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S C Ng

Publications and source records attributed to S C Ng.

At least 19 recordsLinked to original sources
PubMed1992

Sequential 1H and 15N NMR assignments and secondary structure of a recombinant anti-digoxin antibody VL domain.

A uniformly 15N-labeled recombinant light-chain variable (VL) domain from the anti-digoxin antibody 26-10 has been investigated by heteronuclear two-dimensional (2D) and three-dimensional (3D) NMR spectroscopy. Complementary homonuclear 2D NMR studies of the unlabeled VL domain were also performed. Sequence-specific assignments for 97% of the main-chain and 70% of the side-chain proton resonances have been obtained. Patterns of nuclear Overhauser effects observed in 2D NOESY, 3D NOESY-HSQC, and 3D NOESY-TOCSY-HSQC spectra afford a detailed characterization of the VL domain secondary structure in solution. The observed secondary structure--a nine-stranded antiparallel beta-barrel--corresponds to that observed crystallographically for VL domains involved in quaternary associations. The locations of slowly exchanging amide protons have been discerned from a 2D TOCSY spectrum recorded after dissolving the protein in 2H2O. Strands B, C, E, and F are found to be particularly stable. The possible consequences of these results for domain-domain interactions are discussed.

Amino Acid Sequence↗
PubMed1992

Synthesis of 2-(p-chlorobenzyl)-3-aryl-6-methoxybenzofurans as selective ligands for antiestrogen-binding sites. Effects on cell proliferation and cholesterol synthesis.

A series of nonsteroidal compounds, 2-(p-chlorobenzyl)-3-aryl-6- methoxybenzofurans derived from the 2-(p-chlorobenzyl)-6-methoxy-3(2H)-benzofuranones has been synthesized. The key steps in the synthesis were reactions of 2-(p-chlorobenzyl)-6-methoxy-3(2H)-benzofuranones with the arylorganometallic reagents followed by dehydration of the resulting carbinols. The benzofurans are ligands for antiestrogen-binding sites (AEBS) and display no significant interaction with the estrogen receptor (ER). All bind to AEBS with equivalent or greater affinity than tamoxifen. These compounds decrease [3H]thymidine incorporation in AEBS-containing EL4 lymphoid cells and MCF7 breast cancer cells in a concentration-dependent manner between 10(-8) and 10(-6) M and are generally more inhibitory than tamoxifen. In contrast, they have no effect on [3H]thymidine incorporation by an AEBS-deficient variant of the MCF7 cell line, RTx6. The present findings of (1) selective and high affinity binding of the benzofurans to AEBS, (2) their concentration-dependent inhibition of [3H]thymidine incorporation in AEBS-containing cells, and (3) their lack of antiproliferative effect in an AEBS-deficient cell line suggest a functional role for AEBS in mediating the antigrowth effect of these compounds. Two of the more active benzofuran compounds also significantly inhibited de novo cholesterol biosynthesis in EL4 cells which lack ER. This effect could be obtained after 5 h of treatment and preceded significant loss of cell viability. This is the first demonstration that selective ligands of AEBS (other than the known nonsteroidal antiestrogens) interfere with cholesterol biosynthesis-an action that may contribute to their antigrowth effect.

Benzofurans↗
PubMed1992

Chromatographic resolution of the chiral isomers of several beta-blockers over cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase.

The optical resolution of seven beta-blockers which have in common the N-isopropyl-3-aryloxy-2-hydroxypropylamine moiety was carried out by HPLC using the cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase to quantitatively characterize the enantioselectivity of these compounds. The capacity factors and separation factors at different column temperature were determined with some qualitative trends derived. A compensation effect was observed for these compounds where there exists an approximately linear relationship between the enantiomeric differences in enthalpic and entropic energies.

Adrenergic beta-Antagonists↗
PubMed1992

In vitro decondensation of mammalian sperm and subsequent formation of pronuclei-like structures for micromanipulation.

In this study, we describe an efficient protocol for the formation of in vitro developed pronuclei for micromanipulation techniques. Our approach involved incubation of demembranated or permeabilized mammalian sperm in a phosphate buffer supplemented with heparin and beta-mercaptoethanol. Under the prevailing conditions, we achieved a uniform and reliable synchronous decondensation of sperm nuclear DNA. This initial decondensation facilitated the removal of mammalian protamines upon subsequent incubation in an amphibian egg extract. The interchange of protamines for histones to stabilize the DNA structure is recognized as a prerequisite for pronuclear formation. Furthermore, immunocytochemical studies have revealed that pronuclear development is accompanied by the formation of a nuclear lamina with corresponding DNA synthesis. The method described gave a high yield of nuclei during pronuclear formation. Ultimately, our aim is to transfer the in vitro-developed pronuclei into mammalian oocytes by micromanipulation. This novel procedure may prove useful in alleviating severe male factor problems especially in oligozoospermic cases in our in vitro fertilization center.

Animals↗
PubMed1992

Embryonic behavior of two-cell mouse embryos frozen by the one- and two-step ultrarapid techniques.

