Biomedical subjects

S Miao

Publications and source records attributed to S Miao.

At least 19 recordsLinked to original sources

PubMed2006

Inhibition of the growth of human gastric carcinoma in vivo and in vitro by swainsonine.

In Europe, swainsonine has been studied widely for prevention of metastasis and cancer therapy. In order to investigate the effects and mechanisms of swainsonine on the human gastric carcinoma SGC-7901 cell, we carried out in vivo and in vitro experiments. After treatment with swainsonine, an effective dose and IC50 value of swainsonine for SGC-7901 cells were examined by MTT assay. Cell-cycle distribution and apoptotic rates were analyzed using FCM, and [Ca2+]i was measured using LSCM. The expression of p53, c-myc and Bcl-2 were determined using an immunocytochemical method. Simultaneously, 50 mice were divided randomly into five groups. Three groups were administrated swainsonine at dose of 3, 6 and 12 mg/kg body wt., two control groups were administrated N.S. 20 ml/kg body wt. and 5-Fu 20 mg/kg body wt., respectively, by intraperitoneal injection. The inhibition rate was calculated and pathological sections were observed. The growth of SGC-7901 cell is inhibited by swainsonine in vitro, with an IC50 value at 24 h of 0.84 microg/ml, and complete inhibition concentration is 6.2 microg/ml. After treatment with swainsonine at the concentrations of 0.5, 1.5 and 4.5 microg/ml for 24 h, the expression of apoptosis inhibiting gene p53 and bcl-2 decreases, and the apoptotic trigger gene c-myc increases markedly (p<0.05), as well as [Ca2+]i overloading, SGC-7901 cell is induced to apoptosis in the end. It is also found that the percentages of S phase are 38.8%, 39.7% and 29.6%, respectively (20.0% in control group and 23.2% in 5-Fu group). The rates of inhibition were 13.2%, 28.9%, 27.3%, respectively, when the nude mice were administered swainsonine (p<0.05 or 0.01). The structure of the tumor showed hemorrhage, necrosis and inflammatory cell infiltration. We therefore conclude that swainsonine could inhibit cell proliferation in vitro and the growth of human gastric carcinoma in vivo. The mechanisms of swainsonine-induced apoptosis may relate to [Ca2+]i overloading and the expression of apoptosis-related genes.

Animals↗

PubMed2006

Discovery of a novel series of inhibitors of human cytomegalovirus primase.

Infection by human cytomegalovirus (hCMV) remains a potent threat to susceptible people throughout the world. We have discovered a series of imidazolyl-pyrimidine compounds, which were found to be irreversible inhibitors of the hCMV UL70 primase based on results from radiolabeling and SAR studies. Two promising analogs are described that rival ganciclovir and cidofovir in antiviral potency and possess improved cytotoxicity profiles.

Antiviral Agents↗

PubMed2006

The diversity of the DnaJ/Hsp40 family, the crucial partners for Hsp70 chaperones.

DnaJ/Hsp40 (heat shock protein 40) proteins have been preserved throughout evolution and are important for protein translation, folding, unfolding, translocation, and degradation, primarily by stimulating the ATPase activity of chaperone proteins, Hsp70s. Because the ATP hydrolysis is essential for the activity of Hsp70s, DnaJ/Hsp40 proteins actually determine the activity of Hsp70s by stabilizing their interaction with substrate proteins. DnaJ/Hsp40 proteins all contain the J domain through which they bind to Hsp70s and can be categorized into three groups, depending on the presence of other domains. Six DnaJ homologs have been identified in Escherichia coli and 22 in Saccharomyces cerevisiae. Genome-wide analysis has revealed 41 DnaJ/Hsp40 family members (or putative members) in humans. While 34 contain the typical J domains, 7 bear partially conserved J-like domains, but are still suggested to function as DnaJ/ Hsp40 proteins. DnaJA2b, DnaJB1b, DnaJC2, DnaJC20, and DnaJC21 are named for the first time in this review; all other human DnaJ proteins were dubbed according to their gene names, e.g. DnaJA1 is the human protein named after its gene DNAJA1. This review highlights the progress in studying the domains in DnaJ/Hsp40 proteins, introduces the mechanisms by which they interact with Hsp70s, and stresses their functional diversity.

