Biomedical subjects

G T Westermark

Publications and source records attributed to G T Westermark.

At least 37 records · Page 2Linked to original sources

PubMed1994

Expression of cone transducin, Gz alpha, and other G-protein alpha-subunit messenger ribonucleic acids in pancreatic islets.

The G-proteins are a family of heterotrimeric guanine nucleotide-binding proteins that play important roles in signal transduction and whose expression is regulated in a tissue-specific manner. Here we have surveyed the expression of G-protein alpha-subunits in mouse pancreatic islets. Degenerate oligonucleotide primers corresponding to conserved primary sequences in known G alpha-subunits were used in a reverse transcriptase-polymerase chain reaction, and the amplified complementary DNA (cDNA) fragments were subcloned and sequenced. Over 100 clones were analyzed, from which we determined that islet cells express at least seven G alpha-subunits: G8 alpha, Gi1 alpha or Gi3 alpha, Gi2 alpha, G11 alpha, G14 alpha, Gz alpha, and Gt2 alpha (cone transducin). In particular, the identification of Gz alpha and Gt2 alpha was of interest in that previous studies had indicated that the expression of Gz alpha was restricted mainly to the brain, retina, and adrenal gland, whereas Gt2 alpha was expressed predominantly in retinal cone photoreceptors. By Western blot analysis, we estimated that the amount of Gz alpha protein present in mouse islets was about 40% of that in retina. To further investigate the expression of Gt2 alpha, mouse Gt2 alpha cDNA was cloned from a retinal library and sequenced. The cDNA was used as a probe for Northern blot analysis, and the results confirmed that mouse islets contained a substantial level of Gt2 alpha messenger RNA (mRNA), albeit less than that found in retina (approximately 5-fold lower). Gt2 alpha mRNA was also shown to be present in a clonal mouse pancreatic alpha-cell line (alpha TC1-6) as well as in adrenal gland, pituitary, and a clonal mouse anterior pituitary cell line (AtT20). In situ hybridization revealed that Gt2 alpha mRNA was expressed essentially throughout the islet, suggesting that it is normally expressed in the abundant islet beta-cells and possibly others. In situ analysis also showed that Gt2 alpha mRNA expressed in the pituitary was limited to the intermediate and anterior lobes. We conclude that islet cells express multiple G-proteins, including several that are normally expressed at high levels in certain neuronal cells.

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

Islet amyloid polypeptide: demonstration of mRNA in human pancreatic islets by in situ hybridization in islets with and without amyloid deposits.

Islet amyloid polypeptide which is normally coexpressed with insulin in beta cells, forms amyloid deposits especially in islets of Type 2 (non-insulin-dependent) diabetic subjects. Occurrence of islet amyloid is paradoxically associated with loss of islet amyloid polypeptide immunoreactivity in beta cells. The present study was undertaken to examine whether the islet amyloid polypeptide gene is expressed in islets with decreased islet amyloid polypeptide immunoreactivity. Pancreatic tissue from 14 patients, 7 with Type 2 diabetes and 7 non-diabetic, were obtained at autopsy or surgery and studied for islet amyloid polypeptide expression by in situ hybridization and for presence of insulin and islet amyloid polypeptide by immunohistochemistry. Six of the specimens from the diabetic and three of those from the non-diabetic patients had varying degrees of islet amyloid polypeptide-derived islet amyloid. Amyloid deposits were associated with decreased numbers of beta cells with islet amyloid polypeptide immunoreactivity despite an apparent normal frequency of insulin-containing cells. This discrepancy might reflect an alteration in islet amyloid polypeptide production or processing at a transcriptional or post-transcriptional level. In contrast to the varying immunohistochemical patterns, islets of all categories showed strong labelling using an islet amyloid polypeptide probe for in situ hybridization. It is concluded that islet amyloid polypeptide production is not altered at the transcriptional level. The following possibilities remain: (1) islet amyloid polypeptide production may be altered at a post-transcriptional level or (2) that islet amyloid polypeptide production is normal but the reduced immunoreactivity of the cells reflects a reduced storage of IAPP in secretory granules.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1993

Human G(olf) alpha: complementary deoxyribonucleic acid structure and expression in pancreatic islets and other tissues outside the olfactory neuroepithelium and central nervous system.

