Coding Sequence Of cDNA Research Articles

Anti-cytoskeleton Immunoscreening of Trypanosoma brucei Expression Library Reveals Novel Immunogenic Conserved Putative Proteins

Background: The overall shape of the trypanosome is defined by an internal cytoskeleton consisting of a network of microtubules that are cross linked both to each other and the inner face of the plasma membrane. However, the total compliment and identity of the trypanosome cytoskeleton proteins are not yet fully determined despite the fact that some of them may be good targets for diagnostics, drugs and/or vaccines discovery.
 Methods: In this study, rabbit anti-Trypanosoma brucei detergent insoluble cytoskeleton sera were produced in vivo and used to probe a T. brucei expression library. The picked plaques were made clonal by a series of library screening followed by PCR amplification, cDNA sequencing and identification of the proteins coded by these sequences using BLAST.
 Results: The previously well-known cytoskeleton proteins (paraflagella rod protein and histone H2B), putative cytoskeleton proteins (Dynein light chain and nucleoporin), conserved hypothetical protein (Tb10.61.2430) and novel cytoskeleton protein coding cDNA sequences (not in the sequenced and published T. brucei genome) were identified in this study.
 Conclusion: This approach is therefore, useable in the search for novel proteins whose utility in the design and development of diagnostics, drugs and/or vaccines can further be studied.

Tartary Buckwheat FtMYB31 Gene Encoding an R2R3-MYB Transcription Factor Enhances Flavonoid Accumulation in Tobacco

The R2R3-MYB transcription factors play an important role in regulating secondary metabolism biosynthesis and abiotic stress in plants. In this article, we report the identification of the transcription factor gene, FtMYB31, from the popular Eurasian crop tartary buckwheat (Fagopyrum tataricum) that enhances accumulation of the nutritionally beneficial compound rutin in transgenic tobacco leaves. The FtMYB31 complete cDNA coding sequence was isolated from the leaves of tartary buckwheat, and multiple protein sequence alignments and conserved domain analysis showed it contained a typical R2R3 MYB domain. Subcellular location experiments showed the FtMYB31 protein is localized in nucleus. The phylogenetic tree clustered FtMYB31 with VvMYBPA1 from Vitis vinifera, and AtMYB123 from Arabidopsis thaliana, belonging to the Subgroup 5 cluster. Comparison by qRT-PCR of FtMYB31 transcripts and those from rutin synthesis-related genes showed a relationship between FtMYB31, Ft4CL, and FtUFGT transcripts and rutin content in different tissues of F. tataricum, with correlation coefficients of − 0.68, 0.69, and 0.47, respectively. Transgenic experiments indicated that FtMYB31 upregulated CHS, F3H, and FLS genes in transgenic tobacco and enhanced the accumulation of rutin and total flavonols. These results suggest that FtMYB31 encodes an R2R3-MYB transcription factor that positively regulates flavonol biosynthesis in tartary buckwheat and tobacco and is a possible target for genetically modifying tartary buckwheat to enhance the content of beneficial compounds such as rutin.

Molecular characterization, cellular localization, and light-enhanced expression of Beta-Na+/H+ Exchanger-like in the whitish inner mantle of the giant clam, Tridacna squamosa, denote its role in light-enhanced shell formation

The fluted giant clam, Tridacna squamosa, lives in symbiosis with photosynthetic zooxanthellae, and can engage in light-enhanced growth and shell formation. Light-enhanced shell formation necessitates the elimination of excess H+ from the extrapallial fluid adjacent to the shell. This study aimed to clone Na+/H+Exchanger (NHE) from the whitish inner mantle adjacent to the extrapallial fluid of T. squamosa, to determine its cellular and subcellular localization, and to evaluate the effect of light exposure on its mRNA expression level and protein abundance therein. The complete coding cDNA sequence of NHE obtained was identified as a homolog of beta NHE (βNHE-like). It consisted of 2925 bp, encoding for a polypeptide of 974 amino acids and 107.1 kDa, and was expressed predominantly in the inner mantle. There, βNHE-like was localized in the apical membrane of the seawater-facing epithelium by immunofluorescence microscopy. After exposure to light for 12 h, the seawater-facing epithelium of the inner mantle displayed consistently stronger immunostaining than that of the control exposed to 12 h of darkness. Western blotting confirmed that light exposure significantly enhanced the protein abundance of βNHE-like in the inner mantle. These results denote that some of the excess H+ generated during light-enhanced shell formation can be excreted through the light-dependent βNHE-like of the seawater-facing epithelium to minimize the impact on the whole-body pH. Importantly, the excreted H+ could dehydrate exogenous HCO3−, and facilitate the absorption of inorganic carbon through the seawater-facing epithelium dedicated for light-enhanced shell formation due to its close proximity with the shell-facing epithelium. Nucleotide symbol combinationsPairs: R = A/G; W = A/T; Y = C/T.Triples: D = A/G/T.