Amino Acid Sequence↗

PubMed2001

Expression and function of the HSD-3.8 gene encoding a testis-specific protein.

The nucleotide sequence of the full length HSD-3.8 cDNA (accession number AF311312), encoding a human sperm component, was determined to consist of 3818 bp with a reading frame of 2778 bp encoding a deduced polypeptide composed of 926 amino acids. A 0.7 kb fragment containing three immunological epitopes of HSD-3.8 cDNA was prepared and used to construct recombinant expression vectors. The constructs were transformed into E.coli BL-21, and the fusion proteins were expressed, isolated and purified. Using the polyclonal antibodies raised against the purified expressed fusion proteins, positive immunostaining occurred over the surface of the postacrosomal zone of human spermatozoa and of germ cells within the seminiferous epithelium of human testis. Intense staining of large pachytene primary spermatocytes occurred. The capacity of the recombinant protein to reduce fertility as an immunogen in adult female rats was assessed. Immunized animals were infertile or exhibited marked reduction in their fertility. Analysis of the deduced HSD-3.8 polypeptide revealed the presence of a tetratricopeptide repeat (TPR) motif, a P-loop sequence that acts as a binding site for ATP/GTP and phosphorylation sites for PKC, CK2 and cAMP/cGMP-dependent protein kinases. A blot overlay assay with [alpha-(32)P]GTP showed that the polypeptide encoded by the 0.7 kb fragment of HSD-3.8 is a GTP binding protein. It was also shown to possess GTPase activity and to be phosphorylated by PKC in vitro. In conclusion, HSD-3.8 is a GTP binding protein and its activity may be regulated by phosphorylation.

Adenosine Triphosphate↗

PubMed2001

Characteristics of patients at a Taipei summer rock concert festival.

BACKGROUND: Rock concerts are popular mass gatherings in Taiwan. Millions of fans participate in rock concerts in Taiwan each year. However, there were no reports on the characteristics of the patients seen in rock concerts in Taiwan. METHODS: Medical care for a summer rock concert festival held in an outdoor stadium in Taipei was coordinated by emergency physicians of a medical center. About 50,000 attendees participated in the two-night concert. Three stations were set up to provide advanced medical care. A standardized form was used to collect information about patients. RESULTS: A total of 28 cases visited the medical stations, fourteen cases each day. They were aged from 13 to 40 years, with an average of 20.8 +/- 6.4. Twenty-one cases were female and seven were male. Twenty-two (79%) were spectators, five (18%) were on-duty staff, and one was a by-stander. Based on an estimation of totally 50,000 participants in the stadium for this two-night festival, the medical use rate was roughly 5.6 PPTT (patients per ten thousand attendees). The most common major problem was fainting which accounted for 13 cases (46%). Of these 13 cases, three cases (23%) lost consciousness and 12 cases (92%) were female. Sixteen cases (57%) were classified as requiring ALS (advanced life support) and 12 cases (43%) as requiring BLS (basic life support). Most cases improved and were discharged after onsite treatment. Only one case was transferred by ambulance due to persistent chest pain. However, she recovered several hours later. CONCLUSIONS: By this preliminary data, first reported in Taiwan, we found that the most common problem was fainting. More than half of the cases seen at the concert required advanced life support. A well-designed emergency medical service (EMS) system is mandatory to provide services for these events.

Adolescent↗

PubMed2000

Expression and characterization of the human YWK-II gene, encoding a sperm membrane protein related to the alzheimer betaA4-amyloid precursorprotein.