G(olf) alpha is a G-protein originally believed to mediate signal transduction exclusively within the olfactory neuroepithelium and subsequently found to be a major stimulatory G-protein in the basal ganglia. Here we present evidence that G(olf) alpha is expressed in several other tissues. The human isoform of G(olf) alpha was isolated from two human insulinoma cDNA libraries. Comparison of the human sequence with rat G(olf) alpha shows 91% nucleotide identity (within the coding region) and 99% identity at the amino acid level. Northern and reverse transcriptase-polymerase chain reaction analyses indicated that G(olf) alpha is expressed in all human insulinomas examined thus far as well as in normal pancreatic islets. G(olf) alpha mRNA was also detected in testis, retina, brain, and liver. Western blot analysis of various mouse tissues demonstrated that the level of G(olf) alpha protein in islets is lower than that in the olfactory neuroepithelium and other parts of the brain; its expression in retina, lung, and spleen was moderately higher than that in islets, and its expression in testis approached that in olfactory neuroepithelium. G(olf) alpha was also detected by immunohistochemistry in mouse islets, human insulinomas, the epithelial lining of mouse epididymis, photoreceptor cells of mouse retina, and mouse lung alveoli. These findings suggest a role for G(olf) alpha in a diverse population of cells located outside the olfactory neuroepithelium and central nervous system.

Amino Acid Sequence↗

PubMed1992

The N-terminal segment of protein AA determines its fibrillogenic property.

The amyloid fibril protein AA consists of a varying long N-terminal part of the precursor protein serum AA. By using synthetic peptides corresponding to human and murine protein AA segments and cyanogen bromide fragments of human protein AA, we show evidence that the amyloidogenic part of the molecule is the first 10-15 amino acid long segment. Amino acid substitutions in this part of the molecule may explain why only one of the two mouse SAA isoforms is amyloidogenic.

Amino Acid Sequence↗

PubMed1992

Seminal vesicle amyloid: the first example of exocrine cell origin of an amyloid fibril precursor.

Amyloid fibrils have been extracted from seminal vesicles, and a dominant 14 kD amyloid fibril protein has been identified. An antiserum to this protein reacted both with amyloid and with epithelial cells in some normal seminal vesicles. These reactions were blocked with seminal vesicle secretion and seminal vesicle amyloid fibril protein, but not by degraded amyloid fibrils or fibril protein from other types of amyloid. It is concluded that seminal vesicle amyloid is derived from secretory proteins of the seminal vesicles. As such, it is the first amyloid described which appears to be the product of an exocrine secretory cell.

Aged↗

PubMed1992

The complete amino acid sequence of bovine serum amyloid protein A (SAA) and of subspecies of the tissue-deposited amyloid fibril protein A.

Bovine serum amyloid A (SAA) was isolated from the acute phase high density lipoprotein (HDL) fraction of a cow suffering from acute mastitis. The elucidated primary structure revealed a protein consisting of 112 amino acid residues. Compared with SAA proteins from other species, the bovine protein was shown to have an insertion of nine amino acid residues between positions 69 and 70. No microheterogeneity could be observed in the protein. Amyloid fibrils extracted from the kidneys were found to contain at least three subspecies of protein AA, consisting of 68, 81 and about 110 amino acid residues. The amino acid sequences established for the protein AA subspecies revealed no microheterogeneity, and were identical to that elucidated for protein SAA.

Amino Acid Sequence↗

PubMed1991

Fibronectin and basement membrane components in renal amyloid deposits in patients with primary and secondary amyloidosis.

