Analysis of aquaporin-mediated diffusional water permeability by coherent anti-stokes Raman scattering microscopy

Water can pass through biological membranes via two pathways: simple diffusion through the lipid bilayer, or water-selective facilitated diffusion through aquaporins (AQPs). Although AQPs play an important role in osmotic water permeability (P_f), the role of AQPs in diffusional water permeability remains unclear because of the difficulty of measuring diffusional water permeability (P_d). Here, we report an accurate and instantaneous method for measuring the P_d of a single HeLa S3 cell using coherent anti-Stokes Raman scattering (CARS) microscopy with a quick perfusion device for H₂O/D₂O exchange. Ultra-high-speed line-scan CARS images were obtained every 0.488 ms. The average decay time constant of CARS intensities (τ_CARS) for the external solution H₂O/D₂O exchange was 16.1 ms, whereas the intracellular H₂O/D₂O exchange was 100.7 ± 19.6 ms. To evaluate the roles of AQP in diffusional water permeability, AQP4 fused with enhanced green fluorescent protein (AQP4-EGFP) was transiently expressed in HeLa S3 cells. The average τ_CARS for the intracellular H₂O/D₂O exchange in the AQP4-EGFP-HeLa S3 cells was 43.1 ± 15.8 ms. We also assessed the cell volume and the cell surface area to calculate P_d. The average P_d values for the AQP4-EGFP-HeLa S3 cells and the control EGFP-HeLa S3 cells were 2.7 ± 1.0 × 10⁻³ and 8.3 ± 2.6 × 10⁻⁴ cm/s, respectively. AQP4-mediated water diffusion was independent of the temperature but was dependent on the expression level of the protein at the plasma membrane. These results suggest the possibility of using CARS imaging to investigate the hydrodynamics of single mammalian cells as well as the regulation of AQPs.     

Genetic variation of the white-spotted longicorn beetle <Emphasis Type="Italic">Anoplophora</Emphasis> spp. (Coleoptera: Cerambycidae) in Japan detected by mitochondrial DNA sequence

Genetic variation of the white-spotted longicorn beetle Anoplophora spp., distributed on the Japanese mainland and the Ryukyu Islands, was examined using nucleotide sequences of mitochondrial DNA. Two fragments, a 1.2-kb-long fragment containing portions of the cytochrome oxidase subunit I and II genes and a 1.4-kb-long fragment containing portions of 16S and 12S rDNAs, were sequenced. In phylogenetic analyses based on the sequences, 294 individuals collected at 75 localities were divided into two major groups (groups A and B) and then split into seven subgroups (A1–A4 and B1–B3). In group A, the closely related subgroups A1, A2, and A3 were mainly distributed on the Japanese mainland and were roughly separated among geographic areas, although the range of A3 spread significantly from Kyushu to the Central Ryukyu Islands. The sequence of A4, detected from one individual collected in eastern Honshu, was almost the same as that reported for A. chinensis (Forster) on the Chinese continent. In group B, subgroups B1 and B2 were restricted to the Central and Southern Ryukyu Islands, respectively, while B3 was distributed widely in both regions. Based on the results, we discuss the geographic origin of the haplotypes and movement of the insects between geographic areas.     

Sex pheromone of the African armyworm <Emphasis Type="Italic">Spodoptera exempta</Emphasis> (Lepidoptera: Noctuidae): identification of components of the Okinawan population and formulation for population monitoring

In 2010, abrupt outbreaks of the African armyworm, Spodoptera exempta (Walker), occured on the Tarama, Iriomote and Kikai Islands in southwestern Japan. Analysis by gas chromatography-electroantennographic detection (GC-EAD) revealed two EAG-active compounds on male antenna in crude extract of virgin females. These compounds were identified as (9Z)-9-tetradecenyl acetate (Z9-14:Ac) and (9Z,12E)-9,12-tetradecadienyl acetate (Z9E12-14:Ac) in ca. 90:10 ratio by subsequent GC-MS analyses. (11Z)-11-Hexadecenyl acetate (Z11-16:Ac), which had previously been identified as a third component in the Kenyan population, was not detected. Binary blends of Z9-14:Ac and Z9E12-14:Ac at ratios between 99:1 and 90:10 showed a potent attractiveness in the field, superior to that of virgin females and comparable to that of the three-component formulation determined in Kenya. For the population survey, a 98:2 blend was used. In Tarama, only a few moths of S. exempta were captured with a light trap during the night when more than 600 males were captured with synthetic sex pheromone; more S. exempta captures with a light trap had been reported than with sex-pheromone traps in Kenya. This indicates that the Okinawan population has different properties from the Kenyan population in pheromone composition and behavioral response to light.     