The YWK-II cDNA, RSD-2, encoding a sperm membrane protein was isolated from a rat testis cDNA expression library. Using the RSD-2 insert in combination with rapid amplification of cDNA ends (RACE), the corresponding human gene was isolated from a human testis cDNA expression library. The human testis cDNA, HSD-2, is 3654 bp in length and contains an open reading frame of 763 codons. Hydropathicity analysis showed that the deduced polypeptide is a single strand transmembrane protein. The deduced polypeptide has partial homology with the amyloid precursor protein (APP) and high homology with the amyloid precursor homologue, APLP2/APPH. The YWK-II gene was mapped and assigned to human chromosome locus: 11q24-25. Northern blotting of various human tissue RNAs using the HSD-2 cDNA as a probe showed that the gene is transcribed ubiquitously. The cytoplasmic domain of HSD-2 was expressed in Escherichia coli. In-vitro studies showed that the recombinant polypeptide bound to a GTP-binding protein (G(o)) and was phosphorylated by protein kinase C and cdc2 kinase. In mammalian F11 cells, the recombinant polypeptide was found to be coupled to G(o). Thus, the YWK-II component has the characteristics of a G(o)-coupled receptor and may be involved in G(o)-mediated signal transduction pathway. Protein kinase C and cdc2 kinase may regulate this pathway in spermatozoa by phosphorylating the cytoplasmic domain of the YWK-II component.

Alzheimer Disease↗

PubMed1999

Assignment of chromosomal locus and evidence for alternatively spliced mRNAs of a human sperm membrane protein (hSMP-1).

The gene (HSD-1) coding a human sperm membrane protein (hSMP-1) was isolated from a human testis cDNA expression library using antibodies found in the serum of an infertile woman. HSD-1 was localized to a single locus on chromosome 9 and assigned to band 9p12-p13 by fluorescent in situ hybridization (FISH) mapping and DAPI (4,6-diamidino-2-phenylindole) banding, using rat/human somatic cell hybrids and metaphase chromosomes of human lymphocytes. In rescreening a testis lambdagt10 cDNA expression library, the full-length cDNA (HSD-1) and several truncated cDNAs with heterologous regions were isolated from positive clones. The heterology consisted of deletion, insertion and alteration of the 5'-end. These heterologous truncated fragments may be produced by alternative splicing of mRNAs. Two recombinant prokaryotic expression vectors were constructed with one of the heterologous fragment (clone #26) with and without the alternative 5'-end. Escherichia coli transfected with the construct containing the alternative 5'-end failed to produce the recombinant product, whereas those transfected with the vector lacking the 5'-end produced hSMP-1. DNASIS analysis of the structure of #26 mRNA suggests that the 5'-end has a stable secondary configuration that may maintain the mRNA in an inactivated state, whereby hindering its translation and preventing the expression of the gene.

Amino Acid Sequence↗

PubMed1999

Uniparental isodisomy resulting from 46,XX,i(1p),i(1q) in a woman with short stature, ptosis, micro/retrognathia, myopathy, deafness, and sterility.

We report on a 43-year-old woman who was referred for evaluation because of minor facial anomalies, myopathy, sterility, short stature, hearing loss, downward slant of palpebral fissures, bilateral ptosis, severe micro/retrognathia, high arched palate, and scoliosis. Cytogenetic analyses utilizing GTG/CBG bandings showed presence of one i(1p) and one i(1q) without normal chromosome 1 homologues. Fluorescence in situ hybridization analysis showed hybridization to only two chromosomes, consistent with the G-banded interpretation of i(1p) and i(1q). To the best of our knowledge, this is the first case of isochromosomes 1p and 1q replacing the two normal chromosome 1s. Molecular investigations using markers for chromosome 1 showed inheritance of only one set of paternal alleles and absence of any maternal alleles in the patient. The adverse phenotype of the patient may be due to one or more recessive mutations, genomic imprinting, or a combination of both.

