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Biomedical subjects

L Geng

Publications and source records attributed to L Geng.

At least 37 records · Page 2Linked to original sources
PubMed2001

[Application of video laryngoscope in diagnosis and treatment of laryngeal diseases].

OBJECTIVE: To investigate the value of video laryngoscope in diagnosis of laryngeal diseases. To study the effect of video laryngoscope in treatment of laryngeal diseases. METHOD: Pentax VNL-1530T and Olympus BF-240T video laryngoscope were used in diagnosis of 3628 cases of laryngeal diseases and in treatment of 1221 cases of laryngeal diseases. RESULT: 3628 cases were examined by video laryngoscope, and correct diagnosis was made. 768 cases of vocal cord polyp and 249 cases of vocal cord node were excised, 113 cases of foreign bodies in hypopharynx or larynx were removed. 91 cases of other laryngeal diseases were treated under video laryngoscope. CONCLUSION: Video laryngoscope is a new excellent instrument in diagnosis and treatment of laryngeal disease.

Adolescent↗
PubMed2001

DNA typing system for HLA-A2 alleles by polymerase chain reaction with sequence-specific primers.

OBJECTIVE: To establish a PCR-SSP method for discriminating as many HLA-A*02 alleles, which could easily be introduced into a routine laboratory. METHODS: In this study we typed HLA-A*02 polymorphisms by a sequence-specific primer (SSP) method, which involved round 1 and round 2 PCR reactions to detect 17 HLA-A*02 alleles (they are HLA-A*0201-0217 alleles) covering exon 2 and exon 3. RESULTS: We have found that DNA sample concentration and purity were the most important variable in determining the quality of the result. For identifying correct band size, the size marker used was important. We noticed that different PCR machines performed differently. By this method, we detected 20 HLA-A*02 positive genomic DNA samples and found 4 kinds of HLA-A*02 alleles. They were HLA-A*0201, 0203, 0206 and 0210. CONCLUSION: The HLA-A*02 PCR-SSP method was proven to be a reliable and easily applicable typing method. Our results suggest that the SSP described here provides an optimal HLA-A*02 typing technique that may be useful in selecting donor-recipient pairs in bone marrow transplantation between unrelated individuals.

Alleles↗
PubMed2000

Modification of the composition of polycystin-1 multiprotein complexes by calcium and tyrosine phosphorylation.

Mutations in the PKD1 gene are responsible for >85% of autosomal dominant polycystic kidney disease (ADPKD). The protein product of PKD1, polycystin-1, is a large, modular membrane protein, with putative ligand-binding motifs in the extracelluar N-terminal portion, 9-11 transmembrane domains and an intracellular C-terminal portion with phosphorylation sites. A role for polycystin-1 as a cell surface receptor involved in cell-matrix and cell-cell interactions has been proposed. In this study, we have analyzed polycystin-1 and associated protein distribution in normal human epithelial cells and examined the role of cell-matrix versus cell-cell interactions in regulation of the assembly of polycystin-1 multiprotein complexes. Immunocytochemistry, sucrose density gradient sedimentation, co-immunoprecipitation analyses and in vitro binding assays have shown that polycystin-1 associates with the focal adhesion proteins talin, vinculin, p130Cas, FAK, alpha-actinin, paxillin and pp60c-src in subconfluent normal human fetal collecting tubule (HFCT) epithelia when cell-matrix interactions predominate. Polycystin-1 also forms higher S value complexes with the cell-cell adherens junction proteins E-cadherin, beta- and gamma-catenins in confluent cultures when cell-cell interactions are predominant. Polycystin-1 multiprotein complexes can be disrupted by cytochalasin D but not by colchicine, suggesting involvement of the actin cytoskeleton. Although inhibition of tyrosine phosphorylation by tyrphostin inhibits polycystin-1-FAK interactions, E-cadherin interactions are enhanced. High calcium treatment also increases polycystin-1-E-cadherin interactions.

Cadherins↗
PubMed2000

In vivo interaction of the adapter protein CD2-associated protein with the type 2 polycystic kidney disease protein, polycystin-2.