Increased apical sodium-dependent glucose transporter abundance in the ctenidium of the giant clam Tridacna squamosa upon illumination.

Giant clams contain phototrophic zooxanthellae, and live in nutrient-deficient tropical waters where light is available. We obtained the complete cDNA coding sequence of a homolog of mammalian sodium/glucose cotransporter 1 (SGLT1) - SGLT1-like - from the ctenidium of the fluted giant clam, Tridacna squamosaSGLT1-like had a host origin and was expressed predominantly in the ctenidium. Molecular characterizations reveal that SGLT1-like of T. squamosa could transport urea, in addition to glucose, as other SGLT1s do. It has an apical localization in the epithelium of ctenidial filaments and water channels, and the apical anti-SGLT1-like immunofluorescence was stronger in individuals exposed to light than to darkness. Furthermore, the protein abundance of SGLT1-like increased significantly in the ctenidium of individuals exposed to light for 12 h, although the SGLT1-like transcript level remained unchanged. As expected, T. squamosa could perform light-enhanced glucose absorption, which was impeded by exogenous urea. These results denote the close relationships between light-enhanced glucose absorption and light-enhanced SGLT1-like expression in the ctenidium of T. squamosa Although glucose absorption could be trivial compared with the donation of photosynthates from zooxanthellae in symbiotic adults, SGLT1-like might be essential for the survival of aposymbiotic larvae, leading to its retention in the symbiotic stage. A priori, glucose uptake through SGLT1-like might be augmented by the surface microbiome through nutrient cycling, and the absorbed glucose could partially fulfill the metabolic needs of the ctenidial cells. Additionally, SGLT1-like could partake in urea absorption, as T. squamosa is known to conduct light-enhanced urea uptake to benefit the nitrogen-deficient zooxanthellae.

Caligus rogercresseyi acetylcholinesterase types and variants: a potential marker for organophosphate resistance

BackgroundControl of the sea louse Caligus rogercresseyi in the Chilean salmonid industry is reliant on chemical treatments. Azamethiphos was introduced in 2013, although other organophosphates were previously used. In 2014, reduced sensitivity to azamethiphos was detected in the Los Lagos Region using bioassays. The main target of organophosphates is the enzyme acetylcholinesterase (AChE). Mutations in the AChE gene are the main cause of organophosphate resistance in arthropods, including other sea lice. In the present study, we aimed to characterize C. rogercresseyi AChE(s) gene(s) and to study the association between AChE variants and azamethiphos resistance in this sea louse species.MethodsSamples of adult male and female C. rogercresseyi were collected in the Los Lagos Region in 2014. Twenty-four hour exposure bioassays with azamethiphos were performed to select sensitive and resistant lice. The full-length cDNA coding sequences encoding for two AChEs in C. rogercresseyi were molecularly characterized. One of the AChE genes was screened by direct sequencing in the azamethiphos-selected lice to search for variants. An additional louse sampling was performed before and after an azamethiphos treatment in the field in 2017 to validate the findings.ResultsThe molecular analysis revealed two putative AChEs in C. rogercresseyi. In silico analysis and 3D modelling of the protein sequences identified both of them as invertebrate AChE type 1; they were named C. rogercresseyi AChE1a and 1b. AChE1a had the characteristics of the main synaptic AChE, while AChE1b lacked some of the important amino acids of a typical AChE. A missense change found in the main synaptic AChE (1a), F318F/V (F290 in Torpedo californica), was associated with survival of C. rogercresseyi at high azamethiphos concentrations (bioassays and field treatment). The amino acid change was located in the acyl pocket of the active-site gorge of the protein.ConclusionsThe present study demonstrates the presence of two types of AChE1 genes in C. rogercresseyi. Although enzymatic assays are needed, AChE1a is most probably the main synaptic AChE. The function of AChE1b is unknown, but evidence points to a scavenger role. The AChE1a F/V318 variant is most probably involved in organophosphate resistance, and can be a good marker for resistance monitoring.

