Biomedical subjects

C L Hew

Publications and source records attributed to C L Hew.

At least 37 records · Page 2Linked to original sources

PubMed1999

Secretory expression and site-directed mutagenesis studies of the winter flounder skin-type antifreeze polypeptides.

Winter flounder contains both liver-type, extracellular antifreeze polypeptides (wflAFPs) and less active skin-type, intracellular antifreeze polypeptides (wfsAFPs). The lower activity of wfsAFPs might be due to their lack of complete ice-binding motifs '-K-DT-'. In order to test the functional role of this putative ice-binding motif, mutations were introduced into the N-terminal or C-terminal regions of wfsAFP-2, which lack any presumptive ice-binding motifs. The wild-type and mutant wfsAFP-2 were secreted in Escherichia coli culture media as mature antifreeze proteins and purified to homogeneity. Surprisingly, the antifreeze activity decreased with the introduction of ice-binding motifs. However, there was a corresponding decrease in alpha-helical content as well as thermal stability and this would suggest a compromise in retaining helical structure with the presence of ice-binding motifs. These studies have brought new definitions of the roles of ice-binding motif residues in type I antifreeze proteins.

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 skin-type antifreeze protein gene intron of the winter flounder is a ubiquitous enhancer lacking a functional C/EBPalpha binding motif.

The winter flounder antifreeze protein (AFP) intron contains a liver-specific enhancer (Element B) which was shown earlier to bind CCAAT/enhancer binding protein (C/EBP)alpha. In contrast, as demonstrated in the present studies, the intron of the skin-type AFP gene acted as a ubiquitous enhancer and contained a TA insertion at similar region to Element B (Element S) which destroyed its interaction with C/EBPalpha. Furthermore, a TA insertion of Element B by site-directed mutagenesis decreased its liver enhancer activity. The presence or absence of C/EBPalpha binding motifs in Element B and Element S, respectively, may provide a mechanism for their differential expression.

Animals↗

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 the ice-binding surface on a type III antifreeze protein with a "flatness function" algorithm.

Antifreeze proteins (AFPs) adsorb to surfaces of growing ice crystals, thereby arresting their growth. The prevailing hypothesis explains the nature of adsorption in terms of a match between the hydrophilic side chains on the AFP's ice-binding surface (IBS) and the water molecules on the ice surface. The number and spatial arrangement of hydrogen bonds thus formed have been proposed to account, respectively, for the binding affinity and specificity. The crystal structure of a type III AFP from ocean pout (isoform HPLC-3) has been determined to 2.0-A resolution. The structure reveals an internal dyad motif formed by two 19-residue, loop-shaped elements. Based on of the flatness observed on the type I alpha-helical AFP's IBS, an automated algorithm was developed to analyze the surface planarity of the globular type III AFP and was used to identify the IBS on this protein. The surface with the highest flatness score is formed by one loop of the dyad motif and is identical to the IBS deduced from earlier mutagenesis studies. Interestingly, 67% of this surface contains nonpolar solvent-accessible surface area. The success of our approach to identifying the IBS on an AFP, without considering the presence of polar side chains, indicates that flatness is the first approximation of an IBS. We further propose that the specificity of interactions between an IBS and a particular ice-crystallographic plane arises from surface complementarity.

Algorithms↗

PubMed1998

Two rainbow trout (Oncorhynchus mykiss) albumin genes are differentially regulated.

Two distinct albumin cDNAs (rtALB1 and rtALB2) were isolated from the rainbow trout (Oncorhynchus mykiss) liver cDNA library. The rtALB1 cDNA (2761 bp) contains a 69 bp 5' untranslated region (UTR), a 1821 bp reading region, and a long 3' UTR of 872 bp. The rtALB2 cDNA (2250 bp) contains a 78 bp 5' UTR, a 1824 bp coding region, and a 348 bp 3' UTR. The two albumins are 81.5% and 77.5% identical in their nucleotides and protein sequences, respectively. Both rtALB1 and rtALB2 genes are expressed only in the liver. The albumin mRNA was first detected in 5-week-old embryos and was tissue-specific. The two albumin genes were differentially expressed, with the rtALB1 transcripts being 3 to 10 times more abundant than the rtALB2 transcripts. This differential expression was partially regulated at the transcriptional level. Promoter analysis showed that the rtALB1 gene had a typical albumin promoter structure. However, the rtALB2 promoter was abnormal in the TATA box region and was less effective in activating the reporter gene in the mammalian cell lines. These variations in rainbow trout albumin promoter sequences might account for their differences in transcriptional efficiency.

