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Comp Biochem Physiol B Biochem Mol Biol [journal]
- The adhesive skin exudate of Notaden bennetti frogs (Anura: Limnodynastinae) has similarities to the prey capture glue of Euperipatoides sp. velvet worms (Onychophora: Peripatopsidae). [JOURNAL ARTICLE]
- Comp Biochem Physiol B Biochem Mol Biol 2013 May 7.
The dorsal adhesive secretion of the frog Notaden bennetti and the prey-capture "slime" ejected by Euperipatoides sp. velvet worms look and handle similarly. Both consist largely of protein (55-60% of dry weight), which provides the structural scaffold. The major protein of the onychophoran glue (Er_P1 for Euperipatoides rowelli) and the dominant frog glue protein (Nb-1R) are both very large (260-500 kDa), and both give oddly "turbulent" bands. Both major proteins, which are rich in Gly (16-17 mol%) and Pro (7-12 mol%) and contain 4-hydroxyproline (Hyp, 4 mol%), have the composition of intrinsically unstructured proteins. Their propensities for elastomeric or amyloid structures are discussed in light of Er_P1's large content of intrinsically disordered long tandem repeats. The low carbohydrate content of both glues is consistent with conventional protein glycosylation, which in the N. bennetti adhesive was explored by 2D PAGE. The N-linked sugars of Nb-1R appear to prevent inappropriate self-aggregation. Some peptide sequences from Nb-1R are presented. Overall, there are enough similarities between the frog and the velvet worm glues to suspect that they employ related mechanisms for setting and adhesion. A common paradigm is proposed for amphibian and onychophoran adhesives, which, if correct, points to convergent evolution.
- Olive flounder (Paralichthys olivaceus) cystatin B: Cloning, tissue distribution, expression and inhibitory profile of piscine cystatin B. [JOURNAL ARTICLE]
- Comp Biochem Physiol B Biochem Mol Biol 2013 May 3.
Among the cystatin superfamily, cystatin B, also known as stefin B, is an intracellular inhibitor that regulates the activities of cysteine proteases, such as papain and cathepsins. In this study, the 536bp cystatin B cDNA (referred to hereafter as PoCystatin B) was cloned from olive flounder (Paralichthys olivaceus) using a combination of the rapid amplification of cDNA ends (RACE) approach and olive flounder cDNA library screening. To determine the tissue distribution of PoCystatin B mRNA, the expression of PoCystatin B in normal and lipopolysaccharide (LPS)-stimulated flounder tissues were compared with that of the inflammatory cytokines interleukin (IL)-1β, IL-6, and IL-8 by reverse transcription (RT)-polymerase chain reaction (PCR). The results of the RT-PCR analysis revealed ubiquitous PoCystatin B expression in normal and LPS-stimulated tissues. To characterize the enzymatic activity of PoCystatin B protein, recombinant PoCystatin B protein was overexpressed in Escherichia coli BL21(DE3) cells in the pCold™ TF DNA expression vector as a soluble fusion protein of 67-kDa. PoCystatin B inhibited papain cysteine protease, bovine cathepsin B, and fish cathepsins F and X to a greater extent, whereas fish cathepsins L, S, and K were inhibited to a lesser extent. These results indicate that the enzymatic characteristics of the olive flounder cystatin B are similar to those of mammalian cystatin B proteins, and provide a better understanding of the mechanisms of regulation of cathepsins and cystatins in marine organisms.
- Blood vitamin D3 metabolite concentrations of adult female bearded dragons (Pogona vitticeps) remain stable after ceasing UVb exposure. [JOURNAL ARTICLE]
- Comp Biochem Physiol B Biochem Mol Biol 2013 May 3.
Vitamin D deficiency can lead to several health problems collectively called metabolic bone disease (MBD). One commonly kept reptile species prone to develop MBD if managed incorrectly is the bearded dragon (Pogona vitticeps). This study aimed to determine the extent to which adult female bearded dragons fed a diet low in vitamin D can use stored vitamin D and its metabolites to maintain plasma 25(OH)D3 and 1,25(OH)2D3 concentrations after discontinuing UVb exposure. Blood samples of healthy adult female bearded dragons, exposed to UVb radiation for over 6months were collected (day 0) after which UVb exposure was discontinued for 83days and blood was collected. Blood plasma was analysed for concentrations of total Ca, total P, ionized Ca, uric acid, 25(OH)D3 and 1,25(OH)2D3. There was no significant change in plasma 25(OH)D3 and 1,25(OH)2D3 concentrations during the study. While total Ca and P in whole blood was found to significantly decrease over time (P<0.0088 and 0.0016, respectively), values were within the reference range. Plasma ionized Ca tended (P=0.0525) to decrease during the study. Adult female bearded dragons, previously exposed to UVb, are able to maintain blood vitamin D metabolite concentrations when UVb exposure is discontinued for a period of up to 83days.
