Localization of calcitonin gene-related peptide and islet amyloid polypeptide in the rat and mouse pancreas

Summary It was previously demonstrated that the two chemically related peptides calcitonin gene-related peptide (CGRP) and islet amyloid polypeptide (IAPP) both occur in the pancreas. We have now examined the cellular localization of CGRP and IAPP in the rat and the mouse pancreas. We found, in both the rat and the mouse pancreas, CGRP-immunoreactive nerve fibers throughout the parenchyma, including the islets, with particular association with blood vessels. CGRP-immunoreactive nerve fibers were regularly seen within the islets. In contrast, no IAPP-immunoreactive nerve fibers were demonstrated in this location. Furthermore, in rat islets, CGRP immunoreactivity was demonstrated in peripherally located cells, constituting a major subpopulation of the somatostatin cells. Such cells were lacking in the mouse islets. IAPP-like immunoreactivity was demonstrated in rat and mouse islet insulin cells, and, in the rat, also in a few non-insulin cells in the islet periphery. These cells seemed to be identical with somatostatin/CGRP-immunoreactive elements. In summary, the study shows (1) that CGRP, but not IAPP, is a pancreati neuropeptide both in the mouse and the rat; (2) that a subpopulation of rat somatostatin cells contain CGRP; (3) that mouse islet endocrine cells do not contain CGRP; (4) that insulin cells in both the rat and the mouse contain IAPP; and (5) that in the rat, a non-insulin cell population apparently composed of somatostatin cells stores immunoreactive IAPP. We conclude that CGRP is a pancreatic neuropeptide and IAPP is an islet endocrine peptide in both the rat and the mouse, whereas CGRP is an islet endocrine peptide in the rat.     

The pathogenesis of maturity-onset diabetes mellitus: Is there a link to islet amyloid polypeptide?

The discovery of a novel polypeptide (Islet Amyloid Polypeptide: IAPP) isolated from human and cat islet amyloid and from amyloid of a human insulinoma is reviewed. Structurally, IAPP from the human and cat resembles calcitonin gene-related peptide (CGRP). The structural similarities between the neuropeptide CGRP and IAPP support the premise that IAPP is hormonal in nature. Our immunohistochemical studies also indicate that normal islet B-cells of several mammalian species (including man and cat) give strong immunoreactivity with antiserum directed to a synthetic peptide segment of IAPP. The fact that IAPP is deposited as amyloid in the pancreatic islets of type 2 (noninsulin-dependent) diabetics strongly supports an important but yet unknown link between IAPP and the development of this disease.     

The complete sequence of the mitochondrial genome of the Chinook salmon, Oncorhynchus tshawytscha

The complete sequence of the mitochondrial genome of Chinook salmon, Oncorhynchus tshawytscha, has been determined. The circular genome consisting of 16,644 base pairs encodes thirteen proteins, the 12S and 16S ribosomal RNAs, and 22 transfer RNAs. These genes are ordered in the same way as most other vertebrates. The nucleotide and amino acid sequences of the ribosomal RNAs and the thirteen protein-coding genes were compared with those of other salmonids such as Oncorhynchus mykiss, Salmo salar, Salvelinus fontinalis, Salvelinus alpinus and Coregonus lavaretus. The sequence features of the control region (D-loop), the origin of L-strand replication and a putative peptide codified by the 16S mitochondrial RNA are described and discussed.     

The complete mitochondrial DNA sequence of the Atlantic salmon, Salmo salar.

The complete sequence of the Atlantic salmon (Salmo salar) mitochondrial genome has been determined. The entire sequence is 16665 base pairs (bp) in length, with a gene content (13 protein-coding, two ribosomal RNA [rRNA] and 22 transfer RNA [tRNA] genes) and order conforming to that observed in most other vertebrates. Base composition and codon usage have been detailed. Nucleotide and derived amino acid sequences of the 13 protein-coding genes from Atlantic salmon have been compared with their counterparts in rainbow trout. A putative structure for the origin of L-strand replication (O(L)) is proposed, and sequence features of the control region (D-loop) are described.     

