Convergent gene losses and pseudogenizations in multiple lineages of stomachless fishes.

The regressive evolution of independent lineages often results in convergent phenotypes. Several teleost groups display secondary loss of the stomach, and four gastric genes, atp4a, atp4b, pgc, and pga2 have been co-deleted in agastric (stomachless) fish. Analyses of genotypic convergence among agastric fishes showed that four genes, slc26a9, kcne2, cldn18a, and vsig1, were co-deleted or pseudogenized in most agastric fishes of the four major groups. kcne2 and vsig1 were also deleted or pseudogenized in the agastric monotreme echidna and platypus, respectively. In the stomachs of sticklebacks, these genes are expressed in gastric gland cells or surface epithelial cells. An ohnolog of cldn18 was retained in some agastric teleosts but exhibited an increased non-synonymous substitution when compared with gastric species. These results revealed novel convergent gene losses at multiple loci among the four major groups of agastric fish, as well as a single gene loss in the echidna and platypus.

Adsorption Characteristics of Allura Red AC onto Sawdust and Hexadecylpyridinium Bromide-Treated Sawdust in Aqueous Solution.

The Allura red AC (ARAC) dye adsorption onto natural sawdust (NSD) and hexadecylpyridinium bromide-treated sawdust (MSD) was investigated in aqueous solution as a function of contact time, solution pH, particle size, adsorbent dosage, dye concentration, temperature, and ionic strength. The adsorbents were characterized by Fourier transform infrared spectroscopy and X-ray diffraction crystallography. The dye adsorption onto both adsorbents was confirmed by field emission scanning electron microscopy and energy-dispersive X-ray spectroscopy. The maximum dye adsorption was found within 120 min at pH 2.0 for NSD and pH 3.0 for MSD, respectively, with a particle size of 0-75 µm and an adsorbent dosage of 0.07 g/50 mL ARAC dye solution (50 µmol/L). The batch adsorption kinetic data were followed by the pseudo-second-order kinetic model rather than the pseudo-first-order and Elovich kinetic models. Equilibrium adsorption isotherms were explained by the Langmuir isotherm model, and the maximum extent of adsorption was found to be 52.14 µmol/g for NSD and 151.88 µmol/g for MSD at 55 °C. The values of activation energy (E a) and thermodynamic parameters (ΔG ⧧, ΔH ⧧, ΔS ⧧, ΔG°, ΔH° and ΔS°) proved that the ARAC dye adsorption onto both adsorbents NSD and MSD is a spontaneous-endothermic physisorption process. ARAC (98-99%) was released from dye-loaded adsorbents in aqueous solution (pH ≥ 12) within 120 min. The adsorbents NSD and MSD were reused for a second time without significant loss of their adsorption efficiency.

Investigation of the Antibacterial Activity and in vivo Cytotoxicity of Biogenic Silver Nanoparticles as Potent Therapeutics.

Biogenic nanoparticles are the smartest weapons to deal with the multidrug-resistant "superbugs" because of their broad-spectrum antibacterial propensity as well as excellent biocompatibility. The aqueous biogenic silver nanoparticles (Aq-bAgNPs) and ethanolic biogenic silver nanoparticles (Et-bAgNPs) were synthesized using aqueous and ethanolic extracts of Andrographis paniculata stem, respectively, as reducing agents. Electron microscopic images confirmed the synthesis of almost spherical shaped biogenic silver nanoparticles (bAgNPs). The zeta potentials of the nanoparticles were negative and were -22 and -26 mV for Aq-bAgNPs and Et-bAgNPs, respectively. The antibacterial activity of bAgNPs was investigated against seven pathogenic (i.e., enteropathogenic Escherichia coli, Salmonella typhi, Staphylococcus aureus, Vibrio cholerae, Enterococcus faecalis, Hafnia alvei, Acinetobacter baumannii) and three nonpathogenic (i.e., E. coli DH5α, E. coli K12, and Bacillus subtilis) bacteria at different time points (i.e., 12, 16, 20, and 24 h) in a dose-dependent manner (i.e., 20, 40, and 60 µg) through broth dilution assay, disk diffusion assay, CellToxTM Green uptake assay, and trypan blue dye exclusion assay. The lowest minimum inhibitory concentration value for both the bAgNPs was 0.125 µg. Et-bAgNPs showed the highest antibacterial activity against S. aureus at 60 µg after 16 h and the diameter of inhibited zone was 28 mm. Lipid peroxidation assay using all the bacterial strains revealed the formation of malondialdehyde-thiobarbituric acid adduct due to the oxidation of cell membrane fatty acids by bAgNPs. The bAgNPs showed excellent hemocompatibility against human as well as rat red blood cells. Furthermore, there was no significant toxicity observed when the levels of rat serum ALT, AST, γ-GT (i.e., liver function biomarkers), and creatinine (i.e., kidney function biomarker) were determined.