The colorful mantle of the giant clam, Tridacna squamosa, expresses a light-dependent manganese superoxide dismutase to ameliorate oxidative stresses due to its symbiotic association with zooxanthellae

Giant clams harbor extracellular symbiotic zooxanthellae in a tubular system that pervades mainly the fleshy and colorful outer mantle. During insolation, the symbiotic zooxanthellae conduct photosynthesis and donate photosynthates to the host clam. The host could encounter hyperoxia because photosynthesizing zooxanthellae also release O2. Hence, the host tissues, particularly the outer mantle, would need to prevent plausible oxidative damages caused by the increased production of reactive oxygen species (ROS), including superoxide radical (O 2 •− ), during insolation. Superoxide dismutases (SODs) are enzymes which scavenge intracellular O 2 •− . The complete cDNA coding sequence of a Manganese SOD (MnSOD) had been cloned from the outer mantle of the fluted giant clam, Tridacna squamosa. It comprised 678 bp, encoding for a 226 amino acid polypeptide of 24.9 kDa, with a mitochondrial targeting sequence. Its host origin was confirmed through phenogramic and sequence similarity analyses. After 12 h of light exposure, the transcript level and protein abundance of MnSOD/MnSOD increased significantly in the outer mantle as compared with the control kept in darkness for 12 h, which was unrelated to circadian rhythm. In light, the outer mantle could have augmented metabolic rate with increased mitochondrial ROS production due to increases in the supply of substrates to the zooxanthellae for photosynthesis and the receipt of O2 and photosynthates from the symbionts. Therefore, the light-dependable expression of MnSOD/MnSOD in the outer mantle of T. squamosa could be an antioxidative response acquired by the host to detoxify excess mitochondrial O 2 •− and prevent oxidative damage attributable to its symbiotic association with autotrophic zooxanthellae.

Light-enhanced expression of Carbonic Anhydrase 4-like supports shell formation in the fluted giant clam Tridacna squamosa

Giant clams represent symbiotic associations between a host clam and its extracellular zooxanthellae. They are able to grow in nutrient-deficient tropical marine environments and conduct light-enhanced shell formation (calcification) with the aid of photosynthates donated by the symbiotic zooxanthellae. In light, there is a high demand for inorganic carbon (Ci) to support photosynthesis in the symbionts and light-enhanced calcification in the host. In this study, we cloned and characterized a host Carbonic Anhydrase 4 homolog (CA4-like) from the whitish inner mantle of the giant clam Tridacna squamosa. The full cDNA coding sequence of CA4-like consisted of 1002 bp, encoding for 334 amino acids of 38.5 kDa. The host CA4-like was phenogramically distinct from algal CAs. The transcript level of CA4-like in the inner mantle was ~3-fold higher than those in the colorful outer mantle and the ctenidium. In the inner mantle, CA4-like was immunolocalized in the apical membrane of the seawater-facing epithelial cells, but absent from the shell-facing epithelium. Hence, CA4-like was positioned to catalyze the conversion of HCO3− to CO2 in the ambient seawater which would facilitate CO2 uptake. The absorbed CO2 could be converted back to HCO3− by the cytoplasmic CA2-like. As the protein abundance of CA4-like increased in the inner mantle after 6 or 12 h of light exposure, there could be an augmentation of the total CA4-like activity to increase Ci uptake in light. It is plausible that the absorbed Ci was allocated preferentially for shell formation due to the close proximity of the seawater-facing epithelium to the shell-facing epithelium in the inner mantle that contains only few zooxanthellae.

The ctenidium of the giant clam, Tridacna squamosa, expresses an ammonium transporter 1 that displays light-suppressed gene and protein expression and may be involved in ammonia excretion

