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Biomedical subjects

M Wan

Publications and source records attributed to M Wan.

At least 19 recordsLinked to original sources
PubMed2000

Inhibitory activity of flavonoids and tannins against HIV-1 protease.

Twenty-nine flavonoids and six hydrolyzable tannins were studied for their inhibitory activity against human immunodeficiency virus (HIV)-1 protease using fluorescence and HPLC assays. Among the flavonoids, flavones, flavanones, flavonols, catechols and chalcones, the flavonols were the most active category while flavanones and catechols displayed low activity. Quercetin was the most potent inhibitor of the target enzyme with an IC50 value of 58.8 microM, while butein and luteolin showed moderate activity. Of the hydrolyzable tannins tested, three ellagitannins which contain a hexahydroxvdiphenoyl (HHDP) unit linked to the O-3 and 0-6 positions of the sugar, were found to strongly inhibit HIV-1 protease. The IC50 values of corilagin and repandusinic acid on HIV-1 protease were 20.7 and 12.5 microM, respectively.

Flavonoids↗
PubMed1999

Suppression of tumorigenicity in human ovarian cancer cell lines is controlled by a 2 cM fragment in chromosomal region 6q24-q25.

Multiple distinct regions of chromosome 6 are frequently affected by losses of heterozygosity in primary human ovarian carcinomas. We introduced a normal human chromosome 6 into HEY and SKOV-3 ovarian carcinoma cell lines using microcell-mediated chromosome transfer techniques to further investigate the role of this chromosome in ovarian tumorigenesis. The exogenous chromosome was stably propagated in the recipient cells based on fluorescence in situ hybridization (FISH) analyses with a chromosome 6 painting probe. The tumorigenicity of HEY and SKOV-3 cells was completely suppressed after transfer of chromosome 6, but not after transfer of a chromosome 11q13-qter fragment used as control. Using 46 polymorphic microsatellite markers, the region bounded by D6S1649 and D6S1564 was found to be commonly deleted in HEY: chromosome 6 tumorigenic revertant clones. The boundaries of the commonly deleted region could be further narrowed down to a 2 cM (based on the Whitehead genetic map) or 0.36 megabase (based on gdb mapping data) region between D6S1637 and D6S1564 after transferring the exogenous chromosome from revertants into mouse L cells and performing allelic deletion mapping studies against this mouse background. We conclude that this region contains a tumor suppressor gene important for the control of ovarian tumor development.

Animals↗
PubMed1999

Variant antibody identification by peptide mapping.

During development of CGP56901, a monoclonal antibody (MAb) specific for a unique epitope on human IgE, the protein A-purified IgG from one of the candidate production cell lines, showed an additional minor heavy chain (H-chain) band with a molecular weight slightly lower than that of the principal H-chain band on SDS-PAGE. The N-terminal amino acid sequence of this minor H-chain species indicated that at least the first 30 amino acids were identical to those of the antibody light-chain (L-chain) variable domain. More detailed studies using peptide mapping and amino acid sequencing analysis confirmed a crossover event between the V genes of the antibody. The position is between Arg108 of the L chain and Ala124 of the H chain. This crossover resulted in a variant H chain, which had 16 fewer amino acid residues than the normal CGP56901 H chain. These results show that peptide mapping is a useful "first-line" analytical tool in the characterization of the quality of the monoclonal antibody.

Amino Acid Sequence↗
PubMed1999

Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.

