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Biomedical subjects

M S Lan

Publications and source records attributed to M S Lan.

At least 19 recordsLinked to original sources
PubMed2006

The insulinoma-associated 1: a novel promoter for targeted cancer gene therapy for small-cell lung cancer.

The insulinoma-associated 1 (INSM1) gene is expressed exclusively during early embryonal development, but has been found re-expressed at high levels in neuroendocrine tumors. The regulatory region of the INSM1 gene is therefore a potential candidate for regulating expression of a therapeutic gene in transcriptionally targeted cancer gene therapy against neuroendocrine tumors. We analyzed expression of a reporter gene from a 1.7 kb region of the INSM1 promoter in a large number of small-cell lung cancer (SCLC) cell lines. This INSM1 promoter region showed very high levels of expression in most of the SCLC cell lines and expression was absent in cell lines of non-neuroendocrine origin. Inclusion of the general transcriptional enhancer from SV40 compromised the specificity of the promoter and did not enhance transcription in most of the SCLC cell lines. For comparison, the region of the gastrin releasing peptide (GRP) previously suggested for SCLC gene therapy was analyzed in a similar manner. High expression was observed for a number of cell lines, but unlike for the INSM1 promoter, reporter gene expression from the GRP promoter did not correlate to the relative GRP mRNA levels, demonstrating that this region may not contain all necessary regulatory elements. Expression of the suicide gene herpes simplex virus thymidine kinase (HSV-TK) from the INSM1 promoter in combination with treatment with the prodrug ganciclovir (GCV) caused a significant increase in GCV sensitivity specifically in INSM1-expressing cell lines. The INSM1 promoter is therefore a potential novel tool for transcriptionally targeted gene therapy for neuroendocrine tumors.

Antiviral Agents↗
PubMed2003

Identification of three genomic haplotypes 5' to the human CD1D gene and their distribution in four ethnic groups.

CD1d presents lipid antigen to a conserved population of natural killer (NK) T cells, which participate in host immune defense, tumor cell rejection and suppression of autoimmunity. The levels of human CD1d expression vary significantly between individuals. To understand such variation, we sequenced the region up to 1.7 kb 5' upstream of the translation start site and partially through exon 2 in 44 white Americans. We also studied two tagged single nucleotide polymorphisms (SNP) in 112 white Americans, 60 African-Americans, 88 Europeans, and 84 Chinese people from the region. Six SNP present in the region (-836C-->T, -773C-->T, -764C-->G, -713A-->T, -365A-->G and +363A-->G) were found to be in a complete linkage disequilibrium and comprised three haplotypes. Haplotype 1 had -836C, -773C, -764C, -713A, -365A and +363A. Haplotype 2 had -836C, -773T, -764C, -713A, -365A and +363A. Haplotype 3 had -836T, -773C, -764G, -713T, -365G and +363G. -773C-->T and -764C-->G can serve as the tagged SNP to differentiate the three haplotypes. The frequency of haplotype 1 was significantly higher in African Americans than in the other three ethnic groups, whereas the frequency of haplotype 3 was significantly higher in the Chinese people than those in the other three groups. The finding of the three haplotypes provides a genetic marker for CD1d and facilitates the study of the functional role of the genetic variations in human CD1d expression and regulation.

Antigens, CD1↗
PubMed2001

The pancreas-specific protein disulphide-isomerase PDIp interacts with a hydroxyaryl group in ligands.

Using a cross-linking approach, we have recently demonstrated that radiolabelled model peptides or misfolded proteins specifically interact in vitro with two members of the protein disulphide- isomerase family, namely PDI and PDIp, in a crude extract from sheep pancreas microsomes. In addition, we have shown that tyrosine and tryptophan residues within a peptide are the recognition motifs for the binding to PDIp. Here we examine non-peptide ligands and present evidence that a hydroxyaryl group is a structural motif for the binding to PDIp; simple constructs containing this group and certain xenobiotics and phytoestrogens, which contain an unmodified hydroxyaryl group, can all efficiently inhibit peptide binding to PDIp. To our knowledge this is the first time that the recognition motif of a molecular chaperone or folding catalyst has been specified as a simple chemical structure.