We identified a developmentally regulated gene from mouse kidney whose expression is up-regulated in metanephrogenic mesenchyme cells when they are induced to differentiate to epithelial cells during kidney organogenesis. The deduced 70.5-kDa protein, originally named METS-1 (mesenchyme-to-epithelium transition protein with SH3 domains), has since been cloned as a CD2-associated protein (CD2AP). CD2AP is strongly expressed in glomerular podocytes, and the absence of CD2AP in mice results in congenital nephrotic syndrome. We have found that METS-1/CD2AP (hereafter referred to as CD2AP) is expressed at lower levels in renal tubular epithelial cells in the adult kidney, particularly in distal nephron segments. Independent yeast two-hybrid screens using the COOH-terminal region of either CD2AP or polycystin-2 as bait identified the COOH termini of polycystin-2 and CD2AP, respectively, as strong interacting partners. This interaction was confirmed in cultured cells by co-immunoprecipitation of endogenous polycystin-2 with endogenous CD2AP and vice versa. CD2AP shows a diffuse reticular cytoplasmic and perinuclear pattern of distribution, similar to polycystin-2, in cultured cells, and the two proteins co-localize by indirect double immunofluorescence microscopy. CD2AP is an adapter molecule that associates with a variety of membrane proteins to organize the cytoskeleton around a polarized site. Such a function fits well with that hypothesized for the polycystin proteins in renal tubular epithelial cells, and the present findings suggest that CD2AP has a role in polycystin-2 function.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Two-dimensional fluorescence correlation in capillary electrophoresis for peak resolution and species identification.

A new spectroscopic dimension-fluorescence intensity correlation--is introduced to enhance peak resolution and species identification in capillary electrophoresis. In two-dimensional correlation CE, a conventional electropherogram is spread into two dimensions through cross-correlation analysis of fluorescence time response. A laser that is sinusoidally modulated in intensity is used as the excitation source. Three channels of information are collected during a CE run: the steady-state intensity, the ac amplitude, and the phase-resolved fluorescence intensity. The correlation between two chosen channels is then evaluated. A two-dimensional correlation electropherogram consists of a plot of the correlation intensity versus two axes of migration time. Through correlation analysis, species discrimination and peak resolution are significantly enhanced without having to physically separate the solutes. Two-dimensional correlation CE showed complete resolution between two overlapping sample peaks with a resolution of 0.28 in the conventional one-dimensional electropherogram. In separations of polycyclic aromatic hydrocarbons by micellar electrokinetic chromatography (MEKC), two-dimensional correlation analysis resolved all overlapping elution peaks unseparable by one-dimensional MEKC, demonstrating the utility of 2D correlation in separation method development. The capability of 2D correlation CE in species identification is demonstrated with a sequence of 39 consecutively injected peaks containing four fluorescent dyes. Species identification in sequencing is achieved without complex data treatment in two-dimensional correlation CE.

Electrophoresis, Capillary↗
PubMed2000

Cardiac defects and renal failure in mice with targeted mutations in Pkd2.

PKD2, mutations in which cause autosomal dominant polycystic kidney disease (ADPKD), encodes an integral membrane glycoprotein with similarity to calcium channel subunits. We induced two mutations in the mouse homologue Pkd2 (ref.4): an unstable allele (WS25; hereafter denoted Pkd2WS25) that can undergo homologous-recombination-based somatic rearrangement to form a null allele; and a true null mutation (WS183; hereafter denoted Pkd2-). We examined these mutations to understand the function of polycystin-2, the protein product of Pkd2, and to provide evidence that kidney and liver cyst formation associated with Pkd2 deficiency occurs by a two-hit mechanism. Pkd2-/- mice die in utero between embryonic day (E) 13.5 and parturition. They have structural defects in cardiac septation and cyst formation in maturing nephrons and pancreatic ducts. Pancreatic ductal cysts also occur in adult Pkd2WS25/- mice, suggesting that this clinical manifestation of ADPKD also occurs by a two-hit mechanism. As in human ADPKD, formation of kidney cysts in adult Pkd2WS25/- mice is associated with renal failure and early death (median survival, 65 weeks versus 94 weeks for controls). Adult Pkd2+/- mice have intermediate survival in the absence of cystic disease or renal failure, providing the first indication of a deleterious effect of haploinsufficiency at Pkd2on long-term survival. Our studies advance our understanding of the function of polycystin-2 in development and our mouse models recapitulate the complex human ADPKD phenotype.