PURPOSE: A modified two-step ultrarapid freezing technique was compared to the one-step ultrarapid freezing technique. Two-cell mouse embryos were frozen-thawed using the two freezing protocols, and postthaw cryoprotectant removal was carried out in either a single- or a multiple-step procedure. RESULTS: Statistically similar cryosurvival (96.95-100%) and blastocyst formation rates (87.95-91.47%) were obtained with both freezing groups. In addition, the method of cryoprotectant removal did not have any significant effect on the survival and development of the frozen-thawed embryos in both groups. Blastocysts formed following single-step cryoprotectant removal had significantly lower inner cell mass counts in the one-step than in the two-step group (26.14 and 27.59, respectively; P < 0.05). Embryo transfer studies showed that the implantation and fetal formation rates of embryos frozen by the two-step technique (61.67 and 60.0%, respectively) were similar to those of embryos frozen by the one-step technique (74.12 and 71.76%, respectively). CONCLUSION: These results demonstrate that the ultrarapid two-step technique is as effective in cryopreserving two-cell mouse embryos as the ultrarapid one-step technique.

Animals↗
PubMed1992

Production of stable anti-digoxin Fv in Escherichia coli.

We have created a bacterial expression-export system and have used it to express (14 mg l-1) the variable region fragment (Fv) of an anti-digoxin antibody (26-10) in Escherichia coli. The expression-export plasmid contains a T7 promoter and the E. coli signal sequences ompA [Movva et al., J. biol. Chem. 255, 27-29 (1980)] and phoA [Inouye et al., J. Bacteriol. 149, 434-439 (1982)] fused to heavy chain (VH) and light chain (VL) variable region sequences to generate an artificial cistron. The 26-10 Fv protein made using this system was soluble, unlike many other expression systems which produce insoluble proteins in the form of inclusion bodies. The 26-10 VH and VL proteins were cleaved at their mature N-termini and exported into the bacterial periplasm where they could be easily extracted and affinity purified on ouabain-Sepharose. 26-10 Fv bound to digoxin with similar affinity and specificity as the whole 26-10 antibody (Ka for Fv, 1.3 x 10(9) M-1, Ka for IgG, 7 x 10(9) M-1). 26-10 Fv appears to be remarkably stable in comparison with other Fv fragments. The half-life for chain dissociation of 26-10 Fv was 48 hr compared to the reported 1.5 hr half-life of McPC603 Fv. We present the proton NMR spectra of the 26-10 Fv as preliminary evidence that this expression-export system can be used to facilitate the analysis of the solution structure of 26-10 Fv by NMR.

Amino Acid Sequence↗
PubMed1992

Fertilization, cleavage, and cytogenetics of 48-hour zona-intact and zona-free human unfertilized oocytes reinseminated with donor sperm.

OBJECTIVE: To examine the fertilization rates of 48-hour unfertilized oocytes inseminated with fertile donor sperm and to evaluate the cleavage and cytogenetics of ensuing embryos. DESIGN: Prospective. SETTING: Assisted reproductive technology (ART) program. PATIENTS: Four hundred ninety-seven unfertilized oocytes from 97 ART patients were categorized into four groups. A (zona-intact) and B (zona-free) were from patients with partial fertilization failure, whereas C (zona-intact) and D (zona-free) were total fertilization failures. RESULTS: Fertilization rates in groups A and B were significantly higher than C and D (33.2% to 60.9% versus 20.0% to 48.1%; P less than 0.01). Zona-free oocytes had higher fertilization rates than zona-intact oocytes (48.1% to 60.9% versus 20.0% to 32.2%). Multiple pronuclei were high in zona-free oocytes (33.1% to 41.3%). Forty-eight to 54% of embryos generated after donor insemination had chromosome anomalies (mosaicism, aneuploidy, pulverization). CONCLUSIONS: One cause of total fertilization failure appears to lie in intrinsic oocyte problems confined to the zona and oolemma. The fertilization of 48-hour unfertilized oocytes may be of some value in diagnosing fertilization failure in ART patients.

Cell Division↗
PubMed1992

Improved pregnancy rate after transfer of embryos grown in human fallopian tubal cell coculture.

OBJECTIVE: To evaluate the embryonic behavior in vitro and the pregnancy and implantation rates of embryos grown in a human ampullary cell coculture system. DESIGN: In a prospective study, two pronuclei embryos were cultured on human ampullary feeder layers up to the two to six-cell and blastocyst stages and replaced either as tubal, uterine, or sequential transfers. SETTING: Assisted reproductive technology program in a university-based hospital. PATIENTS: Fifty women with a mean age of 35.6 years who went through a single coculture cycle. Thirty of the patients were admitted for in vitro fertilization (IVF) and 20 for tubal embryo transfer (TET). RESULTS: The overall clinical pregnancy rate (PR) for all 50 patients was 44% per cycle (IVF, 37%; TET, 55%) and the implantation rate was 31.8% (IVF, 31.0%; TET, 32.6%). Sixty-eight percent of pregnant patients were over 35 years, and 68% had two previously failed assisted reproduction cycles. Five of 9 patients who received sequential transfers became pregnant. Three of the 22 pregnancies aborted (2 after sequential transfer), and there was one ectopic. Overall, 88% of two to six-cell stage embryos were of good quality. CONCLUSIONS: The human ampullary coculture system produces better quality embryos, increased numbers of blastocysts with improved PRs and implantation rates. The beneficial effects of the feeder layer may be through the release of embryotrophic factors and detoxification of the medium by the cells. Coculture is a new concept in assisted reproduction and has tremendous potential in boosting conception rates by mimicking the in vivo environment.

