Polycystic Kidney Disease in the Medaka (Oryzias latipes) pc Mutant Caused by a Mutation in the Gli-Similar3 (glis3) Gene

Polycystic kidney disease (PKD) is a common hereditary disease in humans. Recent studies have shown an increasing number of ciliary genes that are involved in the pathogenesis of PKD. In this study, the Gli-similar3 (glis3) gene was identified as the causal gene of the medaka pc mutant, a model of PKD. In the pc mutant, a transposon was found to be inserted into the fourth intron of the pc/glis3 gene, causing aberrant splicing of the pc/glis3 mRNA and thus a putatively truncated protein with a defective zinc finger domain. pc/glis3 mRNA is expressed in the epithelial cells of the renal tubules and ducts of the pronephros and mesonephros, and also in the pancreas. Antisense oligonucleotide-mediated knockdown of pc/glis3 resulted in cyst formation in the pronephric tubules of medaka fry. Although three other glis family members, glis1a, glis1b and glis2, were found in the medaka genome, none were expressed in the embryonic or larval kidney. In the pc mutant, the urine flow rate in the pronephros was significantly reduced, which was considered to be a direct cause of renal cyst formation. The cilia on the surface of the renal tubular epithelium were significantly shorter in the pc mutant than in wild-type, suggesting that shortened cilia resulted in a decrease in driving force and, in turn, a reduction in urine flow rate. Most importantly, EGFP-tagged pc/glis3 protein localized in primary cilia as well as in the nucleus when expressed in mouse renal epithelial cells, indicating a strong connection between pc/glis3 and ciliary function. Unlike human patients with GLIS3 mutations, the medaka pc mutant shows none of the symptoms of a pancreatic phenotype, such as impaired insulin expression and/or diabetes, suggesting that the pc mutant may be suitable for use as a kidney-specific model for human GLIS3 patients.     

Olfactory Proteins Mediating Chemical Communication in the Navel Orangeworm Moth,Amyelois transitella

Background

The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control — like pheromone-based approaches — are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins.

Methodology

By analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components.

Conclusion

We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.     

<Emphasis Type="Italic">Hirsutella proturicola</Emphasis> sp. nov. isolated from a proturan, <Emphasis Type="Italic">Baculentulus</Emphasis> densus (Protura,Hexapoda)

A new species of Hirsutella, H. proturicola, isolated from a subterranean proturan (Baculentulus densus; Protura, Hexapoda), is described and illustrated. Hirsutella proturicola is characterized by producing monoblastic phialides of 24–51.5 × 2.5–5 μm with a slightly roughened neck, fusiform and curved conidia of 9–18 × 2.5–4 μm that have a truncate base and a papillate projection often capped with sheath-like mucilage, and pluricellular, globose to subglobose chlamydospores of 21–48 × 21–41.5 μm. This species is morphologically and phylogenetically close to H. rostrata, an acaropathogenic species, but can be distinguished from the size of the phialides and the size and shape of the conidia.     

Lecanicillium and Verticillium species from Indonesia and Japan including three new species

Forty-six Lecanicillium strains and one Verticillium strain were isolated from subterranean and epiphytic arthropods, soil, and other sources collected in Indonesia and Japan. These strains were identified as nine Lecanicillium and one Verticillium species including six undescribed species based on light microscopy and the sequences of the ITS-1 and ITS-2 regions including 5.8S ribosomal DNA. Four of the ten species (L. araneicola, L. kalimantanense, Lecanicillium sp. 4, and V. indonesiacum) were recovered from Indonesia, five of the ten (L. attenuatum, L. fusisporum, L. psalliotae, Lecanicillium sp. 1, and Lecanicillium sp. 3) were from Japan, and L. saksenae was from both countries. In this article, new species (L. araneicola, L. kalimantanense, and V. indonesiacum) and a new combination (L. saksenae) are proposed from the fungi isolated from epiphytic and subterranean arthropods collected in East Kalimantan.     

