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Biomedical subjects

K W Ng

Publications and source records attributed to K W Ng.

At least 19 recordsLinked to original sources
PubMed1992

Chromatographic analysis of antibiotic materials in food.

The monitoring of food materials for antibiotic residues is an area of increasing concern and importance due to the potential impact on human health. Large-scale screening applications require methods that are rapid, accurate, provide low detection limits and are free from interference. The problem is further complicated by the wide range of chemical functionalities and modes of operation exhibited by the antibiotic materials of physiological significance in use today. As demonstrated, chromatographic methods provide many of the advantages necessary for screening applications. Judicious choice of sample preparation method, separation mode and detection strategy can provide significant immunity from problems associated with the food matrix. Gas chromatography can provide extremely high separation efficiencies, however, only a limited number of antibiotic compounds are inherently volatile enough for direct analysis by gas chromatography. Derivatization to enhance the volatility of the antibiotic is one approach to overcome this limitation. Among the methods available, reversed-phase high-performance liquid chromatography is used extensively for the analysis of many antibiotic systems as it does not require derivatization and it combines relatively high separation efficiencies with low detection limits. The diverse group of properties exhibited by the antibiotic materials in use today suggests that the choice of detection strategy is a key component in the successful development of an analysis technique. Derivatization of the antibiotic material is frequently used to add either a fluorogenic of chromogenic moiety to the antibiotic compound to enhance detection. Derivatization procedures suffer from several limitations which are problematic when making measurements in complicated food matrices. Among the different detection modes utilized for antibiotic analysis, polarimetric detection has the potential to provide extremely selective detection of most antibiotic materials, and this selective response can minimize many of the constraints placed upon the separation system by the sample matrix. Although many of the separation modes used for antibiotic analysis are well developed, separations based on capillary electrophoretic methods have much potential in the field of antibiotic analysis. Future investigations are needed to extend the generality of these techniques and expand their use into the field of food analysis.

Anti-Bacterial Agents↗
PubMed1992

Response of preterm infants to hepatitis B vaccine.

Ninety-nine preterm infants with birth weights < 1750 gm had three doses of hepatitis B vaccine. Fifty-seven received the first dose when they weighed > or = 1000 gm (group 1) and 42 when they weighed > or = 2000 gm (group 2). The final seropositive rates and geometric mean titers of group 1 infants (79%, 61 mIU/ml) and group 2 infants (91%, 262 mIU/ml) were less than that of 43 normal term infants (100%, 679 mIU/ml).

Analysis of Variance↗
PubMed1991

Retinoic acid modulation of mRNA levels in malignant, nontransformed, and immortalized osteoblasts.

Clonal cell lines presumably "arrested" at a particular stage of differentiation are useful models to study the processes of differentiation in osteoblasts. UMR-201 is a presumptive preosteoblastic nontransformed rat clonal cell line with a limited life span in culture. Two immortalized cell lines, UMR-201-10A (10A) and UMR-201-10B (10B), were derived from UMR-201 by stable transfection with simian virus (SV) 40 large T antigen. This study compares the growth and profile of gene expression of the immortalized cell lines with those of UMR-201 and UMR-106-06, a rat clonal cell line with well-defined osteoblast-like phenotypic characteristics. All four cell lines constitutively expressed the mRNA for the gamma, alpha, and beta receptors for retinoic acid (RA), the growth hormone receptor, pro-alpha 1(I) collagen, osteonectin, bone proteoglycan I, and bone morphogenetic proteins (BMP) 1 and 2A. Alkaline phosphatase mRNA was absent in the preosteoblast cell lines but was induced by treatment with 10(-6) M RA, which also increased the steady-state levels of mRNA for osteopontin and BMP1. mRNA for matrix gla protein was constitutively present and further induced by RA in UMR-201 and 10B only. Messenger RNA for bone sialoprotein and bone morphogenetic protein 3 were constitutively expressed in UMR-106-06 and UMR-201 but absent in the immortalized cell lines. None of the cell lines expressed measurable mRNA for bone gla protein or bone proteoglycan II. 10B grew more rapidly than UMR-201, but unlike UMR-201, it was also able to proliferate in serum-free medium and exhibit anchorage-independent growth. In summary, this study identifies novel retinoic acid effects on gene expression in these cells. Differences noted in the expression of mRNAs between UMR-106-06 and the other cell lines may provide some insight into the sequence of expression of these phenotypic characteristics as osteoblasts differentiate.