Ammonium transporters (AMTs) can participate in ammonia uptake or excretion across the plasma membrane of prokaryotic, plant and invertebrate cells. The giant clam, Tridacna squamosa, harbors nitrogen-deficient symbiotic zooxanthellae, and normally conducts light-enhanced ammonia absorption to benefit the symbionts. Nonetheless, it can excrete ammonia when there is a supply of exogenous nitrogen or exposed to continuous darkness. This study aimed to elucidate the role of AMT1 in the ctenidium of T. squamosa by cloning and characterizing the AMT1/AMT1, determining its subcellular localization, and examining changes in its transcript and protein expression levels in response to light exposure. The cDNA coding sequence of AMT1 from T. squamosa consisted of 1527bp and encoded 508 amino acids of 54.6kDa. AMT1-immunofluorescence was detected mainly at the apical epithelium of ctenidial filaments, and it decreased significantly after 12h of exposure to light. By contrast, the epithelial cells surrounding the tertiary water channels in the ctentidium, which are known to exhibit light-enhanced glutamine synthetase expression and take part in the assimilation of exogenous ammonia in light, did not display any AMT1-immunolabelling. Furthermore, the transcript level and protein abundance of ctenidial AMT1/AMT1 decreased significantly at the 6th and 12th h of light exposure. Taken together, these results indicate that AMT1 might participate in ammonia excretion instead of ammonia absorption and assimilation in T. squamosa. It is probable that the expression levels of AMT1/AMT1 need to be down-regulated during light exposure to achieve light-enhanced ammonia uptake.

Molecular Characterization of a Dual Domain Carbonic Anhydrase From the Ctenidium of the Giant Clam, Tridacna squamosa, and Its Expression Levels After Light Exposure, Cellular Localization, and Possible Role in the Uptake of Exogenous Inorganic Carbon.

A Dual-Domain Carbonic Anhydrase (DDCA) had been sequenced and characterized from the ctenidia (gills) of the giant clam, Tridacna squamosa, which lives in symbiosis with zooxanthellae. DDCA was expressed predominantly in the ctenidium. The complete cDNA coding sequence of DDCA from T. squamosa comprised 1,803 bp, encoding a protein of 601 amino acids and 66.7 kDa. The deduced DDCA sequence contained two distinct α-CA domains, each with a specific catalytic site. It had a high sequence similarity with tgCA from Tridacna gigas. In T. squamosa, the DDCA was localized apically in certain epithelial cells near the base of the ctenidial filament and the epithelial cells surrounding the tertiary water channels. Due to the presence of two transmembrane regions in the DDCA, one of the Zn2+-containing active sites could be located externally and the other one inside the cell. These results denote that the ctenidial DDCA was positioned to dehydrate to CO2 in seawater, and to hydrate the CO2 that had permeated the apical membrane back to in the cytoplasm. During insolation, the host clam needs to increase the uptake of inorganic carbon from the ambient seawater to benefit the symbiotic zooxanthellae; only then, can the symbionts conduct photosynthesis and share the photosynthates with the host. Indeed, the transcript and protein levels of DDCA/DDCA in the ctenidium of T. squamosa increased significantly after 6 and 12 h of exposure to light, respectively, denoting that DDCA could participate in the light-enhanced uptake and assimilation of exogenous inorganic carbon.

Molecular characterization, light-dependent expression, and cellular localization of a host vacuolar-type H+-ATPase (VHA) subunit A in the giant clam, Tridacna squamosa, indicate the involvement of the host VHA in the uptake of inorganic carbon and its supply to the symbiotic zooxanthellae

The giant clam, Tridacna squamosa, represents a clam-zooxanthellae association. In light, the host clam and the symbiotic zooxanthellae conduct light-enhanced calcification and photosynthesis, respectively. We had cloned the cDNA coding sequence of a Vacuolar-type Proton ATPase (VHA) subunit A, ATP6V1A, from T. squamosa, whereby the VHA is an electrogenic transporter that actively ‘pumps’ H+ out of the cell. The ATP6V1A of T. squamosa comprised 1866 bp, encoding a protein of 622 amino acids and 69.9 kDa, and had a host-origin. Its gene expression was strong in the ctenidium and the colorful outer mantle, but weak in the whitish inner mantle, corroborating a previous proposition that VHA might have a trivial role in light-enhanced calcification. Light exposure led to significant increases in the gene and protein expression levels of ATP6V1A/ATP6V1A in the ctenidium and the outer mantle. In the ctenidium, the ATP6V1A was localized in the apical epithelia of the filaments and tertiary water channels, indicating that the VHA could participate in the increased excretion of H+ produced during light-enhanced calcification. Additionally, the excreted H+ would augment HCO3– dehydration in the external medium and facilitate the uptake of CO2 by the ctenidium during insolation. In the outer mantle, the ATP6V1A was detected in intracellular vesicles in a type of cells, presumably iridocytes, surrounding the zooxanthellal tubules, and in the apical epithelium of zooxanthellal tubules. Hence, the host VHA could participate in the transfer of inorganic carbon from the hemolymph to the luminal fluid of the tubules by increasing the supply of H+ for the dehydration of HCO3– to CO2 during insolation to benefit the photosynthesizing zooxanthellae.

