Use of calcium channel blockers and risk of suicide. Perscriptions for particular drug are influenced by numerous factors.
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Calcium Channel Blockers↗
Biomedical subjects
Y T Chen
Publications and source records attributed to Y T Chen.
Mutational analysis of the conserved cationic residues of Bacillus stearothermophilus 6-phosphoglucose isomerase.
The importance in catalysis of the conserved arginine (R207) and lysine residues (K144, K294, K356, and K425) of 6-phosphoglucose isomerase from Bacillus stearothermophilus was assessed by site-directed mutagenesis and kinetic analysis. In general mutations had minor effects on the Km for fructose 6-phosphate. More dramatic effects were seen on kcat. The R207A mutant had a five orders of magnitude decrease in kcat relative to the wild-type enzyme. There was a significant recovery, by three orders of magnitude, in the kcat for the R207K mutant. The results suggest that the positive charge provided by R207 plays a critical role in the isomerization reaction. K425 was substituted with alanine, valine, phenylalanine, tryptophan and aspartate. All mutant enzymes at position 425 had kcat decreased in the range of several-hundred-fold. For the other mutants, K294A and K144A, the kcat values were 3.5% and 27% of the wild-type enzyme, respectively. No effects on catalysis were observed for the K356A mutant. The results suggest that R207, K144, K294, and K425 are located in the active site of the enzyme. The active-site location and the catalytic roles of K425 and K294 are supported further by the inhibitory effects of pyridoxal 5'-phosphate on enzymatic activities. The data also confirm the importance of K425 and K144 anticipated by the affinity labeling studies of the corresponding residues by pyridoxal 5'-phosphate in pig muscle phosphoglucose isomerase.
Amino Acid Sequence↗
Mechanisms of epithelial cell-cell adhesion and cell compaction revealed by high-resolution tracking of E-cadherin-green fluorescent protein.
Cadherin-mediated adhesion initiates cell reorganization into tissues, but the mechanisms and dynamics of such adhesion are poorly understood. Using time-lapse imaging and photobleach recovery analyses of a fully functional E-cadherin/GFP fusion protein, we define three sequential stages in cell-cell adhesion and provide evidence for mechanisms involving E-cadherin and the actin cytoskeleton in transitions between these stages. In the first stage, membrane contacts between two cells initiate coalescence of a highly mobile, diffuse pool of cell surface E-cadherin into immobile punctate aggregates along contacting membranes. These E-cadherin aggregates are spatially coincident with membrane attachment sites for actin filaments branching off from circumferential actin cables that circumscribe each cell. In the second stage, circumferential actin cables near cell-cell contact sites separate, and the resulting two ends of the cable swing outwards to the perimeter of the contact. Concomitantly, subsets of E-cadherin puncta are also swept to the margins of the contact where they coalesce into large E-cadherin plaques. This reorganization results in the formation of a circumferential actin cable that circumscribes both cells, and is embedded into each E-cadherin plaque at the contact margin. At this stage, the two cells achieve maximum contact, a process referred to as compaction. These changes in E-cadherin and actin distributions are repeated when additional single cells adhere to large groups of cells. The third stage of adhesion occurs as additional cells are added to groups of >3 cells; circumferential actin cables linked to E-cadherin plaques on adjacent cells appear to constrict in a purse-string action, resulting in the further coalescence of individual plaques into the vertices of multicell contacts. The reorganization of E-cadherin and actin results in the condensation of cells into colonies. We propose a model to explain how, through strengthening and compaction, E-cadherin and actin cables coordinate to remodel initial cell-cell contacts to the final condensation of cells into colonies.
Actins↗
Expression of SSX genes in human tumors.