Single Nucleotide Polymorphism that Accompanies a Missense Mutation (Gln488His) Impedes the Dimerization of Hsp90

A single nucleotide polymorphism (SNP) that causes a missense mutation of highly conserved Gln488 to His of the α isoform of the 90-kDa heat shock protein (Hsp90α) molecular chaperone is observed in Caucasians. The mutated Hsp90α severely reduced the growth of yeast cells. To investigate this molecular mechanism, we examined the domain–domain interactions of human Hsp90α by using bacterial 2-hybrid system. Hsp90α was expressed as a full-length form, N-terminal domain (residues 1–400), or middle (residues 401–617) plus C-terminal (residues 618–732) domains (MC domain/amino acids 401–732). The Gln488His substitution in MC domain did not affect the intra-molecular interaction with N-terminal domain, whereas the dimeric interaction-mediated by the inter-molecular interaction between MC domains was decreased to 32%. Gln488Ala caused a similar change, whereas Gln488Thr, which exceptionally occurs in mitochondrial Hsp90 paralog, fully maintained the dimeric interaction. Therefore, the SNP causing Gln488His mutation could abrogate the Hsp90 function due to reduced dimerization.     

Phosphoproteomic analysis of distinct tumor cell lines in response to nocodazole treatment

Here, we report for the first time a comparative phosphoproteomic analysis of distinct tumor cell lines in the presence or absence of the microtubule‐interfering agent nocodazole. In total, 1525 phosphorylation sites assigned to 726 phosphoproteins were identified using LC‐MS‐based technology following phosphopeptide enrichment. Analysis of the amino acid composition surrounding the identified in vivo phosphorylation sites revealed that they could be classified into two motif groups: pSer‐Pro and pSer‐Asp/Glu. Phosphoproteomic change resulting from nocodazole treatment varied among cell lines in terms of the numbers of total phosphopeptides identified, motif groups, and functional annotation groups; however, the cell lines were equally sensitive to nocodazole. The identified phosphoproteome subset contained major signaling proteins and proteins known to be involved in mitosis, but did not always exhibit the same changes in the tumor cells from nocodazole treatment. In spite of the complex changes observed in the phosphorylation of many of the proteins, possible common features induced by nocodazole were found, including phosphorylation of nucleophosmin (NPM) S254 and coatomer protein complex, subunit α (COPA) S173, suggesting that the events are not cell‐type specific but events generally occurring in mitosis or induced by a microtubule‐interfering agent. Further, temporal analysis of phosphoproteome change revealed that phosphorylation of NPM S254 and COPA S173 was observed from the early (6 h) and late (24 h) time point after nocodazole treatment, respectively, suggesting that NPM S254 may be involved in the induction of M‐phase arrest by nocodazole, whereas COPA S173 may be caused as a result of M‐phase arrest.     

A comprehensive analysis of Trypanosoma brucei mitochondrial proteome

The composition of the large, single, mitochondrion (mt) of Trypanosoma brucei was characterized by MS (2‐D LC‐MS/MS and gel‐LC‐MS/MS) analyses. A total of 2897 proteins representing a substantial proportion of procyclic form cellular proteome were identified, which confirmed the validity of the vast majority of gene predictions. The data also showed that the genes annotated as hypothetical (species specific) were overpredicted and that virtually all genes annotated as hypothetical, unlikely are not expressed. By comparing the MS data with genome sequence, 40 genes were identified that were not previously predicted. The data are placed in a publicly available web‐based database (www.TrypsProteome.org). The total mitochondrial proteome is estimated at 1008 proteins, with 401, 196, and 283 assigned to the mt with high, moderate, and lower confidence, respectively. The remaining mitochondrial proteins were estimated by statistical methods although individual assignments could not be made. The identified proteins have predicted roles in macromolecular, metabolic, energy generating, and transport processes providing a comprehensive profile of the protein content and function of the T. brucei mt.     