Abnormalities, Multiple↗

PubMed1999

Chloride dependency of renal brush-border membrane phosphate transport.

In our present study, we examined the effect of Cl(-) on rabbit renal brush-border membrane (BBM) phosphate (P(i)) uptake. It was found that the Na(+)-dependent BBM (32)P uptake was significantly inhibited by Cl(-) replacement in the uptake solution with other anions, or by Cl(-) transport inhibitors, including DIDS, SITS, diphenylamine-2-carboxylate (DPC), niflumic acid (NF), and 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB). Intravesicular formate or Cl(-) increased BBM (36)Cl(-) uptake but did not affect BBM (32)P uptake. BBM (22)Na(+) uptake was lowered by Cl(-) replacement in the uptake solution but not by Cl(-) transport inhibitors. Changes in transmembrane electrical potential altered BBM (36)Cl(-) and (32)P uptake in directions consistent with a net inward movement of negative and positive charges, respectively. However, the Cl(-)-dependent BBM P(i) uptake was not affected by changes in transmembrane electrical potential. Finally, a similar Cl(-) dependency of P(i) uptake was also found with BBM derived from rat and mouse kidneys. In summary, our study showed that a component of Na(+)-dependent P(i) uptake was also Cl(-) dependent in rabbit, rat, and mouse renal BBM. The mechanism underlying this Cl(-) dependency remains to be identified.

Animals↗

PubMed1999

[Tissue specificity and expression of a human sperm protein's gene BSD-2.4].

OBJECTIVE: To investigate the tissue specificity and expression in spermatogenic stage of BSD-2.4 cDNA which might be related with fertility and its sequence was received by GenBank. METHODS: Dot blot, Northern blot, and in situ hybridization were used. RESULTS: Northern blot analysis of sixteen human tissues showed that BSD-2.4 cDNA had one transcript only with testis. BSD-2.4 mRNA was detected in germ cells of human testis. CONCLUSIONS: BSD-2.4 is a testis-specific protein. The cDNA was expressed throughout the stages of the spermatogenic cycle.

Animals↗

PubMed1999

[The characterization of a novel testis-specific nucleoporin gene].

OBJECTIVE: To investigate the structure and function of testis-specific gene and spermatogenesis in human. METHODS: Screening cDNA expression library, 5' rapid amplification of cDNA ends, Northern blot and fluorescent in situ hybridization(FISH) were used. Gene expressing, purified of expressed protein by affinity chromatography and SDS-PAGE as well as phosphorylation of expressed protein in vitro by PKC and p34cdc2 were observed. RESULTS: A cDNA designated as BS-63 was isolated and found to consist of 2,209 bp with an open reading frame of 2,100 bp and assigned the accession number U64675 by GenBank. The deduced polypeptide consisted of 700 amino acid residues containing XFXFG or FG motifs that were characteristic of nuclear pore complex (NPC) protein and acted as potential binding sites for Ran. The N-terminal region had high homology with Ran BP2/Nup 358, a nucleoporin component, showing that BS-63 was a member of the NPC family. Northern blot analysis of mRNA prepared from various human tissues showed that BS-63 gene was transcribed in two forms: 6.0 and 8.5 kb. The 8.5 kb transcript was present in low amounts in several somatic tissues; whereas the 6.0 kb transcript was expressed only in testis. Analysis by FISH method mapped the BS-63 gene in 2q11.2-12. A protein band with an estimated Mr of 80,000 was detected with E. coli BL21 (DE3) transfected with recombinant plasmid pET30a (+)-BS-63. In vitro phosphorylation test indicated the BS-63 recombinant protein could be phosphorylated by PKC and p34cdc2. CONCLUSIONS: The study was the first demonstration that the BS-63 gene encoding a nucleoporin-related protein with Ran binding sites was expressed in germ cells of human testis.

Amino Acid Sequence↗

PubMed1998

Integrated YAC/STS physical and genetic map of 22.5 Mb of human Xq24-q26 at 56-kb inter-STS resolution.

