Search PubMed⌕ Search

Biomedical subjects

S J Chan

Publications and source records attributed to S J Chan.

At least 37 records · Page 2Linked to original sources
PubMed1995

A transgene coding for a human insulin analog has a mitogenic effect on murine embryonic beta cells.

We have investigated the mitogenic effect of three mutant forms of human insulin on insulin-producing beta cells of the developing pancreas. We examined transgenic embryonic and adult mice expressing (i) human [AspB10]-proinsulin/insulin ([AspB10]ProIN/IN), produced by replacement of histidine by aspartic acid at position 10 of the B chain and characterized by an increased affinity for the insulin receptor; (ii) human [LeuA3]insulin, produced by the substitution of leucine for valine in position 3 of the A chain, which exhibits decreased receptor binding affinity; and (iii) human [LeuA3, AspB10]insulin "double" mutation. During development, beta cells of AspB10 embryos were twice as abundant and had a 3 times higher rate of proliferation compared with beta cells of littermate controls. The mitogenic effect of [AspB10]ProIN/IN was specific for embryonic beta cells because the rate of proliferation of beta cells of adults and of glucagon (alpha) cells and adrenal chromaffin cells of embryos was similar in AspB10 mice and controls. In contrast to AspB10 embryos, the number of beta cells in the LeuA3 and "double" mutant lines was similar to the number in controls. These findings indicate that the [AspB10]ProIN/IN analog increased the rate of fetal beta-cell proliferation. The mechanism or mechanisms that mediate this mitogenic effect remain to be determined.

Amino Acid Sequence↗
PubMed1995

Proprotein convertases in amphioxus: predicted structure and expression of proteases SPC2 and SPC3.

SPC2 and SPC3 are two members of a family of subtilisin-related proteases which play essential roles in the processing of prohormones into their mature forms in the pancreatic B cell and many other neuroendocrine cells. To investigate the phylogenetic origins and evolutionary functions of SPC2 and SPC3 we have identified and cloned cDNAs encoding these enzymes from amphioxus (Branchiostoma californiensis), a primitive chordate. The amino acid sequence of preproSPC2 contains 689 aa and is 71% identical to human SPC2. In contrast, amphioxus prproSPC3 consists of 774 aa and exhibits 55% identity to human SPC3. These results suggest that the primary structure of SPC2 has been more highly conserved during evolution than that of SPC3. To further investigate the function(s) of SPC2 and SPC3 in amphioxus, we have determined the regional expression of these genes by using a reverse transcriptase-linked polymerase chain reaction (RT-PCR) assay. Whole amphioxus was dissected longitudinally into four equal-length segments and RNA was extracted. Using RT-PCR to simultaneously amplify SPC2 and SPC3 DNA fragments, we found that the cranial region (section 1) expressed equal amounts of SPC2 and SPC3 mRNAs, whereas in the caudal region (section 4) the SPC2-to-SPC3 ratio was 5:1. In the mid-body sections 2 and 3 the SPC2-to-SPC3 ratio was 1:5. By RT-PCR we also determined that amphioxus ILP, a homologue of mammalian insulin/insulin-like growth factor, was expressed predominately in section 3. These results suggest that the relative levels of SPC2 and SPC3 mRNAs are specifically regulated in various amphioxus tissues. Furthermore, the ubiquitous expression of these mRNAs in the organism indicates that they are involved in the processing of other precursor proteins in addition to proILP.

Amino Acid Sequence↗
PubMed1995

Proteolytic processing mechanisms in the biosynthesis of neuroendocrine peptides: the subtilisin-like proprotein convertases.

The recent discovery of a novel family of precursor processing endoproteases has greatly accelerated progress in understanding the complex mechanisms underlying the maturation of prohormones, neuropeptides, and many other precursor-derived proteins. At least six members of this family have been found thus far in mammalian species, several having alternatively spliced isoforms, and related enzymes have been identified in many invertebrates, including molluscs, insects, nematodes, and coelenterates. The proprotein convertases are all dependent on calcium for activity and all possess highly conserved subtilisin-like domains with the characteristic catalytic triad of this serine protease (ordered Asp, His, and Ser along the polypeptide chain). Two members of this family, PC2 (SPC2) and PC1/PC3 (SPC3), appear to play a preeminent role in neuroendocrine precursor processing. Both convertases are expressed only in the brain and in the extended neuroendocrine system, while another important family member--furin/PACE (SPC1)--is expressed more ubiquitously, in almost all tissues, and at high levels in liver. SPC2 and SPC3 exhibit acidic pH optima and other properties which enhance their activity in the acidic, calcium-enriched environment of the dense-core secretory granules of the regulated pathway in neuroendocrine cells, while furin has a neutral pH optimum and is localized predominantly to the trans Golgi network where it is retained by a C-terminal transmembrane domain. Furin processes a wide variety of precursors in the constitutive pathway, such as those of growth factors, receptors, coagulation factors, and viral glycoproteins. Recent findings on the processing of proopiomelanocortin, proinsulin, proglucagon, and several other neuroendocrine precursors by SPC2 and SPC3 are discussed, along with information on the structure, properties, evolution, developmental expression, and regulation of the convertases. An inherited defect in the fat/fat mouse which affects the processing of proinsulin, and probably also many other prohormones, due to a point mutation in carboxypeptidase E has recently been identified and has begun to provide new insights into the functional integration of the individual processing steps.