Alkaline Phosphatase↗
PubMed1991

Growth hormone (GH) receptors in clonal osteoblast-like cells mediate a mitogenic response to GH.

GH receptors have not hitherto been demonstrated or characterized on osteoblasts. In this study we report the characterization of functional GH receptors on the clonal rat osteoblast-like cell line UMR 106.06. The receptors have a typical somatogenic specificity, with high affinity for human GH, 10-fold lower affinity for rat GH, and very poor affinity for rat PRL. The affinity for rat GH is 1.2 +/- 0.4 x 10(9) M-1, and there are approximately 9000 receptors per cell. GH binding increased over several hours when incubations were carried out in serum free minimal essential medium, but binding reduced rapidly when incubations were carried out in Tris-NaCl-Mg++, HEPES, or bicarbonate buffer, suggesting a critical dependence of receptor expression on nutritional factors. Rat GH stimulated proliferation of UMR 106.06 cells in a dose-dependent fashion with a maximum 43 +/- 2% stimulation above control and half-maximal effect at a final hormone concentration of 15 +/- 3 ng/ml. A proliferative response was not observed at low cell density, suggestive of a requirement for a threshold concentration of autocrine mediators or density-dependent receptor expression. A monoclonal antibody (MAb 263) which blocks GH binding to a subset (type 1) of rat hepatic GH receptors did not block binding to osteoblast GH receptors, and did not block the proliferative response. Thus, the proliferative response appears to be mediated by a class of GH receptors not blocked by MAb 263 and possibly related to the type 2 hepatic GH receptors. RNA was extracted from UMR 106.06 cells and a second osteoblast-like cell line UMR 201. Hybridization to a 32P-labeled complementary DNA probe to the rabbit hepatic growth hormone receptor revealed two major labelled bands (3.5 and 1.2 kilobases) and one minor band (2.4 kilobases) in both cell types. In summary, GH receptors are present in clonal osteoblast-like cells, and the receptors mediate a proliferative response to GH. The UMR 106.06 cells provide a valuable model system for studying the mechanism of GH action.

Animals↗
PubMed1990

Insulinlike growth factor 1 regulates mRNA levels of osteonectin and pro-alpha 1(I)-collagen in clonal preosteoblastic calvarial cells.

A nontransformed rat clonal cell line (UMR-201) with phenotypic characteristics of osteoblastic precursor cells was found to respond to insulinlike growth factor 1 (IGF-1) by increased osteonectin and pro-alpha 1(I)-collagen mRNA expression. Cells were treated for 24 h with insulin, growth hormone, or IGF-1 to study the regulation of messenger RNA for osteonectin and pro-alpha 1(I)-collagen using Northern blot hybridization. UMR-201 cells possess specific high-affinity receptors for growth hormone, although there were no significant effects of growth hormone (10(-9)-10(-7) M) or insulin (10(-9)-10(-6) M) on mRNA species for osteonectin or pro-alpha 1(I)-collagen. However, IGF-1 increased both mRNA species from a concentration of 10(-9) M. The effect on osteonectin mRNA expression was likely due to increased transcription; when 5' flanking osteonectin (ON) genomic fragments were linked to the bacterial reporter gene chloramphenicol acetyltransferase (CAT) and introduced by transfection into UMR-201 cells, the transcriptional activity of the ON-CAT construct was increased 235 and 270% by 10(-8) and 10(-7) M IGF-1, respectively. In contrast, growth hormone did not change the transcriptional activity of the ON-CAT construct. In confirmation of other work, transforming growth factor beta (TGF-beta, 0.1-2.5 ng/ml) increased mRNA for osteonectin and pro-alpha 1(I)-collagen in a dose-dependent manner. Transforming growth factor alpha (TGF-alpha) at 0.1-10 ng/ml had no consistent effects in repeated experiments on osteonectin and pro-alpha 1(I)-collagen mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Osteoblasts display receptors for and responses to leukemia-inhibitory factor.