Identification and lateral membrane localization of cyclin M3, likely to be involved in renal Mg2+ handling in seawater fish.

The kidney of marine teleosts is the major site of Mg(2+) excretion and produces urine with a high Mg(2+) concentration. However, the transporters involved in Mg(2+) excretion are poorly understood. The cyclin M (Cnnm; also known as ancient conserved domain protein) family comprises membrane proteins homologous to the bacterial Mg(2+) and Co(2+) efflux protein, CorC. To understand the molecular mechanism of Mg(2+) homeostasis in marine teleosts, we analyzed the expression of the Cnnm family genes in the seawater (SW) pufferfish, torafugu (Takifugu rubripes), and the closely related euryhaline species, mefugu (Takifugu obscurus). Database mining and phylogenetic analysis indicated that the Takifugu genome contains six members of the Cnnm family: two orthologs of Cnnm1, one of Cnnm2, one of Cnnm3, and two of Cnnm4. RT-PCR analyses indicated that Cnnm2, Cnnm3, and Cnnm4a are expressed in the kidney, whereas other members are mainly expressed in the brain. Renal expression of Cnnm3 was upregulated in SW mefugu, whereas renal expression of Cnnm2 was upregulated in freshwater (FW) mefugu. No significant difference was observed in renal expression of Cnnm4a between SW and FW mefugu. In situ hybridization and immunohistochemical analyses of the SW mefugu kidney revealed that Cnnm3 is expressed in the proximal tubule, and its product localizes to the lateral membrane. When Cnnm3 was expressed in Xenopus laevis oocytes, whole cellular Mg(2+) content and free intracellular Mg(2+) activity significantly decreased. These results suggest that Cnnm3 is involved in body fluid Mg(2+) homeostasis in marine teleosts.

Identification and proximal tubular localization of the Mg²âº transporter, Slc41a1, in a seawater fish.

The second most abundant cation in seawater (SW), Mg²âº, is present at concentrations of ~53 mM. Marine teleosts maintain plasma Mg²âº concentration at 1-2 mM by excreting Mg²âº into the urine. Urine Mg²âº concentrations of SW teleosts exceed 70 mM, most of which is secreted by the renal tubular epithelial cells. However, molecular mechanisms of the Mg²âº secretion have yet to be clarified. To identify transporters involved in Mg²âº secretion, we analyzed the expression of fish homologs of the Slc41 Mg²âº transporter family in various tissues of SW pufferfish torafugu (Takifugu rubripes) and its closely related euryhaline species mefugu (Takifugu obscurus). Takifugu genome contained five members of Slc41 genes, and only Slc41a1 was highly expressed in the kidney. Renal expression of Slc41a1 was markedly elevated when mefugu were transferred from fresh water (FW) to SW. In situ hybridization analysis and immunohistochemistry at the light and electron microscopic levels revealed that Slc41a1 is localized to vacuoles in the apical cytoplasm of the proximal tubules. These results suggest that pufferfish Slc41a1 is a Mg²âº transporter involved in renal tubular transepithelial Mg²âº secretion by mediating Mg²âº transport from the cytosol to the vacuolar lumen, and support the hypothesis that Mg²âº secretion is mediated by exocytosis of Mg²âº-rich vacuoles to the lumen.

O2-filled swimbladder employs monocarboxylate transporters for the generation of O2 by lactate-induced root effect hemoglobin.