The HOM-MEL-40 antigen which is encoded by the SSX-2 gene was originally detected as a tumor antigen recognized by autologous IgG antibodies in a melanoma patient. Expression analysis demonstrated that SSX-2 is a member of the recently described cancer/testis antigen (CTA) class as it is expressed in a variety of different human neoplasms, but not in normal tissues with the exception of testis and a weak expression in the thyroid. Further studies demonstrated that SSX-2 belongs to a gene family consisting of at least 5 homologous genes. We now report the analysis of the expression of all 5 SSX genes in 325 specimens of human neoplasms from various histological origins, using reverse transcription polymerase chain reaction (RT-PCR). SSX-1, -2, and -4 were found to be expressed in 8%, 15% and 15%, of the tumors, respectively, while the expression of the SSX-5 gene was rare (7/325), and SSX-3 expression was not detected. For defined tumor types, expression of at least one of the SSX family members was most frequently observed in head and neck cancer (75%), followed by ovarian cancer (50%), malignant melanoma (43%), lymphoma (36%), colorectal cancer (27%) and breast cancer (23%), while leukemias and the few cases of leiomyosarcomas, seminomas and thyroid cancers were found not to express any SSX gene.
Antigens, Neoplasm↗
Identification of multiple cancer/testis antigens by allogeneic antibody screening of a melanoma cell line library.
Cancer/testis (CT) antigens-immunogenic protein antigens that are expressed in testis and a proportion of diverse human cancer types-are promising targets for cancer vaccines. To identify new CT antigens, we constructed an expression cDNA library from a melanoma cell line that expresses a wide range of CT antigens and screened the library with an allogeneic melanoma patient serum known to contain antibodies against two CT antigens, MAGE-1 and NY-ESO-1. cDNA clones isolated from this library identified four CT antigen genes: MAGE-4a, NY-ESO-1, LAGE-1, and CT7. Of these four, only MAGE-4a and NY-ESO-1 proteins had been shown to be immunogenic. LAGE-1 is a member of the NY-ESO-1 gene family, and CT7 is a newly defined gene with partial sequence homology to the MAGE family at its carboxyl terminus. The predicted CT7 protein, however, contains a distinct repetitive sequence at the 5' end and is much larger than MAGE proteins. Our findings document the immunogenicity of LAGE-1 and CT7 and emphasize the power of serological analysis of cDNA expression libraries in identifying new human tumor antigens.
Amino Acid Sequence↗
Characterization of human colon cancer antigens recognized by autologous antibodies.
The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) has proven to be a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. In the present study, 48 distinct antigens (NY-CO-1-NY-CO-48) reactive with autologous IgG were identified by SEREX analysis in 4 patients with colon cancer. Sequencing analysis showed that 17 of the cDNA clones were previously uncharacterized molecules and 31 represented known gene products. The individual cDNA clones were analyzed in the following manner: a search for mutations or other structural changes; an analysis of mRNA expression in a panel of normal tissues; and a frequency analysis of the antibody response to the expressed product in the sera of colon cancer patients and normal individuals. The initial analysis showed NY-CO-13 to be a mutated version of the p53 tumor suppressor gene. Three of the 48 antigens showed a differential pattern of mRNA expression, with NY-CO-27 (galectin-4) expressed primarily in gastrointestinal tract, and NY-CO-37 and -38 showing a pattern of tissue-specific isoforms. With regard to immunogenicity, 20 of the 48 antigens were detected by allogeneic sera; 14 of these were reactive with sera from both normal donors and cancer patients, and 6 other clones (NY-CO-8, -9, -13, -16, -20 and -38) reacted exclusively with sera from colon cancer patients (ranging from 14% to 27%). Our results on colon cancer illustrate both the complexity and the potential of the SEREX approach for analysis of the humoral immune response against human cancer.
Adult↗
New paths in human cancer serology.
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Antigens, Neoplasm↗
A survey of the humoral immune response of cancer patients to a panel of human tumor antigens.
