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Biomedical subjects

A J Chen

Publications and source records attributed to A J Chen.

At least 19 recordsLinked to original sources
PubMed2005

Progesterone primes zona pellucida-induced activation of phospholipase A2 during acrosomal exocytosis in guinea pig spermatozoa.

We investigated, using guinea-pig spermatozoa as a model, whether phospholipase A2 (PLA2) is involved in progesterone or zona pellucida (ZP)-stimulated acrosomal exocytosis, if progesterone enhances ZP-induced activation of PLA2, and mechanisms underlying PLA2 regulation. Spermatozoa were capacitated and labeled in low Ca2+ medium with [14C]choline chloride or [14C]arachidonic acid, washed, and then exposed to millimolar Ca2+ and progesterone and/or ZP. Each agonist stimulated decrease of phosphatidylcholine (PC) and release of arachidonic acid and lysoPC, indicative of PLA2 activation. Aristolochic acid (a PLA2 inhibitor) abrogated lipid changes and exocytosis, indicating that these lipid changes are essential for exocytosis. Exposure of spermatozoa to submaximal concentrations of both progesterone and ZP resulted in a synergistic increase of arachidonic acid and lysoPC releases, and exocytosis, suggesting that, under natural conditions, both agonists interact to bring about acrosomal exocytosis. Progesterone-induced PLA2 activation appears to be mediated by a GABA(A)-like receptor, because bicuculline (a GABA(A) receptor antagonist) blocked arachidonic acid release and exocytosis. In agreement with this, GABA mimicked progesterone actions. ZP-induced activation of PLA2 seemed to be transduced via G(i) proteins because pertussis toxin blocked arachidonic acid release and acrosomal exocytosis. PLA2 may be regulated by PKC because progesterone- or ZP-induced release of arachidonic acid was blocked by the PKC inhibitors staurosporine or chelerythrine chloride. PLA2 could also be regulated by the cAMP-PKA pathway; inclusion of the PKA inhibitor 14-22 amide or H-89 led to a reduction in arachidonic acid release or exocytosis after progesterone or ZP. Taken together, these results suggest that PLA2 plays an essential role in progesterone or ZP-stimulated exocytosis with progesterone priming ZP action.

Acrosome↗
PubMed2001

[Cloning of VEGF receptor KDR and its expression in insect cells].

The cDNA fragment of the first 3 loops of VEGF receptor, KDR, was cloned by PCR and inserted into a baculovirus expression plasmid pFASTBACI. The competent E. coli DH10BAC cell, which contain another plasmid with baculovirus genome in it, was transformed with pFASTBACI-KDRn3. Homologous recombination in the prokaryotic cells resulted in a recombinant plasmid containing KDRn3 in baculovirus genome. Transfection of the insect cell SF-9 with above plasmid generated a recombinant baculorvirus contain target gene fragment. SDS-PAGE and Western blot analysis of the supernatant of the infected SF-9 cell showed that KDRn3 was secreted in the medium. The recombinant protein was verified with Western blot and tested for their binding activity with VEGF. Its anti-angiogenic activity was assayed on chorionic allantoic membrane(CAM) of fertilized egg. The results showed that the recombinant protein could inhibit new vessel formation on CAM of fertilized eggs.

Angiogenesis Inhibitors↗
PubMed2001

Expression of human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop cDNA in Pichia pastoris, purification of the expressed product and detection of its biological activity.

OBJECTIVE: To express human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop cDNA in Pichia. pastroris, and to purify the expressed product and detect its biological activity. METHODS: By inserting human Flt-1 (1-3 loop) cDNA coding 316 amino acid residues into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and the sequences of alpha secreting signal peptides, a recombinant expression plasmid pPIC9K/Flt-1 (1-3) was constructed and transformed to yeast host strain GS115, then His+ Muts phenotype transformant was screened out and cultured in flasks, and Flt-1 (1-3) was expressed under the induction of 1% methanol. RESULTS: SDS-PAGE showed that after being induced with 1% methanol for 4d, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed. Western blot showed good antigenicity and specificity of expressed product. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, the purity of the expressed product reached above 90%. Biological assay proved that the expressed product could bind to hVEGF165 and inhibit the proliferation of HUVEC stimulated by hVEGF165. CONCLUSION: Human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop was successfully expressed. The study lays a foundation for further application of the expressed product in the treatment of vasoformation related diseases, such as tumor and diabetic retinopathy.

Base Sequence↗
PubMed2001

Expression of human vascular endothelial growth factor (VEGF165) in Pichia pastoris and its biological activity.