Specific binding of leukemia-inhibitory factor (LIF) to osteoblasts, but not multinucleated osteoclasts, was demonstrated by receptor autoradiography by using cells isolated from newborn rat long bones. The clonal rat osteogenic sarcoma cells, UMR 106-06, which have several phenotypic properties of osteoblasts, expressed 300 LIF receptors per cell, with an apparent KD of 60 pM. Treatment of calvarial osteoblasts or UMR 106-01 cells with LIF resulted in a dose-dependent inhibition of plasminogen activator (PA) activity. Both calvarial osteoblasts and osteogenic sarcoma cells were shown by Western blotting and reverse fibrin autography to produce plasminogen activator inhibitor-1 (PAI-1), the production of which was increased by LIF treatment. Northern blot analysis revealed that LIF treatment resulted in a rapid (peak 1 hour), dose-dependent increase in mRNA for PAI-1. LIF treatment of the preosteoblast cell line, UMR 201, enhanced the alkaline phosphatase response of these cells to retinoic acid. Each of the osteoblast-like cell types (calvarial osteoblasts, UMR 106-06, and UMR 201) was shown to produce LIF by bioassay and, by using the polymerase chain reaction (PCR), was shown to express low levels of mRNA for LIF. These data establish that cells of the osteoblast lineage are targets for LIF action. The reported anabolic effects of this cytokine on bone formation in vivo could be related to inhibition of protease activity. LIF may be an important paracrine modulator in bone, or perhaps an autocrine one, based on the evidence for its production by osteoblasts and osteoblast-like cells.

Alkaline Phosphatase↗
PubMed1990

Humoral hypercalcemia of malignancy.

Studies on humoral hypercalcemia of malignancy have shown that tumors produce a protein that acts through the parathyroid hormone (PTH) receptor but is immunologically distinct from PTH. We have recently purified and cloned a parathyroid hormone-related protein (PTHrP) from a human lung cancer cell line. Full length cDNA clones were isolated and found to encode a prepropeptide of 36 amino acids and a mature protein of 141 amino acids. Eight of the first 13 amino terminal residues are identical with human PTH, although antisera directed at the amino terminus of PTHrP do not recognize PTH. A 34-amino acid synthetic peptide, PTHrP(1-34), was several times more potent than bovine or human PTH(1-34) in bioassays promoting the formation of cAMP and plasminogen activity in osteogenic sarcoma cells and activation of adenylate cyclase in chick kidney membranes. PTHrP(1-34) was also more potent than PTH(1-34) in stimulating cAMP and phosphate excretion and reducing calcium excretion in the isolated perfused rat kidney. PTHrP has been consistently demonstrated by immunohistochemistry in squamous cell carcinomas and in keratinocytes present in normal skin, but not in normal or hyperplastic parathyroid tissues or other tumors. PTHrP-like activity has been extracted from ovine placenta and fetal parathyroid tissue, suggesting that PTHrP may play a role in fetal calcium homeostasis.

Amino Acid Sequence↗
PubMed1989

Structure of the 5' flanking region of the gene encoding human parathyroid-hormone-related protein (PTHrP).

We have characterized a human genomic clone that contains the 5' coding and 5' flanking sequences of the human parathyroid hormone-related protein gene (PTHrP). The 5' end of the gene contains three exons separated by two small introns of 60 and 165 bp, respectively. The coding region of the PTHrP gene exhibits significant structural homology to the human parathyroid hormone gene (PTH), including the position of at least two introns. However, there is no significant nucleotide sequence homology to the PTH gene within the intragenic region nor in the flanking genomic sequences. The PTHrP gene has been localized, by chromosomal in situ hybridization to bands p11 or p12, on human chromosome 12. Analysis of the 5'-noncoding DNA reveals a complex, putative regulatory region, with multiple potential transcription start points. Nucleotide sequence analysis shows the position of one consensus TATA sequence, at -514 bp, from the start of translation whereas the other regulatory domain is located at least 1 kb further 5' to this consensus TATA sequence. Evidence from the structure of a number of cDNA clones, as well as S1 nuclease and primer extension studies supports the hypothesis that the PTHrP gene contains at least two mRNA transcription start points that define two putative regulatory domains. The result of expression from these different promoters combined with an alternative splicing event would be to produce multiple forms of PTHrP mRNA that differ in the 5'-untranslated region. This analysis of the human PTHrP gene is the first report of a PTHrP gene for any species.

Base Sequence↗
PubMed1989

Opposing influences of glucocorticoid and retinoic acid on transcriptional control in preosteoblasts.