Animals↗
PubMed2001

Defective morphogenesis and functional maturation in fetal islet-like cell clusters from OLETF rat, a model of NIDDM.

A failure in the compensate proliferation of pancreatic beta-cells, as the primary pathogenic event, has been reported in OLETF rat, a model of NIDDM. The aim of the present study is to define whether the beta-cell defect is attributed to the fetal stage islet development, if so, whether the defect involves down regulation of PDX-1 protein expression. Morphological changes, beta-cell function, and the expression of PDX-1 protein were examined in the cultured fetal islet-like cell clusters (ICCs) from OLETF rats along with their diabetes-resistant control counterpart LETO rats in the presence of 5.5 or 11.1mM glucose for 48, 72, 96, and 120-hr, respectively. We have observed four abnormalities in the ICCs of OLETF rats. First, a defective morphogenesis was noted during the 72 to 120-hr ICC culture, a period characterized by a dramatic increase in both beta-cell and non-beta-cell (alpha, delta, and PP) populations in control rats. This defective morphogenesis was demonstrated by a growth retardation of epithelial stratification and poor development of both beta-cell and non-beta-cell masses along with a parallel decline in relevant islet hormone contents. Second, a functional defect was characterized by failure to response to glucose during the 96 to 120-hr-cultured ICCs. Third, the ultrastructural analysis revealed a significant reduction in the number of secretory granules. Four, Western blot analysis showed a significant decrease of PDX-1 protein expression in the OLETF ICCs cultured in 11.1mM glucose for 48 to 72-hr and in 5.5mM glucose for 120-hr. Therefore, we concluded that during the fetal stage of islet development, OLETF rats exhibit both morphological and functional defects.

Animals↗
PubMed2001

L-Arginine regulates the expression of the T-cell receptor zeta chain (CD3zeta) in Jurkat cells.

L-Arginine is a versatile amino acid that plays a central role in the normal function of several organ systems including the immune system. Its availability is tightly controlled and varies significantly in different organs and tissues in the body. L-Arginine plays an important role in supporting T-cell proliferation. Its depletion in certain disease states results in a diminished T-cell response. The main purpose of this study was to determine the effect of the depletion of L-arginine on the expression of the T-cell receptor (TCR) proteins. When the helper T-cell line Jurkat was cultured in arginine-free medium, there was a preferential decrease in the expression of the TCR zeta chain (CD3zeta). The reduced expression of CD3zeta was observed within 24 h of culture in L-arginine-free medium and was completely reversed with the replenishment of L-arginine. Furthermore, the absence of L-arginine blocked the normal re-expression of the TCR that had been internalized after antigen stimulation. There also was a significant decrease in proliferation of Jurkat cells in the absence of L-arginine; however, L-arginine depletion did not prevent the up-regulation of the interleukin 2 receptor chains upon stimulation, nor did it significantly diminish the production of interleukin 2. The changes in the expression of CD3zeta chain were not induced by apoptosis. Thus, the availability of L-arginine in the microenvironment may play a significant role in regulating the expression of the TCR.

Apoptosis↗
PubMed2000

The human leukocyte antigen HLA DRB3*020/DQA1*0501 haplotype is associated with Graves' disease in African Americans.