Evidence is growing for both humoral and cellular immune recognition of human tumor antigens. Antibodies with specificity for antigens initially recognized by cytotoxic T lymphocytes (CTLs), e.g., MAGE and tyrosinase, have been detected in melanoma patient sera, and CTLs with specificity for NY-ESO-1, a cancer-testis (CT) antigen initially identified by autologous antibody, have recently been identified. To establish a screening system for the humoral response to autoimmunogenic tumor antigens, an enzyme-linked immunosorbent assay (ELISA) was developed using recombinant NY-ESO-1, MAGE-1, MAGE-3, SSX2, Melan-A, and tyrosinase proteins. A survey of sera from 234 cancer patients showed antibodies to NY-ESO-1 in 19 patients, to MAGE-1 in 3, to MAGE-3 in 2, and to SSX2 in 1 patient. No reactivity to these antigens was found in sera from 70 normal individuals. The frequency of NY-ESO-1 antibody was 9.4% in melanoma patients and 12.5% in ovarian cancer patients. Comparison of tumor NY-ESO-1 phenotype and NY-ESO-1 antibody response in 62 stage IV melanoma patients showed that all patients with NY-ESO-1(+) antibody had NY-ESO-1(+) tumors, and no patients with NY-ESO-1(-) tumors had NY-ESO-1 antibody. As the proportion of melanomas expressing NY-ESO-1 is 20-40% and only patients with NY-ESO-1(+) tumors have antibody, this would suggest that a high percentage of patients with NY-ESO-1(+) tumors develop an antibody response to NY-ESO-1.
Antibodies, Neoplasm↗
Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein.
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein localization in a variety of systems. Applications using GFP reporters have expanded greatly due to the availability of mutants with altered spectral properties, including several blue emission variants, all of which contain the single point mutation Tyr-66 to His in the chromophore region of the protein. However, previously described "BFP" reporters have limited utility, primarily due to relatively dim fluorescence and low expression levels attained in higher eukaryotes with such variants. To improve upon these qualities, we have combined a blue emission mutant of GFP containing four point mutations (Phe-64 to Leu, Ser-65 to Thr, Tyr-66 to His, and Tyr-145 to Phe) with a synthetic gene sequence containing codons preferentially found in highly expressed human proteins. These mutations were chosen to optimize expression of properly folded fluorescent protein in mammalian cells cultured at 37 degreesC and to maximize signal intensity. The combination of improved fluorescence and higher expression levels yield an enhanced blue fluorescent protein that provides greater sensitivity and is suitable for dual color detection with green-emitting fluorophores.
Fluorescence↗
Human lung cancer antigens recognized by autologous antibodies: definition of a novel cDNA derived from the tumor suppressor gene locus on chromosome 3p21.3.
Serological analysis of a recombinant lung cancer cDNA expression library with the autologous patient serum led to the isolation of 20 clones representing 12 different genes: 4 of these were known genes, and the other 8 were previously unknown genes. Of the four known genes, aldolase A (NY-LU-1), previously shown to be overexpressed in lung cancer, was most frequently isolated. The other three genes were annexin XI, human HIV Rev-interacting protein Rip-1, and the human homologue of the ATP-binding arsA component of the bacterial arsenite transporter, all of which are known to be widely expressed in human tissues. Among the eight unknown genes, of most interest was NY-LU-12. Cloning of full-length NY-LU-12 showed that this cDNA was derived from the same gene as g16, a partially sequenced gene that mapped to the lung cancer tumor suppressor gene locus on chromosome 3p21. The reported g16 sequence, however, was significantly shorter (2433 versus 3591 bp). As a result of alternate splicing and subsequent frameshift, the reported g16 protein is 603 amino acids shorter than the NY-LU-12 product (1123 residues) at its COOH terminus and would therefore lack the epitopes recognized by the autologous serum. Analysis of the putative NY-LU-12 protein sequence predicted that it is a nuclear zinc finger protein with two RNA-binding domains, and Southern analysis showed that this gene is partially deleted in the lung cancer line NCI-H740 but not in nine other lung cancer lines. Screening of normal and cancer patient sera showed anti-NY-LU-12 seroreactivity in 2 of 21 allogeneic lung cancer patients but not in 24 patients with other tumors or in 16 sera from healthy donors. Comparison of NY-LU-12 cDNA from Lu15 tumor and normal lung tissue by DNA sequencing and/or single-strand conformation polymorphism analysis showed no evidence of mutation. Considering the high frequency of 3p21 alterations in lung cancer and the fact that the tumor suppressor gene or genes in this locus have not been identified, additional studies on the NY-LU-12 gene and its product are warranted.