Membrane-bound acid pyrophosphatase from Sulfolobus tokodaii, a thermoacidophilic archaeon: heterologous expression of the gene and characterization of the product

Membranes of Sulfolobus tokodaii, a thermoacidophilic archaeon that grows optimally at pH 2–3, 75–80°C, show the ability to hydrolyze PPi with an optimum pH of 2–3. This acid PPase is proposed to be a dolicholpyrophosphatase that participates in glycoprotein biosynthesis. In the present study, the archaeal membranes hydrolyzed isopentenylpyrophosphate and geranylpyrophosphate, compounds related to dolicholpyrophosphate, at pH 3. However, the dolicholpyrophosphate-binding antibiotic bacitracin failed to inhibit the acid PPase. To investigate further the function and structure of the acid PPase, the gene was cloned and heterologously expressed in Escherichia coli. The membranes from recombinant E. coli showed PPase activity with similar pH and temperature dependence, substrate specificity, and kinetic parameters to those reported for Sulfolobus membranes. The acid PPase was solubilized and purified to electrophoretic homogeneity from the recombinant E. coli. The purified enzyme showed similar K _m values for PPi, ATP, and ADP to the membrane-bound enzyme. Lipids from the Sulfolobus membranes enhanced the activity to about threefold. Studies involving deletion mutants indicated that basic amino acids in the N-terminal (Arg2 and Lys3), as well as the residues (4th–69th) possibly twice-spanning the membrane, are essential for integration of the enzyme into membranes.     

Direct Observation of ATP-Induced Conformational Changes in Single P2X4 Receptors

The ATP-gated P2X₄ receptor is a cation channel, which is important in various pathophysiological events. The architecture of the P2X₄ receptor in the activated state and how to change its structure in response to ATP binding are not fully understood. Here, we analyze the architecture and ATP-induced structural changes in P2X₄ receptors using fast-scanning atomic force microscopy (AFM). AFM images of the membrane-dissociated and membrane-inserted forms of P2X₄ receptors and a functional analysis revealed that P2X₄ receptors have an upward orientation on mica but lean to one side. Time-lapse imaging of the ATP-induced structural changes in P2X₄ receptors revealed two different forms of activated structures under 0 Ca²⁺ conditions, namely a trimer structure and a pore dilation-like tripartite structure. A dye uptake measurement demonstrated that ATP-activated P2X₄ receptors display pore dilation in the absence of Ca²⁺. With Ca²⁺, the P2X₄ receptors exhibited only a disengaged trimer and no dye uptake was observed. Thus our data provide a new insight into ATP-induced structural changes in P2X₄ receptors that correlate with pore dynamics.     

Network Properties of Robust Immunity in Plants

Two modes of plant immunity against biotrophic pathogens, Effector Triggered Immunity (ETI) and Pattern-Triggered Immunity (PTI), are triggered by recognition of pathogen effectors and Microbe-Associated Molecular Patterns (MAMPs), respectively. Although the jasmonic acid (JA)/ethylene (ET) and salicylic acid (SA) signaling sectors are generally antagonistic and important for immunity against necrotrophic and biotrophic pathogens, respectively, their precise roles and interactions in ETI and PTI have not been clear. We constructed an Arabidopsis dde2/ein2/pad4/sid2-quadruple mutant. DDE2, EIN2, and SID2 are essential components of the JA, ET, and SA sectors, respectively. The pad4 mutation affects the SA sector and a poorly characterized sector. Although the ETI triggered by the bacterial effector AvrRpt2 (AvrRpt2-ETI) and the PTI triggered by the bacterial MAMP flg22 (flg22-PTI) were largely intact in plants with mutations in any one of these genes, they were mostly abolished in the quadruple mutant. For the purposes of this study, AvrRpt2-ETI and flg22-PTI were measured as relative growth of Pseudomonas syringae bacteria within leaves. Immunity to the necrotrophic fungal pathogen Alternaria brassicicola was also severely compromised in the quadruple mutant. Quantitative measurements of the immunity levels in all combinatorial mutants and wild type allowed us to estimate the effects of the wild-type genes and their interactions on the immunity by fitting a mixed general linear model. This signaling allocation analysis showed that, contrary to current ideas, each of the JA, ET, and SA signaling sectors can positively contribute to immunity against both biotrophic and necrotrophic pathogens. The analysis also revealed that while flg22-PTI and AvrRpt2-ETI use a highly overlapping signaling network, the way they use the common network is very different: synergistic relationships among the signaling sectors are evident in PTI, which may amplify the signal; compensatory relationships among the sectors dominate in ETI, explaining the robustness of ETI against genetic and pathogenic perturbations.