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Y Ning

Publications and source records attributed to Y Ning.

At least 55 records · Page 3Linked to original sources
PubMed1997

A microfabricated device for rapid protein identification by microelectrospray ion trap mass spectrometry.

Nanoelectrospray mass spectrometry, the infusion at low flow rates of unseparated peptide mixtures representing protein proteolytic digests into an electrospray ionization mass spectrometer (MS), has been shown to be a suitable method for the analysis of small amounts of proteins. However, the current technique is time consuming, tedious, and difficult to automate. We used microfabrication technologies to construct a device for the sequential infusion of different peptide samples into an electrospray ionization MS without the need for sample manipulation. In this device, etched sample and buffer reservoirs are connected via etched channels to microelectrospray ion source. Peptide samples, typically unseparated tryptic digests of proteins, are applied to different reservoirs. A flow of liquid originating from a specific reservoir is generated and selectively directed toward the microsprayer and the MS by electroosmotic pumping. The analyte proteins are identified by searching sequence databases with the information contained in the collision-induced spectra of selected peptides. With this system, we have achieved a limit of detection in the low femtomoles per microliter range for peptide standards. We also show that samples deposited in different reservoirs can be sequentially mobilized without cross-contamination and that proteins can be conclusively identified at the low femtomoles per microliter level. The successful coupling online of microfabricated devices to an electrospray ionization MS represents an essential step toward the construction of automated, high-throughput, and high-sensitivity analytical systems.

Amino Acid Sequence↗
PubMed1997

Transcriptional regulation of surfactant proteins SP-A and SP-B by phorbol ester.

Phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) activate protein kinase C and have been previously shown to down-regulate surfactant proteins SP-A and SP-B in H441 adenocarcinoma cells. We used H441 cells and human fetal lung to further study the mechanism of TPA action and to examine physiologic relevance. In H441 cells, TPA (10 nM) treatment for 24 h decreased SP-A mRNA content to approximately 5% of control cells, with half-maximal effect at approximately 0.5 nM, and reduced SP-A gene transcription rate to 28% of control after 8 h exposure. In cells cultured in the presence of dexamethasone, which increases the low basal level of SP-B expression, TPA decreased both SP-B mRNA content (approximately 8% of control) and rate of transcription (7% of control). In cultured human fetal lung explants, TPA decreased SP-A and SP-B protein and mRNA in a time- and dose-dependent fashion, with half-maximal effect on mRNAs at approximately 3 nM and approximately 50% inhibition after 24 h of exposure, and similarly reduced SP-A and SP-B gene transcription (approximately 55% of control at 8-24 h). We conclude that TPA acts primarily at the level of gene transcription to down-regulate both SP-A and SP-B in H441 and fetal lung cells, and we speculate that inflammatory and other agents that act through PKC may modulate expression of the surfactant proteins and alter surfactant function in vivo.

Culture Techniques↗
PubMed1997

Inhibition of aflatoxin B1-induced initiation of hepatocarcinogenesis in the rat by green tea.

Several studies have demonstrated that green tea (GT) inhibits various chemically induced cancers in experimental animals. In the present study, effect of GT has been examined on the initiation of aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rat. Young male Fischer rats were given AIN-76A diet with or without 0.5% instant GT powder in their drinking water for 2 or 4 weeks. Initiation was examined by hepatic AFB1-DNA binding in vivo, AFB1 metabolism in vitro and by the appearance of AFB1-induced glutathione S-transferase placental form (GST-P)-positive hepatocytes detected by immunohistochemical method. There was no influence of GT feeding on microsome-mediated AFB1 binding to exogenous DNA. However, GT feeding enhanced microsome-mediated formation of non-toxic hydroxylated metabolites of AFB1 by 2-3-fold. Hepatic nuclear AFB1-DNA binding in vivo was significantly inhibited by about 20-30% in animals pretreated with GT: AFB1-induced GST-P positive single hepatocytes were inhibited significantly by 60-70% in rats pretreated with GT. These results suggest that feeding of GT inhibits initiation of AFB1-induced hepatocarcinogenesis in the rat by modulation of AFB1 metabolism, thereby inhibiting AFB1-DNA binding and AFB1-induced GST-P-positive hepatocytes.

