Biomedical subjects

L Meng

Publications and source records attributed to L Meng.

At least 55 records · Page 3Linked to original sources

PubMed1999

In vitro maturation of human preovulatory oocytes reconstructed by germinal vesicle transfer.

OBJECTIVE: To describe a micromanipulation-electrofusion procedure for transferring germinal vesicles (GVs) between immature human oocytes. DESIGN: Pilot study to assess oocyte maturation after an invasive micromanipulation procedure. SETTING: Research laboratory at a university medical center. PATIENT(S): Immature oocytes were discarded from intracytoplasmic sperm injection (ICSI)-IVF cycles of patients 23-48 years of age. INTERVENTION(S): Initially, GV removal and transfer were performed on the same oocyte; these "self-reconstructed" oocytes were then cultured in vitro for up to 50 hours and examined periodically for maturation as judged by the extrusion of the first polar body. In a second study, GVs from oocytes of "old" patients (>38 years old) were successfully transferred into enucleated immature oocytes of "young" patients (<31 years old). MAIN OUTCOME MEASURE(S): Extrusion of the first polar body was monitored in "reconstructed" and control oocytes; karyotypes also were analyzed at meiosis II. RESULT(S): From 48 oocytes from old patients, 12 GVs were successfully removed, transferred, and fused into previously enucleated oocytes from young patients. After in vitro culture, 7 of these "reconstructed" oocytes matured to meiosis II, a maturation rate not significantly different from that observed in nonmanipulated controls. A normal, second meiotic metaphase chromosome complement was observed in 4 of 5 reconstructed oocytes. CONCLUSION(S): Normal meiosis can occur after the transfer of a GV into an enucleated host oocyte. Germinal vesicle transfer may be a valuable research procedure that generates cell models to characterize the cytoplasmic-nuclear interplay for cell cycle regulation, maturation, and fertilization in the human oocyte; it also may be a potentially attractive alternative to oocyte donation.

Adult↗

PubMed1999

Nuclear transfer in the rhesus monkey: practical and basic implications.

In early 1997, the birth of a lamb after transfer of the nucleus from an adult mammary gland cell into an enucleated oocyte, along with the production of rhesus monkeys by nuclear transfer of embryonic cells, marked a reemergence of the field of mammalian cloning. Clonally derived rhesus monkeys would be invaluable in biomedical research, and the commercial interests in transgenic sheep and cattle propagated by cloning are substantial. Nuclear transfer technology is under consideration in human in vitro fertilization clinics to overcome infertility secondary to advanced maternal age or mitochondrial-based genetic disease. Nuclear transfer involves preparing a cytoplast as a recipient cell, in most cases a mature metaphase II oocyte from which the chromosomes have been removed. A donor nucleus cell is then placed between the zona and the cytoplast, and fusion, as well as cytoplast activation, is initiated by electrical stimulation. Successful reprogramming of the donor cell nucleus by the cytoplast is critical--a step that may be influenced by cell cycle stage. Embryos produced by nuclear transfer are cultured in vitro for several cell divisions before cryopreservation or transfer to the oviduct or uterus of a host mother. The efficiency of producing live young by nuclear transfer in domestic species is low, with a high frequency of developmental abnormalities in both preterm and term animals. However, a number of pregnancies have now been established using fetal cells as the source of donor nuclei. The use of cell lines not only allows large clone sizes but also supports the ability to genetically manipulate cells in vitro before nuclear transfer. Ongoing research focused on the production of clonally derived rhesus monkeys using fetal fibroblasts and embryonic stem cells as the source of donor nuclei will be reviewed.

Animals↗

PubMed1999

A pilot study of mammographic density patterns among Japanese women.

