body weight, 5 times per week), which was lower than the in vitro cytotoxic level, caused a loss of long coarse hairs, which were replaced by short fine (vello) hairs. Histological architecture of the skin appeared normal except for the confinement of hair follicles to the dermis without any extension into the hypodermis.

Animals↗
PubMed1992

Extracellular ATP induces rapid cell rounding in cultured human Chang liver cells.

Extracellular ATP (7 microM-14 mM) induced monolayer Chang liver cells (ATCC CCL 13) to retract and round up in dose-dependent and time-dependent manners. ATP-induced rounding was concomitant with raised intracellular pH (7.3 to 7.8) and suppressed (a) in sodium-free medium (250 mM sucrose), (b) with the addition of 1 mM quinidine, an inhibitor of Na+/H+ exchanges, and (c) with the addition of 10 nM staurosporine, a potent inhibitor of protein kinase C that is known to activate Na+/H+ exchanges. These findings suggest that ATP-induced cell rounding is due to activation of Na+/H+ antiporters.

Adenosine Triphosphate↗
PubMed1992

Distinctive uptake of neutral red by mitotic cancer cells.

Neutral red stains both normal and cancer mitotic cells, but uptake by living mitotic cancer cells is distinctly higher than in normal cells. This new approach to cancer cell identification is demonstrated in 4 established tumorigenic cancer cell lines: human skin epidermoid carcinoma A431, mouse Cloudman malignant melanoma, human oral epidermoid carcinoma and rat hepatoma. Human Chang liver cells served as normal controls. With epidermal growth factor (EGF) prepulse, neutral red uptake is dramatically enhanced. The possibility of a causal relationship with M-phase specific phosphorylation is discussed.

Animals↗
PubMed1992

Microvillus form of focal anchorage in human Chang liver cells rounded by antiporter activation: scanning electron microscopy profiles and evidence of traction origin.

Na+/H+ antiporter activation in human Chang liver cells produces a flat-to-round (FTR) change in cell shape with gross reduction in cell profile area. Scanning electron microscopy (SEM) vividly displays a third phenomenon, viz., the development of focal microvillus anchors. Reduction in cell profile area concomitant with the development of this microvillus form of focal anchorage is quantitated by on-line image analysis during SEM examination. The reduction in profile area is corroborated by spectrophotometric digitization in light microscopy. Transmission electron microscopy (TEM) of rounded cells shows large endocytic channels and endosomes consistent with the observation of internalization of fluoresceinated-dextrans (FDx) of a diverse range of sizes, from 4,400 to 2,000,000 molecular weight, with cell rounding. Concomitant endocytosis of this magnitude indicates massive plasma membrane internalizations which could explain the very considerable profile area reduction and suggest that the microvillus anchors are probably traction processes. Antiporter mediated rounding (AMR) provides a highly reproducible and simple model for the production of anchoring microvilli ('filopodia') whereby they can be further explored.

Carrier Proteins↗
PubMed1992

Inheritance of susceptibility: Lamarckism revisited.

The familial nature of susceptibility to rheumatic fever has been known for nearly three quarters of a century but even after massive ascertainments of affected families in 5 major cities, viz Toronto, Belfast, London, Glasgow and New York, a consensus on the exact mode of inheritance could not be reached. Reduced penetrance was suggested 40 years ago and still cited today even though the fit is poor. However with the sampling bias of the observed data resolved in the recently formulated geometric continuum v(affected-1) x P(sibship) (where O less than v----infinity) all those published ascertainments clearly show a unilocal Mendelian recessive mode of inheritance. Since rheumatic fever is clearly associated with streptococcal sore throat, I have therefore demonstrated the inheritance of an acquired trait. This Larmarckian concept is explained using simple numerical examples.

Genetic Predisposition to Disease↗
PubMed1991

Expression of a high-affinity form of UDP-glucuronosyltransferase in human foetal liver cells in culture on exposure to mercuric chloride.