Molecular characterization of a novel algal glutamine synthetase (GS) and an algal glutamate synthase (GOGAT) from the colorful outer mantle of the giant clam, Tridacna squamosa, and the putative GS-GOGAT cycle in its symbiotic zooxanthellae

Giant clams harbor symbiotic zooxanthellae (Symbiodinium), which are nitrogen-deficient, mainly in the fleshy and colorful outer mantle. This study aimed to sequence and characterize the algal Glutamine Synthetase (GS) and Glutamate Synthase (GLT), which constitute the glutamate synthase cycle (or GS-GOGAT cycle, whereby GOGAT is the protein acronym of GLT) of nitrogen assimilation, from the outer mantle of the fluted giant clam, Tridacna squamosa. We had identified a novel GS-like cDNA coding sequence of 2325 bp, and named it as T. squamosa Symbiodinium GS1 (TSSGS1). The deduced TSSGS1 sequence had 774 amino acids with a molecular mass of 85 kDa, and displayed the characteristics of GS1 and Nucleotide Diphosphate Kinase. The cDNA coding sequence of the algal GLT, named as T. squamosa Symbiodinium GLT (TSSGLT), comprised 6399 bp, encoding a protein of 2133 amino acids and 232.4 kDa. The zooxanthellal origin of TSSGS1 and TSSGOGAT was confirmed by sequence comparison and phylogenetic analyses. Indeed, TSSGS1 and TSSGOGAT were expressed predominately in the outer mantle, which contained the majority of the zooxanthellae. Immunofluorescence microscopy confirmed the expression of TSSGS1 and TSSGOGAT in the cytoplasm and the plastids, respectively, of the zooxanthellae in the outer mantle. It can be concluded that the symbiotic zooxanthellae of T. squamosa possesses a glutamate synthase (TSSGS1-TSSGOGAT) cycle that can assimilate endogenous ammonia produced by the host clam into glutamate, which can act as a substrate for amino acid syntheses. Thus, our results provide insights into why intact giant clam-zooxanthellae associations do not excrete ammonia under normal circumstances.

Carbonic anhydrase 2-like in the giant clam, Tridacna squamosa: characterization, localization, response to light, and possible role in the transport of inorganic carbon from the host to its symbionts.

The fluted giant clam, Tridacna squamosa, lives in symbiosis with zooxanthellae which reside extracellularly inside a tubular system. Zooxanthellae fix inorganic carbon (Ci) during insolation and donate photosynthate to the host. Carbonic anhydrases catalyze the interconversion of CO 2 and HCO3−, of which carbonic anhydrase 2 (CA2) is the most ubiquitous and involved in many biological processes. This study aimed to clone a CA2 homolog (CA2‐like) from the fleshy and colorful outer mantle as well as the thin and whitish inner mantle of T. squamosa, to determine its cellular and subcellular localization, and to examine the effects of light exposure on its gene and protein expression levels. The cDNA coding sequence of CA2‐like from T. squamosa comprised 789 bp, encoding 263 amino acids with an estimated molecular mass of 29.6 kDa. A phenogramic analysis of the deduced CA2‐like sequence denoted an animal origin. CA2‐like was not detectable in the shell‐facing epithelium of the inner mantle adjacent to the extrapallial fluid. Hence, CA2‐like is unlikely to participate directly in light‐enhanced calcification. By contrast, the outer mantle, which contains the highest density of tertiary tubules and zooxanthellae, displayed high level of CA2‐like expression, and CA2‐like was localized to the tubule epithelial cells. More importantly, exposure to light induced significant increases in the protein abundance of CA2‐like in the outer mantle. Hence, CA2‐like could probably take part in the increased supply of inorganic carbon (Ci) from the host clam to the symbiotic zooxanthellae when the latter conduct photosynthesis to fix Ci during light exposure.

Na+/H+ Exchanger 3 Is Expressed in Two Distinct Types of Ionocyte, and Probably Augments Ammonia Excretion in One of Them, in the Gills of the Climbing Perch Exposed to Seawater.