Mammographic density patterns, which refer to the distribution of fat, connective, and epithelial tissue in the healthy female breast, have been shown to be related to breast cancer risk. We used a quantitative method to assess mammographic densities in 41 mammograms from women in Japan without a diagnosis of breast cancer. Information about reproductive behavior and family history for breast cancer was available from a questionnaire. The statistical analysis applied Spearman correlation coefficients and multiple linear regression. The breast size as measured on the cranio-caudal view of the mammogram was approximately 12% larger, the size of the dense areas was 20% smaller, and the mean percent mammographic densities were 30% greater among premenopausal than among postmenopausal women. We found a strong relation between age at menarche and mammographic densities in premenopausal women and significant associations for age, family history of breast cancer, and age at menopause with mammographic densities in postmenopausal women. These preliminary data will be used to plan a future study that will compare mammographic density patterns and the relative importance of dietary, reproductive, and anthropometric factors between women in Japan and in the United States.

Adult↗

PubMed1999

Frequency and pattern of p53 gene mutation in a cohort of Spanish women with node-negative breast cancer.

Ethnic, racial and regional differences in the frequency and pattern of p53 gene mutations have been well documented. Some of these differences have been shown to have an impact on the survival of patients with breast cancer. In this study we explored the frequency and pattern of p53 abnormality in a cohort of Spanish women with node-negative breast cancer using PCR, subcloning and DNA sequencing of archival tumors. One hundred and seventy-eight cases of breast cancer diagnosed between 1981 and 1986 at the University of Oviedo Hospital in Oviedo, Spain were subjects of this study. Sequence analysis of exons 5 through 8 of p53 was performed on subcloned PCR-amplified DNA, extracted from formalin-fixed, paraffin-embedded tumors. Appropriate positive, negative, PCR, and polymerase controls were utilized and evaluated. Duplicate samples of the genomic DNA were re-evaluated on all cases showing more than one mutation. One hundred and five out of 178 breast cases (59%) carried one or more p53 gene mutations. Mutations were distributed randomly from codon 128 to 305. There were 123 (88%) transition, 10 (7%) transversion, 5 (3.5%) splice junction mutations, and 2 (1.5%) deletions. Eighty-three cases (61.5%) had missense mutation, 45 (33.5%) silent, 5 (3.5%) nonsense and 2 (1.5%) frameshifts. Eighty (75%) of 120 transitions were G:C to A:T, 11 (25%) of which occurred at CpG sites. Sixteen mutations were in novel codons not reported in breast cancers previously. Codons with the highest frequency of mutations in this group were 278, 273, 213 and 227. We also detected 27 tumors with more than one mutation within a single exon or in different exons in the same patient. These findings suggest that the frequency and pattern of p53 mutations in this group of Spanish women with breast cancer is different than those reported in the United States and Northern Europe.

Breast Neoplasms↗

PubMed1999

The expression of p53 tumor suppressor gene in breast cancer cells is down-regulated by cytokine oncostatin M.

Previously (J. Liu, et al., Cell Growth Differ., 8: 667-676, 1997), we showed that oncostatin M (OM), a cytokine produced by activated T cells and macrophages, inhibited the proliferation of breast cancer cells derived from solid tumors and malignant effusions. OM-treated cells showed reduced growth rates and differentiated phenotypes. Because the p53 tumor suppressor protein plays an important role in cellular proliferation, we examined p53 protein expression in three OM-responsive breast cancer cell lines, MCF-7, MDA-MB231, and H3922. Western blot analysis showed that p53 protein levels in all three of the cell lines were decreased by OM treatment. Reduction of p53 protein was detected after 1 day of OM treatment and reached maximal suppression of 10-20% of control after 3 days in H3922 and 40% of control after 4 days in MCF-7 cells. A comparison of p53 mRNA in OM-treated cells versus untreated control cells showed that exposure to OM reduced the steady-state levels of p53 mRNA transcripts to an extent similar to that of the p53 protein levels. This observation suggests that the effect of OM on p53 protein expression does not occur at the posttranslational level. Nuclear run-on assays verified that OM decreased the number of actively transcribed p53 mRNAs, which suggests a transcriptional regulatory mechanism. The effect of OM on p53 expression seems to be mediated through the extracellular signal-regulated kinase (ERK) pathway, inasmuch as the inhibition of ERK activation with a specific inhibitor (PD98059) to the ERK upstream kinase mitogen/extracellular-regulated protein kinase kinase abrogated the OM inhibitory activity on p53 expression in a dose-dependent manner. In addition to OM, we showed that the p53 protein expression in MCF-7 cells was also decreased by phorbol 12-myristate 13-acetate treatment (PMA). Because both OM and PMA induce MCF-7 cells to differentiate, our data suggest that p53 expression in breast cancer cells is down-regulated during the differentiation process.

