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Biomedical subjects

G L Fletcher

Publications and source records attributed to G L Fletcher.

At least 19 recordsLinked to original sources
PubMed2006

Cardiorespiratory modifications, and limitations, in post-smolt growth hormone transgenic Atlantic salmon Salmo salar.

In recent years, there has been a great deal of interest in how growth hormone (GH) transgenesis affects fish physiology. However, the results of these studies are often difficult to interpret because the transgenic and non-transgenic fish had very different environmental/rearing histories. This study used a stable line of size-matched GH Atlantic salmon (Salmo salar) that were reared in a shared tank with controls (at 10 degrees C, for approximately 9 months) to perform a comprehensive examination of the cardiorespiratory physiology of GH transgenic salmon, and serves as a novel test of the theory of symmorphosis. The GH transgenic salmon had a 3.6x faster growth rate, and 21 and 25% higher values for mass-specific routine and standard oxygen consumption (M(O(2))), respectively. However, there was no concurrent increase in their maximum M(O(2)), which resulted in them having an 18% lower metabolic scope and a 9% reduction in critical swimming speed. This decreased metabolic capacity/performance was surprising given that the transgenics had a 29% larger heart with an 18% greater mass-specific maximum in situ cardiac output, a 14% greater post-stress blood haemoglobin concentration, 5-10% higher red muscle and heart aerobic enzyme (citrate synthase or cytochrome oxidase) activities, and twofold higher resting and 1.7x higher post-stress, catecholamine levels. However, gill surface area was the only cardiorespiratory parameter that was not enhanced, and our data suggest that gill oxygen transfer may have been limiting. Overall, this research: (1) shows that there are significant metabolic costs associated with GH transgenesis in this line of Atlantic salmon; (2) provides the first direct evidence that cardiac function is enhanced by GH transgenesis; (3) shows that a universal upregulation of post-smolt (adult) GH transgenic salmon cardiorespiratory physiology, as suggested by symmorphosis, does not occur; and (4) supports the idea that whereas differences in arterial oxygen transport (i.e. cardiac output and blood oxygen carrying capacity) are important determinants of inter-specific differences in aerobicity, diffusion-limited processes must be enhanced to achieve substantial intra-specific improvements in metabolic and swimming performance.

Animals↗
PubMed2005

Vitrification assays with embryos from a cold tolerant sub-arctic fish species.

Pseudopleuronectes americanus is a Northern teleost species that produces antifreeze proteins (AFPs) to protect them from freezing during the winter. These AFPs bind to ice crystals to inhibit their growth, and they also protect cell membranes at low temperatures. In this study, vitrification trials were done with fish embryos at three different developmental stages, using two different protocols for incorporating the vitrifying solutions. Toxicity of the cryoprotectants and permeability to dimethyl sulfoxide were analyzed. Embryos were vitrified in 0.5 ml straws by direct immersion in liquid nitrogen, and their morphology and development analyzed following thaw. The embryos responded well to vitrification as evidenced by the high percentage that exhibited good morphology following thaw. Although none of the embryos hatched, a small percentage (0.92%) of them showed active movements within the chorion and continued to develop for a number of days following thaw. This is the first record of post-thaw development of vitrified fish embryos.

Animals↗
PubMed2001

Efficacy of antifreeze protein types in protecting liposome membrane integrity depends on phospholipid class.

Antifreeze proteins have been reported to be capable of maintaining the membrane integrity of cold sensitive mammalian cells when exposed to hypothermic temperatures. However the mechanism(s) whereby these proteins exert this protective effect is unknown. The present study used liposomes as a model system to examine the nature of the interactions between four antifreeze (glyco)protein types (AFP I, II, III and AFGP) and albumin, with lipid membranes. Fluorescein isothiocyanate labelling indicated that all of the proteins bound to the three liposome types (dielaidoylphosphatidylcholine (DEPC), dielaidoylphosphatidylethanolamine (DEPE) and dielaidoylphosphatidylglycerol (DEPG)). AFGP was found to be highly effective at preventing leakage from all three liposome compositions as they were cooled through their phase transition temperatures. This was not the case for the other proteins. All four antifreeze types prevented zwitterionic DEPC liposomes from leaking as they were cooled through their phase transition temperature. However, albumin was equally as effective, indicating that this capacity was not unique to antifreeze proteins. All of the proteins, except AFGP, induced the negatively charged DEPG liposomes to leak prior to cooling, and were less effective than AFGP in preventing phase transition leakage from DEPE liposomes. It is proposed that many proteins, including antifreeze proteins, can protect zwitterionic liposomes, such as DEPC, by binding to the lipid bilayer thereby maintaining the ordered structure of the membrane during phase transition. However, when the membrane contains a negatively charged polar group, such as with DEPE and DEPG, proteins, although bound to them, may not be able to maintain sufficient membrane organization to prevent leakage during phase transition or, they may gain entry into the lipid bilayer, disrupt the structure and induce leakage. These results imply that the efficacy of antifreeze proteins in the cold protection of mammalian cells will not only depend on protein structure, but also on the lipid composition of the cell membrane.