Effects of mixed cropping on population densities and parasitism rates of the diamondback moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis> (Lepidoptera: Plutellidae)

Effects of mixed cropping and barrier crops on the population density and parasitism of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), were evaluated in field plots of cabbage grown in Bali, Indonesia. The densities of P. xylostella at larval and pupal stages, as well as the overall density at larval plus pupal stages, were significantly lower in cabbage/coriander mixed cropping subplots than in cabbage monoculture subplots. Parasitism of P. xylostella by the larval parasitoid Diadegma semiclausum (Hellen) (Hymenoptera: Ichneumonidae) was not significantly different between the mixed and monocultural cropping systems. These results do not support the so-called enemies hypothesis, but suggest that disruption of the host searching behavior of female moths by neighboring non-host plants is the mechanism behind the associational resistance observed in the coriander mixed cropping system. The inclusion of a Napier grass barrier between mixed crop and monoculture subplots did not affect the influence of mixed cropping on larval and pupal densities. Therefore, Napier grass, which is used locally as a fence for preventing livestock invasion of fields, would not obstruct the pest-reducing effect of coriander/cabbage mixed cropping.     

Presence of allochronic populations of the white grub beetle <Emphasis Type="Italic">Dasylepida ishigakiensis</Emphasis> (Coleoptera: Scarabaeidae) on Ishigaki Island,Japan

To investigate the seasonal prevalence of Dasylepida ishigakiensis Niijima et Kinoshita (Coleoptera: Scarabaeidae), synthetic sex pheromone traps were placed on Ishigaki Island in 2008 and 2009. One population is known to occur in the forest and fly for mating from early March to early April. The trap data revealed a second population in the farmland that flew for mating much earlier (mid-January to late February) than did the other population, indicating the presence of allochronic populations. Adults of the farmland population were caught throughout the sugarcane-grown area on the island, whereas those of the forest population appeared to be limited to the forest area. The temporal pattern of trap catches of each population was generally the same between 2008 and 2009. Relatively large trap catches were observed in the west central plane area and a small northern area of the northeastern peninsula of the island. These results suggest that, although it has not caused any damage to agricultural crops on this island, this beetle might be a potentially serious pest of sugarcane crops in the future, as reported on another island of Okinawa.     

Mortality of coho salmon (Oncorhynchus kisutch) associated with burdens of multiple parasite species

Multiple analytical techniques were used to evaluate the impact of multiple parasite species on the mortality of threatened juvenile coho salmon (Oncorhynchus kisutch) from the West Fork Smith River, Oregon, USA. We also proposed a novel parsimonious mathematical representation of macroparasite distribution, congestion rate, which (i) is easier to use than traditional models, and (ii) is based on Malthusian parameters rather than probability theory. Heavy infections of Myxobolus insidiosus (Myxozoa) and metacercariae of Nanophyetus salmincola and Apophallus sp. occurred in parr (subyearlings) from the lower mainstem of this river collected in 2007 and 2008. Smolts (yearlings) collected in 2007–2010 always harboured fewer Apophallus sp. with host mortality recognised as a function of intensity for this parasite. Mean intensity of Apophallus sp. in lower mainstem parr was 753 per fish in 2007 and 856 per fish in 2008, while parr from the tributaries had a mean of only 37 or 13 parasites per fish, respectively. Mean intensity of this parasite in smolts ranged between 47 and 251 parasites per fish. Over-dispersion (variance to mean ratios) of Apophallus sp. was always lower in smolts compared with all parr combined or lower mainstem parr. Retrospective analysis based on smolt data using both the traditional negative binomial truncation technique and our proposed congestion rate model showed identical results. The estimated threshold level for mortality involving Apophallus sp. was at 400–500 parasites per fish using both analytical methods. Unique to this study, we documented the actual existence of these heavy infections prior to the predicted mortality. Most of the lower mainstem parr (approximately 75%) had infections above this level. Heavy infections of Apophallus sp. metacercariae may be an important contributing factor to the high over-wintering mortality previously reported for these fish that grow and develop in this section of the river. Analyses using the same methods for M.insidiosus and N. salmincola generally pointed to minimal parasite-associated mortality.     