Blotting, Western↗

PubMed1999

Exploring the feasibility and effects of a high-fruit and -vegetable diet in healthy women.

Based on reports that fruits and vegetables may protect against breast cancer, this randomized intervention study tested the feasibility of increasing fruit and vegetable intake among healthy women to 9 daily servings through individual dietary counseling and group activities. Adherence to the dietary recommendations was monitored by 24-h food recalls, log sheets, and plasma carotenoid assessments. To explore possible cancer protective mechanisms of fruits and vegetables, we investigated the treatment effect on plasma phenol levels and on thiobarbituric acid-reactive substances measured as malondialdehyde equivalents, a possible marker of oxidative damage. At baseline, women in the intervention (n = 13) and control (n = 16) group reported an average daily consumption of 3.3 and 3.2 fruit and vegetable servings, respectively. After 3 and 6 months of intervention, intake in the intervention group had increased to 8.3 and 7.4 servings, whereas the control group reported an average of 4.2 and 4.1 daily servings. An increase of plasma carotenoid levels from 1249 microg/liter at baseline to 1854 and 1827 microg/liter after 3 and 6 months confirmed compliance with the dietary recommendations in the intervention group. Plasma carotenoid levels among controls changed slightly from 1165 to 1231 and 1291 microg/liter Whereas total phenol levels did not respond according to our hypothesis, malondialdehyde levels decreased slightly in the intervention group. These results suggest that motivated women can substantially increase their fruit and vegetable intake, which leads to a notable increase in plasma carotenoid levels.

Adult↗

PubMed1999

[Preparation of monoclonal antibodies to human vascular endothelial growth factor(VEGF121) and identification of its expression on gastric carcinoma cell line MGC803].

OBJECTIVE: To further elucidate the source of VEGF in solid tumors. METHODS: Traditional hybridoma technology was used to prepare VEGF monoclonal antibodies (McAb); it's activity was measured by 3H-TdR incorporation on human umbilical vein endothelial cells (HUVEC). VEGF gene was amplified by PCR with cDNA as template from MGC803 cell lines. The PCR products were cloned into fusion protein prokaryotic expression vector PGEX2T which expressed in E. coli XL-1 blue. Western blot analysis was used to identify expression of VEGF. RESULTS: The McAbs could specifically bind to VEGF in ELISA. One of the McAbs(5C5) could neutralize the mitogenic activity of VEGF121 on HUVEC in a dose-dependent manner. The VEGF gene fragment obtained from RT-PCR cloned to PGEX2T vector expressed a protein product which reacted specifically with McAb 5C5. CONCLUSION: Gastric carcinoma is shown to express VEGF. It may be a major source of VEGF in human solid tumor. Monoclonal antibody against VEGF may be of value in the treatment of cancer.

Animals↗

PubMed1999

[Separation of p-aminobenzenearsonic acid and its oxide by ion-pair reversed-phase high performance liquid chromatography].

To study the separation of p-aminobenzenearsonic acid (PABAA) and its oxide, p-aminophenylarsine oxide (PAPAO), both the absorption spectra were scanned at the wavelengths from 200 nm to 380 nm. PABAA had absorption maximum at 254 nm and PAPAO 258 nm. The effects of salt concentration, column temperature, methanol and ion-pair agent concentrations on the capacity factor were investigated. Compounds of high polarity showed almost no retention on reversed-phase column; as the volume fraction of the methanol decreased from 90% to 10%, the retention time of PABAA gradually increased with broad peak, and partially eluted when methanol volume fraction being below 20%. With temperature rising, the retention time of PABAA was decreased. But PABAA capacity factor can be increased by selecting an appropriate salt concentration for the mobile phase. The cetyltrimethyl and tetrabutyl ammonium ions were separately added as ion-pair agents to the mobile phase containing methanol in phosphate buffer of 10 mmol/L, the changes of retention time were observed. The mechanism of retention based on reversed phase ion-pair model is proposed. Besides, the retention behaviour is also influenced by size exclusion in stationary phase as well as polar interactions with residual silanol group on the silica surface.