Rett syndrome (RTT, MIM 312750) is a progressive neurodevelopmental disorder and one of the most common causes of mental retardation in females, with an incidence of 1 in 10,000-15,000 (ref. 2). Patients with classic RTT appear to develop normally until 6-18 months of age, then gradually lose speech and purposeful hand use, and develop microcephaly, seizures, autism, ataxia, intermittent hyperventilation and stereotypic hand movements. After initial regression, the condition stabilizes and patients usually survive into adulthood. As RTT occurs almost exclusively in females, it has been proposed that RTT is caused by an X-linked dominant mutation with lethality in hemizygous males. Previous exclusion mapping studies using RTT families mapped the locus to Xq28 (refs 6,9,10,11). Using a systematic gene screening approach, we have identified mutations in the gene (MECP2 ) encoding X-linked methyl-CpG-binding protein 2 (MeCP2) as the cause of some cases of RTT. MeCP2 selectively binds CpG dinucleotides in the mammalian genome and mediates transcriptional repression through interaction with histone deacetylase and the corepressor SIN3A (refs 12,13). In 5 of 21 sporadic patients, we found 3 de novo missense mutations in the region encoding the highly conserved methyl-binding domain (MBD) as well as a de novo frameshift and a de novo nonsense mutation, both of which disrupt the transcription repression domain (TRD). In two affected half-sisters of a RTT family, we found segregation of an additional missense mutation not detected in their obligate carrier mother. This suggests that the mother is a germline mosaic for this mutation. Our study reports the first disease-causing mutations in RTT and points to abnormal epigenetic regulation as the mechanism underlying the pathogenesis of RTT.

Amino Acid Sequence↗
PubMed1999

Rett syndrome and beyond: recurrent spontaneous and familial MECP2 mutations at CpG hotspots.

Rett syndrome (RTT) is a neurodevelopmental disorder characterized by loss of acquired skills after a period of normal development in infant girls. The responsible gene, encoding methyl-CpG binding protein 2 (MeCP2), was recently discovered. Here we explore the spectrum of phenotypes resulting from MECP2 mutations. Both nonsense (R168X and R255X) and missense (R106W and R306C) mutations have been found, with multiple recurrences. R168X mutations were identified in six unrelated sporadic cases, as well as in two affected sisters and their normal mother. The missense mutations were de novo and affect conserved domains of MeCP2. All of the nucleotide substitutions involve C-->T transitions at CpG hotspots. A single nucleotide deletion, at codon 137, that creates a L138X stop codon within the methyl-binding domain was found in an individual with features of RTT and incontinentia pigmenti. An 806delG deletion causing a V288X stop in the transcription-repression domain was identified in a woman with motor-coordination problems, mild learning disability, and skewed X inactivation; in her sister and daughter, who were affected with classic RTT; and in her hemizygous son, who died from congenital encephalopathy. Thus, some males with RTT-causing MECP2 mutations may survive to birth, and female heterozygotes with favorably skewed X-inactivation patterns may have little or no involvement. Therefore, MECP2 mutations are not limited to RTT and may be implicated in a much broader phenotypic spectrum.

Alleles↗
PubMed1999

In vivo hemodynamic evaluation based on transverse Doppler measurements of blood velocities and vessel diameter.

We have developed a method for simultaneously measuring the blood flow velocity distributions along a line perpendicular to the vessel wall and the instantaneous diameter of the common carotid artery. Thus, the wall shear rates and shear rate distribution across the arterial lumen could be computed accurately. In this method, transverse Doppler technique is combined with ultrasonic A echo-mode, using only one single crystal transducer. In order to satisfy the requirements of high spatial and velocity resolutions, especially extending to a very low velocity range in the case of low signal-to-noise ratio, a combination scheme of 10-MHz operating frequency, sample volume tracking, 12-bit A/D, 512-point fast Fourier transform with 5-kHz sample rate, spectrum average, variable threshold for bandwidth estimation, smoothing of velocity curves, and hardware was carefully designed. The velocity distribution measurements based on transverse Doppler spectrums were also validated under the steady flow conditions using a flow loop system. Results from this study show this ultrasonic system to be capable of measuring the shear rates at wall accurately and shear rate distributions across the common carotid arterial lumen.

Adult↗
PubMed1998

Conserved chromosomal location and genomic structure of human and mouse fatty-acid amide hydrolase genes and evaluation of clasper as a candidate neurological mutation.