The isolation, purification and amino-acid sequence of insulin from the teleost fish Cottus scorpius (daddy sculpin)

Insulin from the principal islets of the teleost fish, Cottus scorpius (daddy sculpin), has been isolated and sequenced. Purification involved acid/alcohol extraction, gel filtration, and reverse-phase high-performance liquid chromatography to yield nearly 1 mg pure insulin/g wet weight islet tissue. Biological potency was estimated as 40% compared to porcine insulin. The sculpin insulin crystallised in the absence of zinc ions although zinc is known to be present in the islets in significant amounts. Two other hormones, glucagon and pancreatic polypeptide, were copurified with the insulin, and an N-terminal sequence for pancreatic polypeptide was determined. The primary structure of sculpin insulin shows a number of sequence changes unique so far amongst teleost fish. These changes occur at A14 (Arg), A15 (Val), and B2 (Asp). The B chain contains 29 amino acids and there is no N-terminal extension as seen with several other fish. Presumably as a result of the amino acid substitutions, sculpin insulin does not readily form crystals containing zinc-insulin hexamers, despite the presence of the coordinating B10 His.     

Parasite fauna of the landlocked Atlantic salmon Salmo salar L. in the basin of Namsen river

The 26 specimens of the landlocked Atlantic salmon Salmo salar L. from Namsen river and its tributary Melingselva river (central Norway) were studied by the method of complete parasitological examination in the period of 1997-1998. The 15 parasite species have been recovered. Ration of parasite with a direct and complex cycle is approximately equal, 6 and 9 respectively. Larval stages of parasites predominate among the parasites with a complex cycle. The parasite fauna fond in the landlocked Atlantic salmon is most similar to that in youngsters of the lake salmon (Salmo salar morpha sebago Girard) and the migrating Atlantic salmon (Salmo salar L.).     

Sequence divergence in a specific region of islet amyloid polypeptide (IAPP) explains differences in islet amyloid formation between species

Amyloid deposits in the islets of Langerhans occur in association with type 2 diabetes mellitus (DM) in humans and cats and consist of a 37-amino-acid polypeptide known as islet amyloid polypeptide (IAPP). In order to find an explanation for the situation that islet amyloid (IA) does not develop in common rodent species, we have deduced the amino acid sequence of the IAPP molecule in mouse, rat and hamster. We find that a specific region of the molecule diverges to a high degree. Synthetic peptides corresponding to this region of human and hamster IAPP were compared for their ability to form amyloid fibrils in vitro. Whereas the human peptide readily formed fibrils with amyloid character, the hamster peptide completely lacked this property. We suggest this to be a likely explanation for the differences in IA formation between humans and rodents and discuss our findings in relation to the type 2 DM syndrome.     

Evolution of the salmonid mitochondrial control region.

To explore the evolutionary nature of the salmonid mitochondrial DNA (mtDNA) control region (D-loop) and its utility for inferring phylogenies, the entire region was sequenced from all eight species of anadromous Pacific salmon, genus Oncorhynchus; the Atlantic salmon, Salmo salar; and the Arctic grayling, Thymallus arcticus. A comparison of aligned sequences demonstrates that the generally conserved sequence elements that have been previously reported for other vertebrates are maintained in these primitive teleost fishes. Results reveal a significantly nonrandom distribution of nucleotide substitutions, insertions, and deletions that suggests that portions of the salmonid D-loop may be under differential selective constraints and that most of the control region of these fishes may evolve at a rate similar to that of the remainder of their mtDNA genomes. Maximum likelihood and Fitch parsimony analyses of 9 kb of aligned salmonid sequence data give evolutionary trees of identical topology. These results are consistent with previous molecular studies of a limited number of salmonid taxa and with more comprehensive, classical analyses of salmonid evolution. Predictions from these data, based on a molecular clock assumption for the mtDNA control region, are also consistent with fossil evidence that suggests that species of Oncorhynchus could be as old as the Middle Pliocene and would have thus given rise to the extant Pacific salmon prior to about 5 or 6 million years ago.     

Mucous cell types in the branchial epithelium of the euryhaline fish Poecilia vivipara

On transfer to sea water for 45 days, the return of appetite was later and growth rates tended to be lower for triploid Atlantic salmon, Salmo salar , reared together with diploid Atlantic salmon. All mortalities comprised of triploid salmon (29%) and were attributable to failed smolt syndrome. No correlation was found between the growth of diploid or triploid fish in fresh water and their subsequent growth on transfer to sea water.     