Animals↗
PubMed2000

[Detection of circulating pemphigus vulgaris antibodies in families of patients with pemphigus vulgaris].

OBJECTIVE: To detect the autoantibodies on 30 first-degree relatives of 12 patients with pemphigus vulgaris (PV), who may share the same susceptible gene with the patients. METHODS: Immunoblot and indirect immunofluorescence (IIF) method were used to detect circulatory PV antibodies in the peripheral blood. RESULTS: PV antibodies were found in 19 of 30 (63%) relatives by both methods, but 10 control serum were all negative by both methods. CONCLUSION: Healthy PV antibody carriers may also exist in high frequency in the first-degree relatives of Chinese PV patients.

Adolescent↗
PubMed1999

Cutting edge: SLP-76 cooperativity with FYB/FYN-T in the Up-regulation of TCR-driven IL-2 transcription requires SLP-76 binding to FYB at Tyr595 and Tyr651.

SLP-76 (Src homology (SH) 2-domain-containing leukocyte protein of 76 kDa) and FYB/SLAP (FYN-T-binding protein/SLP-76-associated protein) are two hemopoietic cell-specific adaptor proteins downstream of TCR-activated protein tyrosine kinases. SLP-76 has been implicated as an essential component in T cell signaling. FYB is selectively phosphorylated by FYN-T, providing a template for the recruitment of FYN-T and SLP-76 SH2 domains. Coexpression of FYN-T, FYB, and SLP-76 can synergistically up-regulate IL-2 production in T cells upon TCR ligation. In this report, we show that two tyrosines, Tyr595 and Tyr651, of FYB are major sites of phosphorylation by FYN-T and mediate binding to SLP-76 in Jurkat T cells. Furthermore, the synergistic up-regulation of IL-2 promoter activity in the FYN-T-FYB-SLP-76 pathway is contingent upon the interaction between FYB and SLP-76, but not the interaction between FYB and FYN-T. These observations define a pathway by which SLP-76 interacts with downstream components in the up-regulation of T cell cytokine production.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Identification of phosphorylation sites in the PKD1-encoded protein C-terminal domain.

The PKD1-encoded protein, "polycystin-1", has a large N-terminal extracellular portion, multiple transmembrane domains, and a short intracellular C-terminal tail with four tyrosine residues and two putative sites for serine phosphorylation. Its function in kidney development and autosomal dominant polycystic kidney disease (ADPKD) is still unknown. We have subcloned the cDNA encoding the polycystin-1 C-terminal domain (PKD1-CTD) into a prokaryotic expression vector, and site-directed mutagenesis was performed to target the four tyrosine residues and four serine residues in two putative phosphorylation sites. In vitro phosphorylation assays were conducted on both wild type and mutant PKD1-CTD fusion proteins. It was found that the wild type PKD1-CTD and all mutant fusion proteins, except S4251G/S4252G, could be phosphorylated by lysates from cultured normal human renal collecting tubule (NHCT) cells, as well as by commercially purified cAMP-dependent protein kinase (PKA). The phosphorylation of the PKD1-CTD fusion protein by NHCT lysates was greatly enhanced by cAMP and its analog 8-Br-cAMP, and inhibited by the specific PKA inhibitors PKI(6-22) and H-89. Activators and inhibitors of protein kinase C (PKC) had no effects on the phosphorylation of the PKD1-CTD fusion protein. Using commercially purified pp60(c-src) (c-src) it was also shown that the PKD1-CTD fusion protein could be phosphorylated by c-src in vitro, and that this phosphorylation could be abolished by a mutation Y4237F. By comparing the amino acid sequence at 4249-4253 (RRSSR) with the consensus sequence for PKA phosphorylation (RRXSX), we suggest that the serine residue at 4252 is the target of phosphorylation by a cAMP-dependent protein kinase in NHCT cell lysates. In addition, we suggest that Y4237 might be phosphorylated by c-src in living cells.

