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S J Chan

Publications and source records attributed to S J Chan.

At least 73 records · Page 4Linked to original sources
PubMed1988

A mutant human proinsulin is secreted from islets of Langerhans in increased amounts via an unregulated pathway.

A coding mutation in the human insulin gene (His-B10----Asp) is associated with familial hyperproinsulinemia. To model this syndrome, we have produced transgenic mice that express high levels of the mutant prohormone in their islets of Langerhans. Strain 24-6 mice, containing about 100 copies of the mutant gene, were normoglycemic but had marked increases of serum human proinsulin immunoreactive components. Biosynthetic studies on isolated islets revealed that approximately 65% of the proinsulin synthesized in these mice was the human mutant form. Unlike the normal endogenous mouse proinsulin, which was almost exclusively handled via a regulated secretory pathway, up to 15% of the human [Asp10]proinsulin was rapidly secreted after synthesis via an unregulated or constitutive pathway, and approximately 20% was degraded within the islet cells. The secreted human [Asp10]proinsulin was not processed proteolytically. However, the processing of the normal mouse and human mutant proinsulins within the islets from transgenic mice was not significantly impaired. These findings suggest that the hyperproinsulinemia of the patients is the result of the continuous secretion of unprocessed mutant prohormone from the islets via this alternative unregulated pathway.

Animals↗
PubMed1988

Use of in vitro DNA amplification to screen family members for an insulin gene mutation.

The DNA polymerase chain reaction can be a powerful tool for amplifying selected segments of genomic DNA for investigation of point mutations that are inaccessible via classic restriction-fragment-length polymorphism analysis. We have applied this method to an analysis of the incidence of heterozygosity for the mutant insulin allele insulin Wakayama (A3 Val----Leu) in two unrelated Japanese families having the hyperinsulinemic mutant insulin syndrome. The results indicate that this method is simple, sensitive, and accurate and should be useful for screening larger (diabetic) populations to detect single-base substitutions in the insulin gene that lead to either altered (pro)insulin structure and/or insulin production.

Amino Acid Sequence↗
PubMed1988

Expression of rat and mouse cathepsin B precursors in Escherichia coli.

Vectors for the expression of mouse preprocathepsin B and rat procathepsin B, under the control of a modified lac operon regulatory region, in plasmid pKK233-2 were constructed. In rapidly growing cells induction of preprocathepsin B was cytotoxic. Immune precipitable cathepsin B, compatible in size with that expected for the unglycosylated proenzyme, was produced in both constructs. However, pepsin digestion of extracts did not produce any cathepsin B activity, indicating either that the recombinant forms are not correctly folded or were produced at such low levels that activity could not be detected.

Animals↗
PubMed1987

Insulin Wakayama: familial mutant insulin syndrome in Japan.

We describe a family from Japan displaying the mutant insulin syndrome with hyperinsulinaemia and an increased insulin: C-peptide molar ratio. Serum insulin isolated from several family members showed reduced in vitro biological activity, and analysis by high performance liquid chromatography revealed a peak co-eluting with human insulin and a second species of increased hydrophobicity co-migrating with the previously reported Insulin Wakayama. The insulin genes from the propositus were cloned and sequenced, revealing one normal allele; the second allele, encoding a leucine for valine amino acid substitution at position 3 of the insulin A chain, was similar to that previously described for Insulin Wakayama. Synthesized [LeuA3] insulin showed 0.14% of receptor binding activity on rat adipocytes and a 10-fold prolonged half-life in a somatostatin-infused dog compared with human insulin. The finding of the same mutant gene in two unrelated Japanese families suggests that Insulin Wakayama may be discovered in additional Japanese families with hyperinsulinaemia and/or diabetes.

Adult↗
PubMed1987

A mutation in the B chain coding region is associated with impaired proinsulin conversion in a family with hyperproinsulinemia.