Rare and fleeting: an example of interspecific recombination in animal mitochondrial DNA

Recombination is thought to occur only rarely in animal mitochondrial DNA (mtDNA). However, detection of mtDNA recombination requires that cells become heteroplasmic through mutation, intramolecular recombination or 'leakage' of paternal mtDNA. Interspecific hybridization increases the probability of detecting mtDNA recombinants due to higher levels of sequence divergence and potentially higher levels of paternal leakage. During a study of historical variation in Atlantic salmon (Salmo salar) mtDNA, an individual with a recombinant haplotype containing sequence from both Atlantic salmon and brown trout (Salmo trutta) was detected. The individual was not an F1 hybrid but it did have an unusual nuclear genotype which suggested that it was a later-generation backcross. No other similar recombinant haplotype was found from the same population or three neighbouring Atlantic salmon populations in 717 individuals collected during 1948-2002. Interspecific recombination may increase mtDNA variability within species and can have implications for phylogenetic studies.     

cDNA cloning and sequence analysis of preproendothelin-1 (PPET-1) from salmon, Oncorhynchus keta

The presence of endothelin (ET)-like immunoreactivity and the cardiovascular effects of mammalian ET-1 in fish have been reported. To identify ET-related peptides in fish, we screened the cDNA library of the salmon (Oncorhynchus keta) stomach by means of rapid amplification of cDNA ends, and we cloned cDNAs encoding an ET-related peptide. The salmon ET-related sequence of 21 amino acids is identical to the trout ET-1 peptide recently purified from kidney specimens of Oncorhynchus mykiss. The deduced amino acid sequence of salmon pre-proET-1 (PPET-1) comprises 244 amino acids, including a putative signal sequence and mature ET-1, as well as big ET-1 and ET-1-like sequences. This precursor, the first reported PPET-1 sequence for Salmoniformes, Teleostei, has low homology with the sequences of human, mouse, frog (Xenopus laevis), and zebrafish (Danio rerio) PPET-1 (26%, 29%, 24%, and 39%, respectively).     

Presence of islet amyloid polypeptide in rat islet B and D cells determines parallelism and dissociation between rat pancreatic islet amyloid polypeptide and insulin content.

The islet amyloid polypeptide (IAPP) immunoreactivity of the adult rat pancreas is located in insulin-containing B cells as well as in somatostatin-containing D cells. In both cell types, the IAPP immunoreactivity is identical to rat synthetic IAPP in terms of its elution position after reversed phase HPLC and its binding to IAPP antibodies. The IAPP content per 10(6) B-cells is more than 100 fold lower than the corresponding insulin content, but comparable to the IAPP content of D cells. After induction of diabetes by streptozotocin, pancreatic IAPP seems predominantly located in somatostatin-containing cells. In normal rats, pancreatic insulin and IAPP content increase 20 fold from birth to 12 weeks of age; beyond week 12, the further rise in pancreatic insulin was not paralleled by an increase in IAPP content.     

Fish gonadotropin-releasing hormone gene and molecular approaches for control of sexual maturation: development of a transgenic fish model.

The prepro-GnRH gene and mRNA primary structure were fully established from Atlantic salmon (Salmo salar) and partially from rainbow trout (Oncorhynchus mykiss). Results show that the GnRH coding region of 30 base pairs is well conserved during evolution. In contrast, the GnRH-associated peptide (GAP) sequence shows very limited homology when the GnRH genes from mammalian and teleost species are compared. A simple method for selecting transgenic fish after transfer of the firefly luciferase gene was developed. The method involves bioluminescent measurement of live animals in a scintillation counter.     

Cholesterol levels in the blood of Atlantic salmonids

Total cholesterol and high density lipids (HDL) were measured in the blood of Atlantic salmon (Salmo salar), brook trout (Salvelinus fontinalis) and rainbow trout (Salmo gairdneri) from a variety of dietary and environmental regimes. At spawning the total cholesterol of cage reared S. salar was high compared to sea ranched S. salar. Starvation of S. salar in freshwater for 6-11 months depressed total blood cholesterol levels, but not to unusually low levels. A cholesterol supplement of 0.02 g/100 g body weight to a chow diet of 0.38 g/100 g body weight had no significant effect on the total blood cholesterol level in Salvelinus fontinalis. The high proportion of HDL in the blood of S. salar is discussed in terms of its possible importance during the atherosclerotic lesion formation that normally accompanies spawning in these salmon.     