Cells, Cultured↗
PubMed1999

Lipofuscin accumulation in iris pigment epithelial cells exposed to photoreceptor outer segments.

Lipofuscin accumulates in retinal pigment epithelial (RPE) cells with increasing age in response to phagocytotic degradation of worn-out disks of photoreceptor outer segments (POS). This study investigates the ability of iris pigment epithelial (IPE) cells to ingest POS and compares the phagocytotic capacity of RPE and IPE cells by measuring their amounts of lipofuscin-specific autofluorescence.IPE and RPE cells were isolated from the same calf eyes. After growing to confluence, primary cultures from 15 eyes (69 wells) were exposed to bovine POS daily, or kept as controls. After 1, 2 and 3 weeks, respectively, the cellular amounts of lipofuscin were quantified by a computerized static cytofluorometer system (Nikon Microphot SA with a Nikon p102 photometer). Lipofuscin-specific fluorescence (550 nm barrier filter eliminating nonspecific autofluorescence) of 100 randomly selected individual cells from each well was measured. Electron microscopy revealed phagosomes (containing POS disk membranes) and lipofuscin-like inclusions in the cytoplasm of RPE and IPE cells. In both IPE and RPE cultures, the POS-challenged cells had significantly higher (P<0. 01) lipofuscin-specific fluorescence than the control cells. The fluorescence levels were higher in POS-challenged RPE cells as compared to IPE cells after 1 (P< 0.001), 2 (P<0.01) and 3 (P<0.001) weeks, respectively. The fluorescence level of POS-fed IPE cells at 3 weeks was 56% of that of RPE cells.IPE cells have the ability to phagocytose POS, resulting in an accumulation of lipofuscin intracellularly. The amount of lipofuscin was lower in IPE cells compared to RPE cells, probably reflecting the lower phagocytotic capacity of IPE.

Animals↗
PubMed1999

Atypical immature metaplasia (AIM) of the cervix: is it related to high-grade squamous intraepithelial lesion (HSIL)?

Atypical immature metaplasia (AIM) is a poorly characterized cervical lesion with uncertain biological and clinical significance. AIM shares some, but not all, morphological features of squamous intraepithelial lesions (SILs). SILs are characterized by human papillomavirus (HPV) positivity and increased cellular proliferation, but these features have not been fully evaluated in AIM. Genomic DNA was extracted from 27 microdissected cervical biopsy specimens diagnosed as AIM. HPV DNA was detected by polymerase chain reaction (PCR), using two different sets of L1 gene consensus primers. HPV types were identified by sequence analysis of PCR products and comparison with published HPV sequences. The cell proliferation index was assessed by immunohistochemical staining for Ki-67 (MIB-1) antigen and expressed as the percentage of Ki-67-positive cells. Comparison groups included normal cervix (n = 10) and low-grade (LSILs, n = 19) and high-grade squamous intraepithelial lesions (HSILs, n = 11). Intermediate- or high-risk HPV DNA was detected in 67% (18 of 27) of AIM cases. Low-risk HPV DNA was not detected in any of the specimens. The Ki-67 index in AIM (mean, 33.0 +/- 20.3; median, 29) was comparable to that of LSILs (mean, 21.4 +/- 4.6; median, 21) and was significantly higher than that of normal cervix (mean, 11.0 +/- 2.1; median, 11) (P< .01) and lower than that of HSILs (mean, 60.4 +/- 13.2; median, 60) (P < .01). Of the cases with available follow-up, HPV-positive AIMs were significantly more likely to have a concurrent or subsequent diagnosis of typical HSIL (12 of 15, 80%) than HPV-negative AIMs (one of six, 45%) (P = .014). The wide range of Ki-67 indices and variable HPV status in AIM suggest that AIM represents a heterogeneous group of lesions including bona fide HSILs (high-risk HPV-positive, high Ki-67 index), antecedents (precursors?) of HSILs (high-risk HPV-positive, low to moderate Ki-67 index), and benign reactive conditions (HPV-negative, variable Ki-67 index). HPV testing may be useful in the assessment of atypical epithelial proliferations of the cervix for which a diagnosis of AIM is considered.