Amino Acid Sequence↗
Clinical and metabolic correction of pompe disease by enzyme therapy in acid maltase-deficient quail.
Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase (GAA), a glycogen degrading lysosomal enzyme. GAA-deficient (AMD) Japanese quails exhibit progressive myopathy and cannot lift their wings, fly, or right themselves from the supine position (flip test). Six 4-wk-old acid maltase-deficient quails, with the clinical symptoms listed, were intravenously injected with 14 or 4.2 mg/kg of precursor form of recombinant human GAA or buffer alone every 2-3 d for 18 d (seven injections). On day 18, both high dose-treated birds (14 mg/kg) scored positive flip tests and flapped their wings, and one bird flew up more than 100 cm. GAA activity increased in most of the tissues examined. In heart and liver, glycogen levels dropped to normal and histopathology was normal. In pectoralis muscle, morphology was essentially normal, except for increased glycogen granules. In sharp contrast, sham-treated quail muscle had markedly increased glycogen granules, multi-vesicular autophagosomes, and inter- and intrafascicular fatty infiltrations. Low dose-treated birds (4.2 mg/kg) improved less biochemically and histopathologically than high dose birds, indicating a dose-dependent response. Additional experiment with intermediate doses and extended treatment (four birds, 5.7-9 mg/kg for 45 d) halted the progression of the disease. Our data is the first to show that an exogenous protein can target to muscle and produce muscle improvement. These data also suggest enzyme replacement with recombinant human GAA is a promising therapy for human Pompe disease.
Animals↗
Development of Th1 and Th2 populations and the nature of immune responses to hepatitis B virus DNA vaccines can be modulated by codelivery of various cytokine genes.
In this study, we provide direct evidence that the magnitude and nature of the immune response to a DNA vaccine can be differentially regulated by codelivery of various mouse cytokine genes. Mice immunized with a hepatitis B virus (HBV) DNA vaccine and the IL-12 or IFN-gamma gene exhibited a significant enhancement of Th1 cells and increased production of anti-HBV surface IgG2a Ab, as well as a marked inhibition of Th2 cells and decreased production of IgG1 Ab. In contrast, coinjection of the IL-4 gene significantly enhanced the development of specific Th2 cells and increased production of IgG1 Ab, whereas Th1 differentiation and IgG2a production were suppressed. Coinjection of the IL-2 or the granulocyte-macrophage-CSF gene enhanced the development of Th1 cells, while the development of Th2 cells was not affected, and the production of IgG1 and IgG2a Ab were both increased. The CTL activity induced by HBV DNA vaccination was most significantly enhanced by codelivery of the IL-12 or IFN-gamma gene, followed by the IL-2 or granulocyte-macrophage-CSF gene, whereas codelivery of the IL-4 gene suppressed the activity. When challenged with HBV surface Ag (HBsAg)-expressing syngeneic tumors, significant reduction of tumor growth was observed in mice that were coadministered the IL-12 gene but not the IL-4 gene. Taken together, these results demonstrate that application of a cytokine gene in a DNA vaccine formulation can influence the differentiation of Th cells as well as the nature of an immune response and may thus provide a strategy to improve its prophylactic and therapeutic efficacy.
Adjuvants, Immunologic↗
Simultaneous humoral and cellular immune response against cancer-testis antigen NY-ESO-1: definition of human histocompatibility leukocyte antigen (HLA)-A2-binding peptide epitopes.