Animals↗
PubMed1995

Amyloid formation in response to beta cell stress occurs in vitro, but not in vivo, in islets of transgenic mice expressing human islet amyloid polypeptide.

BACKGROUND: Human, but not mouse, islet amyloid polypeptide (IAPP) is amyloidogenic. Transgenic mice overexpressing human IAPP in the beta cells of the islets of Langerhans should be useful in identifying factors important for the deposition of IAPP as insoluble amyloid fibrils. MATERIALS AND METHODS: Transgenic mice expressing human IAPP were examined using several experimental models for the production of persistent hyperglycemia, as well as for the overstimulation and/or inhibition of beta cell secretion. Obesity was induced by aurothioglucose. Persistent hyperglycemia was produced by long-term administration of glucocorticosteroids or by partial pancreatectomy. Inhibition of normal beta cell exocytosis by diazoxide administration, with or without concurrent dexamethasone injections, was carried out to increase crinophagy of secretory granules. The human IAPP gene was also introduced into the ab and ob mouse models for diabetes. Finally, isolated islets cultivated in vitro at high glucose concentration were also examined. RESULTS: No amyloid deposits were found in the pancreata of any of the animals, either by light microscopy after Congo red staining or by electron microscopy after immunogold labeling with antibodies specific for human IAPP. Aurothioglucose treatment resulted in increased numbers of granules in the beta cell and the appearance of large lysosomal bodies without amyloid. However, islets from db and ob mice expressing human IAPP cultivated in vitro in the presence of glucocorticosteroid and/or growth hormone, were found to contain extracellular amyloid deposits reacting with antibodies to human IAPP. CONCLUSIONS: Oversecretion of human IAPP or increased crinophagy are not sufficient for amyloid formation. This indicates that other factors must influence amyloid deposition; one such factor may be the local clearance of IAPP.

Amyloid↗
PubMed1994

Nucleotide sequence and analysis of the mouse SPC3 promoter region.

Insulin is converted from the higher molecular weight proprotein, proinsulin by highly specific proteolytic cleavage at two dibasic amino acid sites. SPC3 and SPC2, two recently identified prohormone convertase that are specifically expressed in beta cells and other neuroendocrine cells, appear to be responsible for those cleavages. We have sequenced the 5'-upstream region of the SPC3 gene and examined its promotor/enhancer activity and most of several deletion mutants in several cell lines. This region contains no CAAT box but has several non-functional TATA-like sequences and several putative transcriptional regulatory elements, including AP-1, Sp1 and cAMP response elements. These features are not unlike those of the human SPC2 upstream region. In beta TC3 insulinoma cells, the sequence between the EcoRI (620 bp) and NsiI (702 bp) sites seems to be important for gene expression, while the sequence between the NsiI and DraI (775 bp) sites may contain strong enhancer element(s).

Amino Acid Sequence↗
PubMed1994

High-potency hybrid compounds related to insulin and amphioxus insulin-like peptide.

