Redox state-dependent and sorbitol accumulation-independent diabetic albuminuria in mice with transgene-derived human aldose reductase and sorbitol dehydrogenase deficiency.

AIMS/HYPOTHESIS: We investigated the role played by sorbitol accumulation in the kidney in the development of diabetic albuminuria. METHODS: We created mice ( hAR-Tg:SDH null) with transgene-derived human aldose reductase and sorbitol dehydrogenase (SDH) deficiency, and analysed (i). the contribution of accumulated sorbitol to urinary albumin excretion rate, and (ii). the effect of the aldose reductase inhibitor, epalrestat, on the diabetic redox state, including decreased renal reduced glutathione concentrations or increased lactate to pyruvate ratios in the diabetic kidney. RESULTS: Compared to littermates, non-diabetic transgenic mice had a 2.6-fold increase in aldose reductase mRNA. In a diabetic group, aldose reductase mRNA in hAR-Tg mice was 2.7-fold higher than in littermates. In the diabetic and non-diabetic groups, hAR-Tg:SDH null mice had the highest sorbitol content among all four genetic types including hAR-Tg:SDH null, SDH null, hAR-Tg and littermates. The urinary albumin excretion rate in non-diabetic groups was similar in the four genetic types of mouse. In diabetic groups it was greater than in non-diabetic groups, but did not correlate with the sorbitol content among the four genetic types of mouse. When aldose reductase inhibitor and streptozotocin were given simultaneously at 6 weeks of age, epalrestat prevented diabetic increases in urinary albumin excretion rate and completely prevented diabetic decreases in reduced glutathione concentrations and diabetic increases in lactate to pyruvate ratios, even in the presence of transgenic aldose reductase. CONCLUSIONS/INTERPRETATION: The degree of diabetic albuminuria in genetically modified mice is dependent on the redox state and independent of polyol accumulation; aldose reductase inhibitor can prevent diabetic albuminuria by normalising diabetic redox changes.

Diabetes and pancreatic tumours in transgenic mice expressing Pa x 6.

AIMS/HYPOTHESIS: Both endocrine and exocrine cells of the pancreas differentiate from epithelial cells of primitive pancreatic ducts, and four types of pancreatic islet cells (alpha, beta, delta, and PP cells) are derived from the common pluripotent precursor cells. Although Pa x 6 is expressed in all islet cells, Pa x 4 is detected only in beta cells. In homozygous Pa x 4-null mice, beta cells are absent, whereas the number of alpha cells is increased. Therefore, we hypothesized that the balance of Pa x 4 and 6 is one of the determinants by which the common progenitor cells differentiate into alpha or beta cells. METHODS: To change this balance, we generated transgenic mice overexpressing Pa x 6 driven by the insulin promoter or the PDX1 promoter. RESULTS: In both types of transgenic mice, normal development of beta cells was disturbed, resulting in apoptosis of beta cells and diabetes. In Insulin/Pa x 6-Tg mice, beta cells were specifically affected, whereas in PDX/Pa x 6-Tg mice, developmental abnormalities involved the whole pancreas including hypoplasia of the exocrine pancreas. Furthermore, PDX/Pa x 6-Tg mice experienced proliferation of both ductal epithelia and islet cells and subsequent cystic adenoma of the pancreas. CONCLUSION/INTERPRETATION: These findings suggest that Pa x 6 promotes the growth of ductal epithelia and endocrine progenitor cells and that the suppression of Pa x 6 is necessary for the normal development of beta cells and the exocrine pancreas.

Expression of somatostatin receptor subtypes and growth inhibition in human exocrine pancreatic cancers.