Arsanilic Acid↗

PubMed1999

[INDO/CI studies on the electronic spectra of tetraphenylporphyrin and its derivatives].

MNDO calculations and group theory were carried out on tetraphenylporphyrine (TPP) to obtain its optimum molecular geometry. We employed an INDO/CI method to study the electronic structures of tetraphenylporphyrine and its derivatives and identify their main electonic absorption bands. Five-orbital model was advanced to explain the typical characteristics of electronic spectra of H2TPP and its derivatives. The calculated wavelengths were in good agreement with the experimental values. The effects of the substitution of variant group were discussed and some helpful conclusions were drawn.

Models, Molecular↗

PubMed1998

The antiproliferative agent didemnin B uncompetitively inhibits palmitoyl protein thioesterase.

Dynamic protein palmitoylation has been proposed to regulate GTP-binding proteins by controlling their membrane association and thus their access to key signaling proteins. While the palmitoyl protein thioesterase(s) responsible for depalmitoylation of plasma membrane-associated signaling proteins has (have) not been identified, the lysosomal palmitoyl protein thioesterase 1 (PPT1) has proven useful in in vitro studies of membrane localization requirements of GTP-binding proteins. We have previously reported the binding of the antiproliferative cyclic depsipeptide didemnin B to PPT1. To investigate the nature of this binding and its possible effects on PPT1 enzymatic activity, human PPT1 was expressed in an insect cell baculoviral system, and inhibition assays were performed using both [3H]palmitoyl Ha-Ras and myristoyl-CoA as PPT1 substrates. Didemnin B was shown to inhibit recombinant human PPT1 with a Ki of 92 nM. Kinetic analysis of this inhibition revealed that didemnin B inhibits PPT1 uncompetitively. Providing biochemical support for an uncompetitive mode of inhibition, in vitro binding studies of PPT1 and didemnin indicate that the natural product binds preferentially to the enzyme-substrate complex PPT1-palmitoyl-CoA. As the first described inhibitor of PPT1, didemnin B may prove to be a useful tool in the investigation of protein palmitoylation regulation.

Acyl Coenzyme A↗

PubMed1998

Expression of the tumor metastatic suppressor gene in mouse melanoma model: inverse association to metastatic potential.

Data obtained in experimental cutaneous melanomas have suggested that the nm23 gene may function as a metastasis suppressor gene. The nm23 level in 8 human cutaneous melanoma cell lines and 2 murine melanoma cell lines were examined. Each melanoma cell line was transplanted subcutaneously into the flank of nude mice, and the metastatic behavior was evaluated by counting lung tumor foci and by determining host survival time. It was found that expression of nm23 mRNA in human melanomas is correlated closely with reduced metastatic behavior in experimental animals and may serve as a sensitive prognostic indicator of malignancy and survival in patients with melanomas.

Animals↗

PubMed1998

Eponemycin analogues: syntheses and use as probes of angiogenesis.

Derivatives of the epoxy-beta-aminoketone containing natural product eponemycin have been prepared in order to study the molecular mode of action of this anti-angiogenic compound. Synthesis and use of a biotinylated dihydroeponemycin analogue demonstrated that dihydroeponemycin forms a covalent adduct with at least two intracellular proteins in human endothelial cells. Pretreatment of cells with a five equivalent excess of dihydroeponemycin precluded biotin-dihydroeponemycin binding indicating a specific interaction between natural product and the target proteins. This biotin-dihydroeponemycin derivative will prove useful in the purification and identification of eponemycin receptors.

Amides↗

PubMed1998

Recent progress in mammalian cloning.