OBJECTIVE: To express human vascular endothelial growth factor (hVEGF165) cDNA in Pichia pastroris, purify the expressed product and detect the biological activity of it. METHODS: By inserting hVEGF165 cDNA coding 165 amino acid residues into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and the sequences of alpha secreting signal peptides, a recombinant expression plasmid pPIC9K/hVEGF165 was constructed and transformed to yeast host strain KM71, then multiple-copy insert transformants were screened out and cultured in flasks, and hVEGF165 was expressed under the induction of 1% methanol. RESULTS: SDS-PAGE showed that after being induced with 1% methanol for 4 d, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed. Western blot showed good antigenicity and specificity of expressed product. After being purified by Heparin-Sepharose CL6B affinity chromatography, the purity of expressed product reached above 90%. Biological assays proved that the expressed product could stimulate the proliferation of HUVEC. CONCLUSION: hVEGF165 was successfully expressed. The study opened up a wide prospect for the application of VEGF165 in the prevention and treatment of ischemic heart disease and other tissue ischemic diseases such as secondary arterial occlusion in limbs.

Base Sequence↗
PubMed2000

Acupuncture modulates the limbic system and subcortical gray structures of the human brain: evidence from fMRI studies in normal subjects.

Acupuncture, an ancient therapeutic technique, is emerging as an important modality of complementary medicine in the United States. The use and efficacy of acupuncture treatment are not yet widely accepted in Western scientific and medical communities. Demonstration of regionally specific, quantifiable acupuncture effects on relevant structures of the human brain would facilitate acceptance and integration of this therapeutic modality into the practice of modern medicine. Research with animal models of acupuncture indicates that many of the beneficial effects may be mediated at the subcortical level in the brain. We used functional magnetic resonance imaging (fMRI) to investigate the effects of acupuncture in normal subjects and to provide a foundation for future studies on mechanisms of acupuncture action in therapeutic interventions. Acupuncture needle manipulation was performed at Large Intestine 4 (LI 4, Hegu) on the hand in 13 subjects [Stux, 1997]. Needle manipulation on either hand produced prominent decreases of fMRI signals in the nucleus accumbens, amygdala, hippocampus, parahippocampus, hypothalamus, ventral tegmental area, anterior cingulate gyrus (BA 24), caudate, putamen, temporal pole, and insula in all 11 subjects who experienced acupuncture sensation. In marked contrast, signal increases were observed primarily in the somatosensory cortex. The two subjects who experienced pain instead of acupuncture sensation exhibited signal increases instead of decreases in the anterior cingulate gyrus (BA 24), caudate, putamen, anterior thalamus, and posterior insula. Superficial tactile stimulation to the same area elicited signal increases in the somatosensory cortex as expected, but no signal decreases in the deep structures. These preliminary results suggest that acupuncture needle manipulation modulates the activity of the limbic system and subcortical structures. We hypothesize that modulation of subcortical structures may be an important mechanism by which acupuncture exerts its complex multisystem effects.

Acupuncture Therapy↗
PubMed2000

Functions and functional domains of the GTPase Cdc42p.

Cdc42p, a Rho family GTPase of the Ras superfamily, is a key regulator of cell polarity and morphogenesis in eukaryotes. Using 37 site-directed cdc42 mutants, we explored the functions and interactions of Cdc42p in the budding yeast Saccharomyces cerevisiae. Cytological and genetic analyses of these cdc42 mutants revealed novel and diverse phenotypes, showing that Cdc42p possesses at least two distinct essential functions and acts as a nodal point of cell polarity regulation in vivo. In addition, mapping the functional data for each cdc42 mutation onto a structural model of the protein revealed as functionally important a surface of Cdc42p that is distinct from the canonical protein-interacting domains (switch I, switch II, and the C terminus) identified previously in members of the Ras superfamily. This region overlaps with a region (alpha5-helix) recently predicted by structural models to be a specificity determinant for Cdc42p-protein interactions.

Amino Acid Sequence↗
PubMed2000

[Expression and characterization of human vascular endothelial growth factor receptor Flt-1 extracellular domain in Pichia pastoris].

By inserting VEGF receptor Flt-1(1-3 loop) cDNA into Pichia pastoris expression vector pPIC9K containing AOX1 promotor and the sequences of alpha secreting signal peptides, the expression plasmid pPIC9K/Flt-1(1-3) was constructed and transformed into GS115. The multi-copy insert transformants were selected and cultivated in flasks. After 4 days of 1% methanol induction, the expressed Flt-1(1-3) accumulated up to 30% of total proteins in supernatant. The expressed Flt-1(1-3) was further proved with good antigenicity and high specificity by ELISA and Western blot. They can bind to VEGF and inhibit HUVEC proliferation stimulated by VEGF.