A growing number of human tumor antigens have been described that can be recognized by cytotoxic T lymphocytes (CTLs) in a major histocompatibility complex (MHC) class I-restricted fashion. Serological screening of cDNA expression libraries, SEREX, has recently been shown to provide another route for defining immunogenic human tumor antigens. The detection of antibody responses against known CTL-defined tumor antigens, e.g., MAGE-1 and tyrosinase, raised the question whether antibody and CTL responses against a defined tumor antigen can occur simultaneously in a single patient. In this paper, we report on a melanoma patient with a high-titer antibody response against the "cancer-testis" antigen NY-ESO-1. Concurrently, a strong MHC class I-restricted CTL reactivity against the autologous NY-ESO-1-positive tumor cell line was found. A stable CTL line (NW38-IVS-1) was established from this patient that reacted with autologous melanoma cells and with allogeneic human histocompatibility leukocyte antigen (HLA)-A2(-), NY-ESO-1-positive, but not NY-ESO-1-negative, melanoma cells. Screening of NY-ESO-1 transfectants with NW38-IVS-1 revealed NY-ESO-1 as the relevant CTL target presented by HLA-A2. Computer calculation identified 26 peptides with HLA-A2-binding motifs encoded by NY-ESO-1. Of these, three peptides were efficiently recognized by NW38-IVS-1. Thus, we show that antigen-specific humoral and cellular immune responses against human tumor antigens may occur simultaneously. In addition, our analysis provides a general strategy for identifying the CTL-recognizing peptides of tumor antigens initially defined by autologous antibody.
Aged↗
Direct and regulated interaction of integrin alphaEbeta7 with E-cadherin.
The cadherins are a family of homophilic adhesion molecules that play a vital role in the formation of cellular junctions and in tissue morphogenesis. Members of the integrin family are also involved in cell to cell adhesion, but bind heterophilically to immunoglobulin superfamily molecules such as intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, or mucosal addressin cell adhesion molecule (MadCAM)-1. Recently, an interaction between epithelial (E-) cadherin and the mucosal lymphocyte integrin, alphaEbeta7, has been proposed. Here, we demonstrate that a human E-cadherin-Fc fusion protein binds directly to soluble recombinant alphaEbeta7, and to alphaEbeta7 solubilized from intraepithelial T lymphocytes. Furthermore, intraepithelial lymphocytes or transfected JY' cells expressing the alphaEbeta7 integrin adhere strongly to purified E-cadherin-Fc coated on plastic, and the adhesion can be inhibited by antibodies to alphaEbeta7 or E-cadherin. The binding of alphaEbeta7 integrin to cadherins is selective since cell adhesion to P-cadherin-Fc through alphaEbeta7 requires >100-fold more fusion protein than to E-cadherin-Fc. Although the structure of the alphaE-chain is unique among integrins, the avidity of alphaEbeta7 for E-cadherin can be regulated by divalent cations or phorbol myristate acetate. Cross-linking of the T cell receptor complex on intraepithelial lymphocytes increases the avidity of alphaEbeta7 for E-cadherin, and may provide a mechanism for the adherence and activation of lymphocytes within the epithelium in the presence of specific foreign antigen. Thus, despite its dissimilarity to known integrin ligands, the specific molecular interaction demonstrated here indicates that E-cadherin is a direct counter receptor for the alphaEbeta7 integrin.
Amino Acid Sequence↗
Prenatal diagnosis and carrier detection for glycogen storage disease type III using polymorphic DNA markers.
Deficiency of glycogen debranching enzyme gene (AGL) causes glycogen storage disease type III (GSD-III), an autosomal recessive disease. Prenatal diagnosis and carrier detection using enzymatic methods are technically difficult and have limited ability to distinguish a carrier from an affected patient. Mutations in the AGL gene can be used for these purposes. However, the mutations identified thus far account for less than half of the total mutant alleles, and no common mutations have been detected except in North African Jews and in a rare subtype of the disease (GSD-IIIb). Our recent identification of three highly informative DNA polymorphic markers in the AGL gene allowed us to perform prenatal diagnosis and carrier detection in two GSD-III families with unknown mutations, using the polymerase chain reaction (PCR) and restriction analysis. In one family, a fetus was diagnosed to be a GSD-III carrier and his carrier status was confirmed postnatally. A newborn in the second family was postnatally diagnosed with the disease.