Antifreeze Proteins↗
PubMed2001

Isolation and characterization of type I antifreeze proteins from Atlantic snailfish (Liparis atlanticus) and dusky snailfish (Liparis gibbus).

Antifreeze proteins (AFPs) were isolated from the blood plasma of Atlantic snailfish Liparis atlanticus and dusky snailfish Liparis gibbus, which belong to the Teleost family Cyclopteridae, a close relative of sculpins. Using a combination of gel filtration chromatography and reversed-phase HPLC, proteins were purified to individual peaks. Atlantic snailfish plasma contained two different proteins (MW=9344, 9415) while dusky snailfish plasma contained five protein isoforms (MW=9514-9814), as determined by mass spectrometry. Further characterization revealed that these proteins are rich in alanine (>50 mol%), and have alpha-helical secondary structure that can undergo reversible thermal denaturation. Thermal hysteresis activities of these proteins were similar to each other but lower than the major type I AFPs from winter flounder. Results of this study have indicated that although the AFPs from snailfish are significantly larger than previously described type I AFPs, they share enough characteristics to be classified in this group.

Amino Acid Sequence↗
PubMed2001

Isolation and characterization of skin-type, type I antifreeze polypeptides from the longhorn sculpin, Myoxocephalus octodecemspinosus.

The antifreeze polypeptides (AFPs) are found in several marine fish and have been grouped into four distinct biochemical classes (type I-IV). Recently, the new subclass of skin-type, type I AFPs that are produced intracellularly as mature polypeptides have been identified in the winter flounder (Pleuronectes americanus) and the shorthorn sculpin (Myoxocephalus scorpius). This study demonstrates the presence of skin-type AFPs in the longhorn sculpin (Myoxocephalus octodecemspinosus), which produces type IV serum AFPs. Using polymerase chain reaction-based methods, a clone that encoded for a type I AFP was identified. The clone lacked a signal sequence, indicating that the mature polypeptide is produced in the cytosol. A recombinant protein was produced in Escherichia coli and antifreeze activity was characterized. Four individual Ala-rich polypeptides with antifreeze activity were isolated from the skin tissue. One polypeptide was completely sequenced by tandem MS. This study provides the first evidence of a fish species that produces two different biochemical classes of antifreeze proteins (type I and type IV), and enforces the notion that skin-type AFPs are a widespread biological phenomenon in fish.

Amino Acid Sequence↗
PubMed2001

Antifreeze glycoproteins: relationship between molecular weight, thermal hysteresis and the inhibition of leakage from liposomes during thermotropic phase transition.

Antifreeze glycoproteins (AFGP) were isolated and purified from the blood plasma of rock cod (Gadus ogac), using DEAE-Bio-gel ion exchange chromatography, followed by high performance liquid chromatography (HPLC). The purified proteins were analyzed using polyacrylamide gel electrophoresis (PAGE), and electrospray mass spectrometry. The results indicated that rock cod synthesize seven size classes of glycoproteins, ranging from 2.6 to 24 kDa, with each size class containing multiple isoforms. Antifreeze activity, as determined by thermal hysteresis, indicated that the AFGP could be separated into two groups, with the larger size classes (molecular mass>13 kDa) having approximately 3-4 times the activity of the smaller, proline containing, size classes (molecular mass<10 kDa). All of the AFGP size classes prevented leakage from dielaidoylphosphatidylcholine (DEPC) liposomes as they were cooled through their phase transition temperature, with the larger size classes being approximately 4 times as effective as the smaller ones. It is hypothesized that AFGP prevent liposomes from leaking as they pass through the phase transition temperature by binding to the phospholipid membrane.

Animals↗
PubMed2001

Antifreeze proteins of teleost fishes.