Structural Similarities and Differences between Amyloidogenic and Non-Amyloidogenic Islet Amyloid Polypeptide (IAPP) Sequences and Implications for the Dual Physiological and Pathological Activities of These Peptides

IAPP, a 37 amino-acid peptide hormone belonging to the calcitonin family, is an intrinsically disordered protein that is coexpressed and cosecreted along with insulin by pancreatic islet β-cells in response to meals. IAPP plays a physiological role in glucose regulation; however, in certain species, IAPP can aggregate and this process is linked to β-cell death and Type II Diabetes. Using replica exchange molecular dynamics with extensive sampling (16 replicas per sequence and 600 ns per replica), we investigate the structure of the monomeric state of two species of aggregating peptides (human and cat IAPP) and two species of non-aggregating peptides (pig and rat IAPP). Our simulations reveal that the pig and rat conformations are very similar, and consist of helix-coil and helix-hairpin conformations. The aggregating sequences, on the other hand, populate the same helix-coil and helix-hairpin conformations as the non-aggregating sequence, but, in addition, populate a hairpin structure. Our exhaustive simulations, coupled with available peptide-activity data, leads us to a structure-activity relationship (SAR) in which we propose that the functional role of IAPP is carried out by the helix-coil conformation, a structure common to both aggregating and non-aggregating species. The pathological role of this peptide may have multiple origins, including the interaction of the helical elements with membranes. Nonetheless, our simulations suggest that the hairpin structure, only observed in the aggregating species, might be linked to the pathological role of this peptide, either as a direct precursor to amyloid fibrils, or as part of a cylindrin type of toxic oligomer. We further propose that the helix-hairpin fold is also a possible aggregation prone conformation that would lead normally non-aggregating variants of IAPP to form fibrils under conditions where an external perturbation is applied. The SAR relationship is used to suggest the rational design of therapeutics for treating diabetes.     

Sequence of a cDNA clone encoding an Atlantic salmon ribosomal protein

We report here the nucleotide sequence of a cDNA clone encoding a salmon (Salmo salar) ribosomal (r) protein. The encoded protein shows 62.3% and 62% similarity with the L14 and L18 r-proteins in Xenopus laevis and rat, respectively.     

Design of peptide-based inhibitors of human islet amyloid polypeptide fibrillogenesis

Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in the pancreas of over 90% of all cases of type-2 diabetes. We have generated a series of overlapping hexapeptides to target an amyloidogenic region of IAPP (residues 20-29) and examined their effects on fibril assembly. Peptide fragments corresponding to SNNFGA (residues 20-25) and GAILSST (residues 24-29) were strong inhibitors of the beta-sheet transition and amyloid aggregation. Circular dichroism indicated that even at 1:1 molar ratios, these peptides maintained full-length IAPP (1-37) in a largely random coil conformation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP resulted in the formation of only semi-fibrous aggregates and loss of the typical high density and morphology of IAPP fibrils. This inhibitory activity, particularly for the SNNFGA sequence, also correlated with a reduction in IAPP-induced cytotoxicity as determined by cell culture studies. In contrast, the peptide NFGAIL (residues 22-27) enhanced IAPP fibril formation. Conversion to the amyloidogenic beta-sheet was immediate and the accompanying fibrils were more dense and complex than IAPP alone. The remaining peptide fragments either had no detectable effects or were only weakly inhibitory. Specificity of peptide activity was illustrated by the fragments, SSNNFG and AILSST. These differed from the most active inhibitors by only a single amino acid residue but delayed the random-to-beta conformational change only when used at higher molar ratios. This study has identified internal IAPP peptide fragments which can regulate fibrillogenesis and may be of therapeutic use for the treatment of type-2 diabetes.     

Comparative susceptibility of Atlantic salmon (Salmo salar) to the enteric redmouth bacterium and Aeromonas salmonicida

The bacterium causing enteric redmouth (ERM) and Aeromonas salmonicida were found to be equally pathogenic for fingerling Atlantic salmon (Salmo salar). Injection of 5 x 10(5) cells of ERM or A. salmonicida killed all salmon within 96 h. After a 30 min exposure to water-borne cells of the two test bacteria about one-half of the test salmon died within 14 days. Both ERM and A. salmonicida were transmitted horizontally. Results indicate that efforts should be made to prevent introduction of ERM into watersheds where Atlantic salmon occur.     

N-Terminal Sequence and Main Characteristics of Atlantic Salmon (Salmo salar) Albumin

Atlantic salmon (Salmo salar) serum albumin was purified from plasma and its N-terminal sequence determined. Atlantic salmon albumin is the predominant plasma protein, negatively charged, at pH 8.6. Albumin was purified to >95% purity which yielded a single band on SDS-PAGE and agarose gel electrophoresis. The molecular weight of the purified albumin was approximately 6,5 kDa. The N-terminal sequence of Atlantic chinook salmon albumin was consistent with that predicted from its previously determined cDNA sequence and was identical to that of salmon (Oncorhynchus tshawytscha) albumin through the first 15 residues. However, the fact that the actual N-terminus was different from that predicted from cDNA sequence indicates that Atlantic salmon albumin, like chinook salmon albumin, lacks a propeptide.