Information on genetic susceptibility to Graves' disease in African Americans is limited. We studied DRB1, DQB1, DRB3 subtypes, DQA1*0501, DQA1*0201, and CTLA-4 polymorphisms in 49 African American patients with adult onset Graves' disease and 47 racially-matched controls using PCR-based sequence-specific priming methods. There were no significant differences in DRB1 or DQB1 allelic frequencies or CTLA-4 polymorphisms between patients and controls. However, we found that the frequency of DRB3 was significantly increased in the patients (75.5% vs. 57.4%, P = 0.006, X2 = 3.52), especially for the DRB3*0202 subtype (53.1% vs. 23.4, P = 0.003, X2 = 8.91). In this one respect, the finding was in concordance with our previous observations in Caucasian patients with adult-onset Graves' disease. In addition, whereas the frequency of DQA1*0501 was increased (P = 0.018, X2 = 5.63) in our patients, the haplotype of DRB3/DQA1*0501, or DRB3*0202/DQA1*0501 was found to be more strongly associated (P = 0.008, X2 = 7.0; P = 0.0008, X2 = 11.34, respectively). These data suggest that DRB3*0202, particularly when found with DQA1*0501 in a haplotype is a susceptible gene(s) for Graves' disease in adult African Americans. Considering these data with those in Caucasian patients, our results would suggest that the primary Graves susceptible locus is likely DRB3 and not DRB1.

Abatacept↗
PubMed1998

Genomic structure and promoter sequence of the insulin-dependent diabetes mellitus autoantigen, IA-2 (PTPRN).

IA-2 is a transmembrane protein tyrosine phosphatase, expressed in neuroendocrine cells, and a major autoantigen in insulin-dependent diabetes mellitus. In the present study we elucidated the structure of the IA-2 gene (HGMW-approved symbol PTPRN) and its promoter sequence. A 40-kb genomic clone covering the whole IA-2 coding sequence and 4 kb proximal 5'-upstream sequence was isolated and mapped. The IA-2 gene encompasses approximately 20 kb with 23 exons ranging from 34 bp to more than 650 bp. The extracellular domain is encoded by exons 1-12, the transmembrane region by exon 13, and the intracellular domain by exons 14-23. The transcriptional start site(s) of the IA-2 gene was mapped by 5' rapid amplification of cDNA ends to 97 bp upstream of the translational start site. A 3-kb 5'-upstream region was sequenced, revealing a GC-rich region and TATA-less sequence containing several potential transcription-regulating sites (i.e., Sp1, CREB, GATA-1, and MZF). Functional promoter activity was confirmed by transient transfection of U87MG cells with deletion mutants linked to a luciferase reporter gene.

Autoantibodies↗
PubMed1998

IA-2 and IA-2beta: the immune response in IDDM.

Pancreatic islet cell autoantigens associated with insulin-dependent diabetes mellitus (IDDM) include a recently identified family of protein tyrosine phosphatase-like molecules, notably IA-2 and IA-2beta. IA-2 is a 979 amino acid transmembrane protein located on human chromosome 2q35, whereas IA-2beta is 986 amino acids long located on human chromosome 7q36. Comparison of human IA-2 and IA-2beta showed 74% identity within the intercellular domains, but only 27% indentify within the extracellular domains. These IA-2 molecules are expressed predominantly in cells of neuroendocrine origin, particularly pancreatic islets and brain. Radioimmunoprecipitation with recombinant IA-2 and IA-2beta has been used to measure autoantibodies to these molecules and their intracellular fragments. Autoantibodies to IA-2 are detected in the majority (60% to 80%) of newly diagnosed IDDM patients and in less than 2% of controls. The major antigenic determinants of both IA-2 and IA-2beta reside within the C-terminus of their intracellular domains. In first-degree relatives of IDDM patients, the presence of autoantibodies to IA-2 is predictive of IDDM and in combination with autoantibodies to glutamic acid decarboxylase (GAD) the positive predictive value is in the 50% range. The role of IA-2 and IA-2beta in the pathogenesis of IDDM is still unclear. Identification of these antigens has extended our ability to predict the disease and may be valuable in the search for antigen-specific therapies to prevent IDDM.

Animals↗
PubMed1998

The gene responsible for autoimmune polyglandular syndrome type 1 maps to chromosome 21q22.3 in US patients.