Albumins↗

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

Presence of distinct cis-acting elements on gonadotropin gene promoters in diverse species dictates the selective recruitment of different transcription factors by steroidogenic factor-1.

The nuclear receptor steroidogenic factor-1 (SF-1) regulates the cell-specific expression of the pituitary gonadotropin subunit genes. Several potential DNA-binding sites for SF-1, estrogen receptor (ER) and the immediate-early transcription factor NGFI-A are found in LHbeta genes from many species. In this study, we have examined the action and interaction of these transcription factors on LHbeta gene promoters from two representative vertebrate species, i.e. rat and salmon. Cotransfection studies in COS-1 cells have shown that the action of SF-1 on salmon gonadotropin IIbeta (sGTHIIbeta) gene promoter was dramatically enhanced when combined with ER. The rat LHbeta promoter was activated by SF-I or ER individually, but these two factors, however, were unable to act in synergism on this promoter. In contrast, NGFI-A, specifically in cooperation with SF-1, transactivated the rat LHbeta gene expression but was ineffective on the sGTHIIbeta gene. Gel shift experiments showed that this lack of activation was due to the low affinity of the salmon NGFI-A-responsive element for its binding protein. In conclusion, our studies demonstrate that differential recruitment of distinct transcription factors by SF-1 might be a common mechanism to activate the cell-specific gonadotropin gene expression in different species.

Animals↗

PubMed1997

Rapid identification and isolation of zebrafish cDNA clones.

A fast and economical approach, referred to as cDNA clone tagging, was adapted to identify and isolate zebrafish cDNA clones. The basic approach was to partially sequence the coding region of size selected cDNA clones and the partial sequences were then used as tags for identifying the clones through homology search. To benefit maximally from the tagging approach, two cDNA libraries, derived from embryonic and adult fish poly(A)+ RNAs, respectively, were constructed by unidirectional cloning; conceptually, they have the potential to represent all expressed zebrafish genes. A total of 1084 clones were sequenced from the two libraries, and 511 clones were identified, based on sequence homology. These identified clones were derived from at least 261 genes, encoding 48 translational machinery proteins, 47 cytosolic proteins, 43 cytoskeletal proteins, 41 nuclear proteins, 32 membrane proteins, 22 secreted proteins, 20 mitochondrial proteins and 8 proteins with an unknown location. Of the 261 distinct cDNA clones identified, 254 were isolated for the first time in the zebrafish. These tagged cDNA clones, identified and unidentified, provide rich resources for developmental analysis as well as mapping of zebrafish genome. The long-term objective of this study is to establish a tagged zebrafish gene library that can be accessed both by hybridization screening against the plasmid DNAs and by electronic screening using the sequence information.

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↗

PubMed1997

Teleost FTZ-F1 homolog and its splicing variant determine the expression of the salmon gonadotropin IIbeta subunit gene.

Steroidogenic factor 1, a member of the fushi tarazu factor 1 (FTZ-F1) subfamily of nuclear receptors, is a key regulator in mammalian reproduction. From an embryonic complementary DNA library, the zebrafish homolog of FTZ-F1 (zFF1A) and an alternatively spliced variant (zFF1B) were isolated. zFF1B represented a C-terminally truncated version of zFF1A. Whole mount in situ hybridization and reverse transcriptase-PCR analysis revealed that both zFF1A and B transcripts were present in the developing pituitaries, adult fish brain, gonads, and liver, albeit zFF1B messenger RNA was absent in testis. Comparison of the primary sequences of zFF1 with those of other FTZ-F1 subfamily members showed a close structural relationship between the mouse liver receptor homolog, which activated the alpha1-fetoprotein gene in rodent liver. However, similar to mouse steroidogenic factor 1, zFF1A regulated chinook salmon gonadotropin IIbeta subunit gene expression. On the contrary, zFF1B, which could bind a consensus gonadotrope-specific element with an affinity similar to that of zFF1A, lacked both the trans-activation function and synergistic interaction with the estrogen receptor. Furthermore, cotransfection studies in HeLa cells showed that zFF1B was a strong competitor for the action of zFF1A on the chinook salmon gonadotropin IIbeta subunit gene promoter. Our investigation suggests that 1) zFF1 represents an ancestor protein of the vertebrate FTZ-F1 homologs; 2) the antagonistic relationship between zFF1A and -B may dictate the expression of the FTZ-F1 target genes in a variety of tissues, including the pituitary; and 3) the naturally occurring zFF1B provides evidence that the C-terminal portion of zFF1A (80 amino acid residues) contains a major trans-activation function and a protein-protein interface.