The antiproliferative effects of somatostatin and its analogs on human pancreatic cancers were studied: (1) by evaluating the gene expression of somatostatin receptor (sstr) subtypes in human pancreatic cancer cell lines and cancer tissue specimens, (2) by evaluating the antiproliferative effects of somatostatin analogs, and (3) by evaluating the effect of sstr-2 cDNA transduction. Using a reverse transcriptase polymerase chain reaction (RT-PCR), the gene expression of five sstr subtypes (sstr-1 to -5) was examined in eight cell lines, and in ten pancreatic cancer tissues and in the normal surrounding pancreatic tissues. The antiproliferative effects of somatostatin (SS) -14 and its two analogs (SMS 201-995, RC-160) were examined by means of an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (thiazolyl blue)) assay on three cell lines and Panc-1 transfectants with human sstr (hsstr)-2A cDNA. Sstr-2 was expressed in all samples tested. All examined cell lines simultaneously expressed sstr-2 and -5, while most of the examined pancreatic cancer tissues did not express both of these subtypes simultaneously. Somatostatin analogs inhibited epidermal growth factor (EGF)-stimulated pancreatic cancer cell proliferation. The cell proliferation was further and significantly inhibited by 14% in stable transfectants of Panc-1 cells with hsstr-2A. Based on these findings, it is concluded that somatostatin analogs with their antiproliferative effects mediated by sstr-2 could be potentially useful in the treatment of pancreatic cancers.

Apoptosis and remodelling of beta cells by paracrine interferon-gamma without insulitis in transgenic mice.

AIMS/HYPOTHESIS: To examine whether interferon-gamma destroys islet beta cells directly or indirectly through lymphocyte activation, or whether direct action of interferon-gamma on beta cells by itself induces diabetes without insulitis. METHODS: To avoid possible nonspecific breakdown of beta cells by transgenic overexpression of interferon-gamma by the insulin promoter, we generated transgenic mice expressing interferon-gamma under the control of rat glucagon promoter (RGP-IFN-gamma-Tg mice). RESULTS: The absence of insulitis in RGP-IFN-gamma-Tg mice enabled us to investigate the direct effects of paracrine interferon-gamma. In RGP-IFN-gamma-Tg mice, serum concentrations of interferon-gamma and tumour necrosis factor-alpha (TNF-alpha) were 50 and 6 times higher than those in their littermates, respectively, and glucose-responsive insulin secretion decreased to one-half the level of that in the littermates. Transgenic interferon-gamma induced remodelling of beta cells where apoptosis of many beta cells was compensated by their vigorous regeneration and diabetes did not occur in most of the RGP-IFN-gamma-Tg mice. CONCLUSION/INTERPRETATION: Interferon-gamma alone is insufficient for the complete destruction of beta cells in vivo, and factors other than interferon-gamma including activated lymphocytes or other cytokines, are necessary in addition to interferon-gamma for the development of Type I (insulin-dependent) diabetes mellitus.

Hypoplasia of pancreatic islets in transgenic mice expressing activin receptor mutants.

Activin, a member of the TGF-beta superfamily, regulates the growth and differentiation of a variety of cell types. Based on the expression of activin in pancreatic rudiments of rat embryos and stimulation of insulin secretion from adult rat pancreatic islets by activin, activin is implicated in the development and function of islets. To examine the significance of activin signaling in the fetal and postnatal development of islets, transgenic mice expressing a dominant negative form of activin receptor (dn-ActR) or a constitutively active form of activin receptor (ActR-T206D) in islets were generated together with the transgenic mice expressing intact activin receptor (intact ActR) as a negative control. Transgenic mice with both dn-ActR and ActR-T206D showed lower survival rates, smaller islet area, and lower insulin content in the whole pancreas with impaired glucose tolerance when compared with transgenic mice with intact ActR or littermates, but they showed the same alpha cell/beta cell ratios as their littermates. In addition to islet hypoplasia, the insulin response to glucose was severely impaired in dn-ActR transgenic mice. It is suggested that a precisely regulated intensity of activin signaling is necessary for the normal development of islets at the stage before differentiation into alpha and beta cells, and that activin plays a role in the postnatal functional maturation of islet beta cells.

Absence of germ-line mutations of the multiple endocrine neoplasia type 1 (MEN1) gene in familial pituitary adenoma in contrast to MEN1 in Japanese.