Adolescent↗
Quantitative analysis of antibiotics by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Comparative studies of the matrix-assisted laser desorption/ionization (MALDI) of 30 antibiotics were made using alpha-cyano-4-hydroxycinnamic acid (HCCA), 2,5-dihydroxybenzoic acid (DHB), 5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphyrin and meso-tetra(N-methyl-4-pyridyl)porphyrin matrices. Most antibiotics generated intense protonated molecules in HCCA and DHB matrices, and sodium or potassium adduct ions in porphyrin matrices. Using sulfonamide antibiotics as model compounds, DHB was found to be the most suitable matrix for quantitative analysis. Detection limits were about 1 picomole. Linear correlation (R2 > 0.97), between the sample quantity over the range 1 to 100 picomole and the signal response, was obtained when ratios of the sum of peak areas of protonated molecules and sodium adduct ions, with reference to that of a structurally analogous internal standard (acetaminophen), were measured. The precision was found to be in the range of 4 to 32% relative standard deviation, dependent on the type and concentration of the analyte. A simple acylation derivatization process was developed to confirm the presence of suspected antibiotic residues. It is demonstrated that MALDI is a viable technique for the quantitative analysis of low molecular weight antibiotics.
Anti-Bacterial Agents↗
Angiotensinogen gene polymorphism is associated with insulin resistance in nondiabetic men with or without coronary heart disease.
BACKGROUND: Variants of the angiotensinogen gene may increase the risk of having arterial hypertension and coronary heart disease (CHD), but their effect on insulin resistance remains unknown. METHODS: We determined M235 and T174 allele status and fasting plasma glucose, insulin, and lipids values in nondiabetic men with CHD documented on angiography (n = 102) and in a control group (n = 145). Plasma glucose and insulin responses to 75 gm oral glucose tolerant test and insulin resistance as measured by an insulin suppression test were also carried out in 46 (45%) patients with CHD and in 73 (50%) members of a control group. RESULTS: We found no association between M235T status and blood pressure, fasting plasma glucose, insulin, most of the lipids values, and insulin resistance in patients with CHD and normal subjects. Nevertheless, compared with individuals with homozygotes T174, subjects with heterozygotes T174M were associated with greater glucose and insulin response to the oral glucose tolerance test and insulin resistance indicated by higher steady state plasma glucose concentrations in patients with CHD (14.7+/-0.9 vs 11.3+/-0.7 mmol/L, p < 0.04). Similar findings were found in the control group, with higher steady-state plasma glucose values in individuals with heterozygotes T174M than in those with homozygotes T174 (10.1+/-1.4 vs 7.7+/-0.4 mmol/L, p < 0.05). CONCLUSION: We suggest that the angiotensinogen T174M allele might be associated with insulin resistance in nondiabetic men with and without CHD.
Alleles↗
Two new mutations in the 3' coding region of the glycogen debranching enzyme in a glycogen storage disease type IIIa Ashkenazi Jewish patient.
Glycogen storage disease type III (GSD III) is an autosomal recessive disease caused by the deficiency of glycogen debranching enzyme (AGL). We report the finding of two new mutations in a GSD IIIa Ashkenazi Jewish patient. Both mutations are insertion of an adenine into a stretch of 8 adenines towards the 3' end of the coding region, one at position 3904 (3904insA) in exon 30, the second at position 4214 (4214insA) in exon 32. The mutations cause frameshifts and premature terminations of the glycogen debranching enzyme, the first causing a frameshift at amino acid 1304, the second causing a frameshift at amino acid 1408 of the total of 1532. These mutations demonstrate the importance of the 125 amino acids at the carboxy-terminus of the debrancher enzyme for its activity and support the suggestion that the putative glycogen binding domain is located in the carboxy-terminus of the AGL. The mutations cause distinctive single-strand conformation polymorphism (SSCP) patterns enabling easy detection.
Child, Preschool↗