Amino Acid Sequence↗

PubMed1996

An immune response to ice crystals in North Atlantic fishes.

In mammals, the presence of crystals composed of small organic molecules, including urate and related compounds, has been shown to trigger an inflammatory response and the subsequent production of specific immunoglobulins (Ig's). Many fishes that are exposed to ice crystals in cold temperate and polar oceans may harbour ice crystals internally. Here, we report evidence for a specific immune response to ice crystals in cold-ocean marine fishes. Using ice nucleation activity as an assay, anti-ice Ig's were detected in the sera of the cold-ocean marine fish species, ocean pout (Macrozoarces americanus) and Atlantic herring (Clupea harengus harengus), but not in the sera of species that are not exposed to ice. Purified Ig's isolated from ocean pout serum using two different protocols showed ice nucleation activity, thus demonstrating the presence of ice binding specificity among these Ig's.

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↗

PubMed1996

Induction of chinook salmon growth hormone promoter activity by the adenosine 3',5'-monophosphate (cAMP)-dependent pathway involves two cAMP-response elements with the CGTCA motif and the pituitary-specific transcription factor Pit-1.

In this study, the functional role of two cAMP-response elements (CRE) in the promoter of the chinook salmon GH gene and their interactions with the transcription factor Pit-1 in regulating GH gene expression were examined. A chimeric construct of the chloramphenicol acetyltransferase (CAT) reporter gene with the CRE-containing GH promoter (pGH.CAT) was transiently transfected into primary cultures of rainbow trout pituitary cells. The expression of CAT activity was stimulated by an adenylate cyclase activator forskolin as well as a membrane-permeant cAMP analog 8-bromo-cAMP. Furthermore, these stimulatory responses were inhibited by a protein kinase A inhibitor H89, suggesting that these CREs are functionally coupled to the adenylate cyclase-cAMP-protein kinase A cascade. This hypothesis is supported by parallel studies using GH4ZR7 cells, a rat pituitary cell line stably transfected with dopamine D2 receptors. In this cell line, D2 receptor activation is known to inhibit adenylate cyclase activity and cAMP synthesis. Stimulation with a nonselective dopamine agonist, apomorphine, or a D2-specific agonist, Ly171555, suppressed the expression of pGH.CAT in GH4ZR7 cells, and this inhibition was blocked by simultaneous treatment with forskolin. These results indicate that inhibition of the cAMP-dependent pathway reduces the basal promoter activity of the CRE-containing pGH.CAT. The functionality of these CREs was further confirmed by deletion analysis and site-specific mutagenesis. In trout pituitary cells, the cAMP inducibility of pGH.CAT was inhibited after deleting the CRE-containing sequence from the GH promoter. When the CRE-containing sequence was cloned into a CAT construct with a viral thymidine kinase promoter, a significant elevation of cAMP inducibility was observed. This stimulatory response, however, was abolished by mutating the core sequence, CGTCA, in these CREs, suggesting that these cis-acting elements confer cAMP inducibility to the salmon GH gene. The interactions between CREs and the transcription factor Pit-1 in mediating GH gene expression were also examined. In HeLa cells, a human cervical cancer cell line deficient in Pit-1, both basal and cAMP-induced expression of pGH.CAT were apparent only with the cotransfection of a Pit-1 expression vector. These results taken together indicate that the two CREs in the chinook salmon GH gene are functionally associated with the cAMP-dependent pathway and that their promoter activity is dependent on the presence of Pit-1

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Steroidogenic factor 1 and estradiol receptor act in synergism to regulate the expression of the salmon gonadotropin II beta subunit gene.