Kidney biopsies from one patient with primary (AL) and three with secondary (AA) amyloidosis were used for an ultrastructural study of the collocalization of basement membrane proteins and the extracellular matrix protein fibronectin within amyloid deposits. Antibodies against amyloid P component, laminin, and heparan sulphate proteoglycan core protein all reacted with the basement membranes and the amyloid depositions in AA and AL amyloidosis. Monoclonal and polyclonal antibodies against collagen type IV reacted only with the basement membranes. Anti-fibronectin reaction was found in association with the basement membranes in all four cases, while labelling of amyloid depositions was found only in one of the AA amyloid cases and in the AL amyloid depositions. It is concluded that basement membrane components may be of importance for the formation of amyloid fibrils.

Amyloidosis↗

PubMed1990

Islet amyloid polypeptide: pinpointing amino acid residues linked to amyloid fibril formation.

Islet amyloid polypeptide (IAPP), a putative polypeptide hormone, is a product of pancreatic beta-cells and the major constituent of the amyloid deposits seen mainly in islets of type 2 diabetic humans and diabetic cats. The connection between IAPP amyloid formation and diabetes is unknown, but a limited segment of the IAPP molecule, positions 20-29, seems responsible for the aggregation to fibrils. Differences in the amino acid sequence of this region probably determine whether or not islet amyloid can develop in a particular species. Amyloid fibril formation can be mimicked in vitro with the aid of synthetic peptides. With this technique we show that peptides corresponding to IAPP positions 20-29 of human and cat, species that develop IAPP-derived islet amyloid, form amyloid-like fibrils in vitro. The corresponding IAPP segment from three rodent species that do not develop IAPP-derived amyloid did not give rise to fibrils. Substitution of the human IAPP-(20-29) decapeptide with one or two amino acid residues from species without islet amyloid generally reduced the capacity to form fibrils. We conclude that the sequence Ala-Ile-Leu-Ser-Ser, corresponding to positions 25-29 of human IAPP, is strongly amyloidogenic and that a proline-for-serine substitution in position 28, as in several rodents, almost completely inhibits formation of amyloid fibrils.

Amino Acid Sequence↗

PubMed1990

AA-amyloidosis. Tissue component-specific association of various protein AA subspecies and evidence of a fourth SAA gene product.

Protein AA, the major repetitive protein subunit present in fibrils deposited in AA-amyloidosis, is an N-terminal cleavage product of a 104-amino acid precursor, serum amyloid A (SAA). Protein AA subspecies varying between 45 and 94 amino acids in length have been described. In this study it is shown that the different protein AA subspecies are not evenly distributed in amyloid deposits and that in single patients, certain subspecies of protein AA are deposited in specific tissue component sites. Thus larger protein AA subspecies occur in lower concentration in amyloid in the glomeruli compared to other sites and are especially found in amyloid in vessel walls. Three different SAA forms have been predicted from genomic and complementary DNA studies. The existence of a fourth type has been suspected from amino acid sequence studies of purified SAA. Protein AA derived from this fourth type of SAA is now shown to be present in amyloid fibrils in one of the patients studied in this paper.

Amino Acid Sequence↗

PubMed1989

Islet amyloid polypeptide (IAPP) and pro-IAPP immunoreactivity in human islets of Langerhans.

Islet amyloid polypeptide (IAPP) is a 37-amino-acid putative hormone which is expressed by islet B-cells and most probably is co-released with insulin. IAPP is synthesized as an 89-amino-acid prepropeptide in which IAPP is flanked by two short peptides. The two short peptides are ultimately cleaved off at basic residues. In the present study, we used antisera to three different synthetic peptides corresponding to positions 18-30, 40-50 and 53-62 of prepro-IAPP. The two latter peptides fall within the mature IAPP molecule while the first peptide corresponds to the N-terminal flanking peptide. We demonstrate that normal B-cells and islet amyloid both react immunohistochemically with all of these antisera. Using the immunogold labelling technique, we also demonstrate electron microscopically that both the IAPP immunoreactivity and the pro1-IAPP immunoreactivity in amyloid deposits are confined to the amyloid fibrils per se. These data indicate that not only mature IAPP but also the N-terminal flanking peptide is present in islet amyloid deposits. It remains to be shown if the propeptide segments are involved in the pathogenesis of these amyloid depositions.