Cell Division↗
PubMed1999

Cell culture of sporadic hepatitis E virus in China.

The isolation and identification of the 87A strain of epidemic hepatitis E virus (HEV) by means of cell culturing have been described previously. This paper reports the successful isolation of a sporadic HEV strain (G93-2) in human lung carcinoma cell (A549) cultures. The etiology, molecular and biological properties, and serological relationship of this new strain to other, epidemic HEV strains are described. The propagation of both sporadic and epidemic HEV strains in a cell culture system will facilitate vaccine research.

Acids↗
PubMed1999

[Cloning the 5 ' end fragment of ST13 cDNA by nested PCR].

OBJECTIVE: To clone and sequence the 5' -end fragment of ST13 cDNA. METHODS: The 5' -end fragment of the ST13cDNA was amplified directly from cDNA library by nested PCR method, and cloned into pGEM-T. easy vector, then the sequencing of the inserted PCR product was performed. RESULTS: After primary and secondary PCR, two PCR products obtained with the size of about 550bp and 480bp, respectively, were both proved to be the 5' -end of ST13 cDNA by sequencing. CONCLUSION: the method presented is very simple and effective for cloning and sequencing the 5'-end of a target gene.

Cloning, Molecular↗
PubMed1999

The PKD1 gene product, "polycystin-1," is a tyrosine-phosphorylated protein that colocalizes with alpha2beta1-integrin in focal clusters in adherent renal epithelia.

Mutations in the PKD1 gene are responsible for autosomal dominant polycystic kidney disease (ADPKD). Although PKD1 has been cloned and shown to be expressed at high levels in the fetal ureteric bud and ADPKD cystic epithelia in the human kidney, the function of its encoded protein, "polycystin-1" is unknown. In this study we used primary and immortalized human renal epithelial cell lines derived from normal fetal, adult, and ADPKD kidneys, that endogenously express PKD1, to study the biologic function of the polycystin-1 protein. ADPKD renal epithelial cells expressed high levels of polycystin-1 protein and showed increased adhesion to type I collagen by comparison with normal adult human renal epithelia that expressed little polycystin. Adherent ADPKD cells also expressed high levels of alpha2beta1-integrin and their attachment was inhibited by a functional monoclonal antibody to alpha2-integrin. Double labeling and confocal microscopy as well as coimmunoprecipitation analysis showed overlapping colocalization of polycystin-1 with alpha2beta1-integrin as well as with the focal adhesion proteins vinculin and paxillin in multiprotein clusters localized to focal areas of cell membrane contact with type I collagen matrix after short periods of attachment. Immunoprecipitation and Western immunoblot studies also showed that polycystin-1 was posttranslationally modified by tyrosine phosphorylation. These studies suggest that the PKD1-encoded protein is part of a large multiprotein complex in epithelial cells that functions in the regulation of extracellular matrix interactions with the plasma membrane and cell cytoskeleton.

Amino Acid Sequence↗
PubMed1999

[Relationship between the bone marrow cell proliferation and the prognosis in childhood acute lymphoblastic leukemia].