Germ-line mutations of the MEN1 gene were analyzed in five cases of familial and four cases of sporadic multiple endocrine neoplasia type 1 (MEN-1), six cases in three independent pedigrees of familial pituitary adenoma without MEN-1, and three cases of familial isolated primary hyperparathyroidism (FIHP) in Japanese. Eight different types of germ-line mutations in all nine cases of MEN-1 were distributed in exons 2, 3, 7, and 10 and intron 7 of the MEN1 gene. Loss of heterozygosity (LOH) on 11q13 was detected in all nine tumors of these cases with microsatellite analysis. No germ-line mutation of the MEN1 gene was detected in three pedigrees of familial pituitary adenoma and three cases of FIHP. LOH on 11q13 was detected in two cases in one pedigree of familial pituitary adenoma, and one of them showed a heterozygous somatic mutation of the MEN1 gene. No LOH on 11q13 was detected in three cases of FIHP. Based on these, we conclude that the loss of function of menin is etiological for familial or sporadic MEN-1, but not for FIHP or most familial pituitary adenoma without MEN-1.

Amidophosphoribosyltransferase limits the rate of cell growth-linked de novo purine biosynthesis in the presence of constant capacity of salvage purine biosynthesis.

Factors controlling relative flux rates of the de novo and salvage pathways of purine nucleotide biosynthesis during animal cell growth are not fully understood. To examine the relative role of each pathway for cell growth, three cell lines including CHO K1 (a wild-type Chinese hamster ovary fibroblast cell line), CHO ade -A (an auxotrophic cell line deficient of amidophosphoribosyltransferase (ATase), a presumed rate-limiting enzyme of the de novo pathway), and CHO ade -A transfected with human ATase cDNA (-A+hATase) resulting in 30-350% of the ATase activity of CHO K1, were cultured in purine-rich or purine-free media. Based on the enzyme activities of ATase and hypoxanthine phosphoribosyltransferase, the metabolic rate of the de novo and salvage pathways, the rate of cell growth (growth rate) in three cell lines under various culture conditions, and the effect of hypoxanthine infusion on the metabolic rate of the de novo pathway in rat liver, we concluded the following. 1) In -A+hATase transfectants, ATase activity limits the rate of the de novo pathway, which is closely linked with the growth rate. 2) Purine nucleotides are synthesized preferentially by the salvage pathway as long as hypoxanthine, the most essential source of purine salvage, can be utilized, which was confirmed in rat liver in vivo by hypoxanthine infusion. The preferential usage of the salvage pathway results in sparing the energy expenditure required for de novo synthesis. 3) The regulatory capacity of the de novo pathway (about 200%) was larger than that of the salvage pathway (about 20%) with constant hypoxanthine phosphoribosyltransferase activity.

Prevention of adoptively transferred diabetes in nonobese diabetic mice with IL-10-transduced islet-specific Th1 lymphocytes. A gene therapy model for autoimmune diabetes.

Four pancreatic islet-specific CD4+ helper T (Th) 1 (Th1) clones and two Th1 clones transduced with an SRalpha promoter-linked murine IL-10 (mIL-10) cDNA of 2.0-6.0 x 10(6) cells were adoptively transferred to nonobese diabetic (NOD) mice at age 8 d. Cyclophosphamide (CY) was administered at age 37 d (plus CY), and the incidence of diabetes and the histological grade of insulitis were examined at age 47 d. After the adoptive transfer of IL-10-transduced Th1 cells, polymerase chain reaction (PCR) and reverse-transcription (RT)-PCR detected the neo gene and the retrovirus vector-mediated IL-10 mRNA in situ in recipient islets, respectively. RT-PCR detected the decrease of IFN-gamma mRNA relative to IL-10 mRNA in IL-10-transduced Th1 clones in vitro and also in recipient islets. All four wild type Th1 clones plus CY induced the insulitis grade of 2.75 and diabetes in 66% of recipient NOD mice. IL-10-transduced two Th1 clones plus CY induced periinsulitis with the grade of 1.43 and diabetes in 8.0%. The 1:1 mixture of wild type Th1 cells and IL-10-transduced Th1 cells plus CY induced periinsulitis with the grade of 1.85 and diabetes in 20%. The suppression of diabetes through decreasing IFN-gamma mRNA by the tissue-specific delivery of IL-10 to pancreatic islets with IL-10-transduced Th1 cells affords us the starting basis to develop the gene therapy for autoimmune diabetes.