Marine teleosts at high latitudes can encounter ice-laden seawater that is approximately 1 degrees C colder than the colligative freezing point of their body fluids. They avoid freezing by producing small antifreeze proteins (AFPs) that adsorb to ice and halt its growth, thereby producing an additional non-colligative lowering of the freezing point. AFPs are typically secreted by the liver into the blood. Recently, however, it has become clear that AFP isoforms are produced in the epidermis (skin, scales, fin, and gills) and may serve as a first line of defense against ice propagation into the fish. The basis for the adsorption of AFPs to ice is something of a mystery and is complicated by the extreme structural diversity of the five antifreeze types. Despite the recent acquisition of several AFP three-dimensional structures and the definition of their ice-binding sites by mutagenesis, no common ice-binding motif or even theme is apparent except that surface-surface complementarity is important for binding. The remarkable diversity of antifreeze types and their seemingly haphazard phylogenetic distribution suggest that these proteins might have evolved recently in response to sea level glaciation occurring just 1-2 million years ago in the northern hemisphere and 10-30 million years ago around Antarctica. Not surprisingly, the expression of AFP genes from different origins can also be quite dissimilar. The most intensively studied system is that of the winter flounder, which has a built-in annual cycle of antifreeze expression controlled by growth hormone (GH) release from the pituitary in tune with seasonal cues. The signal transduction pathway, transcription factors, and promoter elements involved in this process are just beginning to be characterized.

Animals↗
PubMed2000

The rat ortholog of the presumptive flounder antifreeze enhancer-binding protein is a helicase domain-containing protein.

The expression of winter flounder liver-type antifreeze protein (wflAFP) genes is tissue-specific and under seasonal and hormonal regulation. The only intron of the major wflAFP gene was demonstrated to be a liver-specific enhancer in both mammalian cell lines and flounder hepatocytes. Element B, the core enhancer sequence, was shown to interact specifically with a liver-enriched transcription factor, CCAAT/enhancer-binding protein alpha (C/EBPalpha), as well as a presumptive antifreeze enhancer-binding protein (AEP). In this study, the identity of the rat AEP ortholog was revealed via its DNA-protein interaction with element B. It is a helicase-domain-containing protein, 988 amino acids in length, and is homologous to mouse Smubp-2, hamster Rip-1 and human Smubp-2. The specific binding between element B and AEP was confirmed by South-Western analysis and gel retardation assays. Residues in element B important to this interaction were identified by methylation interference assays. Mutation on one of the residues disrupted the binding between element B and AEP and its enhancer activity was significantly reduced, suggesting that AEP is essential for the transactivation of the wflAFP gene intron. The rat AEP is ubiquitously expressed in various tissues, and the flounder homolog is present as shown by genomic Southern analysis. The potential role of AEP in regulating the flounder AFP gene expression is discussed.

Amino Acid Sequence↗
PubMed1998

Skin-type antifreeze protein from the shorthorn sculpin, Myoxocephalus scorpius. Expression and characterization of a Mr 9, 700 recombinant protein.

A cDNA clone encoding a presumptive antifreeze protein was isolated from a skin library from shorthorn sculpin, Myoxocephalus scorpius. The clone encodes a 92-residue mature polypeptide (sssAFP-2) without any signal and prosequence, which suggests an intracellular localization. It is the largest alanine-rich, alpha-helical type I antifreeze protein known. A recombinant fusion protein containing an N-terminal-linked His-tag was produced and purified from Escherichia coli. This protein is alpha-helical at 0 degreesC and exhibits significant antifreeze activity. Northern blot and reverse transcription-polymerase chain reaction analyses indicate that sssAFP-2 mRNA has limited tissue distribution and is present in peripheral tissues such as skin and dorsal fin, but is notably absent in the liver. These studies reinforce recent evidence that indicate that the external tissues of cold water marine fishes are major organs for antifreeze protein synthesis and are likely the first line of defense against the threat of freezing.

Amino Acid Sequence↗
PubMed1998

The ice-binding site of Atlantic herring antifreeze protein corresponds to the carbohydrate-binding site of C-type lectins.

The type II antifreeze proteins (AFPs) of smelt and Atlantic herring are homologous to the carbohydrate-recognition domains (CRDs) of Ca2+-dependent (C-type) animal lectins and, like these lectins, acquire a stable and active structure upon binding Ca2+ ions. In the C-type lectin CRD, the carbohydrate-binding site is located at a Ca2+-binding site. Site-directed mutagenesis was used to test the hypothesis that the ice-binding site of the type II AFP corresponds to the carbohydrate-binding site of the lectins. To disrupt this site in the herring AFP without perturbing the Ca2+-dependent protein fold, a double mutant was constructed that changed the Ca2+- and carbohydrate-binding motif from the galactose-type of wild-type AFP containing the sequence Gln-Pro-Asp to a mannose-type that has the sequence Glu-Pro-Asn and is also known to bind Ca2+. The mutant AFP exhibited proper Ca2+ binding, folding, and stability as demonstrated by ruthenium red staining, proteolysis protection assays, and CD spectroscopy. However, it showed no antifreeze activity (thermal hysteresis) and did not alter ice crystal morphology to form bipyramidal crystals as does the active wild-type AFP. These results demonstrate that the ice-binding site of the herring type II AFP corresponds to the carbohydrate-binding site of the C-type lectin CRDs and further suggest that this ice-binding function evolved from the carbohydrate-binding site of a preexisting C-type lectin.