Aged↗

PubMed1989

Co-localization of islet amyloid polypeptide and insulin in the B cell secretory granules of the human pancreatic islets.

Islet amyloid polypeptide is a novel 37 amino-acid-residues polypeptide which has been isolated from amyloid deposits in an insulinoma, and in human and cat islets of Langerhans. The molecule has 46% homology with the calcitonin gene-related peptide. Light microscopy examination of the pancreas shows that islet amyloid polypeptide immunoreactivity is restricted to the islet B cells. The present study utilized a rabbit antiserum against a synthetic peptide corresponding to positions 20-29 of islet amyloid polypeptide, a sequence without any amino-acid identity with calcitonin gene-related peptide. By applying the immunogold technique at the ultrastructural level, it was shown that both insulin and islet amyloid polypeptide immunoreactivity occurs in the central granular core of the human B cell secretory granules, while the A cells remain unlabelled. The demonstration that islet amyloid polypeptide is a granular protein of the B cells may indicate that it is released together with insulin. Further studies are necessary to evaluate the functional role of islet amyloid polypeptide.

Aged↗

PubMed1989

Islet amyloid polypeptide (IAPP):cDNA cloning and identification of an amyloidogenic region associated with the species-specific occurrence of age-related diabetes mellitus.

We have cloned and sequenced a human islet amyloid polypeptide (IAPP) cDNA. A secretory 89 amino acid IAPP protein precursor is predicted from which the 37 amino acid IAPP molecule is formed by amino- and carboxyterminal proteolytic processing. The IAPP peptide is 43-46% identical in amino acid sequence to the two members of the calcitonin gene-related peptide (CGRP) family. Evolutionary conserved proteolytic processing sites indicate that similar proteases are involved in the maturation of IAPP and CGRP and that the IAPP amyloid polypeptide is identical to the normal proteolytic product of the IAPP precursor. A synthetic peptide corresponding to a carboxyteminal fragment of human IAPP is shown to spontaneously form amyloid-like fibrils in vitro. Antibodies against this peptide cross-react with IAPP from species that develop amyloid in pancreatic islets in conjunction with age-related diabetes mellitus (human, cat, racoon), but do not cross-react with IAPP from other tested species (mouse, rat, guinea pig, dog). Thus, a species-specific structural motif in the putative amyloidogenic region of IAPP is associated with both amyloid formation and the development of age-related diabetes mellitus. This provides a new molecular clue to the pathogenesis of this disease.

Amino Acid Sequence↗

PubMed1989

Amino acid sequence variations in protein AA of cats with high and low incidences of AA amyloidosis.

1. Amyloid isolated from the liver of a domestic short-haired (DSH) cat was dissolved and purified by gel filtration for amino acid sequence analysis. 2. Sequences of two major peptides corresponding to positions 18-23 and 25-75 of human amyloid protein AA were obtained when cyanogen bromide-cleaved protein was applied to an amino acid sequenator. 3. Comparison of these regions of amyloid protein from the Abyssinian cat (high incidence of AA amyloidosis) and DSH cat (low incidence of AA amyloidosis) revealed three amino acid differences, two of which occurred within regions that are completely conserved in the Abyssinian cat and all other species. 4. Secondary prediction plots showed less potential for amyloidogenicity (i.e., less beta-sheet conformation) in protein AA of the DSH cat as compared to the Abyssinian cat and other animal species. 5. These differences in protein AA of the DSH cat may, therefore, be linked to the comparatively uncommon occurrence of AA amyloidosis in the DSH cat as compared to the Abyssinian cat and other animals species.

Amino Acid Sequence↗

PubMed1989

Massive vascular AA-amyloidosis: a histologically and biochemically distinctive subtype of reactive systemic amyloidosis.