Bridged to Fused Ring Interchange. Methodology for the Construction of Fused Cycloheptanes and Cyclooctanes. Total Syntheses of Ledol, Ledene, and Compressanolide.

The type two intramolecular Diels-Alder reaction (T2IMDA) is an efficient method for the formation of medium rings. The methodology is particularly effective for the construction of seven- and eight-membered rings. A strategy for the synthesis of functionalized cycloheptanes and cyclooctanes has been developed that involves a bridged to fused ring interchange. The T2IMDA provides a synthesis for rigid bridged bicyclic molecules that can be stereoselectively elaborated before ozonolysis of the bridgehead double bond. Following oxidative cleavage, aldol condensation provides fused bicyclic ring systems that otherwise are difficult to synthesize. This methodology is amenable to the synthesis of terpene natural products. This is demonstrated here through total syntheses of (+/-)-ledol and (+/-)-ledene and a formal synthesis of (+/-)-compressanolide.

Molecular cloning of a human cDNA encoding a trifunctional enzyme of carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase in de Novo pyrimidine synthesis.

A human CAD cDNA encoding a trifunctional enzyme of carbamoylphosphate synthetase-aspartate transcarbamoylase-dihydroorotase, which catalyzes the first three steps of de novo pyrimidine nucleotide biosynthesis, was cloned from a human fibroblast cell line of TIG-1-20 by polymerase chain reaction (PCR). The predicted open reading frame encodes a protein of 2,225 amino acids with a deduced molecular weight (Mr) OF 242,913. The deduced amino acid sequence exhibits 95.3 and 76.1% identity with the CAD sequences of hamster and Squalus acanthias. The DNA fragment of 6,679 bp containing the full-length coding sequence was amplified by nested PCR using the first-strand cDNA of human cell lines of TIG-1-20 and COLO205 as a template. Southern blot analysis suggested that the CAD gene exists as a single copy in the human genome.

Transgenic expression of IL-10 in pancreatic islet A cells accelerates autoimmune insulitis and diabetes in non-obese diabetic mice.

To study the paracrine effect of IL-10 on autoimmune insulitis and diabetes, we produced IL-10 transgenic non-obese diabetic (NOD) mice (NOD-IL-10) in which murine IL-10 was expressed in pancreatic islet A cells under the control of a rat glucagon promoter without directly manipulating pancreatic islet B cells. Among 11 founder mice, four of four males and three of seven females developed diabetes by 10 weeks of age. Histological analysis of six NOD-IL-10 revealed severe insulitis and prominent ductal proliferation. NOD-IL-10 also showed spotty lymphocytic infiltration in the lung and liver in four of six founder mice. The onset of diabetes in NOD-IL-10 was remarkably earlier than that of 14 weeks of age at the earliest in female non-transgenic NOD mice. When the NOD-IL-10 mouse was backcrossed to C57BL/6 mice, none of the resulting F1, B-N2 or B-N3 generation toward C57BL/6 mice showed diabetes even at 39 weeks of age, in spite of the presence of peri-insulitis and prominent ductal proliferation, while two of four mice of the N-N2 generation toward NOD mice showed early-onset diabetes. Thus, transgenic paracrine expression of IL-10 in situ in the NOD genetic background enhances autoimmune insulitis and diabetes in their onset and severity, ignoring gender difference. Because expression of IL-10 was detected by polymerase chain reaction in pancreatic islets of non-transgenic NOD mice after 5 weeks of age, IL-10 secreted in situ is regarded to enhance cell-mediated autoimmune diabetes, in spite of established in vitro anti-Th1 activity of IL-10.

An end-trimming method to amplify adjacent cDNA fragments by PCR.