We describe the synthesis and biological evaluation of five two-chain, insulin-like compounds structurally related both to insulin and to a putative insulin like peptide (ILP) whose sequence was deduced from a cDNA cloned from Branchiostoma californiensis (amphioxus), a primitive vertebrate [Chan, S. J., Cao, Q.-P., & Steiner, D. F. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9319-9323]. The present compounds feature an A-chain corresponding to the A-domain of the putative amphioxus ILP A-domain, except that amino acid substitutions have been made at positions A2, A3, A5, and/or A8, linked via disulfide bonds to the B-chain of bovine insulin. Amphioxus ILP [2 Ile] A/insulin B, amphioxus ILP [2 Ile, 8 His] A/insulin B, amphioxus ILP [2 Ile, 5 Gln, 8 His] A/insulin B, and amphioxus ILP [2 Ile, 3 Ile, 5 Gln, 8 His] A/insulin B all display insulin-like metabolic activity and growth-promoting activity (mitogenesis) equal to or greater than that of natural insulin. Amphioxus ILP [8 His] A/insulin B shows activity in these assays greater than that of its parent compound, but not as high as compounds featuring Ile rather than Leu at position A2. In contrast, the parent compound of the present analogues, i.e., amphioxus ILP A/insulin B, displays potencies ranging from 4.0 to 9.8% relative to insulin in insulin receptor binding and lipogenesis assays, respectively. This parent compound displayed activity in growth factor assays too low for exact quantitation [Chu, Y.-C., Hu, S. Q., Zong, L., Burke, G. T., Gammeltoft, S., Chan, S. J., Steiner, D. F., & Katsoyannis, P. G. (1994) Biochemistry (in press)].(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1994

Insulin-like compounds related to the amphioxus insulin-like peptide.

Three insulin-like compounds consisting of two disulfide-linked polypeptide chains have been synthesized. The A-chains of these compounds correspond either to the A- or to the A + D-domain of the putative amphioxus insulin-like peptide (amphioxus ILP), and their B-chains correspond either to the B-chain of insulin or to a slightly modified (i.e., [1-Thr]) B-domain of amphioxus ILP. The biological potency of these compounds was evaluated in mammalian cells or cell fractions containing either human or rat insulin receptors or human or mouse insulin-like growth factor I (IGF-I) receptors, with respect to binding affinity, insulin-like metabolic activity (lipogenesis), and growth factor activity (mitogenesis). Amphioxus ILP A/bovine insulin B and amphioxus ILP A + D/bovine insulin B exhibited potencies ranging from 2.0 to 9.8% relative to natural insulin, and both compounds were full agonists in lipogenesis assays, stimulating lipogenesis to the same maximal extent as seen with natural insulin. Amphioxus ILP A/amphioxus ILP [1-Thr]B stimulated lipogenesis with a potency of 0.01% relative to natural insulin. We consider this compound also likely to be a full agonist. In assays measuring binding to IGF-I receptors and stimulation of mitogenesis, these compounds displayed some activity although the activity was too low for exact quantification. These results suggest that amphioxus ILP has retained an overall structural similarity to mammalian insulin and IGF-I but has also accumulated substantial mutations which markedly reduce its ability to bind and activate their cognate receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1994

Transcriptional regulation of cathepsin B expression in B16 melanomas of varying metastatic potential.

The highly metastatic B16a melanoma has been shown to express higher levels of cathepsin B (CB) mRNA when compared to the less metastatic variants, B16-F1 and B16-F10, and with normal mouse tissues. This increased expression is now shown to be due to increased gene transcription by nuclear run-off assays and measurements of mRNA stability. Transient expression assays, using promoter fragments from the mouse and human CB genes, demonstrated that both promoters were more active in B16a than in the less metastatic melanomas, B16-F1 and B16-F10. The differential gene expression did not depend on the presence of multiple Sp1 sites in both promoters. A Gel shift assay revealed a specific CB promoter binding protein whose levels are correlated with CB expression and the metastatic potential of the three B16 melanoma variants. These results indicate that the increased expression of CB in the B16a melanoma is due to a specific increase in the amount or activity of a transcriptional activator of the CB gene. The ability of the human CB promoter to activate gene expression in B16a melanoma cells suggests similarities in the regulation of CB expression in tumors from humans and mice.

Animals↗
PubMed1994

Altered gene structure and tissue expression of islet amyloid polypeptide in the chicken.

In mammals, islet amyloid polypeptide (IAPP) is a putative pancreatic peptide hormone that is coproduced and cosecreted with insulin in the beta-cells. However IAPP is also structurally and functionally similar to calcitonin gene-related peptide (CGRP), a 37-amino acid peptide that is expressed predominantly in neurones, and it has been suggested that these peptides arose from a common ancestral gene. In the present study we have characterized an avian IAPP cDNA and gene and have analyzed their expression in various tissues. The cloned chicken IAPP cDNA encodes a 135-amino acid (aa) precursor in which the mature 37-residue IAPP is 80% identical to human IAPP. However, the N-terminal propeptide of chicken proIAPP (55 aa) is considerably longer than that found in the mammalian proIAPPs (9-12 aa) and is comparable in length to that of chicken proCGRP (52 aa). Most of this additional peptide material was found to be encoded in an exon of the cloned chicken IAPP gene that is homologous to exon 3 in the CGRP gene. This exon is absent in the human IAPP gene and thus the exon-intron organization of the chicken IAPP gene more closely resembles that of mammalian CGRP genes. Northern blot analyses demonstrated that chicken IAPP mRNA is expressed predominantly in intestine and brain but at a much lower level in pancreas. The pancreas and intestine contained a single 0.7 kilobase (kb) IAPP transcript while two transcripts, 0.7 kb and 0.9 kb, were detected in brain. Densitometric analysis indicated that IAPP transcripts were 11 times more abundant in brain and intestine than pancreas.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Human islet amyloid polypeptide transgenic mice as a model of non-insulin-dependent diabetes mellitus (NIDDM).