UMR 201 is a nontransformed rat clonal cell line derived from neonatal calvaria with phenotypic characteristics of preosteoblasts. Retinoic acid strongly induces expression of alkaline phosphatase and its mRNA in these cells. Dexamethasone substantially reduced the retinoic acid-induced expression of alkaline phosphatase. This apparent interaction between dexamethasone and retinoic acid effects raised the possibility that interactions may extend to other osteoblast-related phenotypic characteristics in UMR 201 cells. Treatment with dexamethasone resulted in a decrease in the expression of mRNA for pro-alpha 1(I) collagen, but upon coincubation with 1 microM retinoic acid for 24 h, the decrease in mRNA for pro-alpha 1(I) collagen was abrogated. Dexamethasone (Dex) treatment caused a dose-dependent increase in osteonectin mRNA, half maximally effective between 1 nM and 10 nM Dex. One micromolar of retinoic acid alone led to a small increase in expression of osteonectin mRNA but prevented any further increase when Dex was added to retinoic acid-treated cells. To study transcriptional control, osteonectin genomic fragments were linked to the bacterial reporter gene, chloramphenicol acetyltransferase, and introduced by transfection into UMR 201 cells. Dexamethasone increased the transcriptional activity of an osteonectin-chloramphenicol acetyltransferase construct; 100 nM Dex resulted in a 3-fold increase over control cells which was attenuated when 1 microM retinoic acid was added to the incubation, while retinoic acid alone resulted in a 2-fold increase in transcriptional activity. Finally, it was noted that coincubation with retinoic acid and Dex stimulated the proliferation of UMR 201 cells when compared with either treatment alone. This study shows the potential importance of hormonal interactions in the expression of osteoblast function.

Alkaline Phosphatase↗
PubMed1989

Retinoic acid and tumour necrosis factor-alpha act in concert to control the level of alkaline phosphatase mRNA.

Retinoic acid has a specific role in cellular differentiation and is believed to act by regulating the transcription of specific genes. In the present work, evidence is provided to show that alkaline phosphatase (ALP) gene expression is mediated by retinoic acid in a model clonal cell line (UMR 201) derived from rat neonatal calvaria. These cells have the characteristics of relatively undifferentiated mesenchymal cells with a very low basal ALP activity which is dramatically increased by retinoic acid. Messenger RNA for ALP was clearly demonstrated when the cells were treated with 1 microM retinoic acid for 24 h. Recombinant human tumour necrosis factor-alpha (recombinant TNF-alpha) interacted with retinoic acid to potentiate the rise in ALP activity, although recombinant TNF-alpha alone had no effect. The potentiation of retinoic acid-induced ALP activity was correlated with an increased amount of mRNA for ALP with the combined treatment. By observing the rate of decay of mRNA for actin and ALP, we were able to demonstrate that the interaction between retinoic acid and recombinant TNF-alpha modulated the steady state of ALP mRNA. The mode of action of recombinant TNF-alpha may serve as a model for other paracrine regulators of cell function.

Alkaline Phosphatase↗
PubMed1989

Bone cell physiology.

Physiologic regulation of bone cell function is achieved by the combined actions of some circulating hormones and several locally produced cytokines. Interactions among these agents are important, and the regulated generation of proteolytic activities is likely to play a major role in determining net effects on bone cell function. It is likely that the most important local factors in regulating osteoclast formation and activation have yet to be chemically characterized.

Animals↗
PubMed1988

Regulation of alkaline phosphatase expression in a neonatal rat clonal calvarial cell strain by retinoic acid.

A clonal cell strain, UMR 201, was established from a culture of rat calvarial cells by the process of limiting dilution on a collagen substratum. One-day-old neonatal rat calvaria stripped of periosteum were placed on collagen in alpha-MEM with 10% fetal bovine serum (FBS). Cells that grew out from the calvaria were passaged eight times to select cells with the ability to proliferate in culture before cloning was attempted. Cells from the clonal strain were homogeneous in appearance with a doubling time in culture of about 24 hours. The UMR 201 cells formed predominantly type 1 collagen. When treated with retinoic acid (RA), all cells showed an intense staining for alkaline phosphatase (ALP). This effect of RA on the expression of ALP activity was reversible and was time and dose dependent. The earliest change was observed within 6 hours. In contrast, single and isolated clumps of untreated cells stained positively for ALP only when they were confluent. Coincubation with dactinomycin up to 3 hours after the addition of RA completely prevented the expression of ALP, whereas dactinomycin became progressively less effective when added at later times. This is interpreted as indicating a regulatory role of RA on the gene expression of ALP. Other hormones acting on bone, such as 1,25(OH)2 vitamin D3 and dexamethasone, also modulate ALP activity. The cells showed morphologic evidence of senescence after passage 12. Our preliminary studies showed that the UMR 201 cells had the characteristics of relatively undifferentiated mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1987

Hormonal influences on bone cells.