Autoimmune polyglandular syndrome type 1 [APS-1] comprises multiple organ-specific autoimmunities such as acquired hypoparathyroidism and autoimmune Addison's disease, and a predisposition to certain infections such as chronic mucocutaneous candidiasis. An APS-1 candidate gene was assigned to chromosome 21q22.3 by linkage analyses in patients with APS-1 from Finland. To examine the influence of ethnic and geographic differences on the location of the candidate gene locus, we studied 24 US patients with APS-1 by microsatellite marker typing, using five microsatellite markers, D21S49, PFKL, D21S171, D21S1903 and CD18, selected from chromosome 21q22.3. By allelic association analyses, the frequencies of allele number 5 for D21S171 and allele number 8 for D21S1903 were significantly higher in the 24 patients with APS-1 than in 33 controls (33/48 vs. 31/66, P = 0.0207, X2 = 5.35; 12/48 vs. 7/66, P = 0.0418, X2 = 4.15 respectively). The frequency of homozygosity for allele number 5 of D21S171 was also significantly higher in the patients than in controls, 15/24 vs. 9/33 (P = 0.0078, X2 = 7.07). Maximum lod scores detected for the five markers in nine families (containing 15 of the patients with APS-1) were: 2.384 for D21S49, 3.144 for PFKL, 3.506 for D21S171, 4.329 for D21S1903, and 1.130 for CD18. These results confirm the linkage of the candidate APS-1 gene to 21q22.3 in US APS-1 patients, and suggest that the candidate gene is located near the D21S1903 marker. The demonstration of the location of the APS-1 candidate gene to 21q22.3 in an out-bred heterogeneous patient population should promote the physical mapping of the responsible gene.

Alleles↗
PubMed1998

Immunological heterogeneity in type I diabetes: presence of distinct autoantibody patterns in patients with acute onset and slowly progressive disease.

Type I diabetes mellitus may represent a heterogeneous disorder with a distinct pathogenesis in patients with young and adult onset of the disease. To investigate whether serological markers directed to different autoantigens have the potential to distinguish acute onset from slowly progressive Type I diabetes we analysed antibodies to tyrosine phosphatases IA-2/ICA512 (IA-2A) and IA-2beta/phogrin (IA2betaA), antibodies to GAD65 (GADA) and cytoplasmic islet cell antibodies (ICA) in a non-selected group of diabetic patients clinically classified as having Type I or Type II diabetes at diagnosis. Both IA-2A and IA-2betaBA were found to be positively associated with onset before the age of 20 years and the presentation of classical features of Type I diabetes. In Type I diabetes 56 % (112/200) of patients were positive for IA-2A and 38 % (76/200) for IA-2betaA. In contrast, only 1 of 785 (0.1 %) patients with Type II diabetes had IA-2A and all of them were negative for IA-2betaA (p < 0.001). Among the patients with Type II diabetes 7.6% (n = 60) were ICA positive and 2.8% (n = 22) had GADA suggesting the presence of slowly progressive Type I diabetes. GADA were found in 8 of 60 (13.3 %) ICA positive subjects which was lower than the percentage detected in patients with acute onset of diabetes (115/157 73.2%) (p < 0.001). Blocking of double antibody positive sera showed that only 3 of 8 (37.5 %) patients with slowly progressive diabetes had ICA restricted to GAD or IA-2 whereas ICA were completely inhibited in 12 of 20 (60.0 %) patients with Type I diabetes. Among 193 patients with Type II diabetes available for follow-up, 35 % of ICA positives, 58 % of GADA positives and 60 % of those positive for both markers required insulin by 3 years. However, using strict criteria for the switch to insulin treatment the corresponding sensitivity of each marker was only low (9%, 10% and 5%). We show that clinical subtypes of Type I diabetes are associated with distinct humoral autoimmunity. IA-2A and GADA were associated with classical features of Type I diabetes whereas GADA and an uncharacterized ICA subspecificity indicate slowly progressive disease.

Acute Disease↗
PubMed1998

Expression, characterization, processing and immunogenicity of an insulin-dependent diabetes mellitus autoantigen, IA-2, in Sf-9 cells.