A yeast artificial chromosome sequence-tagged site-based (YAC/STS) physical map of 22.5 Mb of the Xq24-q26 cytogenetic band region of the human X chromosome has been assembled. DNA coverage includes 857 large-insert clones formatted with 405 STSs to provide ninefold depth of DNA. At five points, no bridging clones have been recovered from 20 X-chromosome equivalents of human DNA in YACs or bacterial clones, but the placement of 25 ("CA")n polymorphic markers permits the ordering of contigs by comparison with the genetic linkage map and radiation hybrid data. The map localizes the X3000 translocation breakpoint and six genes (ANT2, NDUFA1, LAMP2, OCRL, IGSF1, and HDGF) at better than 100-kb resolution. The relatively gene-poor nature of the region is consistent with relatively low uniform 34-42% GC content in STSs across nearly all of the region.

Chromosome Breakage↗

PubMed1997

Identification of Glu-268 as the catalytic nucleophile of human lysosomal beta-galactosidase precursor by mass spectrometry.

Human lysosomal beta-galactosidase catalyzes the hydrolysis of beta-galactosides via a double displacement mechanism involving a covalent glycosyl enzyme intermediate. By use of the slow substrate 2,4-dinitrophenyl-2-deoxy-2-fluoro-beta-D-galactopyranoside, a glycosyl enzyme intermediate has been trapped on the enzyme. This has allowed the catalytic nucleophile to be identified as Glu-268 by peptic and tryptic digestion of the inactivated enzyme followed by high performance liquid chromatography-electrospray ionization tandem mass spectrometry of the peptide mixture. This glutamic acid is fully conserved in a sequence-related family of enzymes (Family 35), consistent with its essential role.

Amino Acid Sequence↗

PubMed1996

Neuronal differentiation in PC12 cells is accompanied by diminished inducibility of Hsp70 and Hsp60 in response to heat and ethanol.

The stress response of PC12 cells was characterized by evaluating the production of heat shock proteins of the 70 kDa (Hsp70), 60 kDa (Hsp60) and 90 kDa (Hsp90) families by western blot analysis. Induction of Hsp synthesis was elicited by brief exposure to elevated temperatures or by addition of ethanol to the cultures. Normal PC12 cells responded to stress with rapid up-regulation of Hsp70 and Hsp60 production. However, fully differentiated PC12 cells (induced by nerve growth factor, NGF) failed to produce Hsp70 or Hsp60 in response to heat or ethanol treatment. The disappearance of the heat shock response of the cells was directly related to the extent of neuronal differentiation. The cellular levels of the constitutive proteins, Hsc70 and Hsp90, were not altered by differentiation of the cells. Production of Hsps was restored in the differentiated cells by removal of NGF which coincided with the loss of neurite expression and retraction of processes.

Animals↗

PubMed1996

[Analysis of the A/C polymorphic site within the phenylalanine hydroxylase gene].

An A/C polymorphism was identified at nucleotide-11 from the acceptor site of IVS3 of the phenylalanine hydroxylase gene. This polymorphism can be detected easily by means of single strand conformation polymorphism analysis. The frequency for the majior allele (A) was 0.656 and 0.344 for the minor allele (C) with the PIC being 0.351 in Chinese population. There was no linkage disequilibrium between this site and the STR polymorphic site located upstream within the same intron of the gene. The ejjicincy of prenatal gene diagnosis fro PKU was estimated to be 78.1% by haplotype linkage analysis using these two polymorphic sits.

Humans↗

PubMed1996

[Screening for mutations in the PAH gene in Chinese: a new splice and a novel polymorphic mutation].