The activity of UDP-glucuronosyltransferase (UDPGT, EC 2.4.1.17) in human foetal liver cells in culture was measured with two acceptor substrates, namely harmol and 1-naphthol. There was a dose-dependent increase of about 10-400% in UDPGT activity when the cells were exposed to 1-30 microM-HgCl2. Above a critical concentration of 30 microM-HgCl2, the heavy metal ion was toxic to the cells. Kinetic studies of the glucuronidation reaction with harmol and 1-naphthol showed that Hg2+ ions seemed to induce the expression of a high-affinity form of UDPGT, which was absent from the normal controls. The dramatic increase in specific activity in UDPGT was accompanied by a parallel increase in Vmax. measured with harmol and UDP-glucuronic acid. The significance of a possible induction of UDPGT in human foetal liver cells by HgCl2 is discussed.

Cells, Cultured↗
PubMed1991

Sulphate induces very fast cell rounding and detachment.

Within a few minutes of incubation with SO4(2-), cultured monolayer cells retract into round shapes with drastically reduced surface area. Concomitant elevation of phosphoinositide second messenger levels, viz, 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), is observed. A causal relationship with sulphation seems to be suggested by finding (a) sulphation of an added acceptor, 4-methylumbelliferone, (b) sulphation of endogenous glycosaminoglycan (GAG) polymers, (c) inhibition by phenol sulphotransferase inhibitor, DCNP (2,6-dichloro-4-nitrophenol). DCNP also inhibits the second messenger production and cell rounding. Reduced surface area appears to be caused by massive plasma membrane internalization in a distinctive endocytosis which is also seen in cell rounding from directly imposed ionic gradients. Reducing the surface area would reduce the adhesive or attachment sites. Besides demonstrating a highly efficient cell detachment potential, huge macromolecules appear to be readily internalized. The association of sulphation, signal transduction and cell detachment is novel.

Cell Line↗
PubMed1991

Biosynthesis of PAPS in vitro by human liver. Measurement by two independent assay procedures.

The biosynthesis of 3'-phosphoadenosine-5'-phosphosulphate (PAPS) by extracts of human liver from inorganic sulphate and APS was assayed by transferring the "active sulphate" to two different acceptors, namely N-acetyldopamine (NADA) and 4-methylumbelliferone (4-MU). NADA-sulphate was quantified by an HPLC-ECD method while the decrease in 4-MU was monitored continuously by a fluorimetric procedure. The optimum pH was 8.0 for both the PAPS generation and APS-kinase reaction. The apparent Km value for APS determined by the fluorimetric and HPLC-ECD procedures was 17.6 and 16.8 microM, respectively. PAPS-generation measured in 13 samples of human liver showed excellent correlation between the two assay procedures, with correlation coefficients (r) of 0.96 and 0.95 for PAPS generation from inorganic sulphate and APS, respectively. The fluorimetric assay was preferred as it is simple, equally sensitive and more reproducible. There was also a good correlation between the APS-kinase reaction and the two-step PAPS-generation from inorganic sulphate, with r = 0.97 and 0.91, as determined by the fluorimetric and HPLC-ECD procedures. The rate of PAPS generation from inorganic sulphate was only marginally less than that from APS in each of the 13 human liver samples, suggesting that the coupling of ATP-sulphurylase to APS-kinase facilitates "sulphate activation" and releases it from the constraints imposed by the unfavourable ATP sulphurylase reaction.

Adenosine Phosphosulfate↗
PubMed1991

Antiport mediated rounding and endocytosis are enhanced by sulphate.