Amino Acid Sequence↗
PubMed1998

Identification of nuclear proteins interacting with the liver-specific enhancer B element of the antifreeze protein gene in winter flounder.

The intron of the winter flounder antifreeze protein (AFP) gene contains a liver-specific enhancer element B as demonstrated by transient expression in mammalian cells. Element B interacts with rat C/EBPalpha and a novel protein, tentatively designated as the antifreeze enhancer-binding protein (AEP). Present studies revealed that nuclear proteins from the winter flounder liver interact similarly and specifically to element B as shown by footprinting analysis and gel retardation assays. The presence of C/EBP in the flounder liver was confirmed by Western blot analysis. In vitro transcription assays in its homologous system further demonstrated the transactivation activity of the AFP gene intron. The present findings suggest that the mechanisms for regulating liver-specific transcription are evolutionarily conserved.

Animals↗
PubMed1997

The role of CCAAT/enhancer-binding protein alpha and a protein that binds to the activator-protein-1 site in the regulation of liver-specific expression of the winter flounder antifreeze protein gene.

Winter flounder, Pleuronectes americanus, produces antifreeze proteins (AFPs) to avoid freezing during winter. The AFP in the blood is synthesized by the liver and its annual level is regulated by environmental factors and somatotropin. The only intron (+106 to +602) of the AFP gene contains putative binding sites for liver-enriched transcription activators. Due to the lack of an appropriate fish liver cell line, a mammalian model system was used to demonstrate the liver-specific enhancer activity of the intron. Deletion analysis of the intron indicated nucleotide sequences between +192 bp and +334 bp were important for the enhancer activity. Further refinement showed that the element B (+303 to +322 bp) resided in this region interacted with proteins from rat nuclear extracts by DNase I footprinting and mobility shift analysis. Gel retardation with competition and supershift experiments defined element B to include binding sites for CCAAT/enhancer binding protein a (C/EBP alpha), a liver-enriched transcription factor, and a novel activator protein-1 (AP-1) site binding protein, designated as antifreeze enhancer binding protein (AEP). Residues important to DNA-protein interaction in element B were mapped by methylation interference. Mutations in element B by site-directed mutagenesis destroyed the enhancer activity of the intron. Ultraviolet crosslinking experiments using element B revealed the specific binding of two proteins of approximately 43 and 80 kDa. The present findings provide a mechanism(s) by which the tissue specificity of AFP gene expression can be achieved. Integration of the present information with the somatotropin-mediated signal transduction pathways has generated a working model for the hormonal regulation and seasonal expression of the AFP gene.

Animals↗
PubMed1996

Ca2+-dependent antifreeze proteins. Modulation of conformation and activity by divalent metal ions.

The antifreeze proteins (AFPs) are structurally diverse molecules that share an ability to bind to ice crystals and inhibit their growth. The type II fish AFPs of Atlantic herring and smelt are unique among known AFPs in their requirement of a cofactor for antifreeze activity. These AFPs are homologous with the carbohydrate-recognition domains of Ca2+-dependent (C-type) lectins and require Ca2+ for their activity. To investigate the role of metal ions in the structure and function of type II AFPs, the binding of Ca2+ and other divalent cations to herring AFP was investigated. Binding studies using 45Ca2+ demonstrated that the AFP has a single Ca2+-binding site with a Kd of 9 microM. Proteolysis protection studies and measurement of antifreeze activity revealed a conformational change from a protease-sensitive and inactive apoAFP to a protease-resistant active AFP upon Ca2+ binding. Other divalent metal ions including Mn2+, Ba2+, and Zn2+ bind at the Ca2+-binding site and induce a similar change. A saturatable increase in tryptophan emission intensity at 340 nm also occurred upon Ca2+ addition. Whereas antifreeze activity appeared normal when Ca2+ or Mn2+ were bound, it was much lower in the presence of other metal ions. When Ba2+ was bound to the AFP, ice crystals showed a distinct difference in morphology. These studies demonstrate that herring AFP specifically binds Ca2+ and, consequently, adopts a conformation that is essential for its ice-binding activity.