Mutational specificities of brominated DNA adducts catalyzed by human DNA polymerases

Chronic inflammation is known to lead to an increased risk for the development of cancer. Under inflammatory condition, cellular DNA is damaged by hypobromous acid, which is generated by myeloperoxidase and eosinophil peroxidase. The reactive brominating species induced brominated DNA adducts such as 8-bromo-2′-deoxyguanosine (8-Br-dG), 8-bromo-2′-deoxyadenosine (8-Br-dA), and 5-bromo-2′-deoxycytidine (5-Br-dC). These DNA lesions may be implicated in carcinogenesis. In this study, we analyzed the miscoding properties of the brominated DNA adducts generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotides containing a single 8-Br-dG, 8-Br-dA, or 5-Br-dC were used as a template in primer extension reactions catalyzed by human pols α, κ, and η. When 8-Br-dG-modified template was used, pol α primarily incorporated dCMP, the correct base, opposite the lesion, along with a small amount of one-base deletion (4.8%). Pol κ also promoted one-base deletion (14.2%), accompanied by misincorporation of dGMP (9.5%), dAMP (8.0%), and dTMP (6.1%) opposite the lesion. Pol η, on the other hand, readily bypassed the 8-Br-dG lesion in an error-free manner. As for 8-Br-dA and 5-Br-dC, all the pols bypassed the lesions and no miscoding events were observed. These results indicate that only 8-Br-dG, and not 5-Br-dC and 8-Br-dA, is a mutagenic lesion; the miscoding frequency and specificity vary depending on the DNA pol used. Thus, hypobromous acid-induced 8-Br-dG adduct may increase mutagenic potential at the site of inflammation.     

Functional importance of covalent homodimer of reelin protein linked via its central region

Reelin is a 3461-residue secreted glycoprotein that plays a critical role in brain development through its action on target neurons. Although it is known that functional reelin protein exists as multimer formed by interchain disulfide bond(s) as well as through non-covalent interactions, the chemical nature of the multimer assembly has been elusive. In the present study, we identified, among 122 cysteines present in full-length reelin, the single critical cysteine residue (Cys(2101)) responsible for the covalent multimerization. C2101A mutant reelin failed to assemble into disulfide-bonded multimers, whereas it still exhibited non-covalently associated high molecular weight oligomeric states in solution. Detailed analysis of tryptic fragments produced from the purified reelin proteins revealed that the minimum unit of the multimer is a homodimeric reelin linked via Cys(2101) present in the central region and that this cysteine does not connect to the N-terminal region of reelin, which had been postulated as the primary oligomerization domain. A surface plasmon resonance binding assay confirmed that C2101A mutant reelin retained binding capability toward two neuronal receptors apolipoprotein E receptor 2 and very low density lipoprotein receptor. However, it failed to show signaling activity in the assay using the cultured neurons. These results indicate that an intact higher order architecture of reelin multimer maintained by both Cys(2101)-mediated homodimerization and other non-covalent association present elsewhere in the reelin primary structure are essential for exerting its full biological activity.     

The NPC motif of aquaporin-11, unlike the NPA motif of known aquaporins, is essential for full expression of molecular function

The recently identified molecule aquaporin-11 (AQP11) has a unique amino acid sequence pattern that includes an Asn-Pro-Cys (NPC) motif, corresponding to the N-terminal Asn-Pro-Ala (NPA) signature motif of conventional AQPs. In this study, we examined the effect of the mutation of the NPC motif on the subcellular localization, oligomerization, and water permeability of AQP11 in transfected mammalian cells. Furthermore, the effect was also assessed using zebrafish. Site-directed mutation at the NPC motif did not affect the subcellular localization of AQP11 but reduced its oligomerization. A cell swelling assay revealed that cells expressing AQP11 with a mutated NPC motif had significantly lower osmotic water permeability than cells expressing wild-type AQP11. Zebrafish deficient in endogenous AQP11 showed a deformity in the tail region at an early stage of development. This phenotype was dramatically rescued by injection of human wild-type AQP11 mRNA, whereas the effect of mRNA for AQP11 with a mutated NPC motif was less marked. Although the NPA motif is known to be important for formation of water-permeable pores by conventional AQPs, our observations suggest that the corresponding NPC motif of AQP11 is essential for full expression of molecular function.     

Vital roles of the second DNA-binding site of Rad52 protein in yeast homologous recombination

RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a "recombination mediator" to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of Rad52 and Rad51 within the complex are unknown. In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. Rad51-Rad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that Rad52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing.