Biomedical subjects

X Shan

Publications and source records attributed to X Shan.

At least 37 records · Page 2Linked to original sources

PubMed2000

[Relationship between tumor suppressor gene p16 and Rb and early diagnosis of lung cancers].

OBJECTIVES: To study the incidences and mechanisms of tumor suppressor gene p16 and Rb inactivation in lung cancers, and to investigate their relation to biological characters, clinical pathological diagnosis and gene classification diagnosis of lung cancers. METHODS: Immunohistochemistry, double in situ hybridization, PCR, PCR-SSCP and sequencing were applied to study the changes of p16 and Rb genes in 106 lung cancers and 23 lung tissues of benign diseases. RESULTS: Total expression rates of p16 and Rb proteins and mRNAs in lung cancers were lower than those in normal lung tissues and benign lung diseases, and they were specially related to histological type, metastasis and clinical stage of lung cancers. In stage I and II lung cancers, the obvious inactivation of tumor suppressor gene p16 or Rb was examined (32.6% or 28.3%); p16 inactivation was detected mainly in non-small cell lung cancers, and Rb inactivation mainly in small cell lung cancers. There were three mechanisms of homozygous deletions, methylations and mutations for p16 gene inactivation. The deletion rate of p16 exon1 and/or exon2 was 25.8%, mainly took place in p16 protein negative cases of non-small cell lung cancers. 15 cases (16.9%) took part in methylations on SmaI sites of CpG island of p16 gene. PCR-SSCP and sequencing showed that 9 cases had p16 gene mutations. CONCLUSIONS: p16 and Rb genes may play important roles in genesis and progression of lung cancers. Inactivation of p16 or Rb gene may be is an early link of lung canceration, that is very important for early diagnosis of lung cancers. A new gene classification model for lung cancer diagnosis would be set up based on the research of p16 and Rb genes.

Adolescent↗

PubMed2000

[CDKN2/p16 gene cloning and its probe preparation and application in lung cancer].

OBJECTIVES: To clone CDKN2/p16(INK4a) gene, prepare its probe, and to study the change of CDKN2/p16(INK4a) gene in lung cancers. METHODS: Total RNA of normal lung tissue was extracted, CDKN2/p16(INK4a) gene cDNA synthesized, and CDKN2/p16(INK4a) gene recombinant vector, constructed. Southern blot was used to study CDKN2/p16(INK4a) gene in 46 cases of lung cancers, 3 cases of normal lung tissues, 6 cases of lung tissues near cancer, and 3 cases of lymph nodes with lung cancer metastasis. RESULTS: Cloned CDKN2/p16(INK4a) cDNA was proved by enzyme digestion and sequencing. Southern blot showed 4.3 kb band in normal lung tissues and lung tissues near cancers, and deletion of CDKN2/p16(INK4a) gene in cancer tissues and lymph nodes with lung cancer metastasis, with a deletion rate of 17.4% (8/46). CONCLUSION: CDKN2/p16(INK4a) gene may play a role to some extent in progression of lung cancers.

Blotting, Southern↗

PubMed2000

Relations of tumor suppressor gene p16 and RB to early diagnosis of lung cancer.

OBJECTIVES: To study the incidence and mechanism of the tumor suppressor gene p16 and RB inactivation in lung cancer, and to investigate their relations to biological characteristics, clinical pathological diagnosis and gene classification. METHODS: Immunohistochemistry, double in situ hybridization, PCR, PCR-SSCP, and sequencing were used to study the changes in the p16 and RB genes in 106 patients with lung cancer. The specimen of normal lung tissue from the patients with lung cancer and specimens of the 23 patients with benign lung diseases were studied in contrast to those of the same patients. RESULTS: The total expression rate of the p16 and RB genes in the lung cancer tissues of the 106 patients was lower than that in normal lung tissues and tissues of benign lung diseases and was specially related to histological type, metastasis, and clinical stage of lung cancer. In stage I and II lung cancer, the obvious inactivation rate of the tumor suppressor gene p16 or RB was 32.6% or 28.3%. The p16 inactivation appeared largely in non-small cell lung cancer (50.1%), and the RB inactivation largely in small cell lung cancer (88.2%). Homozygous deletion, methylation, and mutation were three mechanisms for the p16 gene inactivation. The deletion rate of the p16 exon 1 and/or exon 2 was 25.8% in the patients with non-small cell lung cancer. Fifteen patients (16.9%) showed methylation at the SmaI sites of CpG island of the p16 gene. PCR-SSCP and sequencing revealed p16 gene mutation in 9 of the 106 patients with lung cancer. CONCLUSIONS: The p16 and RB genes may play an important role in genesis and progression of lung cancer. The inactivation of the p16 or RB gene may be an early sign of lung canceration, that is important for the early diagnosis. A new gene classification model for lung cancer diagnosis would be set up based on the research of the p16 and RB genes.

DNA Methylation↗

PubMed2000

[Thiol reagent thimerosal-induced Ca2+ mobilization in isolated guinea pig cochlear outer hair cells].

OBJECTIVE: To understand mechanism of cochlear outer hair cells (OHCs) intracellular Ca2+ mobilization further. METHODS: Intracellular calcium of isolated guinea pig was investigated using thimerosal, a--SH group oxidizing agent, and fura-2 fluorescence ratio imaging microscopy. RESULTS: In the presence of thimerosal, intracellular Ca2+ concentrations ([Ca2+]i) of OHCs were elevated in a dose-dependent manner. Even in Ca(2+)-free medium, Ca2+ response was still induced. The effects of thimerosal on [Ca2+]i were completely blocked and reversed by (DTT). Neither 1-100 mumol/L ryanodine nor 5-20 mmol/L caffeine altered the effects of thimerosal. Pretreatment with pertussis toxin (PTX) for 30 min did not affect the thimerosal-induced increase in [Ca2+]i The increase in [Ca2+]i when Ca2+ was added during thimerosal application in Ca(2+)-free medium was almost completely blocked by 500 mol/L LaCl3, while nifedipine did not inhibit further increase in [Ca2+]i caused by thimerosal. CONCLUSION: Oxidation of the -SH group of the OHC membrane can induce a Ca2+ release from intracellular Ca2+ stores, which are ryanodine- and caffeine-insensitive, and Ca2+ influx through non-specific Ca2+ channels, but not the nifedipine-sensitive Ca2+ Channels. The possible oxidation of--SH group gated Ca2+ channels in OHCs are worthy of further study.

Animals↗

PubMed1999

Functional characterization of human methylenetetrahydrofolate reductase in Saccharomyces cerevisiae.

Human methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. 5-Methyltetrahydrofolate is a major methyl donor in the remethylation of homocysteine to methionine. Impaired MTHFR can cause high levels of homocysteine in plasma, which is an independent risk factor for vascular disease and neural tube defects. We have functionally characterized wild-type and several mutant alleles of human MTHFR in yeast, Saccharomyces cerevisiae. We have shown that yeast MET11 is a functional homologue of human MTHFR. Expression of the human MTHFR cDNA in a yeast strain deleted for MET11 can restore the strain's MTHFR activity in vitro and complement its methionine auxotrophic phenotype in vivo. To understand the domain structure of human MTHFR, we have truncated the C terminus (50%) of the protein and demonstrated that expressing an N-terminal human MTHFR in met11(-) yeast cells rescues the growth phenotype, indicating that this region contains the catalytic domain of the enzyme. However, the truncation leads to the reduced protein levels, suggesting that the C terminus may be important for protein stabilization. We have also functionally characterized four missense mutations identified from patients with severe MTHFR deficiency and two common missense polymorphisms found at high frequency in the general population. Three of the four missense mutations are unable to complement the auxotrophic phenotype of met11(-) yeast cells and show less than 7% enzyme activity of the wild type in vitro. Both of the two common polymorphisms are able to complement the growth phenotype, although one exhibited thermolabile enzyme activity in vitro. These results shall be useful for the functional characterization of MTHFR mutations and analysis structure/function relationship of the enzyme.

Cell Division↗

PubMed1999

Targeted expression of truncated glued disrupts giant fiber synapse formation in Drosophila.

Glued(1) (Gl(1)) mutants produce a truncated protein that acts as a poison subunit and disables the cytoplasmic retrograde motor dynein. Heterozygous mutants have axonal defects in the adult eye and the nervous system. Here we show that selective expression of the poison subunit in neurons of the giant fiber (GF) system disrupts synaptogenesis between the GF and one of its targets, the tergotrochanteral motorneuron (TTMn). Growth and pathfinding by the GF axon and the TTMn dendrite are normal, but the terminal of the GF axon fails to develop normally and becomes swollen with large vesicles. This is a presynaptic defect because expression of truncated Glued restricted to the GF results in the same defect. When tested electrophysiologically, the flies with abnormal axons show a weakened or absent GF-TTMn connection. In Glued(1) heterozygotes, GF-TTMn synapse formation appears morphologically normal, but adult flies show abnormal responses to repetitive stimuli. This physiological effect is also observed when tetanus toxin is expressed in the GFs. Because the GF-TTMn is thought to be a mixed electrochemical synapse, the results show that Glued has a role in assembling both the chemical and electrical components. We speculate that disrupting transport of a retrograde signal disrupts synapse formation and maturation.

Animals↗

PubMed1999

Itk/Emt/Tsk activation in response to CD3 cross-linking in Jurkat T cells requires ZAP-70 and Lat and is independent of membrane recruitment.

The Tec family tyrosine kinase, Itk has been implicated in T cell antigen receptor (TCR) signaling, yet little is known about Itk regulation. Here, we investigate the role of the tyrosine kinase ZAP-70 in regulating Itk. Whereas Itk was activated in Jurkat T cells in response to CD3 cross-linking, Itk activation was defective in the ZAP-70-deficient P116 Jurkat T cell line. Itk responsiveness to TCR engagement was restored in P116 cells stably transfected with ZAP-70 cDNA. ZAP-70 itself could not directly phosphorylate the Itk kinase domain, indicating an indirect regulation of Itk activity. No role was found for ZAP-70 in regulating Itk recruitment to the plasma membrane, an event that has been suggested to be rate-limiting for the activation of Tec family kinases. Indeed, Itk was found to be constitutively targeted to the membrane fraction in both Jurkat and P116 cells. Lat, a prominent in vivo substrate of ZAP-70 that mediates assembly of multimolecular signaling complexes at the plasma membrane of T cells was also found to be required for TCR-stimulated Itk activation. Itk could not be activated by CD3 cross-linking in a Lat-negative cell line, unless Lat expression was restored. Lat and Itk were observed to co-associate in response to CD3 cross-linking in Jurkat T cells, but not in P116 T cells. The Lat-Itk association correlated with Lat tyrosine phosphorylation, which was deficient in the P116 T cells. These data suggest that ZAP-70 and Lat play important, probably sequential, roles in regulating the activation of Itk following TCR engagement.

Adaptor Proteins, Signal Transducing↗

PubMed1999

[Polymorphism of HLA-DRB1, DQB1 in the Hans of north China]

OBJECTIVE: To understand polymorphism of HLA-DRB1, DQB1 in the Hans of north China and obtain more comprehensive and accurate data on genetics at DNA level. METHODS: Polymerase chain reaction-sequence specific primers (PCR-SSP) were used to determine HLA-DRB1, DQB1 alleles in 107 unrelated healthy Han individuals of north China. RESULTS: The authors determined 14 DRB1, 9 DQB1 alleles, which included not only the allele frequencies that corresponded to the gene frequencies of DR, DQ loci determined by other 9 cooperating labs but also the allele frequencies of DRB1*15, DRB1*16, DQB1*0301, DQB1*0302, DQB1*0303, DQB1*05, DQB1*0601, DQB1*0602, and DQB1*0604 that other serology labs did not report. CONCLUSION: This study has obtained a more comprehensive and accurate data set of the normal allele frequencies and linkage disequilibrium parameters of HLA-DRB1,DQB1 in the Hans of north China, which may be of significance in the studies on population genetics and disease association.

Journal Article↗

PubMed1999

[A DNA probe suitable for the detection of chromosome 21 copy number in human interphase nuclei by fluorescence in situ hybridization].

OBJECTIVE: To prepare DNA probe which can be used to accurately detect chromosome 21 copy number in human interphase nuclei by fluorescence in situ hybridization (FISH). METHODS: A probe was produced by universal primer PCR from YAC clone 881D2 and characterized by FISH to metaphases and interphase nuclei of peripheral lymphocytes from 8 normal individuals and 5 patients suffering from trisomy 21. RESULTS: A DNA probe was produced and its characteristics were :(1) Most of the probes ranged in size from 350bp to 750bp; (2) The hybridization signals of the probes were located near centromere on long arm of human chromosome 21; (3) In 1524 metaphases scored, about 99.95% of hybridization signals were located on chromosome 21; (4) The signals were very bright and recognizable easily in both metaphases and interphases nuclei; (5) The expected copy number of chromosome 21 was detected by FISH with the probe in more than 98.40% of interphase nuclei and 99.60% of metaphases. CONCLUSION: The DNA probe reported here is suitable for the detection of chromosome 21 copy number in interphase nuclei and for the study of segregation of chromosome 21 during mitoses of human lymphocytes in culture.

Cell Nucleus↗

PubMed1999

[Polymorphism of HLA-DRB1, DQB1 in the Hans of north China].

OBJECTIVE: To understand polymorphism of HLA-DRB1, DQB1 in the Hans of north China and obtain more comprehensive and accurate data on genetics at DNA level. METHODS: Polymerase chain reaction-sequence specific primers (PCR-SSP) were used to determine HLA-DRB1, DQB1 alleles in 107 unrelated healthy Han individuals of north China. RESULTS: The authors determined 14 DRB1, 9 DQB1 alleles, which included not only the allele frequencies that corresponded to the gene frequencies of DR, DQ loci determined by other 9 cooperating labs but also the allele frequencies of DRB1*15, DRB1*16, DQB1*0301, DQB1*0302, DQB1*0303, DQB1*05, DQB1*0601, DQB1*0602, and DQB1*0604 that other serology labs did not report. CONCLUSION: This study has obtained a more comprehensive and accurate data set of the normal allele frequencies and linkage disequilibrium parameters of HLA-DRB1, DQB1 in the Hans of north China, which may be of significance in the studies on population genetics and disease association.

Alleles↗

PubMed1999

[Influence of aging and sex on chromosome 21 segregation in human lymphocytes].

OBJECTIVE: To determine the influence of aging and sex on the segregation of chromosome 21. METHODS: Lymphocytes were obtained from healthy donors of different ages, both sexes, and were treated with cytochalasin B. Binucleated cells were hybridized with chromosome 21 specific probe, and simultaneously, chromosome 21 loss and non-disjunction were detected. RESULTS: The coefficients of correlation between age and binucleated cells containing 4, 2 and 6 signals were -0.35(P<0.01), 0.18 and 0.38(P<0.01) respectively. The coefficients of correlation between age and nondisjunction of chromosome 21 and micronuclei were 0.56(P<0.01) and 0.70(P<0.01) respectively. CONCLUSION: There is a significant increase with age in the number of micronuclei and missegregation. Nondisjunction of chromosome 21 is much more frequent than loss in vivo and in vitro. No significant difference in nondisjunctioin between male and female was noted. Age effect is more significant in female than in male.

Adult↗

PubMed1999

[Homozygous deletion of CDKN2/p16 gene in lung cancers].

OBJECTIVE: To study the relationship between CDKN2/p16 gene homozygous deletion and lung cancer progression. METHODS: Improved multiplex PCR technique was applied to detect deletion of CDKN2/p16 gene exon1 and exon2 in 89 cases of lung cancers. RESULTS: Gene deletion rate was increased by the improved PCR technique. Exon 1 and exon 2 deletion rates were 19.1% (17/89) and 22.5% (20/89) respectively, with total rate of exon1 and/or exon2 deletion 25.8% (23/89). Deletion of CDKN2/p16 gene occurred in non-small cell lung carcinoma (NSCLC) and was related to metastasis and progressive stage. CONCLUSION: Abnormality of CDKN2/p16 gene is a genetic factor for NSCLC susceptibility, and may play a role to some extent in NSCLC malignant progression.

Aged↗

PubMed1999

[An extensive matrilineal nonsyndromic sensorineural deafness family and mtDNA 12SrRNA gene mutation].

OBJECTIVE: To investigate the possible cause and molecular genetic mechanism of matrilineal nonsyndromic sensorineural deafness, the authors analyzed an extensive matrilineal nonsyndromic sensorineural deafness family. METHODS: PCR amplification of the nt1555 and nt7445 of the mitochondrial DNA, combined with PCR-SSCP, PCR-RFLP and sequence to analyze the family. RESULTS: The authors found a homoplasmic A to G transition at position 1555(A1555G) of the mitochondrial 12SrRNA gene from all the patients, and four matrilineal relatives of this family, but the mutation was not found in the normal spouses of the family and controls (100 normal persons). CONCLUSION: The A1555G mutation may be one of the major factors that cause deafness in this family.

DNA, Mitochondrial↗

PubMed1999

[Diagnostic value of ENG in occupied lesions of cerbellopontine angle].

OBJECTIVE: To evaluate the diagnostic value of ENG in occupied lesions of cerebellopontine angle. METHOD: Twenty-four cases of pathological confirmed occupied lesions of cerebellopontine angle were examined with ENG before operation. The ENG examination included saccade test, gaze test, ocular pursuit test, optoknetic test, positional test and positioning test. RESULT: ENG tests were compared with the results of operation and pathology. The results showed that abnomal ENG-OKR suggested central lesions. Brun's nystagmus and unilateral canal paresis were of important value in locating the lesions of cerebellopontine angle. The ocular pursuit test and optokinetic test could point out which side the lesions were. CONCLUSION: Patients with cerebellopontine angle lesions smaller than 3 cm in diameter showed unilateral canal paresis in caloric test indicated acoustic neuroma. Patients with cerebellopontine angle lesions showed no canal paresis may be other lesions except acoustic neuroma.

Adult↗

PubMed1998

Evidence for heme-mediated redox regulation of human cystathionine beta-synthase activity.

Human cystathionine beta-synthase catalyzes the first step in the catabolic removal of the toxic metabolite, homocysteine. It is unique in being dependent on both pyridoxal phosphate (PLP) and heme for activity. The reaction involves condensation of serine and homocysteine to give cystathionine. Although the role of PLP can be rationalized in analogy with other PLP-dependent enzymes that catalyze beta-replacement reactions, the role of the heme is unknown. In this study, we have purified and characterized the recombinant human enzyme and have examined the effect of heme oxidation state on enzyme activity. We find that under reducing conditions, generated by addition of titanium citrate, the enzyme exhibits a 1.7-fold lower activity than under oxidizing conditions. Reoxidation of the ferrous enzyme with ferricyanide results in alleviation of inhibition. This redox-linked change in enzyme activity correlates with changes in heme oxidation state monitored by UV-visible spectroscopy. Dithiothreitol, which does not reduce the enzyme-bound heme, does not perturb enzyme activity. These studies provide the first evidence for redox-linked regulation of cystathionine beta-synthase which is heme-dependent.

Cystathionine beta-Synthase↗

PubMed1998

Yeast transcript elongation factor (TFIIS), structure and function. I: NMR structural analysis of the minimal transcriptionally active region.

TFIIS is a general transcription elongation factor that helps arrested RNA polymerase II elongation complexes resume transcription. We have previously shown that yeast TFIIS (yTFIIS) comprises three structural domains (I-III). The three-dimensional structures of domain II and part of domain III have been previously reported, but neither domain can autonomously stimulate transcription elongation. Here we report the NMR structural analysis of residues 131-309 of yTFIIS which retains full activity and contains all of domains II and III. We confirm that the structure of domain II in the context of fully active yTFIIS is the same as that determined previously for a shorter construct. We have determined the structure of the C-terminal zinc ribbon domain of active yTFIIS and shown that it is similar to that reported for a shorter construct of human TFIIS. The region linking domain II with the zinc ribbon of domain III appears to be conformationally flexible and does not adopt a single defined tertiary structure. NMR analysis of inactive mutants of yTFIIS support a role for the linker region in interactions with the transcription elongation complex.

Magnetic Resonance Spectroscopy↗

PubMed1998

Yeast transcript elongation factor (TFIIS), structure and function. II: RNA polymerase binding, transcript cleavage, and read-through.

The transcriptionally active fragment of the yeast RNA polymerase II transcription elongation factor, TFIIS, comprises a three-helix bundle and a zinc ribbon motif joined by a linker region. We have probed the function of this fragment of TFIIS using structure-guided mutagenesis. The helix bundle domain binds RNA polymerase II with the same affinity as does the full-length TFIIS, and this interaction is mediated by a basic patch on the outer face of the third helix. TFIIS mutants that were unable to bind RNA polymerase II were inactive for transcription activity, confirming the central role of polymerase binding in the TFIIS mechanism of action. The linker and zinc ribbon regions play roles in promoting cleavage of the nascent transcript and read-through past the block to elongation. Mutation of three aromatic residues in the zinc ribbon domain (Phe269, Phe296, and Phe308) impaired both transcript cleavage and read-through. Mutations introduced in the linker region between residues 240 and 245 and between 250 and 255 also severely impaired both transcript cleavage and read-through activities. Our analysis suggests that the linker region of TFIIS probably adopts a critical structure in the context of the elongation complex.

Models, Molecular↗

PubMed1998

Three-dimensional solution structure of lactoferricin B, an antimicrobial peptide derived from bovine lactoferrin.

The solution structure of bovine lactoferricin (LfcinB) has been determined using 2D 1H NMR spectroscopy. LfcinB is a 25-residue antimicrobial peptide released by pepsin cleavage of lactoferrin, an 80 kDa iron-binding glycoprotein with many immunologically important functions. The NMR structure of LfcinB reveals a somewhat distorted antiparallel beta-sheet. This contrasts with the X-ray structure of bovine lactoferrin, in which residues 1-13 (of LfcinB) form an alpha-helix. Hence, this region of lactoferricin B appears able to adopt a helical or sheetlike conformation, similar to what has been proposed for the amyloidogenic prion proteins and Alzheimer's beta-peptides. LfcinB has an extended hydrophobic surface comprised of residues Phe1, Cys3, Trp6, Trp8, Pro16, Ile18, and Cys20. The side chains of these residues are well-defined in the NMR structure. Many hydrophilic and positively charged residues surround the hydrophobic surface, giving LfcinB an amphipathic character. LfcinB bears numerous similarities to a vast number of cationic peptides which exert their antimicrobial activities through membrane disruption. The structures of many of these peptides have been well characterized, and models of their membrane-permeabilizing mechanisms have been proposed. The NMR solution structure of LfcinB may be more relevant to membrane interaction than that suggested by the X-ray structure of intact lactoferrin. Based on the solution structure, it is now possible to propose potential mechanisms for the antimicrobial action of LfcinB.

Amino Acid Sequence↗