PURPOSE: Our purpose was to review recent progress in the use of nuclear transfer technology to produce genetically identical mammals. METHODS: A literature review was conducted. RESULTS: The reasons for cloning nonhuman mammals are manyfold including commercial, biomedical, and basic research applications. Individual steps in the nuclear transfer process are itemized, along with a detailed description of the specific approaches used in the production of Dolly, NETI, and DITTO. The potential application of nuclear transfer in the treatment of human infertility is also considered, along with bioethical concerns. Finally, insights are provided concerning the future application of cloning technology in rhesus macaques. CONCLUSIONS: The cloning of a lamb (Dolly) from an adult, mammary gland cell coupled with the successful production of rhesus monkeys (NETI and DITTO) by nuclear transfer of embryonic cells marks the beginning of a "Golden Age" in the development and application of somatic cell cloning technology in mammals.

Animals↗

PubMed1998

The use of a chimera HIV-1/HIV-2 envelope protein for immunodiagnosis of HIV infection: its expression and purification in E. coli by use of a translation initiation site within HIV-1 env gene.

A chimera HIV-1/HIV-2 envelope sequence composed of multiple conserved immunodominant epitopes of HIV-1 envelope protein (HIV-1 IIIB: env482-518 + env548-675) and the HIV-2 gp36 immunodominant epitope (env592-603), was constructed and directly over-expressed in E. coli by using a prokaryotic translation initiation sequence contained within the gene of HIV-1 envelope. The recombinant product was purified and applied in antibody-screening assay. The purified chimera antigen reacted with all the thirty-eight HIV-1 positive serum samples, the two HIV-2 serum samples, and had no cross-reaction with all the eighty-eight normal healthy serum sample. The results indicated that this recombinant chimera HIV-1/HIV-2 envelope protein could be useful for diagnostic purposes of HIV infection.

AIDS Serodiagnosis↗

PubMed1998

Thioredoxin fusion/HIV-1 protease coexpression system for production of soluble human IL6 in E. coli cytoplasm.

In this paper, thioredoxin (TRX) fusion expression system has been modified to produce soluble human IL6 (hIL6) without TRX moiety in E. coli cytoplasm. A novel TRX gene fusion vector was developed that contained at the 3'-end of TRX gene a short DNA sequences encoding a linker peptide '-GSGSGVSQNYPIVQHHHHHH-', serving not only as a specific HIV-1 protease site but also providing six contiguous histidine (His) residues to foreign proteins. The cDNA for hIL6 was cloned into this vector resulting in plasmid pTRX@HISIL6. The cDNA for the HIV-1 protease has been cloned into another compatible plasmid pHMM2, resulting in plasmid pHMM2-PR. Both plasmids were transformed into E. coli strain GI724, and when induced for expression of both proteins, the correct processing of TRX@HISIL6 was obtained, producing hIL6 with His6-tag at the N terminus named HISIL6. A fraction of HISIL6 was found in soluble form and could be purified to homogeneity by Ni-NTA Superflow and ion-exchange chromatography. The biological activity of purified HISIL6 was measured by MTT method in an IL-6-dependent cell line 7TD1 to be 2.1 x 10(8) unit/mg.

Amino Acid Sequence↗

PubMed1998

Deficient 17beta-hydroxysteroid dehydrogenase type 2 expression in endometriosis: failure to metabolize 17beta-estradiol.