- Functional analysis of duplicated genes and N-terminal splice variant of phospholipase C-δ1 in Paralichthys olivaceus. [JOURNAL ARTICLE]
- Comp Biochem Physiol B Biochem Mol Biol 2013 Apr 26.
Phosphoinositide-specific phospholipase C δ (PLC δ) plays an important role in many cellular responses and is involved in the production of second messenger. Here, we describe the presence of novel N-terminal extended alternative splice form of PLC-δ1B in Paralichthys olivaceus, which differs from the reported mammalian PLC-δ1 isoform. The two variants PoPLC-δ1B-Lf and PoPLC-δ1B-Sf share exon 3 (including the PH domain) to exon 16, but differ at the exon 1 (Short form: Sf) and novel exon 2 (Long form: Lf) of the transcript. For the characterization of the novel duplicated gene variant of PLC-δ1B in P. olivaceus, tissue-specific expression with RT-PCR and real-time PCR, and purification and enzymatic characterization of native and recombinant proteins of all the three-types of PLC-δ1 isoforms (PoPLC-δ1A, PoPLC-δ1B-Lf and PoPLC-δ1B-Sf) of P. olivaceus were studied. The PoPLC-δ1A was ubiquitously distributed in gill, kidney and spleen. The PoPLC-δ1B-Lf gene was widely detected in various tissues, especially in the digestive system, while PoPLC-δ1B-Sf was highly expressed in the stomach. The recombinant PoPLC-δ1A, PoPLC-δ1B-Lf and PoPLC-δ1B-Sf proteins were expressed as a histidine-tagged fusion protein in Escherichia coli. The PLC activity of the PoPLC-δ1 isoform proteins showed a concentration-dependent activity to phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). In addition, U73122, the PLC inhibitor, effectively inhibited PLC activities of PoPLC-δ1A, PoPLC-δ1B-Lf and PoPLC-δ1B-Sf proteins. However, PoPLC-δ1A and PoPLC-δ1B-Lf were sensitive at pH7.5, while PoPLC-δ1B-Sf was relatively sensitive at pH7. These results might be useful for the study of phospholipase C-mediated signal transduction in fish.
- Energy sources from the eggs of the wolf spider Schizocosa malitiosa: Isolation and characterization of lipovitellins. [JOURNAL ARTICLE]
- Comp Biochem Physiol B Biochem Mol Biol 2013 Apr 23; 165(3):172-180.
In oviparous species, proteins and lipids found in the vitellus form the lipoproteins called lipovitellins that are the major source of energy for the development, growth, and survival of the embryo. The energy resources provided by the lipovitellins have not yet been investigated in the Order Araneae. Using the wolf spider Schizocosa malitiosa (Lycosidae) as an experimental model, we identified and characterized the lipovitellins present in the cytosol, focusing on the energetic contribution of those lipoprotein particles in the vitellus. Two lipovitellins (LV) named SmLV1 and SmLV2 were isolated. SmLV1 is a high-density lipoprotein with 67% lipid and 3.6% carbohydrate, and SmLV2 is a very high-density lipoprotein with 9% lipid and 8.8% carbohydrate. Through electrophoresis in native conditions we observed that SmLV1 has a molecular mass of 559kDa composed of three apolipoproteins of 116, 87, and 42kDa, respectively. SmLV2 comprised several proteins composed of different proportions of the same subunits (135, 126, 109, and 70kDa). The principal lipids of these lipovitellins are sphingomyelin+lysophosphatidylcholine, esterified sterols, and phosphatidylcholine. Lipovitellin-free cytosol contains abundant phospatidylcholine and triacylglyceride related to the yolk nuclei (the vitellus organizing center). The principal fatty acids of SmLV1 and SmLV2 are 18:2 n-6, 18:1 n-9, and 16:0. Spectrophotometry detected no pigments in either the lipovitellins or the cytosol. The egg caloric content was 92cal/g, at proportions of 59.8% protein, 20.1% carbohydrate, and 19.9% lipid. SmLV1 and SmLV2 provided 19.5% and 17.1% of the calories, respectively. Both lipovitellins contribute mainly with proteins (15.8-18%), with the input of carbohydrates and lipids being lower than 1.3%.