Aflatoxin B1↗
PubMed1997

Spectral karyotyping refines cytogenetic diagnostics of constitutional chromosomal abnormalities.

Karyotype analysis by chromosome banding is the standard method for identifying numerical and structural chromosomal aberrations in pre- and postnatal cytogenetics laboratories. However, the chromosomal origins of markers, subtle translocations, or complex chromosomal rearrangements are often difficult to identify with certainty. We have developed a novel karyotyping technique, termed spectral karyotyping (SKY), which is based on the simultaneous hybridization of 24 chromosome-specific painting probes labeled with different fluorochromes or fluorochrome combinations. The measurement of defined emission spectra by means of interferometer-based spectral imaging allows for the definitive discernment of all human chromosomes in different colors. Here, we report the comprehensive karyotype analysis of 16 samples from different cytogenetic laboratories by merging conventional cytogenetic methodology and spectral karyotyping. This approach could become a powerful tool for the cytogeneticists, because it results in a considerable improvement of karyotype analysis by identifying chromosomal aberrations not previously detected by G-banding alone. Advantages, limitations, and future directions of spectral karyotyping are discussed.

Chromosome Aberrations↗
PubMed1997

Glucocorticoid regulation of surfactant components in immature lambs.

To assess effects of dose and duration of glucocorticoid exposure on maturation of the fetal lung, we administered single or multiple doses of betamethasone (0.5 mg/kg im) to pregnant sheep for 2 or 21 days before preterm delivery at 125 days of gestation. Lung function (compliance, lung volume at 40 cmH2O pressure, and ventilatory efficiency index) was increased after two to four weekly doses of glucocorticoid (2.5- to 4-fold increase) and after 48 h of exposure (1.4- to 2.3-fold). Total protein of lavage fluid decreased similarly with three doses, four doses, and 48 h of treatment. In lambs with long-term exposure to betamethasone, there was a similar, dose-dependent increase in concentrations of saturated phosphatidylcholine and surfactant proteins A (SP-A) and B (SP-B) (maximal 2- to 3-fold in tissue and 10- to 15-fold in lavage fluid). Levels of SP-A and SP-B were closely correlated in lavage fluid. In animals treated for 48 h, only tissue SP-B was increased (2.7-fold). We conclude that 48 h of glucocorticoid treatment improves lung function in the premature lamb without a detectable increase in lavage surfactant components and that longer exposure to antenatal glucocorticoid increases surfactant lipid and proteins in a coordinated fashion. The enhanced response with repetitive dosing indicates that the process of glucocorticoid-induced lung maturation is either reversible and/or gestational age dependent.

Animals↗
PubMed1997

Molecular characterization of a 130-kb terminal microdeletion at 22q in a child with mild mental retardation.

We have analyzed a recently described 22q13.3 microdeletion in a child with some overlapping features of the cytologically visible 22q13.3 deletion syndrome. Patient NT, who shows mild mental retardation and delay of expressive speech, was previously found to have a paternal microdeletion in the subtelomeric region of 22q. In order to characterize this abnormality further, we have constructed a cosmid/P1 contig covering the terminal 150 kb of 22q, which encompasses the 130-kb microdeletion. The microdeletion breakpoint is within the VNTR locus D22S163. The cloning of the breakpoint sequence revealed that the broken chromosome end was healed by the addition of telomeric repeats, indicating that the microdeletion is terminal. This is the first cloned terminal deletion breakpoint on a human chromosome other than 16p. The cosmid/P1 contig was mapped by pulsed-field gel electrophoresis analysis to within 120 kb of the arylsulfatase A gene, which places the contig in relation to genetic and physical maps of the chromosome. The acrosin gene maps within the microdeletion, approximately 70 kb from the telomere. With the distal end of chromosome 22q cloned, it is now possible to isolate genes that may be involved in the overlapping phenotype of this microdeletion and 22q13.3 deletion syndrome.

Cell Line, Transformed↗
PubMed1996

Multicolor spectral karyotyping of human chromosomes.