Aberrant aromatase expression in stromal cells of endometriosis gives rise to conversion of circulating androstenedione to estrone in this tissue, whereas aromatase expression is absent in the eutopic endometrium. In this study, we initially demonstrated by Northern blotting transcripts of the reductive 17beta-hydroxysteroid dehydrogenase (17betaHSD) type 1, which catalyzes the conversion of estrone to 17beta-estradiol, in both eutopic endometrium and endometriosis. Thus, it follows that the product of the aromatase reaction, namely estrone, that is weakly estrogenic can be converted to the potent estrogen, 17beta-estradiol, in endometriotic tissues. It was previously demonstrated that progesterone stimulates the inactivation of 17beta-estradiol through conversion to estrone in eutopic endometrial epithelial cells. Subsequently, 17betaHSD type 2 was shown to catalyze this reaction, and its transcripts were detected in the epithelial cell component of the eutopic endometrium in secretory phase. Because 17beta-estradiol plays a critical role in the development and growth of endometriosis, we studied 17betaHSD-2 expression in endometriotic tissues and eutopic endometrium. We demonstrated, by Northern blotting, 17betaHSD-2 messenger ribonucleic acid (RNA) in all RNA samples of secretory eutopic endometrium (n=12) but not in secretory samples of endometriotic lesions (n=10), including paired samples of endometrium and endometriosis obtained simultaneously from eight patients. This messenger RNA was not detectable in any samples of proliferative eutopic endometrium or endometriosis (n=4) as expected. Next, we confirmed these findings by demonstration of immunoreactive 17betaHSD-2 in epithelial cells of secretory eutopic endometrium in 11 of 13 samples employing a monoclonal antibody against 17betaHSD-2, whereas 17betaHSD-2 was absent in paired secretory endometriotic tissues (n=4). Proliferative eutopic endometrial (n=8) and endometriotic (n=4) tissues were both negative for immunoreactive 17betaHSD-2, except for barely detectable levels in 1 eutopic endometrial sample. Finally, we sought to determine whether deficient 17betaHSD-2 expression in endometriotic tissues is due to impaired progesterone action in endometriosis. We determined by immunohistochemistry the expression of progesterone and estrogen receptors in these paired samples of secretory (n=4) and proliferative (n=4) eutopic endometrium and endometriosis, and no differences could be demonstrated. In conclusion, inactivation of 17beta-estradiol is impaired in endometriotic tissues due to deficient expression of 17betaHSD-2, which is normally expressed in eutopic endometrium in response to progesterone. The lack of 17betaHSD-2 expression in endometriosis is not due to alterations in the levels of immunoreactive progesterone or estrogen receptors in this tissue and may be related to an inhibitory aberration in the signaling pathway that regulates 17betaHSD-2 expression.

17-Hydroxysteroid Dehydrogenases↗

PubMed1998

[The treatment of rat fulminant hepatic failure by auxiliary partial heterotopic liver transplantation: experimental study].

OBJECTIVE: To confirm the effect of auxiliary partial heterotopic liver transplantation (APHLT) in the treatment of fulminant hepatic failure (FHF). METHOD: On 30 rat models of FHF, 62% of partial liver grafts were implanted below the host residual liver with 28 successful operations. The survival rate, biochemical test for liver function, (99m)Tc-HIDA liver scintigraphy and pathological changes of the grafts and host livers were studied. RESULT: The survival rate in 48 hours was 71.4% compared with 0% in 15 FHF animals. 14 days after the operation, the host liver regenerated obviously and the liver function recovered while the graft shrank gradually. 30 days later, the graft became fibrosis completely. CONCLUSION: Depending on the temporary function support of the graft, the native residual liver could regenerate and restore its function while the graft tended to atrophy.

Animals↗

PubMed1998

[Timosaponins E1 and E2].

By means of silica gel chromatography and HPLC, two compounds were isolated from Anemarrhena asphodeloides Bge.. On the basis of chemical and spectral (MS, 1H, 13CNMR, IR) analyses, their structures were elucidated as (25S)-26-O-beta-D-glucopyranosyl-22-hydroxy-5 beta-furost-3 beta, 15 alpha, 26-triol-3-O-beta-D-glucopyranosyl (1-->2)-beta-D-galactopyranoside (1), and (25S)-26-O-beta-D-glucopyranosyl-22-methoxy-5 beta-furost-3 beta, 15 alpha, 26-triol-3-O-beta-D-glucopyranosyl (1-->2)-beta-D-galactopyranoside (2). They are named as timosaponin E1 (1) and timosaponin E2 (2) respectively. The structure of anemarrhenasaponin-I (A) is briefly discussed. Timosaponins are the main active constituents of Anemarrhena asphodeloides, most of them were shown to can inhibit platelet aggregation.

Anemarrhena↗