To model islet amyloidogenesis in NIDDM and explore the glucoregulatory role of islet amyloid polypeptide (IAPP), we have created transgenic mice containing a rat insulin-I promoter-human IAPP fusion gene. Expression of human IAPP was localized to the islets of Langerhans, anterior pituitary and brain in transgenic animals; blood IAPP levels were elevated 5-fold while fasting glucose levels remained normal. Amyloid deposits have not been detected in transgenic islets suggesting that other co-existing abnormalities in NIDDM may be required for the formation of islet amyloid. These animals provide a unique model for exploring this hypothesis and other proposed functions of IAPP.

Amyloid↗
PubMed1993

Cloning and analysis of the gene encoding hummingbird proinsulin.

Because hummingbirds exhibit the highest mass-specific metabolic rates seen among vertebrates and rely on sugars as their main energy source, we have investigated the structure of hummingbird insulin (Selaphorus rufus) to determine whether it possesses structural adaptations that increase its receptor binding affinity (potency). We report here the nucleotide sequence of hummingbird proinsulin determined from hummingbird genomic DNA. The predicted amino acid sequence of the A-chain of hummingbird insulin is identical to that of chickens and the B-chain differs by only one amino acid at a noncritical position, B2 (Val in hummingbird and Ala in chicken). These findings suggest that alterations in secretory and metabolic dynamics of insulin are of greater importance than changes in binding affinity in the adaptation to states of high carbohydrate flux in these very energetic organisms.

Amino Acid Sequence↗
PubMed1993

Characterization of the cathepsin B gene and multiple mRNAs in human tissues: evidence for alternative splicing of cathepsin B pre-mRNA.

We have cloned and characterized multiple messages for cathepsin B that differ in their 5' and 3' untranslated regions (UTRs) from human kidney and the hepatoma cell line HepG2. A comparison of these messages with the cloned human cathepsin B gene reveals that they arise by alternative splicing of a single gene. Processing at a cryptic intron donor site in exon 11 and splicing to exon 12 produces a 4.0-kb message with an alternate 3' UTR in addition to the 2.3-kb message described previously by Chan et al. (1986). Variable removal of exon 2 produces cathepsin B mRNAs which differ by 88 nucleotides in their 5'-UTRs. The ratio of the 2.3-kb to 4.0-kb transcript is about 2:1 in most of the tissues examined, but the ratio of mRNAs with variant 5' UTRs differs widely. Cathepsin B mRNAs lacking exon 2 are predominant in human tumors. In addition, human breast and colon carcinomas and a human melanoma contain a cathepsin B transcript that is also missing exon 3 encoding the signal peptide and 7 residues of the activation propeptide. An in vitro transcription/translation assay was used to demonstrate that this message could be translated from an internal methionine codon (residue 52), producing a 32-kD product lacking the signal peptide and more than half the propeptide. The transcription/translation assay also demonstrated that the variant messages differ in their rates of translation. The relative rates are about 8:2:1 for mRNA lacking exons 2 and 3 compared to mRNA lacking exon 2 and mRNA containing the full-length 5' end, respectively. These results suggest that the expression of cathepsin B in human tissues may be regulated in part at the level of mRNA processing.

Alternative Splicing↗
PubMed1993

Structure of the chum salmon insulin-like growth factor I gene.