OBJECTIVE: to screening for novel mutations in the phenylalanine hydroxylase (PAH) genes in Chines as to investigate the molecular basis of phenylketonuria and to raise its diagnosis rate. METHODS: We applied the polymorase chain reaction (PCR) on amplified DNA fragment harboring exon 4 of the PAH gene. For novel mutations detected, the amplified fragments were sequenced by using ds-DNA cycle-sequencing method. RESULTS: Seven out of 24 cases of PKU had different kinds of band-shifting, which are subjected to two patterns of polymorphism. The first one, only in one case, has one mutation band migrating before the normal bands, while, the second one, which was common among the cases, had both the mutation bands running ahead of the normal bands. By sequence analysis, these polymorphic patterns corresponded to two noval mutations. One was a GT-->AT substitution at the 5' splice donor site of intron 4, and the other is an A to C transversion at position-11 of intron 3. CONCLUSION: The two mutations are both first reported. The 5' donor splicing mutation may result in skipping of the preceeding exon during RNA processing. The A to C transversion, which accounted for about 20% of the Chinese PKU alleles is a polymorphism changes.

DNA Mutational Analysis↗

PubMed1995

Identification of derivatized peptides without radiolabels: tandem mass spectrometric localization of the tagged active-site nucleophiles of two cellulases and a beta-glucosidase.

A new method that uses nonradioactive active site-directed enzyme inactivators and high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESIMS/MS) to identify labeled peptides in a proteolytic digest is described. This method relies upon the fragmentation of labeled peptides in a predictable and reproducible manner in the collision cell of a tandem mass spectrometer. The exoglycanase from Cellulomonas fimi, endoglucanase C from Clostridium thermocellum, and the beta-glucosidase from Agrobacterium faecalis were labeled using 2-deoxy-2-halo-beta-glycosides, digested with pepsin, and subjected to HPLC-ESIMS/MS analysis, scanning in the neutral loss mode. Under these conditions only peptides that lose the (known) mass of the label are detected. Preliminary identification of candidate peptides can be achieved from the mass measured, in combination with the known sequence of the protein. Peptide identity can be confirmed through subsequent sequencing, either via further tandem MS experiments or via the Edman degradation. In all cases the peptides identified in this manner were consistent with those identified by the standard radioactive method. This mass spectrometric method represents a rapid, nonradioisotopic solution to the problem of identifying a modified peptide in a complex mixture. The technique is also sensitive, requiring only picomole amounts of protein.

Bacterial Proteins↗

PubMed1994

Identification of glutamic acid 78 as the active site nucleophile in Bacillus subtilis xylanase using electrospray tandem mass spectrometry.

A new mechanism-based inactivator of beta-1,4-xylanases, 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside, has been synthesized and used to trap the covalent intermediate formed during catalysis by Bacillus subtilis xylanase. Electrospray mass spectrometry confirmed the 1:1 stoichiometry of the incorporation of inactivator into the enzyme. Inactivation of xylanase followed the expected pseudo-first-order kinetic behavior, and kinetic parameters were determined. The intermediate trapped was relatively stable toward hydrolytic turnover (t1/2 = 350 min). However, turnover could be facilitated by transglycosylation following the addition of the acceptor benzyl thio-beta-xylobioside, thus demonstrating the catalytic competence of the trapped intermediate. Reactivation kinetic parameters for this process of kre = 0.03 min-1 and Kre = 46 mM were determined. The nucleophilic amino acid was identified as Glu78 by a tandem mass spectrometric technique which does not require the use of radiolabels. The peptic digest of the labeled enzyme was separated by high-performance liquid chromatography and the eluent fed into a tandem mass spectrometer via an electrospray ionization device. The labeled peptide was identified as one of m/z = 826 (doubly charged) which fragmented in the collision chamber between the mass analyzers with loss of the mass of a 2-fluoroxylobiosyl unit. Confirmation of the peptide identity was obtained both by tandem mass spectrometric sequencing and by Edman degradation of the purified peptide. Glu78 is completely conserved in all members of this xylanase family and indeed is shown to be located in the active site in the recently determined X-ray crystal structure.

Amino Acid Sequence↗