- Characterization of the expression profiles of adipogenesis-related factors, ZNF423, KLFs and FGF10, during preadipocyte differentiation and abdominal adipose tissue development in chickens. [JOURNAL ARTICLE]
- Comp Biochem Physiol B Biochem Mol Biol 2013 Apr 19.
Adipogenesis is controlled by a complicated process involving certain transcriptional events. In chicken adipogenesis, peroxisome proliferator-activated receptor γ (PPARγ) is a key regulator of preadipocyte differentiation and abdominal fat accumulation. However, in a recent study in mammals, some novel factors related to regulation of adipogenesis, including preadipocyte differentiation, were identified in mammals. Therefore, in this study, we aimed to determine the expression profiles of these mammalian adipogenesis-related factors, such as zinc-finger protein 423 (ZNF423), Krüppel-like factor -2, -5, and -15 (KLF-2, -5, -15), and FGF10, in the chicken (Gallus gallus). Specifically, we analyzed their expression in primary preadipocyte differentiation in vitro and also analyzed their tissue distribution and their temporal expression in adipose tissue development in vivo. During chicken adipocyte differentiation, the gene expression of ZNF423, KLF-2, KLF-5 and FGF10 was found to rapidly decrease in the early stage of preadipocyte differentiation. Expression of ZNF423 then increased in the late stage of differentiation. KLF-15 expression increased in a time-dependent manner for 48h. Protein expressions of these factors were reflected by Western blot analysis. High levels of aP2, PPARγ and FGF10 mRNA were found in adipose tissue. In addition, aP2, PPARγ and ZNF423 mRNA levels in the adipose tissue were elevated at days 10 and 20. These expression profiles of the adipogenesis-related factors in chicken are, in part, different from mammalian adipogenesis but this seems to reflect the differences in the regulation of adipogenesis and in adipose tissue functions between avians and mammals.
- Characterization of cDNAs for calmodulin and calmodulin-like protein in the freshwater mussel Hyriopsis cumingii: Differential expression in response to environmental Ca(2+) and calcium binding of recombinant proteins. [JOURNAL ARTICLE]
- Comp Biochem Physiol B Biochem Mol Biol 2013 Apr 18; 165(3):165-171.
Calmodulin and calmodulin-like protein are two crucial calcium regulators in bivalves. However, molecular characteristics and expression patterns of these genes in the freshwater mussel are poorly understood. In this study, two cDNAs encoding novel calmodulin and calmodulin-like protein (HcCaM and HcCaLP) were cloned and characterized from the freshwater pearl mussel Hyriopsis cumingii. The full-length cDNA of HcCaM was 726bp, including a 118-bp 5'-untranslated region (UTR), a 447-bp open reading frame (ORF), and a 161-bp 3'-UTR. The 1217-bp HcCaLP cDNA comprised of a 51-bp 5'-UTR, a 447-bp ORF, and a 716-bp 3'-UTR. The potential phosphorylation sites of, Arg(80) and Phe(100) in deduced HcCaM were mutated to Thr(80) and Tyr(100) in HcCaLP. Tissue-specific expression analysis revealed that HcCaM mRNA was prominently expressed in the gill, mantle center, and foot. In contrast, HcCaLP mRNA was mainly expressed in the mantle edge. The recombinant HcCaM and HcCaLP proteins expressed in Escherichia coli showed the typical Ca(2+) dependent electrophoretic shift characterization as CaM and differed in the calcium binding affinity. The calcium stimulation test that lasted 5weeks implied that HcCaM and HcCaLP had differential expression patterns in response to various environmental Ca(2+) concentrations (0.25-1.25mM). The expression of HcCaM mRNA was up-regulated by low Ca(2+) concentration (0.25mM), and the highest expression of HcCaLP mRNA occurred under Ca(2+) concentration of 1mM.
- Glyceryl trinitrate metabolism in the quail embryo by the glutathione S-transferases leads to a perturbation in redox status and embryotoxicity. [JOURNAL ARTICLE]
- Comp Biochem Physiol B Biochem Mol Biol 2013 Apr 12; 165(3):153-164.