The orphan nuclear receptor steroidogenic factor-1 (SF-1) regulates the expression of several genes involved in the reproductive function and development of the adrenal, the gonads, and the pituitary gonadotropes. It also confers the gonadotrope-specific expression of the glycoprotein hormone a subunit gene by the binding to a gonadotrope-specific element (GSE). In this study, we have shown that SF-1 transactivates the salmon gonadotropin II beta subunit (sGTHII beta) gene expression. SF-1 alone offered a slight but significant enhancement on sGTHII beta promoter activity (7.2 +/- 0.6 fold). However, it stimulated sGTHII beta gene expression dramatically (127 +/- 37 fold) when combined with the estrogen receptor (ER). This synergistic interaction was specific for sGTHII beta promoter as well as for both SF-1 and ER and was estradiol-dose dependent. 5'-Deletion studies of the sGTHII beta promoter identified two putative SF-1 binding sites (GSE) and one previously identified proximal estrogen-responsive element (pERE) at -274 bp involved in this activation. The two GSE sequences located at -354 bp (sGSE(3) and -162 bp (sGSE(2) upstream of the transcription site, although imperfect as compared with the consensus GSE, bound specifically to the in vitro-translated mouse SF-1 protein. 5'-Deletion studies, competition experiments, and site-directed mutagenesis showed that binding to pERE and GSE(2) were necessary for the SF-1/ER synergistic effect. These studies suggest that the synergistic interaction of SF-1 and ER, possibly through cooperative binding or protein-protein interaction, is essential in conferring a cell type-specific expression of the GTHII beta subunit gene.

Animals↗

PubMed1995

Several splicing variants of isl-1 like genes in the chinook salmon (Oncorhynchus tschawytscha) encode truncated transcription factors containing a complete LIM domain.

Several novel cDNA clones have been isolated from a chinook salmon pituitary cDNA library. Sequence analysis of these clones indicates that they are closely related to the rat LIM domain homeobox gene, isl-1. Due to differential splicing, several of the clones encode truncated polypeptides containing a complete copy of the LIM domain without the homeodomain and C-terminal activation domain. The roles of these truncated polypeptides are discussed.

Amino Acid Sequence↗

PubMed1995

Presence of isl-1-related LIM domain homeobox genes in teleost and their similar patterns of expression in brain and spinal cord.

Three novel LIM domain homeobox cDNAs encoding proteins structurally related to the Isl-1 protein were isolated from a chinook salmon pituitary cDNA library. Southern blot analysis of genomic DNA indicate that they are derived from three distinct genes, designated as isl-2a, isl-2b, and isl-3 genes. Nucleotide sequence analysis of reverse transcriptase-polymerase chain reaction amplified products reveal that the isl gene family contains two members (a and b) each of both isl-1 and isl-2 genes, and one member of isl-3 gene in the two tetraploid salmonid species, chinook salmon and rainbow trout, and only one member each of isl-1, isl-2, and isl-3 genes in the diploid zebrafish. The expression of the three isl genes in the rainbow trout were studied by reverse transcriptase-polymerase chain reaction analysis of embryonic and adult RNAs, and by in situ hybridization analysis of 8-week-old hatchlings. The transcripts of all three genes could be detected as early as 4 weeks postfertilization (the eye stage) and increased dramatically in 5-week-old embryos. In the adult, the three isl mRNAs appear to be differentially distributed in various tissues. The level of isl-1 mRNA is generally higher than those of isl-2 and isl-3 mRNAs. In situ hybridization analysis indicates that the transcripts of all three genes are localized in subsets of neurons in the brain and spinal cord. In the retina, isl-1 mRNA could be found in both the ganglion and inner nuclear layers while isl-2 and isl-3 mRNAs could only be detected in the ganglion layer. High level of isl-1 mRNA could also be found in mid-gut and interrenal organ where endocrine cells are densely populated. Based on these observations, we speculate that the three structurally related isl genes may play similar roles in cell determination and differentiation in the developing nervous system.

Aging↗