Amyloid protein AA consists of several subspecies which mainly arise through proteolytic cleavage at various sites of the precursor, serum protein AA. The most common protein AA subspecies (the protein AA prototype) contains 76 amino-acid residues. In previous studies we have shown that distinctive patterns of amyloid infiltration occur in AA-amyloidosis. The amyloid in different patterns of infiltration seems to consist of distinctive protein AA subspecies. In the present study we have analysed protein AA in three patients with a form of AA-amyloidosis with heavy vascular infiltration and show that the amyloid fibrils contain two groups of protein AA subspecies. One, quantitatively predominating, group contains large subspecies of up to 94 amino-acid residues and a second group of protein AA-molecules contains around 50 amino-acid residues. The AA molecules lack the N-terminal arginine residue. It is concluded that AA-amyloidosis with massive vascular infiltration is a distinctive subform with typical clinical and histological appearance and with fibrils containing characteristic protein AA subspecies.

Amino Acid Sequence↗

PubMed1988

Insulin as an amyloid-fibril protein at sites of repeated insulin injections in a diabetic patient.

A patient with Type 1 (insulin-dependent) diabetes mellitus developed localised amyloidosis at the sites of his injections of porcine insulin. A major amyloid fibril protein was extracted and, by means of its amino acid composition and amino acid sequence, it was shown to contain intact insulin molecules. Porcine insulin is the tenth protein and the first foreign protein to be chemically identified in human amyloid fibrils.

Adult↗

PubMed1988

Immunohistochemical identification of heparan sulphate proteoglycan in secondary systemic amyloidosis.

The distribution of proteoglycans in kidneys from patients with secondary (AA) systemic amyloidosis was investigated. Antisera reacting with the protein cores of chondroitin sulphate proteoglycan (CSPG), dermatan sulphate proteoglycan (DSPG) and heparan sulphate proteoglycan (HSPG) were used in conjunction with the peroxidase-antiperoxidase (PAP) method. HSPG was the only proteoglycan found to be specifically localized to the amyloid deposits. The staining was most intense on the endothelial side of the deposits in both the glomeruli and in the vessel walls. No staining was observed after absorption of the HSPG antiserum with a fraction of the amyloid preparations, corresponding in size to that reported for glomerular HSPG. The possible role of HSPG and endothelial cells in the pathogenesis of the amyloid deposits is discussed.

Amyloidosis↗

PubMed1987

Islet amyloid polypeptide-like immunoreactivity in the islet B cells of type 2 (non-insulin-dependent) diabetic and non-diabetic individuals.

A novel peptide, islet amyloid polypeptide (IAPP), with structural resemblance to calcitonin gene-related peptide has recently been purified from amyloid deposits in an insulinoma and from islets of Langerhans. By immunohistochemical methods, using antisera to a synthetic undecapeptide of IAPP and to insulin, we show that freshly fixed islet B cells in man, guinea pig, rat, mouse and hamster exhibit strong IAPP-immunoreactivity while A cells are unreactive. In human autopsy material, all of 11 non-diabetic individuals had IAPP immunoreactivity of the islets. In comparison 8 of the 13 patients with Type 2 (non-insulin-dependent) diabetes had no IAPP immunoreactive cells. The proportion of islet cells having IAPP immunoreactivity exceeded 10% in only 1 of the 5 remaining diabetic patients while in all 13 patients substantially more than 10% of the islet cells contained immunoreactive insulin. IAPP-positive amyloid deposits were found in 20-99% of the islets in 12 of the Type 2 diabetic patients while 6 of 11 non-diabetic subjects had amyloid in 3-11% of their islets. In islets with IAPP-immunoreactive amyloid, very few IAPP-cells were seen despite a strong reaction of the B cells with antiserum to insulin. This study shows that IAPP is a normal islet B cell component and that IAPP immunoreactivity in B cells is diminished in Type 2 diabetes while IAPP is deposited as amyloid fibrils in the islets of Langerhans. Although the function of IAPP is unknown, its occurrence in the islet B cells and its structural relation to calcitonin gene-related peptide makes a hormonal nature probable.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