The swimbladder volume is regulated by O(2) transfer between the luminal space and the blood In the swimbladder, lactic acid generation by anaerobic glycolysis in the gas gland epithelial cells and its recycling through the rete mirabile bundles of countercurrent capillaries are essential for local blood acidification and oxygen liberation from hemoglobin by the "Root effect." While O(2) generation is critical for fish flotation, the molecular mechanism of the secretion and recycling of lactic acid in this critical process is not clear. To clarify molecules that are involved in the blood acidification and visualize the route of lactic acid movement, we analyzed the expression of 17 members of the H(+)/monocarboxylate transporter (MCT) family in the fugu genome and found that only MCT1b and MCT4b are highly expressed in the fugu swimbladder. Electrophysiological analyses demonstrated that MCT1b is a high-affinity lactate transporter whereas MCT4b is a low-affinity/high-conductance lactate transporter. Immunohistochemistry demonstrated that (i) MCT4b expresses in gas gland cells together with the glycolytic enzyme GAPDH at high level and mediate lactic acid secretion by gas gland cells, and (ii) MCT1b expresses in arterial, but not venous, capillary endothelial cells in rete mirabile and mediates recycling of lactic acid in the rete mirabile by solute-specific transcellular transport. These results clarified the mechanism of the blood acidification in the swimbladder by spatially organized two lactic acid transporters MCT4b and MCT1b.

Identification and apical membrane localization of an electrogenic Na⁺/Ca²âº exchanger NCX2a likely to be involved in renal Ca²âº excretion by seawater fish.

Seawater (SW) contains ∼10 mM Ca(2+), yet marine fish must drink seawater as their major water source. Thus marine teleosts fish need to excrete Ca(2+) to maintain whole body Ca(2+) homeostasis. In the intestine, seawater Ca(2+) interreacts with epithelial-secreted HCO(3)(-) by the intestinal epithelium, and the resulting CaCO(3) precipitates, which is rectally excreted. Recently the transporters involved in intestinal HCO(3)(-) secretion were identified. Ca(2+) is also excreted by the kidney, but the protein(s) involved in renal Ca(2+) excretion have not been identified. Here we identified a candidate transporter by using SW pufferfish torafugu (Takifugu rubripes) and its closely related euryhaline species mefugu (Takifugu obscurus), which are becoming useful animal models for studying molecular mechanisms of seawater adaptation. RT-PCR analyses of Na(+)/Ca(2+) exchanger (NCX) family members in various torafugu tissues demonstrated that only NCX2a is highly expressed in the kidney. Renal expression of NCX2a was markedly elevated when mefugu were transferred from freshwater to seawater. In situ hybridization and immunohistochemical analyses indicated that NCX2a is expressed in the proximal tubule at the apical membrane. NCX2a, expressed in Xenopus oocytes, conferred [Ca(2+)](out)- and Na(+)-dependent currents. These results suggest that NCX2a mediates renal Ca(2+) secretion at the apical membrane of renal proximal tubules and has an important role in whole body Ca(2+) homeostasis of marine teleosts.

Differential expression of Na+-Cl- cotransporter and Na+-K+-Cl- cotransporter 2 in the distal nephrons of euryhaline and seawater pufferfishes.

The process of NaCl reabsorption in the distal nephron allows freshwater fishes to excrete hypotonic urine and seawater fishes to excrete urine containing high concentrations of divalent ions; the relevant transporters, however, have not yet been identified. In the mammalian distal nephron, NaCl absorption is mediated by Na(+)-K(+)-Cl(-) cotransporter 2 (NKCC2, Slc12a1) in the thick ascending limb, Na(+)-Cl(-) cotransporter (NCC, Slc12a3) in the distal convoluted tubule, and epithelial sodium channel (ENaC) in the collecting duct. In this study, we compared the expression profiles of these proteins in the kidneys of euryhaline and seawater pufferfishes. Mining the fugu genome identified one NKCC2 gene and one NCC gene, but no ENaC gene. RT-PCR and in situ hybridization analyses demonstrated that NKCC2 was highly expressed in the distal tubules and NCC was highly expressed in the collecting ducts of euryhaline pufferfish (mefugu, Takifugu obscurus). On the other hand, the kidney of seawater pufferfish (torafugu, Takifugu rubripes), which lacked distal tubules, expressed very low levels of NCC, and, in the collecting ducts, high levels of NKCC2. Acclimation of mefugu to seawater resulted in a 2.7× decrease in NCC expression, whereas NKCC2 expression was not markedly affected. Additionally, internalization of NCC from the apical surface of the collecting ducts was observed. These results suggest that NaCl reabsorption in the distal nephron of the fish kidney is mediated by NCC and NKCC2 in freshwater and by NKCC2 in seawater.