Gruppuso et al. [Gruppuso, P.A., Gordon, P., Kahn, C. R., Cornblath, M., Zeller, W. P. & Schwartz, R. (1984) N. Engl. J. Med. 311, 629-634] have recently described a family in which hyperproinsulinemia is inherited in an autosomal dominant pattern, suggesting a structural abnormality in the proinsulin molecule as the basis for this disorder. However, unlike two previous kindreds with a similar syndrome, the serum proinsulin-like material in this family did not appear to be an intermediate conversion product but instead behaved like normal human proinsulin by several criteria. To further characterize this disorder we isolated and sequenced the insulin gene of the propositus. Leukocyte DNA was cloned into lambda-WES and recombinants containing the two insulin alleles, lambda MD41 and lambda MD51, were isolated by plaque hybridization. DNA sequencing of lambda MD51 showed that it contained the normal coding sequence for human preproinsulin. Sequence analysis of lambda MD41, however, revealed a single nucleotide substitution in the codon for residue 10 of proinsulin (CAC----GAC) that predicts the exchange of aspartic acid for histidine in the insulin B chain region. This mutation was also found in an insulin allele cloned from a second affected family member (propositus's father). These results, along with the linkage analysis of Elbein et al. [Elbein, S.C., Gruppuso, P., Schwartz, R., Skolnick, M. & Permutt, M.A. (1985) Diabetes 34, 821-824], strongly implicate this mutation as the cause of the hyperproinsulinemia in this family. Inhibition of the conversion of proinsulin to insulin may be related to altered folding and/or self-association properties of the [Asp10]proinsulin.

Cloning, Molecular↗
PubMed1987

Staphylococcus aureus bacteremia: significance of hyperbilirubinemia.

We studied 233 consecutive episodes of Staphylococcus aureus bacteremia in 230 patients between 1980 and 1984 at a community teaching hospital. Bacteremia was community-acquired in 78 episodes, acquired from nursing homes in 22 episodes and hospital-acquired in 133 episodes. The over-all mortality was 48.9%. Patients greater than or equal to 60 years had higher mortality (62.0%) than patients less than 60 years old (25.3%). Hospital-acquired bacteremia was associated with a higher mortality (59.4%) than community-acquired bacteremia (29.5%). The respiratory tract as the portal of entry of bacteremia was associated with a higher mortality (80.4%), as compared to 53.5% when the portal of entry was undetermined, and 28.1% when the portal of entry was other sources. Increasing serum creatinine levels were associated with increasing mortality: less than 88.4 mumol/l (26.5%), 97.2-168.0 mumol/l (51.1%), and greater than 176.8 mumol/l (67.9%). Increasing serum bilirubin levels were also associated with increasing mortality: less than 17.1 mumol/l (40.6%), 18.8-49.6 mumol/l (57.1%), and greater than 51.3 mumol/l (84.2%). Very high leukocyte counts were associated with higher mortality: greater than 20 X 10(9)/l (73.9%), 10-20 X 10(9)/l (37.4%), and less than 10 X 10(9)/l (45.8%).

Age Factors↗
PubMed1986

Mutations in the guinea pig preproglucagon gene are restricted to a specific portion of the prohormone sequence.

A cDNA clone encoding guinea pig preproglucagon has been isolated from a pancreatic cDNA library. The predicted amino acid sequence of proglucagon is highly conserved in all regions, in comparison to other mammals, except for the C-terminal portion of the 29-residue glucagon region, in which 5 amino acid substitutions have occurred. These changes may serve to offset the reduced receptor-binding potency of the highly mutated insulin in this New World species.

Amino Acid Sequence↗
PubMed1986

Differences in cathepsin B mRNA levels in rat tissues suggest specialized functions.

The tissue distribution of mRNAs encoding two lysosomal proteases, cathepsin B and cathepsin D, was examined using cloned cDNAs to probe Northern and dot blots of RNAs extracted from various rat tissues. Cathepsin B mRNA showed a wide range of variation in expression in the tissues analyzed with the highest concentrations found in spleen and kidney, while the cathepsin D mRNA levels were relatively uniform in these same tissues. Significant quantities of cathepsin B mRNA were detected in total RNA from isolated islets of Langerhans but was not detectable in equivalent amounts of RNA from whole pancreas. The wide variations in tissue levels of cathepsin B mRNA suggest that tissue specific controls may regulate its expression and are compatible with the participation of this protease in specialized cellular functions other than intralysosomal protein degradation.

Animals↗
PubMed1986

Nucleotide and predicted amino acid sequences of cloned human and mouse preprocathepsin B cDNAs.