The methods for establishing osteoblast-rich rat calvarial cell cultures have been described, together with methods for the use of clonal osteogenic sarcoma cells of osteoblast phenotype. The latter clonal lines are useful for several purposes, but all the precautions and quality control measures necessary in the study of clonal lines must be observed. Some of the techniques for studying biochemical responses to hormones in these cells have also been detailed, but clearly others are applicable, including studies of the synthesis of matrix constituents. Osteoclast-like cells have not been considered in this chapter, because osteoclast culture methods have not yet been developed to the degree of purity and reproducibility necessary for this type of biochemical approach.

Adenylyl Cyclases↗
PubMed1987

Plasma levels of immunoreactive melatonin, estradiol, progesterone, follicle stimulating hormone, and beta-human chorionic gonadotropin during pregnancy and shortly after parturition in humans.

Plasma concentrations of immunoreactive melatonin, estradiol, progesterone, follicle stimulating hormone (FSH), and beta-human chorionic gonadotropin (beta hCG) were studied between 1000 and 1230 h in 105 Chinese females during six periods of normal pregnancy and 1-5 min after normal delivery. We have also examined the midday levels of immunoreactive melatonin in the cord blood of fetuses and plasma collected 1-5 min after and 24 h after delivery from their mothers. Concentrations of hormone immunoreactivities were determined by radioimmunoassay, and distinct fluctuations of all hormones were recorded during pregnancy. In the pregnant females, there were significant negative correlations between melatonin and estradiol, melatonin and progesterone, beta hCG and progesterone, and beta hCG and estradiol, and positive correlations between melatonin and FSH and progesterone and estradiol. Furthermore, plasma melatonin levels in the cord blood demonstrated no sex difference and were significantly lower than and correlated positively with the levels in their mothers. Our results suggest that sex steroids may inhibit and FSH may potentiate circulating melatonin levels in gravid women; changes in the levels of melatonin during pregnancy may affect the in utero development of the human embryo; and circulating melatonin in the mother may be the major source of blood melatonin in the fetus before parturition.

Chorionic Gonadotropin↗
PubMed1987

Insulin release from a cloned precursor beta cell line.

This paper describes the establishment in long-term tissue culture of a functional, clonal beta (B) cell line UMR 407/3 derived from neonatal rat pancreas. Immunofluorescence demonstrated specific and uniform staining for insulin. Transmission electron microscopy showed the presence of microvilli and cytoplasmic granules. The doubling time in culture was approximately 60 h in 2% (v/v) fetal calf serum with inhibition of growth at confluence. Biochemical studies demonstrated the incorporation of [3H]leucine into proinsulin and insulin, with insulin comprising 43.6% of the total radioactivity incorporated into immune complexes. When incubated at 37 degrees C for 30 min with Krebs-Ringer bicarbonate buffer (pH 7.4), the amount of insulin released on stimulation by 16.7 mmol glucose/l, 20 mmol DL-glyceraldehyde/l or 20 mmol alpha-ketoisocaproate/l was significantly higher compared with 5.6 mmol glucose/l. The mean insulin content was equivalent to 99 +/- 0.4 fmol (S.E.M.)/5 X 10(5) cells. Regulated insulin release was maintained through at least 15 passages in culture. The cells showed morphological evidence of senescence after passage 26 and this was associated with significant reduction in stimulated insulin release as well as insulin content. The ability of the cells of this clonal line to grow in soft agar suggests that it is a precursor cell line. The clonal B cell lines isolated so far may thus represent variably committed rather than fully differentiated B cells in culture. These clonal non-neoplastic cell lines will be useful models with which to study the regulation of maturation/differentiation of B cells and insulin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