The simultaneous and unequivocal discernment of all human chromosomes in different colors would be of significant clinical and biologic importance. Whole-genome scanning by spectral karyotyping allowed instantaneous visualization of defined emission spectra for each human chromosome after fluorescence in situ hybridization. By means of computer separation (classification) of spectra, spectrally overlapping chromosome-specific DNA probes could be resolved, and all human chromosomes were simultaneously identified.

Animals↗
PubMed1996

Isolation of the human chromosome 22q telomere and its application to detection of cryptic chromosomal abnormalities.

A number of human telomeres have been successfully cloned using a modified yeast artificial chromosome (YAC) vector (half-YAC) cloning strategy, but to date, human chromosome 22q has not been identified by this approach. We used an alternative approach of genomic walking, starting from a subtelomeric sequence, Tel-Bam3.4. present on a number of human chromosomes including 22q. This approach was successful in the development of a cosmid contig representing the terminal 140 kb of human chromosome 22q, providing telomeric closure of the genetic and physical maps for 22q. The most distal region of the contig contains subtelomeric repeats which crosshybridize to a number of chromosomes, while the proximal sequences are unique for 22q. The unique sequence cosmid was used as a 22qter-specific probe for fluorescence in situ hybridization (FISH) analysis, which confirmed that this cosmid was distal to the most telomeric marker previously available for chromosome 22. In addition, this cosmid was used to document a 22q terminal deletion that was not detectable by conventional cytogenetic analysis. Unique telomere-specific FISH probes such as this one will have significant diagnostic value in the detection of cryptic deletions and translocations in patients with unexplained mental retardation and other patient populations.

Cerebroside-Sulfatase↗
PubMed1996

Characterization of familial partial 10p trisomy by chromosomal microdissection, FISH, and microsatellite dosage analysis.

Unbalanced translocations are a frequent cause of multiple congenital anomalies in children. Translocations as small as 2-5 Mb of DNA are detectable by G-banding under optimal conditions. Some of these small translocations are visible but cannot be characterized cytogenetically due to the lack of characteristic banding on Giemsa preparations. We have combined chromosomal microdissection and fluorescence in situ hybridization (FISH) to identify the origin of a small translocated segment in three members of a family with a derivative chromosome 9 and multiple anomalies, including several ophthalmologic anomalies. We microdissected the abnormal region of the derivative 9 chromosome and used this DNA to generate a FISH probe. This probe hybridized to distal 10p on the metaphase spread of the proband, indicating the origin of the translocated segment. A whole 10p FISH probe confirmed the origin by hybridizing to the translocated segment of the derivative chromosome. FISH was then performed with a whole chromosome 9 painting probe and excluded the presence of a reciprocal, balancing translocation. We then studied the chromosome 10 partial duplication with microsatellite markers to better characterize the chromosomal segment that caused these phenotypic features. By examining the involved areas with distal 10p and 9p microsatellite markers, we were able to demonstrate a minimum of 9 Mb of trisomic 10p DNA with a chromosomal breakpoint between 10p14-10p15. We then compared this family's clinical findings to those of individuals with partial 10p trisomy who had been reported in the literature. The clinical phenotypes seen in this family are similar to, but milder than, the phenotypes of persons with the larger partial trisomies of 10p that were diagnosable by cytogenetic analysis alone. This study shows that microdissection and DNA markers can be used to precisely define small translocations that are difficult to identify by conventional G-banded chromosome analysis.

Abnormalities, Multiple↗
PubMed1996

Identification of a novel paternally expressed transcript adjacent to snRPN in the Prader-Willi syndrome critical region.

Small nuclear ribonucleoprotein-associated polypeptide N (snRPN) and an anonymous transcript, PAR-5, are two of the paternally expressed transcripts mapped to the Prader-Willi syndrome critical region. Using long-range PCR, we have isolated the genomic interval between snRPN and PAR-5, identified a novel transcript in this region, and termed it PAR-SN. Northern analysis demonstrates that PAR-SN is expressed in brain, skeletal muscle, and heart. Like snRPN and PAR-5, PAR-SN is expressed exclusively from the paternal homolog in cultured lymphoblasts. Sequence analysis of the transcript revealed no significant open reading frame but did include a polymorphic dinucleotide repeat (CA)17.

Chromosome Mapping↗
PubMed1996

Regulation of surfactant protein D in human fetal lung.