Autoantibodies to a 64-kD protein and a 40-kD tryptic fragment from pancreatic islets have been detected at high frequency in the sera of patients with insulin-dependent diabetes mellitus (IDDM). IA-2, a newly isolated transmembrane protein tyrosine phosphatase, is a major islet cell autoantigen in IDDM and the precursor of a 40-kD tryptic fragment. To express large quantities of recombinant IA-2 protein and analyse post-translational modifications we expressed full-length human IA-2 in baculovirus-infected Sf-9 cells. IA-2 expression was analysed by Western blot and by immunoprecipitation of 35S-methionine-radiolabelled proteins with rabbit antisera or IDDM sera. A 120-kD IA-2 protein was detected during the early, but not the late, phase of the infection. Pulse-chase experiments showed that the 120-kD protein was processed into fragments of 64 kD and smaller fragments of approximately 50 kD, 38 kD and 32 kD. The 64-kD fragment appeared as a doublet. Tunicamycin and PNGase F treatment down-shifted the 120-kD protein and the 64-kD doublet into lower molecular weight bands, suggesting that both were glycosylated. Trypsin treatment converted the 120-kD protein and the 64-kD doublet into a 40-kD fragment. Baculovirus-expressed IA-2 was as sensitive or slightly more sensitive than in vitro translated IA-2 in detecting autoantibodies to IA-2: 66% of sera from newly diagnosed IDDM patients reacted with baculovirus-expressed IA-2 compared with 59% of the same sera which reacted with in vitro translated IA-2. It is concluded that baculovirus-expressed IA-2 is a good source of autoantigen and that a number of lower molecular weight fragments with which IDDM autoantibodies react are derived from the 120-kD full-length IA-2 molecule.

Animals↗
PubMed1998

Profile and differential expression of protein tyrosine phosphatases in mouse pancreatic islet tumor cell lines.

Protein tyrosine phosphatases (PTPs) play important roles in cell growth and differentiation of normal and tumor cells. In this study, we analyzed the PTP profile in two pancreatic islet tumor cell lines. Transcripts were isolated from alphaTC-1 (glucagon-secreting) and betaTC-1 (insulin-secreting) cell lines for templates. A pair of degenerative primers, based on the conserved regions of known PTPs, was used to amplify the transcripts by polymerase chain reaction. A total of 1,620 clones was examined by restriction enzyme analysis and cDNA sequencing. Twenty-one PTPs were identified, including nine cytosolic PTPs (TcPTP, P19PTP, PTP1B, PTPMEG, PTP1C, SYP, PTPH1, PTPL1, and PTPD1), nine transmembrane PTPs (PTPdelta, PTPgamma, PTPkappa, DEP-1, IA-2, LAR, PTPalpha, PTPNE3, and PTPepsilon), and three new PTPs--PTPmu-like PTPkappa-like, and IA-2beta. An RNase protection assay demonstrated that some of these PTPs were expressed predominantly in glucagonoma (i.e., PTPdelta and IA-2) and others in insulinoma (i.e., PTP1C, PTPkappa, and PTPNE3) cells. In this report, we present the first profile of PTPs in alpha and beta tumor cell lines.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and characterization of a highly basic protein, IA-4, expressed in pancreatic islets and brain.

A substraction library was constructed from mouse insulinoma (betaTC-1) and glucagonoma (alphaTC-1) cell lines. Differential screening and sequencing revealed a novel cDNA clone, IA-4, which was expressed in the islets of Langerhans and the brain. IA-4 cDNA is 1,007 bp in length and predicts a protein of 187 amino acids with a molecular mass of 19,940 D. Examination of the amino acid sequence showed a high content of arginine (18.7%), proline (14.4%), alanine (16.0%), leucine (13.4%) and glycine (9.6%). The deduced pI value is 12.5 indicating a highly basic protein. Northern blot analysis revealed a 1-kb mRNA highly expressed in brain, trigeminal ganglia and cell lines of neuroendocrine origin. Rabbit polyclonal antiserum raised against a synthetic IA-4 peptide, designated Pep-1, not only reacted with IA-4 recombinant protein, but also immunostained the islets of Langerhans and large neurons of the hippocampus, cerebral cortex, spinal cord, dorsal ganglia and Purkinje cells of the cerebellum. The high expression of IA-4 protein in neuroendocrine cells and its unique amino acid sequence suggest that IA-4 may have an important, but still undetermined, function in these special cell types.