OBJECTIVE: To study the expression of proliferative antigens in leukemic cells and the relationship between the cell proliferation activity and the prognosis. METHODS: The labeling index (LI) of CD71, Ki-67 and PCNA in normal and leukemic cells were measured by flow cytometry. RESULTS: In normal children, the LI of CD71, Ki-67 and PCNA was (32.18 +/- 16.66)%, (4.82 +/- 9.27)% and (19.69 +/- 25.11)%, respectively, while in ALL children, which was (33.66 +/- 21.52)%, (32.14 +/- 23.59)% and (47.46 +/- 30.96)%, respectively. The PCNA LI of leukemia cells was (47.46 +/- 30.96)% and (27.28 +/- 12.51)% when patients was at presentation and remitted for 3 years, respectively, and was (52.59 +/- 32.00)% and at presentation in 28 CCR children and and (26.94 +/- 14.48)% in 7 high-risk group ALL children (F = 8.877, P < 0.01), respectively. CONCLUSIONS: The proliferation, LI of leukemic cells was higher than that of normal cells, and reduced to normal level after treatment. The higher PCNA proliferation of untreated cells was a favourable marker for prognosis.

Adolescent↗
PubMed1999

[Correlation of conformational change with enzyme activity of glucose isomerase in denaturants monitored with high performance liquid chromatography].

Glucose isomerase (GI) can catalyze in vitro the isomerization of D-glucose to D-fructose. So it is an extremely important industrial enzyme in the commercial conversion of starch to high fructose syrups. In the previous papers, we have purified and characterized the enzyme from streptomyces diastaticus M1033 of China and obtained the crystal structures by X-ray. In this paper, a method for measurement of the dynamic conformational change procedure of glucose isomerase in various concentrations of denaturants by HPLC has been established. At first the relative molecular mass of GI in solution is measured by HPLC on PROTEIN PAK 300SW (7.5 mm i.d. x 30 cm) column. The relative molecular mass of GI is about 150,000. So GI exists as tetramer in the solution without denaturants. In 0-5 mol/L guanidine hydrochloride, incubated at 30 degrees C for 30 min, GI is gradually dissociated into monomer, and at the same time its activity gradually disappears. In various concentrations of urea and incubation at 30 degrees C for 30 min (or 60 degrees C for 1 h), the results are different from that in guanidine, because the monomers peaks of GI is not found. Only in certain concentrations of urea, the small dimer peaks of GI is found, but the activity of GI significantly disappears. Moreover as the increase of the urea concentration, the retention time of tetramer peak is gradually decreases. From the fluorescence spectra, we found the conformation of GI changed in the solution of urea. So perhaps in urea, the conformation of GI become a little unfolded, and the active region is partly damaged, which makes GI partly inactive. Dissociation into inactive monomers and conformation partly unfolding are all the reason of GI inactivation in denaturants.

Aldose-Ketose Isomerases↗
PubMed1998

Widespread expression of an autoantigen-GAD65 transgene does not tolerize non-obese diabetic mice and can exacerbate disease.

Glutamic acid decarboxylase (GAD)65 is a pancreatic beta cell autoantigen implicated as a target of T cells that initiate and sustain insulin-dependent diabetes mellitus (IDDM) in humans and in non-obese diabetic (NOD) mice. In an attempt to establish immunological tolerance toward GAD65 in NOD mice, and thereby to test the importance of GAD in IDDM, we generated three lines transgenic for murine GAD65 driven by a major histocompatibility complex class I promoter. However, despite widespread transgene expression in both newborn and adult mice, T cell tolerance was not induced. Mononuclear cell infiltration of the islets (insulitis) and diabetes were at least as bad in transgenic mice as in nontransgenic NOD mice, and in mice with the highest level of GAD65 expression, disease was exacerbated. In contrast, the same transgene introduced into mouse strain, FvB, induced neither insulitis nor diabetes, and T cells were tolerant to GAD. Thus, the failure of NOD mice to develop tolerance toward GAD65 reflects at minimum a basic defect in central tolerance, not seen in animals not predisposed to IDDM. Hence, it may not be possible experimentally to induce full tolerance toward GAD65 in prediabetic individuals. Additionally, the fact that autoimmune infiltration in GAD65 transgenic NOD mice remained largely restricted to the pancreas, indicates that the organ-specificity of autoimmune disease is dictated by tissue-specific factors in addition to those directing autoantigen expression.

Animals↗