Surfactant protein D (SP-D) is a collagenous glycoprotein, produced by lung type II cells, that has structural and functional similarities with SP-A. In this study we postulated that SP-D and SP-A gene expression are regulated in a similar fashion to provide a coordinated local immune defense response to pulmonary infection. We determined content of SP-D protein and mRNA in second-trimester fetal lung and in postnatal tissue by protein blotting and hybridization analyses. Low levels of SP-D mRNA and protein were detected at 16 wk gestation, before appearance of SP-A, and levels increased during gestation. The content of SP-D did not change during 5 days of explant culture, whereas SP-A increased manyfold. Dexamethasone treatment during culture increased SP-D mRNA and protein about 2-fold with maximal response after 1 to 3 days' exposure to 100 nM steroid; under the same conditions SP-A mRNA content is inhibited. There was no significant change in SP-D mRNA after treatment of explants with adenosine 3',5'-monophosphate (cAMP) analog or interferon-gamma, agents which increase SP-A gene expression, nor after exposure to phorbol ester, tumor necrosis factor-alpha, or lipopolysaccharide at concentrations that reduced levels of SP-A mRNA by approximately 50%. We conclude that SP-D in the human lung is under developmental and glucocorticoid regulation occurring at a pretranslational level. SP-D is not influenced by inflammatory mediators that regulate SP-A, suggesting that these two proteins are not coordinately regulated in response to lung infection.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Detection of a subtle rearrangement of chromosome 22 using molecular techniques.

Conventional cytogenetics is a useful clinical tool that has a lower limit of sensitivity of 2-5 Mb for detection of duplications or deletions. Because the threshold of clinically significant aneusomy is below this range, there is a need for approaches to improve the sensitivity of the detection of aneusomy. We have implemented a system of screening for subtle unbalanced translocations in children with multiple congenital anomalies of unknown cause. Our approach uses subtelomeric microsatellite markers to detect small areas of segmental aneusomy due to unbalanced translocations. Herein we report a patient with severe multiple congenital anomalies and a normal karyotype who was diagnosed by this approach. Microsatellite markers from 41 telomeres were analyzed and were normal with the exception of those on distal chromosome 22. Further analysis with additional microsatellites and fluorescent in situ hybridization confirmed duplication of 22q13.2-qter. We conclude that microsatellite screening can detect subtle unbalanced translocations in children with severe anomalies.

Abnormalities, Multiple↗
PubMed1995

The vascular cell adhesion molecule (VCAM-1) is expressed on a subset of lymphoid and myeloid leukaemias.

The expression of the vascular cell adhesion molecule (VCAM-1), recently identified as cytokine-inducible ligand of the beta 1-integrin VLA-4, was investigated on normal and malignant haemopoietic precursors as well as on haemopoietic cell lines. VCAM-1 was demonstrated on > 20% blasts in 4/22 acute myeloid leukaemia (AML) and 6/10 acute lymphoblastic leukaemia (ALL) specimens but was absent from CD34+ normal bone marrow precursor cells. Interestingly, the VCAM-1+ AMLs classified as M1 and M5 simultaneously expressed N-CAM (CD56), a member of the immunoglobulin family. In ALL, VCAM-1 expression was restricted to Calla+ CD19+ precursors of the c-ALL subtype. VCAM-1 was also found on some cell lines, mainly of the B-lymphocytic type. Furthermore, in 13/20 chronic lymphocytic leukaemia (CLL) samples > 20% of the CD19+ B-lymphocytic precursors carried VCAM-1, which seemed to correlate with more advanced disease. Therefore VCAM-1 expression appeared to characterize leukaemic cells of the B-cell lineage as well as a CD56+ subset of AML. Since its expression was clinically correlated with dermal infiltrates of leukaemic cells in AML and with advanced Rai stages in CLL, VCAM-1 may play a role in enhanced adhesion of the malignant cells to tissues and/or to each other.

Acute Disease↗
PubMed1994

Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen.