Amino Acid Sequence↗
PubMed1998

The relationship between humoral and cellular immunity to IA-2 in IDDM.

Autoantibodies to the neuroendocrine protein insulinoma-associated protein 2 (IA-2), a member of the tyrosine phosphatase family, have been observed in individuals with or at increased risk for IDDM. Because this disease is thought to result from a T-cell-mediated autoimmune destruction of the insulin-producing pancreatic beta-cells, we analyzed humoral and cellular immune reactivity to this autoantigen to further define its role in the pathogenesis of IDDM. Peripheral blood mononuclear cells (PBMC) from individuals with newly diagnosed IDDM or at varying levels of risk for the disease were stimulated in vitro with the entire 42-kDa internal domain of IA-2 (amino acids 603-979), a series of control antigens (glutathionine-S-transferase, tetanus toxoid, Candida albicans, mumps, bovine serum albumin), and a mitogen (phytohemagglutinin). The frequency and mean stimulation index of PBMC proliferation against IA-2 was significantly higher in newly diagnosed IDDM subjects (14 of 33 [42%]; 3.8+/-4.5 at 10 microg/ml) and autoantibody-positive relatives at increased risk for IDDM (6 of 9 [66%]; 3.9+/-3.2) compared with autoantibody-negative relatives (1 of 15 [7%]; 1.8+/-1.0) or healthy control subjects (1 of 12 [8%]; 1.5+/-1.0). The frequencies of cellular immune reactivities to all other antigens were remarkably similar between each subject group. Sera from 58% of the newly diagnosed IDDM patients tested were IA-2 autoantibody positive. Despite investigations suggesting an inverse association between humoral and cellular immune reactivities against islet-cell-associated autoantigens, no such relationship was observed (rs=0.18, P=0.39) with respect to IA-2. These studies support the autoantigenic nature of IA-2 in IDDM and suggest the inclusion of cellular immune responses as an adjunct marker for the disease.

Adolescent↗
PubMed1997

Autoantibodies to IA-2 and IA-2 beta in insulin-dependent diabetes mellitus recognize conformational epitopes: location of the 37- and 40-kDa fragments determined.

IA-2 and IA-2 beta are major autoantigens in insulin-dependent diabetes mellitus (IDDM) and the precursors, respectively, of a 40-and 37-kDa tryptic fragment that reacts with IDDM sera. In the present study, by amino acid sequencing of recombinant IA-2 and IA-2 beta, we determined the tryptic cleavage sites involved in the generation of these fragments. Both cleavage sites are immediately after an arginine residue at position 653 for IA-2 and position 679 for IA-2 beta. The resulting tryptic fragments are 326 and 307 amino acids in length and retain their ability to react with IDDM sera. In contrast to IA-2 and IA-2 beta, other members of the protein tyrosine phosphatase (PTP) family (i.e., RPTP kappa, RPTPmu, NU-3, SHP, and 3CH134) are completely susceptible to digestion by trypsin. Sequence analysis revealed five conserved cysteine residues in IA-2 and IA-2 beta that are not present in other PTPs. Reduction and alkylation of IA-2 and IA-2 beta recombinant proteins resulted in loss of both resistance to digestion by trypsin and reactivity with autoantibodies in IDDM sera. It is concluded that disulfide bond formation plays a critical role in the maintenance of antigenic structure and that the autoantibodies to IA-2/IA-2 beta in IDDM sera recognize conformational epitopes.

Amino Acid Sequence↗