Amino Acid Sequence↗
PubMed1996

Skin antifreeze protein genes of the winter flounder, Pleuronectes americanus, encode distinct and active polypeptides without the secretory signal and prosequences.

Distinct antifreeze polypeptides (AFP) were isolated from the skin of the winter flounder, Pleuronectes americanus, by gel filtration and reverse phase high performance liquid chromatography. In parallel, several cDNA clones were isolated from a skin cDNA library using a liver AFP cDNA probe. Both protein and DNA sequence analyses indicate that flounder skin contains several distinct but homologous alanine-rich AFPs. Although the skin type AFPs contain 11 similar amino acid repeats found in the secretory liver type AFPs, the skin type AFPs are mature polypeptides lacking both the signal and prosequences, indicating that they may function intracellularly. The skin type AFP is significantly less active in thermal hysteretic activity than the liver type AFP. Genomic Southern analysis indicates that like the liver type AFP genes, there are multiple copies (30-40 copies) of skin type AFP. Although the liver type AFP genes are specifically expressed in the liver and to a lesser extent in intestine, the skin type AFP genes are expressed in all tissues examined including the liver and abundantly in exterior tissues, i.e. skin, scales, fin, and gills, suggesting an important protecting role in these exterior tissues.

Alanine↗
PubMed1995

The antifreeze protein genes of the winter flounder, Pleuronectus americanus, are differentially regulated in liver and non-liver tissues.

The synthesis of winter flounder (Pleuronectus americanus) antifreeze protein (AFP) mRNAs in the liver is seasonally regulated by the pituitary gland. With the recent discovery that AFP mRNAs are also present in several non-liver tissues, the aim of the present investigation was to compare the regulatory mechanisms of AFP genes in liver and non-liver tissues. Northern blot analyses indicate that the level of liver AFP mRNA undergoes a several hundred fold difference between the winter and summer months, while AFP mRNAs from gills and kidneys exhibit only a modest 5-10 fold seasonal variation. As expected, the liver AFP mRNA in the hypophysectomized fish was increased by over 40 fold. However, no significant increase was observed for the non-liver AFP mRNAs upon hypophysectomy. These investigations suggest that AFP mRNAs in liver and non-liver tissues are differentially regulated.

Animals↗
PubMed1993

Herring antifreeze protein: primary structure and evidence for a C-type lectin evolutionary origin.

A complementary DNA (cDNA) for a type II antifreeze protein (AFP) was isolated from an Atlantic herring liver cDNA library and sequenced. The predicted protein sequence is homologous to those of the type II fish AFP from smelt and sea raven; it is also homologous to the carbohydrate recognition domains (CRD) of calcium-dependent (C-type) lectins and similar domains in lectin-like proteins. Herring belong to the infradivision Clupeomorpha, which is distinct from the Euteleostei to which all other AFP-producing fish belong. The occurrence of type II AFP in widely divergent fish groups and their homology to C-type lectin CRDs suggest that type II AFPs evolved from these lectins. Amino acid residues forming the hydrophobic cores of rat mannose-binding protein A (MBP-A) that are conserved in character among C-type lectins are also conserved in the herring AFP. The herring AFP also requires Ca2+ for thermal hysteresis activity. These results suggest that herring AFP is structurally and functionally similar to the CRDs of C-type lectins and related domains in other proteins.

Amino Acid Sequence↗
PubMed1992

Structural and functional similarity between fish antifreeze proteins and calcium-dependent lectins.

A cDNA for a type II antifreeze protein was isolated from liver of smelt (Osmerus mordax). The predicted protein sequence is homologous to that from sea raven (Hemitripterus americanus) and both show homology to a family of calcium-dependent lectins. Smelt and sea raven belong to taxonomic orders believed to have diverged prior to Cenozoic glaciation. Thus, type II antifreeze proteins appear to have evolved independently in these fish species from pre-existing calcium-dependent lectins. Sequence alignment of the antifreezes and the lectins suggest that these proteins adopt a similar fold, that the sea raven antifreeze has lost its Ca2+ binding sites, and the smelt antifreeze has retained one site. Experiments show that smelt antifreeze protein activity is responsive to Ca2+ but that of sea raven antifreeze protein is not. These results suggest that the type II fish antifreeze proteins and calcium-dependent lectins share a common ancestry, related folding structures, and functional similarity.

Amino Acid Sequence↗