We describe a method for cDNA cloning by PCR, which we named "an end-trimming method." This method can be used for PCR amplification and cloning of unknown cDNA fragments adjacent to a short stretch of a known sequence by using a combination of a sequence-matched primer with an ATCG sequence added to the 5' end (5'-ATCG-primer) and an adaptor-(dT)17-primer (dT-primer). The fragments amplified by PCR using a 5'-ATCG-primer, which were modified to have a 5'-ATC overhang by blocking the G site, were exclusively cloned into pUC19 with the vector having a 3'-TAG-5' complementary overhang with a confined direction of inserted fragments. Practical application of this method for the determination of rat amidophosphoribosyltransferase cDNA resulted in successful cloning of adjacent cDNA fragments.

Ha-ras codon 12 mutation in papillary tumors of the urinary-bladder - a retrospective study.

This report concerns the retrospective examination of low grade, low stage (Ta/T1) bladder tumors for the Ha-ras codon 12 G-->T mutation. The patients studied had a minimum of 5 years follow-up and were grouped into 3 categories: (i) patients with no recurring tumors, (ii) patients with recurring Ta/T1 tumors and (iii) patients who subsequently developed high grade, high stage tumors. A heminested, non-isotopic, allele-specific polymerase chain reaction (PCR) amplification assay was used. The codon 12 G-->T mutation was found in 10/27 specimens from patients with non-recurring Ta/T1 tumors; in 11/27 initial and 12/23 recurring Ta/T1 tumors, and in 5/8 initial Ta/T1 lesions and 8/12 subsequently developed high grade/stage tumors. Although there was no correlation between disease recurrence and mutation, these results indicate that a relatively large proportion of patients with Ta/T1 tumors of the bladder have cells with the Ha-ras codon 12 G-->T substitution.

The high frequency of TTR M30 in familial amyloidotic polyneuropathy is not due to a founder effect.

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant disease due to mutations in the transthyretin (TTR) gene. Valine30-->methionine (TTR M30) is by far the most common mutation in patients with FAP. In a sample of 11 North American unrelated patients, we previously found that 6 had TTR M30. By utilizing double PASA, we could perform haplotype analysis despite the absence of DNA samples on relatives. The results indicate that at least four of the six patients with TTR M30 have different haplotypes, an observation that is surprising for North American patients in which the ostensible symptoms generally begin after the reproductive years. It is suggested that the most likely explanation is rapid selection against TTR M30 mutations by one of four possible mechanisms.

Molecular cloning of rat amidophosphoribosyltransferase.

The cDNA of rat amidophosphoribosyltransferase (EC 2.4.2.14, ATase), which is the supposed regulatory allosteric enzyme of de novo purine nucleotide biosynthesis, has been cloned by polymerase chain reaction. The predicted open reading frame encodes a protein of 517 amino acids with a deduced molecular weight of 57,436 including a supposed 11-amino acid propeptide. The 16 amino acid residues next to the propeptide were identical to the N-terminal amino acid microsequence of a purified rat liver ATase, which is consistent with the cleavage of the propeptide from the proenzyme in rat liver. The derived amino acid sequence is the first sequence reported for a mammalian ATase and it exhibits 81, 41, 36, and 31% identity with the sequences of chicken, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae ATases, respectively. The molecular weight (M(r)) of 57,436 suggests a tetrameric structure of native ATase with a M(r) of 240,000-248,000. Southern blot analysis suggested that the ATase gene exists as a single copy in the rat genome. Northern blot analysis revealed that ATase is expressed at a high level in brain, heart, liver, and stomach. The ATase mRNA in brain, heart, and stomach was 3.5 kilobases (kb) and in liver the 3.5-kb band was observed as well as an additional band of 4.2 kb. Reverse transcription-polymerase chain reaction analysis showed that ATase is ubiquitously expressed in all tissues examined. Comparison with chicken ATase showed that 2 cysteine residues for an iron-sulfur cluster were conserved. Three conserved and two non-conserved consensus phosphorylation sites for cAMP-dependent protein kinase were found.

Molecular cloning of human amidophosphoribosyltransferase.