Adult↗
PubMed1992

The evaluation of various culture media in combination with dimethylsulfoxide for ultrarapid freezing of murine embryos.

Bicarbonate-buffered HTF medium, Medicult, and T6 are as effective as PB1 medium when used in combination with DMSO in ultrarapid freezing of two-cell mouse embryos. However, the use of phosphate-buffered T6 results in reduced in vitro development and inner cell mass size as compared with bicarbonate- and Hepes-buffered T6 when used with 3.5 M of DMSO. Hence, the use of this media for ultrarapid freezing should be avoided when this concentration of DMSO is used.

Animals↗
PubMed1992

Outcome of 143 pregnancies conceived by assisted reproductive techniques.

One hundred and forty-three pregnancies conceived by assisted reproductive techniques (ART) from October 1985 to June 1989 in the National University Hospital, Singapore, resulted in 66 deliveries and 89 babies. There were 27 (18.7%) biochemical pregnancies, 44 (30.7%) clinical miscarriages and 6 (4.2%) ectopic pregnancies when total pregnancies were considered. Of those who continued pregnancy to second trimester multiple births occurred in 20 (13.3%) patients. A high incidence of vaginal bleeding and hypertension in pregnancy was noted in 32 (48.5%) patients and 18 (27.3%) patients, respectively. Caesarean section was the method of delivery in 48.5% of patients. Twenty-six (29.2%) babies required admission to the neonatal intensive care unit. All babies except one set of twins delivered at 24 weeks of amenorrhoea survived. Fetal abnormality was noted in 2 cases.

Adult↗
PubMed1992

Micro-insemination sperm transfer (MIST) and its application to male subfertility: current strategies to improve results.

It can be difficult to achieve a pregnancy for patients with severe male factor subfertility. Hence, micro-manipulation techniques, have been applied to this problem. Direct deposition of sperm into the oocyte under the zona (micro-insemination sperm transfer, MIST) has improved the fertilisation rates, but pregnancy rates have been very low. This paper describes the possible new strategies to improve the technique currently. They include methods to improve sperm recovery, to improve acrosome reaction, to improve the quality of embryos by using co-cultures and to wait for good cleavage before transfer, and to improve luteal phase support. There are also many new techniques being developed which may contribute to further pregnancy successes. They include the use of electro-fusion, laser-fusion and the Xenopus cell-free system.

Female↗
PubMed1992

Human ampullary co-cultures for blastocyst transfer in assisted reproduction.

Although the assisted reproductive techniques (ART) have contributed significantly over the last decade in alleviating subfertility in the childless couple, the implantation and take-home baby rates have been stubbornly low. A major cause for such low success rates has been the reduced viability of replaced embryos perhaps induced by the suboptimal in vitro conditions used in ART laboratories. One approach to improving embryo viability is to provide the growing embryo with a simulated in vivo environment by replicating the conditions existing in the human fallopian tube in vitro. This requires either the maintenance of an intact fallopian tube in vitro or establishment and maintenance of tubal epithelial cell-lines which could be used as feeder layers for early embryonic growth. The concomitant growth of cells with embryos in vitro has been referred to as co-culture. This paper discusses the in vitro behaviour of human tubal epithelial cells, the fertilisation and growth of embryos in ampullary co-culture, the specificity of co-cultures, the mechanism of action of co-cultures and the methods of screening the human ampullary co-culture system for microbes. The pregnancy and implantation results on 50 patients enrolled for a co-culture clinical trial are presented and the future use of this system discussed.

Cells, Cultured↗
PubMed1992

The first oocyte donation pregnancy home-bred and delivered in Singapore to illustrate a simplified oocyte donation programme--no more embryo freezing, synchronisation or disruption of social life.

The wait for an oocyte donor is highly unpredictable and stressful. The recipients may have to remain contactable at all times. Even when a donor is present, their reproductive cycles may not be in synchrony to enable gamete or embryo replacement, and embryo freezing is required for the transfer of embryos in the subsequent cycle. After the release of the Guidelines for Assisted Reproductive Technique by the Ministry of Health we have established a simplified oocyte donation programme with which the need for synchronisation, embryo freezing and the disruption to the recipient couple's social life are eliminated. To illustrate, we report the first report of oocyte donation pregnancy home bred and delivered in Singapore.

Adult↗