The freshwater climbing perch, Anabas testudineus, is an euryhaline teleost and an obligate air-breather with the ability to actively excrete ammonia. Members of the Na+/H+ exchanger (NHE) family help maintain intracellular pH homeostasis and ionic balance through the electroneutral exchange of Na+ and H+. This study aimed to obtain, from the gills of A. testudineus, the full cDNA coding sequence of nhe3, and to determine the effects of exposure to seawater or 100 mmol l−1 of NH4Cl in fresh water on its mRNA and protein expression levels. Efforts were also made to elucidate the type of ionocyte that Nhe3 was associated with in the branchial epithelium of A. testudineus. The transcript level and protein abundance of nhe3/Nhe3 were very low in the gills of freshwater A. testudineus, but they increased significantly in the gills of fish acclimated to seawater. In the gills of fish exposed to seawater, Nhe3 was expressed in two distinct types of seawater-inducible Na+/K+-ATPase (Nka)-immunoreactive ionocytes. In Nkaα1b-immunoreactive ionocytes, Nhe3 had an apical localization. As these ionocytes also expressed apical Rhcg1 and basolateral Rhcg2, which are known to transport ammonia, they probably participated in proton-facilitated ammonia excretion in A. testudineus during seawater acclimation. In Nkaα1c-immunoreactive ionocytes, Nhe3 was atypically expressed in the basolateral membrane, and its physiological function is uncertain. For A. testudineus exposed to NH4Cl in fresh water, the transcript and protein expression levels of nhe3/Nhe3 remained low. In conclusion, the branchial Nhe3 of A. testudineus plays a greater physiological role in passive ammonia transport and acid-base balance during seawater acclimation than in active ammonia excretion during environmental ammonia exposure.

Molecular characterization of three Rhesus glycoproteins from the gills of the African lungfish, Protopterus annectens, and effects of aestivation on their mRNA expression levels and protein abundance.

African lungfishes are ammonotelic in water. They can aestivate for long periods on land during drought. During aestivation, the gills are covered with dried mucus and ammonia excretion ceases. In fishes, ammonia excretion through the gills involves Rhesus glycoproteins (RhGP/Rhgp). This study aimed to obtain the complete cDNA coding sequences of rhgp from the gills of Protopterus annectens, and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Three isoforms of rhgp (rhag, rhbg and rhcg) were obtained in the gills of P. annectens. Their complete cDNA coding sequences ranged between 1311 and 1398 bp, coding for 436 to 465 amino acids with estimated molecular masses between 46.8 and 50.9 kDa. Dendrogramic analyses indicated that Rhag was grouped closer to fishes, while Rhbg and Rhcg were grouped closer to tetrapods. During the induction phase, the protein abundance of Rhag, but not its transcript level, was down-regulated in the gills, suggesting that there could be a decrease in the release of ammonia from the erythrocytes to the plasma. Furthermore, the branchial transcript levels of rhbg and rhcg decreased significantly, in preparation for the subsequent shutdown of gill functions. During the maintenance phase, the branchial expression levels of rhag/Rhag, rhbg/Rhbg and rhcg/Rhcg decreased significantly, indicating that their transcription and translation were down-regulated. This could be part of an overall mechanism to shut down branchial functions and save metabolic energy used for transcription and translation. It could also be regarded as an adaptive response to stop ammonia excretion. During the arousal phase, it is essential for the lungfish to regain the ability to excrete ammonia. Indeed, the protein abundance of Rhag, Rhbg and Rhcg recovered to the corresponding control levels after 1 day or 3 days of recovery from 6 months of aestivation.

The inner mantle of the giant clam, Tridacna squamosa, expresses a basolateral Na+/K+-ATPase α-subunit, which displays light-dependent gene and protein expression along the shell-facing epithelium.