The cDNA of human amidophosphoribosyltransferase (EC 2.4.2.14, ATase), which is the supposed regulatory allosteric enzyme of de novo purine nucleotide biosynthesis, has been cloned from human hepatoma (HepG2) cDNA library. The predicted open reading frame encodes a protein of 517 amino acids with a deduced molecular weight (Mr) of 57,398, which is consistent with the molecular mass of 56 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the ATase subunit purified from human placenta. The derived amino acid sequence exhibits 93, 82, 41, 37, and 33% identity with the sequences of rat, chicken, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae ATases, respectively. Southern blot analysis suggested that the ATase gene exists as multiple copies. ATase mRNA (3.5 kb) is ubiquitously expressed in various human tissues. Comparison with rat and chicken ATases showed that two cysteine residues for an iron-sulfur cluster were conserved. Four consensus phosphorylation sites for cAMP-dependent protein kinase were found.

From molecular variant to disease: initial steps in evaluating the association of transthyretin M119 with disease.

Traditionally, clinical research has sought to determine the molecular basis of clinical signs and symptoms. Increasingly, the traditional process will be reversed, as many structural protein variants are elucidated as a result of powerful PCR-based methods. Herein we describe a variant of transthyretin (TTR) found by direct genomic sequencing and illustrate the utility of PASA (PCR amplification of specific alleles) in the initial characterization of such variants. TTR is an intriguing protein of unknown function, but deposition of mutant TTR produces familial amyloidotic polyneuropathy (FAP). We identify a carrier of a variant TTR in which threonine119 is changed to methionine (T119----M). T119 is invariant in five mammalian species, suggesting that this residue is important for normal protein function. To determine the frequency of the M119 variant, individuals of northern- and western-European descent were rapidly screened by generating a PASA assay for the sequence change. Four additional individuals were found to be heterozygous for the mutation, for a total of five M119 alleles in 1,666 genes (1/333). Clinical records, initial clinical interviews, and family history of these patients hint at a high frequency of early-onset venous insufficiency and perhaps mild renal dysfunction. Haplotype analysis on the heterozygotes could be performed, despite the absence of samples from relatives, by performing "double PASA." The haplotype data suggest that the M119 variant derives from a common ancestor. The putative functional deficiency caused by TTR M119 should be most marked in the homozygotes, who can be calculated to occur in 1/100,000 conceptions. If viable, these individuals may provide important clues about the physiological role of TTR. Although the nature (if any) of disease caused by TTR M119 remains to be defined, the genetic and clinical data indicate that this mutation does not cause FAP. Future family studies can determine whether the heterozygous state for TTR M119 cosegregates with a disease or trait.

Missense mutations and evolutionary conservation of amino acids: evidence that many of the amino acids in factor IX function as "spacer" elements.

We report 31 point mutations in the factor IX gene and explore the relationship between the level of evolutionary conservation of an amino acid and the probability of a mutation causing hemophilia B. From our total sample of 125 hemophiliacs and from those reported by others, we identify 95 independent missense mutations, 94 of which occur at amino acids that are evolutionarily conserved in the available mammalian factor IX sequences. The likelihood of a missense mutation causing hemophilia B depends on whether the residue is also conserved in the factor IX-related proteases: factor VII, factor X, and protein C. Most of the possible missense mutations in generically conserved residues (i.e., those conserved in factor IX and in all the related proteases) should cause disease. In contrast, missense mutations in factor IX-specific residues (i.e., those conserved in human, cow, dog, and mouse factor IX but not in the related proteases) are sixfold less likely to cause disease. Missense mutations at nonconserved residues are 33-fold less likely to cause disease. At least three models are compatible with these observations. A comparison of sequence alignments from four and nine species of factor IX and an examination of the missense mutations occurring at CpG residues suggest a model in which most residues fall on opposite ends of a spectrum. In about 40% of residues, virtually any missense mutation in a minority of the residues will cause disease, while virtually no missense mutations will cause disease in most of the remaining residues. Thus, many of the residues in factor IX are spacers; that is, the main chains are presumably necessary to keep other amino acid interactions in register, but the nature of the side chain is unimportant.