Fatty-acid amide hydrolase (FAAH) is a membrane-bound enzyme that degrades neuromodulatory fatty acid amides, such as oleamide and anandamide, and is expressed in the mammalian central nervous system. To evaluate FAAH genes as candidates for neurogenetic diseases in humans and mice, we have mapped the loci in both species and have determined their intron-exon structures. The human FAAH gene was mapped to region 1p34-p35, closely linked to D1S197 and D1S443, by using PCR analysis of somatic cell hybrid (SCH) and radiation hybrid mapping panels. Analysis of an SCH mapping panel and a mouse interspecific backcross panel has localized the Faah gene to the conserved syntenic region on mouse chromosome 4, close to the neurological mutation clasper. Faah gene rearrangements were excluded by Southern blot analysis of clasper DNA. No sequence abnormality was detected in PCR products containing the 15 exons and splice junctions of the mouse Faah gene. FAAH protein levels were normal in clasper mouse tissues as determined by enzyme activity assays and Western blotting.

Amidohydrolases↗
PubMed1998

Evaluation of two X chromosomal candidate genes for Rett syndrome: glutamate dehydrogenase-2 (GLUD2) and rab GDP-dissociation inhibitor (GDI1).

The most likely cause of the Rett syndrome (RTT) is an X-linked dominant mutation lethal in hemizygous males. Previous exclusion mapping studies have identified putative regions for the RTT gene on the X chromosome. In the present study, we evaluated two candidate genes, glutamate dehydrogenase-2 (GLUD2) and rab GDP-dissociation inhibitor (GDI1/XAP-4), chosen because of their expression patterns and functions in the central nervous system and their location in the nonexcluded region of Xq. The intronless gene GLUD2, located in Xq25 and expressed in neuronal and testicular tissues, is involved in the metabolism of glutamate, a neurotransmitter reported to be elevated in the spinal fluid of RTT individuals. The GLUD2 gene was screened for mutations by Southern hybridization and by direct sequencing of polymerase chain reaction (PCR) products. The GDI1 gene in Xq28, also known as RABGDIA or XAP-4, encodes a human GDI that is expressed predominantly in neuronal and sensory tissues. All 11 exons and splice junctions of the GDI1 gene were PCR-amplified and sequenced directly or screened by single-strand conformation analysis. No mutation in either of these two genes was found in 22 RTT patients. Therefore, GLUD2 and GDI1 can be excluded as candidate genes for this syndrome.

Blotting, Southern↗
PubMed1998

Detection of ovarian cancer cells: comparison of a telomerase assay and cytologic examination.

BACKGROUND: Telomerase is an enzyme essential for the normal replication of chromosomes. Telomerase activity is absent in most somatic cells in adults, but it is usually expressed in cancer cells, including ovarian carcinoma cells. Our principal goal was to compare the sensitivity of a telomerase assay, i.e., the telomeric repeat amplification protocol (TRAP) assay, with that of cytologic examination in detecting cancer cells in the peritoneal cavity of patients with ovarian carcinoma. METHODS: TRAP assays and cytologic examinations were performed on peritoneal washings and ascitic fluids from 42 patients with active ovarian carcinoma. Control specimens included washings from 29 patients with benign ovarian diseases and ascitic fluids from 14 patients with liver failure. We also evaluated the stability of telomerase in ascitic fluids left unprocessed at room temperature as well as the ability of the TRAP assay to detect cancer cells in mixtures containing large numbers of normal cells. RESULTS: Specimens from 37 (88%) of the 42 patients with ovarian carcinoma tested positive for telomerase. Cytologic examination detected cancer cells in only 27 of the telomerase-positive specimens (i.e., in specimens from 64% of the 42 patients). This difference of 24% (95% confidence interval = 17%-30%) in sensitivity between the two tests was statistically significant (two-sided P = .002). Specimens from five of the patients with ovarian carcinoma were cytologically negative and telomerase negative. All 43 control specimens were cytologically negative, but the TRAP assay detected telomerase in two of them. Telomerase activity was detected in unprocessed samples left at room temperature for 5 days and in mixtures containing a small number of cancer cells and a 2000- to 10000-fold excess of normal cells. CONCLUSIONS: Assaying for telomerase is more sensitive than cytologic examination in detecting cancer cells in the peritoneal cavity of patients with ovarian carcinoma.