Rapid cell detachment concomitant with the flat-to-round (FTR) change that is mediated by an upshifted Na+/H+ antiporter via HCO3(-)-dependent H+ pumping, is significantly enhanced by the addition of Na2SO4 (FTR + SO4): (1) a faster and greater reduction in cell surface area and perimeter, and (2) a higher level of macromolecular internalization which is also amiloride sensitive. At a fixed 1 mg/ml extracellular FITC-dextran (FDx) concentration, the intracellular FDx load is similar irrespective of the particle size, in the range from 4400 to 2 million mol.wt which is a 455-fold diversity. This is inconsistent with entry via limited sized portals which would discriminate against the larger molecular weight species, such as the 2 million mol.wt species that measures up to 5 microns in width. Two million mol.wt FDx loads linearly in direct proportion to the extracellular FDx concentration, simulating simple diffusion. Large-channel endocytosis is considered to be a characteristic of specialized cell types such as phagocytes and macrophages. However, the antiporter mediated endocytosis (AME) shown here is demonstrated in two different cell types which are not known for their endocytic prowess, viz. epitheloid human Chang liver cells (ATCC CCL 13) and human lung fibroblasts (ATCC CCL 202). The rounded cells with internalized FDx start reverting back to their flat and protracted form upon flooding with warm growth medium, a round-to-flat (RTF) change. However the cell surface reversion is not associated with efflux of FDx which are sorted out into 'granular patches', the later stage endosomes without membrane outlines in AME. FDx-loaded cells grow as well as trypsinized cells without FDx loaing and they maintain a significant FDx load even after nearly 4 cell divisions. Toad sperms internalized into Chang cells via antiporter activation are also sorted into granular patches. AME provides (a) distinctive access to large particles, simulating small ion influx, and (b) an alternate membrane recycling capability where granular patches are instrumental in sorting. It appears to be not a simple endocytosis-exocytosis pathway.

Amiloride↗
PubMed1991

Differential oxygen sensitivities in G6PDH activities of cultured keloid and normal skin dermis single cells.

Cell cultures of excised keloids and biopsied normal skin were established from patients of the Plastic Surgery Department of Singapore General Hospital and their single cells quantitated for glucose-6-phosphate dehydrogenase (G6PDH) activity using microspectrophometry. G6PDH has been cited as a transformation-linked discriminant. Analysis of variance shows no difference in overall activity between skin and keloid cells, but under oxygen saturation conditions, keloid G6PDH activity was significantly higher than skin G6PDH activity, although they were almost identical under nitrogen saturation conditions. Differential oxygen sensitivity in G6PDH activities between malignant and non-malignant cells have been reported, but its occurrence between keloid and normal skin cells is novel, especially as the keloid is regarded as a benign tumor with a zero carcinogenicity rate.

Adolescent↗
PubMed1991

Effects of genistein on ATP induced DNA synthesis and intracellular alkalinization in Chang liver cells.

Extracellular ATP (adenosine 5'-triphosphate) produces a dose-dependent elevation of DNA synthesis concomitantly with raised intracellular pH (pHi) in quiescent monolayer Chang liver cells. Genistein produces a dose-dependent suppression of ATP-induced DNA synthesis without affecting cell viability (dead cell index approximately 5%) or pHi elevation. The results suggest that (a) mitogenesis and pHi are not imperative correlates and (b) genistein is effective in inhibiting DNA synthesis whether due to growth factor or "non-growth factor" stimulation.

Adenosine Triphosphate↗
PubMed1991

Rough ER degranulation and swelling resolved as lysosomes in Na+/H+ antiporter mediated rounding of human lung fibroblasts.

Na+/H+ antiporter activation by incubation in bicarbonate buffered saline induces long flat anchored human lung fibroblasts in monolayer culture to retract their cytoplasmic processes and become eventually globular in form. Reduction of cell profile area and perimeter in antiporter mediated rounding (AMR) is quantitated by microspectrophotometry and the roundness form factor (PE) calculated from these parameters. Quantitatively, 2 stages of rounding are discernible. Stage I rounding is a simple retraction phenomenon where PE upshifts marginally. Stage II rounding sees the cell transformed into globular refractile form where PE greater than 0.5. Ultrastructural studies show appreciable swelling of rough ER in Stage I rounding. Stage II rounding is accompanied by enhanced swelling of rough ER and degranulation. A dynamic relationship between rough ER (RER) and smooth ER (SER) in different functional states of a cell is novel. Enlarged SER channels appear resolved in later stages as lysosomal bodies suggesting that degranulated ER membranes are lysosomal targets in autophagocytosis.