Na+/K+-ATPase (NKA) is essential for maintaining the Na+ and K+ gradients, and supporting the secondary active transport of certain ions/molecules, across the plasma membrane of animal cells. This study aimed to clone the NKA α-subunit (NKAα) from the inner mantle adjacent to the extrapallial fluid of Tridacna squamosa, to determine its subcellular localization, and to examine the effects of light exposure on its transcript level and protein abundance. The cDNA coding sequence of NKAα from T. squamosa comprised 3105 bp, encoding 1034 amino acids with an estimated molecular mass of 114 kDa. NKAα had a basolateral localization along the shell-facing epithelium of the inner mantle. Exposure to 12 h of light led to a significantly stronger basolateral NKAα-immunofluorescence at the shell-facing epithelium, indicating that NKA might play a role in light-enhanced calcification in T. squamosa. After 3 h of light exposure, the transcript level of NKAα decreased transiently in the inner mantle, but returned to the control level thereafter. In comparison, the protein abundance of NKAα remained unchanged at hour 3, but became significantly higher than the control after 12 h of light exposure. Hence, the expression of NKAα in the inner mantle of T. squamosa was light-dependent. It is probable that a higher expression level of NKA was needed in the shell-facing epithelial cells of the inner mantle to cope with a rise in Na+ influx, possibly caused by increases in activities of some Na+-dependent ion transporters/channels involved in light-enhanced calcification.

The Whitish Inner Mantle of the Giant Clam, Tridacna squamosa, Expresses an Apical Plasma Membrane Ca2+-ATPase (PMCA) Which Displays Light-Dependent Gene and Protein Expressions.

Giant clams live in symbiosis with extracellular zooxanthellae and display high rates of growth and shell formation (calcification) in light. Light-enhanced calcification requires an increase in the supply of Ca2+ to, and simultaneously an augmented removal of H+ from, the extrapallial fluid where shell formation occurs. We have obtained the complete coding cDNA sequence of Plasma Membrane Ca2+-ATPase (PMCA) from the thin and whitish inner mantle, which is in touch with the extrapallial fluid, of the giant clam Tridacna squamosa. The deduced PMCA sequence consisted of an apical targeting element. Immunofluorescence microscopy confirmed that PMCA had an apical localization in the shell-facing epithelium of the inner mantle, whereby it can actively secrete Ca2+ in exchange for H+. More importantly, the apical PMCA-immunofluorescence of the shell-facing epithelium of the inner mantle increased significantly after 12 h of exposure to light. The transcript and protein levels of PMCA/PMCA also increased significantly in the inner mantle after 6 or 12 h of light exposure. These results offer insights into a light-dependable mechanism of shell formation in T. squamosa and a novel explanation of light-enhanced calcification in general. As the inner mantle normally lacks light sensitive pigments, our results support a previous proposition that symbiotic zooxanthellae, particularly those in the colorful and extensible outer mantle, may act as light-sensing elements for the host clam.

Identification, characterization and modulation of ferritin-H in the sub-Antarctic Notothenioid Eleginops maclovinus challenged with Piscirickettsia salmonis

Ferritin is a major iron storage protein essential not only in the infectious process, but also in any circumstance generating oxidative stress. In this study, the cDNA coding sequence of ferritin-H was obtained from the sub-Antarctic Notothenioid fish Eleginops maclovinus through transcriptomic analysis of the head kidney. This sequence contained a 534 bp open reading frame that coded for a 177 amino acid protein with a molecular weight of 20,786.2 Da and a theoretical pI of 5.56. The protein displayed a region of iron putative response elements in the 5′UTR, two putative ferritin iron-binding region signatures, and seven characteristic amino acids with ferroxidase functions. Phylogenetic analysis related this sequence to ferritin-H sequences of other Antarctic Notothenioid fish, sharing 96.61% similarity. Constitutive gene expression analysis in different organs revealed increased ferritin-H gene expression in the gills, spleen, muscle, and liver. After infection with two bacterial strains of Piscirickettsia salmonis (LF-89 and Austral-005), ferritin-H was differentially expressed depending on bacterial strain and tissue. This study provides relevant information towards understanding the iron metabolism of a sub-Antarctic Notothenioid fish.

Molecular characterization of myostatin from the skeletal muscle of the African lungfish, Protopterus annectens, and changes in its mRNA and protein expression levels during three phases of aestivation.