We here describe a rapid and simple expression screen method that has allowed us to isolate cDNAs coding for inhibitors of DNA synthesis from senescent human diploid fibroblasts. The assay involved transient transcriptional overexpression of a gene product encoded by a cDNA in a proliferating cell, on the assumption that this would be sufficient to block DNA synthesis in a short-term assay using tritiated thymidine autoradiography. Three cDNAs, referred to as senescent cell-derived inhibitors (sdi), that exhibit DNA synthesis-inhibitory activity when introduced into young cycling cells, were successfully identified. Expression of one of the cDNAs, sdi1, increased 10- to 20-fold in senescent compared with young cells and the increase in RNA closely paralleled the onset of the senescent phenotype and loss of cell proliferation. sdi1 expression was also increased in young cells made nondividing (quiescent) by deprivation of growth factors or contact inhibition. Following serum stimulation, RNA levels of sdi1 in quiescent cells were initially increased, but then declined to low levels just prior to the entry of the cells into S phase. In contrast, RNA levels of sdi1 in senescent cells failed to decline, suggesting a role for this gene in maintaining the senescent phenotype. The sdi1 gene has been mapped to the p arm of chromosome 6.

Amino Acid Sequence↗
PubMed1994

[A study of prostaglandine E(2) level in root canal fluid of patients with periapical lesions]

It has been reported that there is close relationship between prostaglandine E(PGE) and the pathogenesis of periapical lesions, In this study the root canal fluid(RCF) of 35 cases with acute,chronic and convalescent periradicular lesions were collected with assessed by the method of radioimmunoassay.The results showed:RCF-PGE(2) levels were increased with the severity of periapical lesion(P<0.01).The PGE level was highest in acute periradicular lesions,middle in chronic,and lowest in convalescent.RCF-PGE(2) levels were significantly different between either two groups of the three groups of patients(P<0.01-0.05).This study repeatedly proved that PGE(2) has certain effect in pathogenesis of periapical lesions and it is meaningful in guiding clinical therapy.It was also pointed out that RCF is one of worthwhile samples in the study of periradicular lesion.

Journal Article↗
PubMed1993

FK506 binding to the 56-kilodalton immunophilin (Hsp56) in the glucocorticoid receptor heterocomplex has no effect on receptor folding or function.

It has recently been reported that the hsp56 component of glucocorticoid receptor heterocomplexes is an immunophilin of the FK506 binding class [Yem, A. W., Tomasselli, A. G., Heinrikson, R. L., Zurcher-Neely, H., Ruff, V. A., Johnson, R. A., & Deibel, M. R. (1992) J. Biol. Chem. 267, 2868-2871; Tai, P. K., Albers, M. W., Chang, H., Faber, L. E., & Schreiber, S. L. (1992) Science 256, 1315-1318]. The existence of binding proteins for these two potent groups of immunosuppressants in the same molecular complex compels us to ask whether FK506 affects glucocorticoid receptor function. We show here that hsp56 is a component of the native L-cell glucocorticoid receptor heterocomplex and that [3H]FK506 binds to the immunopurified, untransformed receptor complex. However, at concentrations in excess of those required to occupy all of its binding sites on hsp56, FK506 does not affect the steroid binding activity of the receptor nor does it stabilize or dissociate the receptor-hsp90 complex. FK506 does not affect steroid-mediated hsp90 dissociation from the receptor in vitro, and it does not affect steroid-mediated nuclear transfer of the receptor or steroid-mediated transcriptional enhancement from a reporter in intact cells. When immunopurified mouse glucocorticoid receptor is reconstituted into a heat shock protein complex by rabbit reticulocyte lysate, hsp56 is present in the reconstituted complex in addition to hsp90 and hsp70. FK506, however, does not affect reconstitution of the complex or return of the receptor to the steroid binding state, a change of conformation that occurs upon receptor association with hsp90.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

"PCR karyotype" of monochromosomal somatic cell hybrids.

Following fusion of human diploid fibroblast-derived microcells with mouse A9 cells, we isolated seven monochromosomal hybrids containing a single human chromosome 2, 3, 5, 12, 15, 20, or 22. Cytogenetic analysis as well as PCR karyotyping (chromosome-specific banding pattern generated by Alu-PCR) was performed on all the hybrids. We here present, for the first time, the specific PCR karyotypes of human chromosomes 2 and 20.

Animals↗