Cathepsin B is a lysosomal thiol proteinase that may have additional extralysosomal functions. To further our investigations on the structure, mode of biosynthesis, and intracellular sorting of this enzyme, we have determined the complete coding sequences for human and mouse preprocathepsin B by using cDNA clones isolated from human hepatoma and kidney phage libraries. The nucleotide sequences predict that the primary structure of preprocathepsin B contains 339 amino acids organized as follows: a 17-residue NH2-terminal prepeptide sequence followed by a 62-residue propeptide region, 254 residues in mature (single chain) cathepsin B, and a 6-residue extension at the COOH terminus. A comparison of procathepsin B sequences from three species (human, mouse, and rat) reveals that the homology between the propeptides is relatively conserved with a minimum of 68% sequence identity. In particular, two conserved sequences in the propeptide that may be functionally significant include a potential glycosylation site and the presence of a single cysteine at position 59. Comparative analysis of the three sequences also suggests that processing of procathepsin B is a multistep process, during which enzymatically active intermediate forms may be generated. The availability of the cDNA clones will facilitate the identification of possible active or inactive intermediate processive forms as well as studies on the transcriptional regulation of the cathepsin B gene.

Amino Acid Sequence↗
PubMed1986

T-cell receptor alpha-chain gene is split in a human T-cell leukemia cell line with a t(11;14)(p15;q11).

Chromosomal rearrangements in malignant T-cell disease frequently involve the chromosome bands containing the T-cell receptor genes. The RPMI 8402 cell line, which was established from the leukemia cells of a patient with T-cell acute lymphoblastic leukemia, is characterized by a translocation involving chromosome 14 (band q11) and chromosome 11 (band p15) [t(11;14)(p15;q11)]. By using in situ chromosomal hybridization and Southern blot analysis to examine RPMI 8402 cells, we determined that the break at 14q11 occurs within the variable region sequences of the T-cell receptor alpha-chain gene (TCRA); the break at 11p15 occurs between the HRAS1 gene and the genes for insulin and the insulin-like growth factor 2. These results suggest that the TCRA sequences activate a cellular gene located at 11p15 in malignant T-cell disorders.

Chromosome Banding↗
PubMed1985

Identification of cDNA clones encoding a precursor of rat liver cathepsin B.

Recent studies have suggested that many lysosomal enzymes, including cathepsin B (EC 3.4.22.1), may be synthesized as larger precursors and proteolytically processed to their mature forms. To determine the structure of the primary translation product of cathepsin B, we have screened a phage cDNA library for clones encoding rat liver cathepsin B. We synthesized two extended DNA oligonucleotides to use as hybridization probes: a 50-mer corresponding to the coding segment for residues 215-231 of mature cathepsin B and a 54-mer corresponding to residues 117-134. After screening 600,000 plaques, five clones were obtained that hybridized to the 32P-labeled 50-mer; of these, two (lambda rCB3 and lambda rCB5) also reacted with the 54-mer. DNA sequence analysis confirmed that lambda rCB3 and lambda rCB5 both encoded rat liver cathepsin B, and the translated sequence is in agreement with the sequence determined [Takio, K., Towatari, T., Katunuma, N., Teller, D. C. & Titani, K. (1983) Proc. Natl. Acad. Sci. USA 80, 3666-3670], except for a tryptophan for glycine substitution at residue 78 and the presence of two amino acids at the junction site of the light and heavy chains. Moreover, the DNA sequence reveals an open reading frame extending beyond the 5' (NH2 terminus), and the predicted COOH terminus of the coding sequence for the mature protein is extended by six amino acids. These results confirm that the biosynthesis of cathepsin B involves a larger precursor form and demonstrate the effectiveness of long oligonucleotide probes for screening to detect rare cloned mRNAs.

Animals↗
PubMed1984

Familial hyperinsulinemia due to a structurally abnormal insulin. Definition of an emerging new clinical syndrome.