Ascitic Fluid↗
PubMed1997

Telomerase activity in benign and malignant epithelial ovarian tumors.

BACKGROUND: Ovarian epithelial tumors include benign lesions lacking invasive and metastatic abilities (cystadenomas) in addition to malignant lesions (carcinomas). An intermediate category, called tumors of low malignant potential (LMP), is also recognized. The merit of this classification is being challenged because the clinical behavior of LMP tumors appears closer to that of cystadenomas than to that of carcinomas. PURPOSE: To verify our hypothesis that the expression of the enzyme telomerase distinguishes these two categories of ovarian epithelial tumors, we examined and compared such expression in ovarian cystadenomas and carcinomas. By examining the expression of telomerase in LMP tumors, we then sought to determine if these tumors were more closely related to cystadenomas or to carcinomas with regard to telomerase expression. METHODS: We examined a total of 64 consecutive ovarian tumors subdivided into 20 carcinomas, 17 LMP tumors, and 27 cystadenomas. We subsequently discarded three of the 27 cystadenomas because of the presence of admixed normal ovarian stroma in those specimens. Tumor subtyping was done without knowledge of the telomerase results, and telomerase assays were likewise interpreted without knowledge of tumor types. Telomerase activity was determined by use of the TRAP (i.e., telomeric repeat amplification protocol) assay. Differences between the proportions of tumors expressing this enzyme in each subgroup were evaluated by use of Fisher's exact test (two-sided). RESULTS: Telomerase activity was detected in all 20 carcinomas and in all 17 LMP tumors examined. In contrast, it was not detected in 19 of the 24 cystadenomas. These differences between rates of telomerase expression in either carcinomas or LMP tumors and those in cystadenomas were statistically significant (P<.0001). All five of the telomerase-positive cystadenomas belonged to a variant called papillary cystadenomas, whereas none of the telomerase-negative cystadenomas belonged to this variant (P<.0001). CONCLUSIONS AND IMPLICATIONS: The presence of telomerase expression in ovarian LMP tumors supports the merit of continuing to separate these tumors from cystadenomas, in spite of their apparent benign clinical course. The finding of telomerase expression in papillary cystadenomas suggests that such tumors may be mechanistically related to LMP tumors and should perhaps be reclassified as variants of LMP tumors. Lack of telomerase expression in ovarian cystadenomas raises questions about the alleged immortality of these tumors because expression of this enzyme is thought to be essential for continuous growth in adult tumors.

Adult↗
PubMed1997

[Causes and influential factors of spectral broadening in Doppler flow signal].

Signals received are the resultant of the blood flow phasors in CW Doppler system and there are spectral broadening phenomena in it, in other words, frequency of signals corresponding to a moving RBC is not single. A model is presented that enables the detailed effects of spectral broadening to be calculated for a CW Doppler system by using geometric boundary argument. Results are given for the circular geometry.

Blood Flow Velocity↗
PubMed1997

[Effects of a high-pass filter on the Doppler blood flow signal].

A high-pass filter is used to remove the large signals scattered or reflected from stationary and slow moving objects in many clinical CW Doppler units. theoretical results are presented that such a filter will lead to overestimate of the mean velocity of Doppler blood flow. As for different frequency signals, high-pass filter have different amplitudes and phase responses, consequently, nonlinear phase shift is produced. It is proved by computer experiment that nonlinear phase shift caused by high-pass filter hardly has any effects on Doppler flow power spectrum if the high-pass filter does not affect the amplitudes of signals.

Blood Flow Velocity↗
PubMed1996

Autoprocessing: an essential step for the activation of HIV-1 protease.