Exposure of stage 9 quail (Coturnix coturnix japonica) embryos to glyceryl trinitrate (GTN) induces malformations that were associated in previous studies with an increase in protein nitration. Increased nitration suggests metabolism of GTN by the embryo. The goals of this study were to characterize the enzymes and co-factors required for GTN metabolism by quail embryos, and to determine the effects of in ovo treatment with N-acetyl cysteine (NAC), a precursor of glutathione (GSH), on GTN embryotoxicity. GTN treatment of quail embryos resulted in an increase in nitrite, a decrease in total GSH, and an increase in the ratio of NADP(+)/NADPH, indicating that redox balance may be compromised in exposed embryos. Glutathione S-transferases (GSTs; EC 126.96.36.199) purified from the whole embryo (Km 0.84mM; Vmax 36μM/min) and the embryonic eye (Km 0.20mM; Vmax 30μM/min) had GTN-metabolizing activity (1436 and 34nmol/min/mg, respectively); the addition of ethacrynic acid, an inhibitor of GST activity, decreased GTN metabolism. Peptide sequencing of the GST isozymes indicated that alpha- or mu-type GSTs in the embryo and embryonic eye had GTN metabolizing activity. NAC co-treatment partially protected against the effects of GTN exposure. Thus, GTN denitration by quail embryo GSTs may represent a key initial step in the developmental toxicity of GTN.
- Seasonal upregulation of catabolic enzymes and fatty acid transporters in the flight muscle of migrating hoary bats, Lasiurus cinereus. [Journal Article]
- Comp Biochem Physiol B Biochem Mol Biol 2013 Jun; 165(2):138-43.
The high energy density of fat, and limited capacity for carbohydrate storage suggest that migrating bats should fuel endurance flights with fat, as observed in migrating birds. Yet, cursorial mammals are unable to support high intensity exercise with fat stores. We hypothesized that migratory bats and birds have converged on similar physiological mechanisms to fuel endurance flight with fat. We predicted bats would seasonally upregulate fatty acid transport and oxidation pathways when migration demands were high. We studied seasonal variation in mitochondrial oxidative enzyme activities and fatty acid transport protein expression in the flight muscle of hoary bats (Lasiurus cinereus). Carnitine palmitoyl transferase, 3-hydroxyacyl-CoA dehydrogenase and citrate synthase activity increased during migration. There were no changes in expression of fatty acid translocase or plasma membrane fatty acid binding protein. Heart-type fatty acid binding protein expression increased 5-fold in migrating females, but did not vary seasonally in males. An aerial insectivore lifestyle, and the coincidence of migration and pregnancy may explain differences in transporter expression compared to previously studied birds. Overall, our results are consistent with seasonal upregulation of lipid metabolism and aerobic capacity, and confirm that migration poses distinct physiological challenges for bats.
- Molecular cloning, characterization and expression analysis of a novel wap65-1 gene from Plecoglossus altivelis. [Journal Article]
- Comp Biochem Physiol B Biochem Mol Biol 2013 Jun; 165(2):144-52.
Warm temperature acclimation associated 65-kDa protein 1 (WAP65-1) is a specific fish plasma glycoprotein that is possibly involved in various physiological or pathological processes. In this study, we obtained the cDNA and genomic DNA sequences of the Plecoglossus altivelis wap65-1 (Pawap65-1) gene. Multiple sequence alignment showed that Pawap65-1 is similar in structure to wap65-1 in fish. Phylogenetic analysis revealed that Pawap65-1 is most closely related to that of a rainbow trout. Pawap65-1 transcripts are present in various tissues and are most abundant in the liver. We expressed recombinant PaWAP65-1 in Escherichia coli and raised antiserum against it in mouse. Western blot analysis revealed that the higher molecular mass of PaWAP65-1 in blood plasma was caused by post-translational N-glycosylation. Quantitative real-time quantitative PCR (qPCR) and Western blot analysis data showed that the hepatic mRNA and blood plasma levels of PaWAP65-1 were both influenced by warm temperature acclimation and cadmium exposure, but not by Listonella anguillarum infection, hypo-osmotic, or cold temperature acclimation. In conclusion, our data reveals that PaWAP65-1 is a stress-related protein, and may play a role in fish acclimation to warm temperature and cadmium exposure.