Bicarbonates↗
PubMed1990

An HPLC-ECD procedure for measuring total phenolsulfotransferase (PST) activity in human liver, platelets and blood.

The phenolsulfotransferase (PST) activity in human liver, platelets and blood was measured under saturating concentrations of the conjugating agent, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Conventional PST assays employ PAP35S at suboptimal concentrations. In addition, the sulfate conjugate formed, namely N-acetyldopamine-sulfate (NADA-sulfate) was quantified directly by high-pressure liquid chromatography cum electrochemical detection (HPLC-ECD). NADA, the biogenic amine acceptor used in this study appeared from kinetic data to be a substrate of both the P and M forms of PST when used in micromolar concentration. Two apparent Km values of 4.2 mumol/l and 22.6 mumol/l were observed. In contrast, only one apparent Km value was evident when the assay was carried out in the presence of 2,6-dichloro-4-nitrophenol (DCNP), a selective inhibitor of the P form of PST or after heat treatment under specified conditions which inactivates the M form of PST. Thus measurement of PST activity with NADA as the acceptor substrate permits the determination of total PST activity and a parallel assay with the inclusion of DCNP would distinguish the two variants of PST, both of which appear to be present in all human tissues.

Arylsulfotransferase↗
PubMed1990

Sulphate conjugation in human liver. Direct measurement of N-acetyldopamine-sulphate.

The overall three-step sulphate conjugating activity of the human liver was determined by the direct measurement of N-acetyldopamine (NADA)-sulphate from ATP and inorganic sulphate. NADA-sulphate was separated from NADA and quantified by HPLC-ECD (high-pressure liquid chromatography-electrochemical detection). The overall sulphate conjugation of NADA showed at pH optimum of 8.0 with apparent Km values for sodium sulphate and NADA of 103 microM and 4.8 microM. respectively. The optimum concentration of ATP was 5 mM and that for Mg2+ ions was 7 mM. The specific activities of 17 samples of human liver, measured by this HPLC-ECD procedure ranged from 940 to 23,128 pmol NADA-sulphate/hr/mg protein. A comparison of these values with those determined by the radiometric method developed previously showed a direct correlation coefficient, r = 0.89. The rates of overall sulphate conjugation of NADA and dopamine measured in these samples by the radiometric assay procedure were also significantly correlated with r = 0.82.

Aged↗
PubMed1990

UDP-glucuronyltransferase activity toward harmol in human liver and human fetal liver cells in culture.

This paper presents a fast HPLC assay for measuring UDP-glucuronyltransferase (UDPGT) activity in extracts of adult human liver and human fetal liver cells in culture. Harmol glucuronide formed was quantitated directly without prior hydrolysis after a simple step of selective extraction of harmol. The method is applicable to crude liver homogenates as well as to partially fractionated preparations. It is several fold more sensitive than the conventional detection of harmol glucuronide by TLC, making it possible to distinguish the low and high affinity forms of UDPGT of adult human liver and to detect the low affinity form in fetal cells. The possible participation of both forms of GT in adults under different conditions and the apparent lack of the high affinity form in the fetal liver is discussed.

Alkaloids↗
PubMed1990

Phenolsulfotransferase (PST) and PAPS in catecholamine metabolism in human tissues: an overview.

Sulfate conjugation in vitro was studied in a number of human tissues, such as the platelets, liver as well as in human foetal liver cells in culture. Essentially, the two enzymes involved in PAPS generation and PST were measured either singly or in combination, representing PAPS generation, PST and the overall three-step sulfate conjugation. These activities in the liver and platelets were comparable in magnitude. In the platelets, PAPS generation and PST activities showed a good correlation to the overall sulfate conjugation. With the expression of these activities in human foetal liver cells in culture, it is anticipated that such cells can be employed as model systems for the study of sulfate conjugation in man.

Adenine Nucleotides↗