Insulin-like growth factor I (IGF-I) plays a major role in development and metabolism. Currently, the cDNA-derived primary structure of IGF-I is known for some mammals and for chicken, frog, and salmon. Additionally, the organization of the human, rat, and chicken IGF-I genes has been established. The investigation of IGF-I gene structure in fish would extend the evolutionary picture for this hormone and facilitate our understanding of the features of the IGF-I gene that are common to all vertebrate species. The cloned chum salmon IGF-I gene appears to be much more compact than the mammalian and avian genes, being less than 20 kb in length. As in other species, however, the mature IGF-I peptide appears to consist of 70 amino acids and is encoded by exons 2 and 3. Intriguingly, exon 1-encoded 5'-untranslated region sequences are highly conserved, while the coding sequences at the 3' end of the same exon are less conserved. The amino terminus of the signal peptide is four amino acids shorter than in the mammalian and avian peptides. The end of the B domain, the C, A, and D domains, and the first part of the E peptide are encoded by exon 3, but the exon 3-encoded E peptide sequence is 27 amino acids longer than in other species. These extra 27 amino acids, encoded by both coho and chum salmon cDNAs, may be deleted by alternative splicing, as suggested from the sequence of a coho salmon IGF-I cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Familial hyperproinsulinemia associated with NIDDM. A case study.

OBJECTIVE: To report studies on an elderly patient with moderate NIDDM associated with marked fasting hyperinsulinemia. RESEARCH DESIGN AND METHODS: The propositus and several family members were studied by a combination of clinical, biochemical, and molecular genetic approaches to define the underlying genetic defect. RESULTS: Fasting levels of contrainsulin hormones were normal, and resistance to exogenous insulin was absent. Gel filtration and reverse-phase high-performance liquid chromatography revealed elevated amounts of a structurally abnormal proinsulin intermediate (AC proinsulin). A study of the family of the propositus showed the same abnormality in 4 of 5 members in 3 successive generations. Genetic analysis revealed a point mutation affecting residue 65 of human proinsulin (Arg-->His) in one allele of the insulin gene in the propositus, a defect similar to that described previously in 3 other apparently unrelated lineages. CONCLUSIONS: This family exhibits a clear-cut relationship between increasing age and metabolic decompensation in all the hyperproinsulinemic members, suggesting that (inherited) metabolic stress and age both contribute to development of diabetes mellitus.

Adolescent↗
PubMed1993

Evolution of insulin-like growth factor binding proteins.

In order to understand more about the origins and evolutionary relationships of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) we have commenced characterization of the IGFBPs in non-mammalian sera. We show by both in vitro labelling with subsequent analytical gel filtration and by ligand blotting that IGFBPs are present in lamprey serum, indicating that the IGFBPs are indeed, very ancient proteins. Furthermore we present evidence that the major serum IGFBP in lamprey is significantly smaller than the high molecular weight IGFBP3 complex in rat serum. In addition, we show that IGFBPs occur widely throughout vertebrates as we detect IGFBPs in ligand blots of serum from marsupials, monotremes, reptiles, birds and turtles.

Animals↗
PubMed1992

Cystatin C and cathepsin B in human colon carcinoma: expression by cell lines and matrix degradation.

Expression of the cysteine proteinase cathepsin B and its physiological inhibitor cystatin C was analyzed in vitro in 1 human fibrosarcoma and 4 human colon carcinoma cell lines. Cystatin C antigen as well as cathepsin B activity were detected in the conditioned media of the 5 cell lines. The corresponding cell extracts expressed high levels of cathepsin B activity, whereas only trace amounts of cystatin C antigen could be found. Northern-blot analysis revealed the presence in the 5 cell lines of a 0.8-kb cystatin C mRNA transcript and 2 cathepsin B transcripts of 2.3 and 4.3 kb. Pepsin treatment of tumor-cell-released cathepsin B induced an average 7.3-fold increase in activity, indicating that the enzyme was mainly present as a latent form in conditioned medium. The pepsin-activated cathepsin B from one colon carcinoma cell line was further characterized using the cysteine proteinase inhibitors E-64, recombinant cystatin C, a cystatin-C-derived peptidyl inhibitor (Z-LVG-CHN2), and cathepsin-B-specific diazomethyl ketone inhibitors (Z-FT(OBzl)-CHN2, Z-FS(OBzl)-CHN2). This activity was totally neutralized by recombinant cystatin C, suggesting a potential for interaction between released extracellular cathepsin B and cystatin C. In vitro assays of degradation of extracellular matrix showed that cysteine proteinase inhibitors could decrease matrix degradation induced by pepsin-activated conditioned media. With colon cells, this inhibition was not observed, indicating a requirement for an extracellular activation of latent cathepsin B. Our data provide evidence that cystatin C and latent cathepsin B are both released extracellularly by colon carcinoma cells in vitro. They suggest that cystatin C and cathepsin B interactions may participate, in an as yet unelucidated way, in the modulation of the invasive phenotype of human colonic tumors.

Carcinoma↗