Humans↗
PubMed1991

Cigarette smoking among school children in Singapore. Part III--Knowledge and attitudes towards smoking.

In the third article in this series which mainly assesses the knowledge of school children on the harmful effects of smoking, findings showed that the majority (90%) of school children surveyed were aware of the harmful effects. Current smokers appeared to be the least knowledgeable (69.4%) whilst non-smokers (89.3%) and experimenters (93.7%) were the most knowledgeable. Lung cancer (49%), smoking is bad for health (25%), smoking is bad for passive smokers (7%) and for heart disease (6%) were the most common responses given by the children on the specific ill effects caused by smoking. Smokers and non-smokers alike agreed that smoking is addictive, the proportions agreeing, once again, being lowest among smokers and highest among non-smokers. This trend was also observed when respondents were asked for their attitudes towards smoking--whether cigarette smoking was harmful and whether smoking made a person popular. The majority of respondents felt that a boy or girl who smoked "looked bad"--39% thought so of boys, while a higher proportion (55%), had the same impression of girls. The main reasons for giving up smoking given by ex-smokers were its harmful effects on health (49%), there being no more "kick" to it (16%), peer pressure (13%) and the disapproval of parents and teachers (13%). Among current smokers, at least half stated that they would like to stop smoking. Most of them had tried to kick the habit before but failed in their attempts. Finally, among the non-smokers, only 0.4% of boys and 0.1% of girls indicated that they might smoke in the future.

Adolescent↗
PubMed1991

Cigarette smoking among school children in Singapore. Part II--Development of the smoking habit.

The second in this series on smoking among school children in Singapore aims to define the characteristics of young smokers. Results show that of respondents who had ever smoked, the median age of experimentation with smoking was 12 years. This was seen among boys as well as girls. The median age at which current smokers went on to smoke regularly was 14 years, approximately two years after trying their first cigarette. Friends and parents were the main sources of the first cigarette for all categories of smokers, whether they be ex smokers, experimenters or current smokers. Among the reasons as to why current smokers smoked, the leading two were "to relax" (46.4%) and "out of sheer curiosity" (22.3%). Boys tend more to smoke with friends of the same sex (40.6%) whilst girls preferred mixed company (45.5%) when they smoked. Fathers played a major role in the development and continuation of smoking among those who had ever smoked before--52.0% of boys and 51.6% of girls currently smoking reported that their fathers smoked as well.

Adolescent↗
PubMed1990

Hemostatic changes in women using a monthly injectable contraceptive for one year.

The effect of consecutively injecting a one-a-month contraceptive (norethisterone enantate 50 mg with estradiol valerate 5 mg) for one year on haematological parameters was evaluated in 42 Chinese women. The healthy volunteers were randomly allocated to either the treatment group (22) or a control group (20). Blood samples were collected in the follicular and luteal phases of a pretreatment cycle, on days 28 +/- 3 after the 1st, 3rd, 6th, 12th injections and in the luteal phase of the post-treatment cycle. The results showed that in both groups, prothrombin time and fibrinogen fluctuated significantly, and leucocyte count was not significantly changed during the whole course. Factor VIII-related antigen and antithrombin III (AT-III) antigen showed minor changes, although in the 3rd treatment cycle, the differences between the two groups in both parameters reached statistical significance. A progressive and significant decrease in Factor X and AT-III functional activity occurred with the monthly injectable treatment, decreasing by about 14% and 20%, respectively, after 12 months of treatment. Haemoglobin levels were increased in the treatment group after the 3rd injection and remained at the higher level during the study period. It is doubtful whether these changes are likely to be of clinical relevance.

Adult↗
PubMed1990

Cigarette smoking among school children in Singapore. Part I--Smoking prevalence.

A cross sectional nationally representative survey of 33,110 school-going children in Singapore aged between 9 and 20 years was carried out to obtain, for the first time, baseline information on smoking among the school-going population in Singapore. The survey was carried out among students attending vocational institutes and public sector schools in Singapore in 1987. The overall smoking prevalence was found to be 2% (3% among boys and 0.2% among girls). Ex-smokers comprised 2% of respondents whilst those who had only experimented with smoking made up 9% of the respondents. Analyses of the data showed that cigarette smoking was more prevalent among Malays, among boys, among older children, and among the less academically inclined. On average, boys smoked a median of 20 cigarettes a week and girls, 12 cigarettes a week. On an international basis, the smoking prevalence among the school-going population in Singapore is significantly lower than that of developed countries like Australia, England and Wales and neighbouring countries such as Malaysia.

Adolescent↗
PubMed1989

The hepatitis B immunization programme in Singapore.

A voluntary immunization programme to prevent perinatal transmission of hepatitis B virus (HBV) infection in Singapore was implemented on 1 October 1985 as an integral component of the national childhood immunization programme. Up to April 1988, a total of 68,845 mothers who attended government maternal and child health clinics were screened for the disease. Of these, 2432 (3.5%) were positive for hepatitis B surface antigen (HBsAg) and 904 (1.3%) for hepatitis B e antigen (HBeAg). Virtually all the babies born to carrier mothers completed the full immunization schedule; and in addition, those of HBeAg-positive mothers were given a dose of hepatitis B immunoglobulin at birth. The hepatitis B immunization programme was extended on 1 September 1987 to cover all newborns. About 90% of the 15,943 babies delivered in government institutions from September 1987 to April 1988 were immunized at birth, with the subsequent doses being administered at maternal and child health clinics at 4-6 weeks and 5 months later. More than 85% of the children given the full course of plasma-derived and yeast-derived hepatitis B vaccine from birth continued to have protective antibody to HBV two years after immunization. The programme is being closely monitored to assess the duration of immunity and the need for booster doses, while seronegative adults are also being encouraged to be vaccinated.

Adolescent↗
PubMed1987

Carrier-free zone electrophoresis, displacement electrophoresis and isoelectric focusing in a high-performance electrophoresis apparatus.

A characteristic feature of high-performance electrophoresis (HPE), the electrophoretic counterpart of high-performance liquid chromatography (HPLC), is that the separation chamber is a thin-walled, narrow-bore (0.05-0.3 mm) glass or fused-silica capillary tube for rapid dissipation of the Joule heat in order to minimize thermal zone deformation even at high field strengths. This paper is centered around the usefulness of HPE for separation in a carrier-free medium (i.e., in buffer alone) and deals with both zone electrophoresis, isoelectric focusing and displacement electrophoresis. Examples are given of analytical and micropreparative separations of inorganic and organic ions, proteins, viruses and bacteria. The run times are 5-30 min. Discontinuous buffer systems have up to now been used exclusively for the separation of proteins by electrophoresis in polyacrylamide gels ("disc electrophoresis"). However, the Ornstein and Davis discontinuous buffer system has been modified to adapt it to carrier-free zone electrophoresis in order to achieve automatic sharpening of the starting zone. Very high resolution of serum proteins was obtained when they were subjected to free high-performance disc electrophoresis in such a modified buffer system. To show that the HPE apparatus permits electrophoresis also in a gel medium, a polyacrylamide electrophoresis in SDS is presented. This experiment illustrates the difference between electropherograms obtained in free solution and in a molecular-sieving medium. Detection can be performed both on- and off-tube. The latter technique permits the rapid identification of the solutes by photodiode array spectrophotometry and the collection of fractions for further studies. The former detection method is simpler but mainly useful for analytical purposes. Non-UV-absorbing ions can be monitored with the aid of an on-tube UV detector if the run is performed in a UV-absorbing buffer.

Blood Proteins↗
PubMed1987

Performance evaluation of non-porous versus porous ion-exchange packings in the separation of proteins by high-performance liquid chromatography.

The performance of a non-porous, anion-exchange packing was evaluated and compared with a number of similar porous high-performance liquid chromatography packings. The non-porous columns were found to be equally efficient for proteins spanning a wide range of molecular weights, while the porous columns exhibited decreasing efficiency as the proteins became larger. The porous materials also exhibited size exclusion effects that were not seen with the non-porous materials, which partially accounts for the loss of efficiency with large proteins. When increasingly steep gradients were employed, the loss of resolution was less with the non-porous materials.

Chromatography, Gel↗
PubMed1987

Noncardioplegic myocardial preservation.

Despite the widespread use of K + cardioplegia, the optimal method of intraoperative myocardial preservation remains controversial. Since 1975, in more than 3,500 patients who have undergone myocardial revascularization at our institutions, intraoperative protection has been provided by a technique of cold-blood perfusion without K + cardioplegia. This report describes our technique, analyzes quantitative ultrastructural changes in four patients before and after bypass to assess cellular damage, and details the clinical and angiographic findings in a subgroup of 157 patients followed postoperatively for up to 6 years. In 100 patients in whom multiple vessels were bypassed, vessels were patent on restudy, thereby excluding technical failure as a cause of impaired left ventricular function. Postoperatively, global left ventricular function was unchanged in 80% and improved in 19%, as compared with preoperative function. Left ventricular function deteriorated in only one patient. We conclude that intraoperative oxygenation by cold-blood perfusion is a successful method of myocardial preservation.

Journal Article↗