We have identified a patient with mild diabetes, marked fasting hyperinsulinemia (89 to 130 microU of insulin per milliliter), and a reduced fasting C-peptide: insulin molar ratio of 1.11 to 1.50 (normal, greater than 4). The patient responded normally to exogenous insulin. However, her endogenous immunoreactive insulin showed reduced biologic activity during a glucose-clamp study with hyperglycemia and a reduced ability to bind to the insulin receptor and stimulate glucose transport in vitro. Family studies showed that five additional relatives in three generations had variable degrees of glucose intolerance, marked hyperinsulinemia, and a reduced peripheral C-peptide:insulin molar ratio. Restriction-endonuclease cleavage of DNA isolated from circulating leukocytes in the patient and in family members with hyperinsulinemia revealed loss of the MboII recognition site in one allele of the insulin gene--consistent with a point mutation at position 24 or 25 in the insulin B chain. Other studies using high-pressure liquid chromatography and detailed gene analysis have identified the defect as a serine for phenylalanine substitution at position 24 of the insulin B chain. The secretion of a structurally abnormal insulin should be considered in patients with hyperinsulinemia who respond normally to exogenous insulin and have a reduced C-peptide:insulin molar ratio. Glucose tolerance may range from relatively normal to overtly diabetic.

Adult↗
PubMed1984

Guinea pig preproinsulin gene: an evolutionary compromise?

We characterized a clone carrying the guinea pig preproinsulin gene, which, in contrast to other mammalian preproinsulin genes, is highly divergent in its regions encoding the B and A chains of mature insulin. Blot hybridization analysis indicates that this gene is present in only one copy in the guinea pig genome and that other normal or mutated preproinsulin genes do not exist in this animal. Moreover, the position of introns in this gene and the homology of its 3' flanking region to the corresponding regions of other sequenced mammalian genes show that it has been derived from the common mammalian stock. The rapid evolution of the region encoding the B and A chains can be interpreted, according to our sequence-divergence analysis, as due to the fixation of both neutral and adaptive mutations.

Amino Acid Sequence↗
PubMed1983

Cloning and nucleotide sequence analysis of the dog insulin gene. Coded amino acid sequence of canine preproinsulin predicts an additional C-peptide fragment.

A 4.0-kilobase HindIII/EcoRI-cleaved dog genomic DNA fragment was shown to contain the dog insulin gene by restriction mapping using a human insulin cDNA probe. This fragment was subsequently cloned in a lambda vector, and the nucleotide sequence of the dog insulin gene was determined. As in several other species, the insulin gene of the dog is interrupted by two intervening sequences, one of 151 base pairs located in the 5' untranslated region and the other of 264 base pairs occurring within the codon of the 7th amino acid of the C-peptide. Translation of the nucleotide sequence in one frame revealed the primary structure of canine preproinsulin. An interesting feature of the coded amino acid sequence is that it predicts a C-peptide of 31 amino acids, 8 residues longer than that reported by Peterson et al. (Peterson, J. D., Nehrlich, S., Oyer, P. E., and Steiner, D. F. (1973) J. Biol. Chem. 247, 4866-4871). The additional octapeptide sequence, Glu-Val-Glu-Asp-Leu-Gln-Val-Arg, is located NH2-terminal to the 23-residue C-peptide sequence described in the earlier report. Its coding sequence is interrupted by the second intervening sequence. The arginine at position 8 suggests that a trypsin-like cleavage may separate the NH2-terminal octapeptide from the remainder of the C-peptide during the post-translational processing of dog proinsulin in the pancreas. The revised C-peptide sequence suggests that the proinsulin C-peptide is more highly conserved in length and overall sequence than was previously supposed.

Amino Acid Sequence↗
PubMed1983

Studies on mutant human insulin genes: identification and sequence analysis of a gene encoding [SerB24]insulin.

Both alleles of the insulin gene of a patient with mild diabetes [maturity-onset-diabetes-of-the-young (MODY)-type syndrome] associated with hyperinsulinemia have been cloned, and the sequences have been determined. One allele contained a mutation (single nucleotide transition) in the coding sequence for the B chain at position 24 (TTC leads to TCC), resulting in the loss of a restriction enzyme (Mbo II) cleavage site in the gene. This mutation results in the substitution of serine for phenylalanine in a critically important region of the insulin molecule that is intimately involved in receptor binding. Both insulin alleles were of the alpha type and, aside from a single nucleotide deletion in the 5' region of the normal allele, their sequences were identical to those previously determined.

Amino Acid Sequence↗