African lungfishes can aestivate and remain torpid without food and water for years, but disuse muscle atrophy is not prominent during aestivation. This study aimed to clone myostatin (mstn/Mstn), a factor associated with disuse muscle atrophy in mammals, from the skeletal muscle of the African lungfish Protopterus annectens, and to determine its mRNA expression level and protein abundance therein during the induction, maintenance, and arousal phases of aestivation. The complete coding cDNA sequence of mstn comprised 1128bp, encoding for 376 amino acids with an estimated molecular mass of 42.9kDa. It was grouped together with Mstn/MSTN of coelacanth and tetrapods in a clade separated from teleost Mstn. After 6 days (the induction phase) of aestivation, the mstn transcript level in the muscle increased significantly, while the protein abundance of Mstn remained comparable to the control. Following that, a significant increase in the expression levels of mstn/Mstn occurred on day 12 (the early maintenance phase) of aestivation. After 6 months of aestivation (the prolonged maintenance phase), the expression levels of mstn/Mstn returned to control levels, indicating the possible impediment of a drastic increase in muscle degradation to prevent muscle atrophy. During 1-3 days of arousal from aestivation, the expression levels of mstn/Mstn in the muscle remained comparable to the control. Hence, tissue reconstruction/regeneration of certain organs might not involve the mobilization of amino acids from the muscle during the early arousal. These results provide insights into how aestivating P. annectens regulates the expression of mstn/Mstn possibly to ameliorate disuse muscle atrophy.

Ammonia exposure affects the mRNA and protein expression levels of certain Rhesus glycoproteins in the gills of climbing perch.

The freshwater climbing perch, Anabas testudineus, is an obligate air-breathing and euryhaline teleost capable of active ammonia excretion and tolerant of high concentrations of environmental ammonia. As Rhesus glycoproteins (RhGP/Rhgp) are known to transport ammonia, this study aimed to obtain the complete cDNA coding sequences of various rhgp isoforms from the gills of A. testudineus, and to determine their mRNA and protein expression levels during 6 days of exposure to 100 mmol l-1 NH4Cl. The subcellular localization of Rhgp isoforms in the branchial epithelium was also examined in order to elucidate the type of ionocyte involved in active ammonia excretion. Four rhgp (rhag, rhbg, rhcg1 and rhcg2) had been identified from the gills of A. testudineus They had conserved amino acid residues for NH4+ binding, NH4+ deprotonation, channel gating and lining of the vestibules. Despite inwardly directed NH3 and NH4+ gradients, there were significant increases in the mRNA expression levels of the four branchial rhgp in A. testudineus at certain time points during 6 days of ammonia exposure, with significant increases in the protein abundances of Rhag and Rhcg2 on day 6. Immunofluorescence microscopy revealed a type of ammonia-inducible Na+/K+-ATPase α1c-immunoreactive ionocyte with apical Rhag and basolateral Rhcg2 in the gills of fish exposed to ammonia for 6 days. Hence, active ammonia excretion may involve NH4+ entering the ionocyte through the basolateral Rhcg2 and being excreted through the apical Rhag, driven by a transapical membrane electrical potential generated by the apical cystic fibrosis transmembrane conductance regulator Cl- channel, as suggested previously.

Voltage-Gated Na+ Channel Isoforms and Their mRNA Expression Levels and Protein Abundance in Three Electric Organs and the Skeletal Muscle of the Electric Eel Electrophorus electricus.

This study aimed to obtain the coding cDNA sequences of voltage-gated Na+ channel (scn) α-subunit (scna) and β-subunit (scnb) isoforms from, and to quantify their transcript levels in, the main electric organ (EO), Hunter’s EO, Sach’s EO and the skeletal muscle (SM) of the electric eel, Electrophorus electricus, which can generate both high and low voltage electric organ discharges (EODs). The full coding sequences of two scna (scn4aa and scn4ab) and three scnb (scn1b, scn2b and scn4b) were identified for the first time (except scn4aa) in E. electricus. In adult fish, the scn4aa transcript level was the highest in the main EO and the lowest in the Sach’s EO, indicating that it might play an important role in generating high voltage EODs. For scn4ab/Scn4ab, the transcript and protein levels were unexpectedly high in the EOs, with expression levels in the main EO and the Hunter’s EO comparable to those of scn4aa. As the key domains affecting the properties of the channel were mostly conserved between Scn4aa and Scn4ab, Scn4ab might play a role in electrogenesis. Concerning scnb, the transcript level of scn4b was much higher than those of scn1b and scn2b in the EOs and the SM. While the transcript level of scn4b was the highest in the main EO, protein abundance of Scn4b was the highest in the SM. Taken together, it is unlikely that Scna could function independently to generate EODs in the EOs as previously suggested. It is probable that different combinations of Scn4aa/Scn4ab and various Scnb isoforms in the three EOs account for the differences in EODs produced in E. electricus. In general, the transcript levels of various scn isoforms in the EOs and the SM were much higher in adult than in juvenile, and the three EOs of the juvenile fish could be functionally indistinct.