論文

Current approaches to the quality control of water are unsatisfying due to either a high cost or the inability to capture all of the relevant information. In this study, near-infrared spectroscopy (NIRS) with aquaphotomics as a novel approach was assessed for the discrimination of natural, processed and aged water samples. Temperature perturbation of water samples was employed to probe the aqueous systems and reveal the hidden information. A radar chart named an aquagram was used to visualize and compare the absorbance spectral patterns of waters at different temperatures. For the spectra acquired at a constant temperature of 30 °C, the discrimination analysis of different water samples failed to produce satisfying results. However, under perturbation by increasing the temperature from 35 to 60 °C, the absorbance spectral pattern of different waters displayed in aquagrams revealed different, water-specific dynamics. Moreover, it was found that aged processed water changed with the temperature, whereas the same processed water, when freshly prepared, had hydrogen bonded structures unperturbed by temperature. In summary, the aquaphotomics approach to the NIRS analysis showed that the water absorbance spectral pattern can be used to describe the character and monitor dynamics of each water sample as a complex molecular system, whose behavior under temperature perturbation can reveal even subtle changes, such as aging and the loss of certain qualities during storage.

Aquaporin (AQP) 6 belongs to the aquaporin water channel family. Unlike other aquaporins, AQP6 functions not as a water channel but as an anion-selective channel. Single-channel analyses have shown AQP6 to flicker rapidly between closed and open status. The atomic structure of AQP1 and amino acid sequence alignments of the mammalian aquaporins reveal two well conserved glycine residues: Gly-57 in transmembrane helix (TM) 2 and Gly-173 in TM5 reside at the contact point where the two helices cross in human AQP1. Uniquely, all known mammalian orthologs of AQP6 have an asparagine residue (Asn-60) at the position corresponding to Gly-57. Here we show that a single residue substitution (N60G in rat AQP6) totally eliminates the anion permeability of AQP6 when expressed in Xenopus oocytes, but the N60G oocytes exhibit significantly higher osmotic water permeability under basal conditions. Replacement of the glycine at this site in AQP0, AQP1, and AQP2 blocked expression of the mutants at the oocyte plasma membrane. We propose that the asparagine residue at the contact point between TM2 and TM5 in AQP6 may function as a teeter board needed for rapid structural oscillations during anion permeation.

The Arabidopsis thaliana AtHKT1 protein, a Na + /K + transporter, is capable of mediating inward Na + currents in Xenopus laevis oocytes and K + uptake in Escherichia coli . HKT1 proteins are members of a superfamily of K + transporters. These proteins have been proposed to contain eight transmembrane segments and four pore-forming regions arranged in a mode similar to that of a K + channel tetramer. However, computer analysis of the AtHKT1 sequence identified eleven potential transmembrane segments. We have investigated the membrane topology of AtHKT1 with three different techniques. First, a gene fusion alkaline phosphatase study in E. coli clearly defined the topology of the N-terminal and middle region of AtHKT1, but the model for membrane folding of the C-terminal region had to be refined. Second, with a reticulocyte-lysate supplemented with dog-pancreas microsomes, we demonstrated that N -glycosylation occurs at position 429 of AtHKT1. An engineered unglycosylated protein variant, N429Q, mediated Na + currents in X. laevis oocytes with the same characteristics as the wild-type protein, indicating that N -glycosylation is not essential for the functional expression and membrane targeting of AtHKT1. Five potential glycosylation sites were introduced into the N429Q. Their pattern of glycosylation supported the model based on the E. coli -alkaline phosphatase data. Third, immunocytochemical experiments with FLAG-tagged AtHKT1 in HEK293 cells revealed that the N and C termini of AtHKT1, and the regions containing residues 135–142 and 377–384, face the cytosol, whereas the region of residues 55–62 is exposed to the outside. Taken together, our results show that AtHKT1 contains eight transmembrane-spanning segments.

Oxidation-reduction (Redox) reactions are crucial for many biological processes, yet there is no method available to evaluate redox states in a non-invasive, continuous manner. Here we introduce a novel approach to distinguish between reduced and oxidized states of glutathione (GSH and GSSG, respectively) using aquaphotomics near-infrared (NIR) spectroscopy and multivariate analysis. We identified clear differences in NIR spectra reflecting not only glutathione itself, but different redox states of glutathione based on the spectral features of water molecular conformations interacting with the reaction site. Molecular dynamic simulations also revealed the difference in water molecule coordination and hydration numbers around the reaction site. This approach not only sheds light on the significance of water molecules in redox reactions but also enables non-destructive, continuous assessment of redox states, with potential applications for bioreactor optimization.