Human immunodeficiency virus type 1 (HIV-1) expresses its structural and functional proteins within Gag-Pol precursor polyproteins. Specific proteolytic processing of the precursors by the viral protease is critical for the maturation and infectivity of viral particles. To observe the influence of autoprocessing on the activation of recombinant HIV-1 protease, we constructed different HIV-1 protease forms, with or without the Phe-Pro bond directly upstream of the protease domain, and expressed them in Escherichia coli systems. We found that the presence of a short upstream sequence of the protease domain, which could generate the original N-terminus of the protease by autoproteolysis of the Phe-Pro bond, resulted in processing of active protease, whereas for a wild-type protease extended only with the initiator methionine, the proteolytic activity was not recovered. Our results suggested that autoprocessing of the direct upstream sequence of the protease domain is an essential step for the activation of recombinant HIV-1 protease in the E. coli expression system. Expression of HIV-1 protease as fusion proteins revealed that the existence of a fusion portion increased the accumulation of expressed protease by affecting its homotypic dimer formation.

Base Sequence↗
PubMed1996

Potential role of the inactivated X chromosome in ovarian epithelial tumor development.

BACKGROUND: Ovarian epithelial tumors can be divided into subcategories often regarded as different stages of neoplastic transformation. Cystadenomas belong to the least aggressive subgroup and are noninvasive and nonmetastatic. Ovarian tumors of low malignant potential (LMP) are intermediate between cystadenomas and carcinomas and show markedly reduced invasive and metastatic abilities. Invasion and metastasis are the hallmarks of carcinomas, which constitute the most aggressive subgroup and can be further subdivided into different grades. PURPOSE: We performed comparative allelotype analyses of ovarian cystadenomas, LMP tumors, and carcinomas, reasoning that such analyses could provide clues about the molecular determinants of their phenotypic differences. Because we realized that allelic losses involving the X chromosome might be associated with LMP tumor development, we determined whether such losses were interstitial and whether they involved the active or the inactive X chromosome. METHODS: Frequencies of loss of heterozygosity (LOH) at specific loci in every chromosomal arm were determined in 16 ovarian cystadenomas, 23 ovarian LMP tumors, 15 low-grade ovarian carcinomas, and 35 high-grade ovarian carcinomas by use of either the polymerase chain reaction (PCR) or Southern blot analyses. We took advantage of the fact that DNA methylation is an important mechanism of X-chromosome inactivation to determine whether losses involving the X chromosome were in the active or the inactive copy. We analyzed the methylation status of retained alleles on the X chromosome by determining whether they could be amplified by PCR after digestion with the methylation-sensitive restriction endonuclease Hpa II. RESULTS: High-grade carcinomas contained frequent(>50%) LOH in four autosomal chromosome arms, i.e., 6q, 13q, 17p, and 17q. Except for 13q, these same chromosomal arms showed frequent LOH in low-grade carcinomas. LOH in autosomal chromosomes was comparatively rare in LMP tumors and was absent in cystadenomas. In contrast, half (eight of 16) of LMP tumors informative for a locus in the proximal portion of chromosome Xq showed LOH at that locus. These losses were the result of interstitial deletions in six of the eight cases and involved the inactive copy of the X chromosome exclusively. Similar losses in the X chromosome were not seen in either cystadenomas or low-grade carcinomas. CONCLUSIONS AND IMPLICATIONS: LOH at multiple loci is associated with the development of ovarian carcinomas but not with the development of cystadenomas and LMP tumors. However, the integrity of a locus in chromosome Xq that possibly escapes X-chromosome inactivation is important for the control of LMP tumor development. The fact that this locus does not appear to be involved in the genesis of low-grade carcinomas suggests that LMP tumors are not precursors of such carcinomas.

Alleles↗
PubMed1996

Anti-HIV triterpene acids from Geum japonicum.

The methanol extract from the whole plant of Geum japonicum was found to inhibit the human immunodeficiency virus (HIV-1) protease. Through bioassay-directed fractionation of the extract, a new triterpene acid along with five known triterpene acids, ursolic acid, epipomolic acid, maslinic acid, euscaphic acid, and tormentic acid, were isolated. The structure of the new compound was determined by spectral means including 1H-1H COSY, HMQC, HMBC, and NOE experiments to be 2 alpha, 19 alpha-dihydroxy-3-oxo-12-ursen-28-oic acid (1). Of these compounds, 1, ursolic acid, and maslinic acid showed potent inhibitory activity against HIV-1 protease.

Amino Acid Sequence↗