Aluminum Resistance in the Arabidopsis Mutant alr-104 Is Caused by an Aluminum-Induced Increase in Rhizosphere pH

A mechanism that confers increased Al resistance in the Arabidopsis thaliana mutant alr-104 was investigated. A modified vibrating microelectrode system was used to measure H⁺ fluxes generated along the surface of small Arabidopsis roots. In the absence of Al, no differences in root H⁺ fluxes between wild type and alr-104 were detected. However, Al exposure induced a 2-fold increase in net H⁺ influx in alr-104 localized to the root tip. The increased flux raised the root surface pH of alr-104 by 0.15 unit. A root growth assay was used to assess the Al resistance of alr-104 and wild type in a strongly pH-buffered nutrient solution. Increasing the nutrient solution pH from 4.4 to 4.5 significantly increased Al resistance in wild type, which is consistent with the idea that the increased net H⁺ influx can account for greater Al resistance in alr-104. Differences in Al resistance between wild type and alr-104 disappeared when roots were grown in pH-buffered medium, suggesting that Al resistance in alr-104 is mediated only by pH changes in the rhizosphere. This mutant provides the first evidence, to our knowledge, for an Al-resistance mechanism based on an Al-induced increase in root surface pH.     

The Role of Chaperone-Mediated Autophagy in Huntingtin Degradation

Huntington Disease (HD) is caused by an abnormal expansion of polyQ tract in the protein named huntingtin (Htt). HD pathology is featured by accumulation and aggregation of mutant Htt in striatal and cortical neurons. Aberrant Htt degradation is implicated in HD pathogenesis. The aim of this study was to investigate the regulatory role of chaperone-mediated autophagy (CMA) components, heat shock protein cognate 70 (Hsc70) and lysosome-associated protein 2A (LAMP-2A) in degradation of Htt fragment 1-552aa (Htt-552). A cell model of HD was produced by overexpression of Htt-552 with adenovirus. The involvement of CMA components in degradation of Htt-552 was determined with over-expression or silencing of Hsc70 and LAMP-2A. The results confirmed previous reports that both macroautophagy and CMA were involved in degradation of Htt-552. Changing the levels of CMA-related proteins affected the accumulation of Htt-552. The lysosomal binding and luminal transport of Htt-552 was demonstrated by incubation of Htt-552 with isolated lysosomes. Expansion of the polyQ tract in Htt-552 impaired its uptake and degradation by lysosomes. Mutation of putative KFERQ motif in wild-type Htt-552 interfered with interactions between Htt-552 and Hsc70. Endogenous Hsc70 and LAMP-2A interacted with exogenously expressed Htt-552. Modulating the levels of CMA related proteins degraded endogenous full-length Htt. These studies suggest that Hsc70 and LAMP-2A through CMA play a role in the clearance of Htt and suggest a novel strategy to target the degradation of mutant Htt.     

Rho激酶抑制剂Y-27632促进大鼠成骨细胞早期分化

已知Rho激酶抑制剂可调控细胞骨架重建,激活相关转录因子,进而促进细胞分化。然而,关于Rho激酶抑制剂对细胞骨架和成骨细胞分化的影响及两者之间关系尚未见报道。本研究旨在阐明Rho激酶抑制剂Y 27632调控细胞骨架重建,促进成骨分化。取新生SD大鼠头盖骨组织体外培养细胞传至第3代,给予Rho激酶抑制剂Y-27632进行干预。采用罗丹明标记的鬼笔环肽对细胞骨架进行细胞化学染色。结果显示,培养1 d和2 d,Rho激酶抑制剂Y-27632处理的细胞呈现多角形,并伴有部分伪足形成。细胞培养4 d和7 d,Y-27632处理的细胞碱性磷酸酶（alkaline phosphatase, ALP）活性明显增加（P<0.01）。实时定量PCR揭示,加Y-27632处理细胞的骨分化相关基因Runx2、Alp、β-catenin(β-cat)、osteopontin(Opn)的mRNA表达水平均显著高于对照细胞（P<0.05或P<0.01）。以上结果证明,Rho激酶抑制剂Y 27632能够影响大鼠成骨细胞的形态,并有促进其分化作用。本研究为骨代谢疾病及组织工程的研究提供了新的线索和启示。     

Rho小G蛋白相关激酶的结构与功能

Rho小G蛋白家族是Ras超家族成员之一,人类Rho小G蛋白包括20个成员,研究最清楚的有RhoA、Rac1和Cdc42。Rho小G蛋白参与了诸如细胞骨架调节、细胞移动、细胞增殖、细胞周期调控等重要的生物学过程。在这些生物学过程的调节中,Rho小G蛋白的下游效应蛋白质如蛋白激酶（p21-activated kinase,PAK）、ROCK（Rho-kinase）、PKN（protein kinase novel）和MRCK（myotonin-related Cdc42-binding kinase）发挥了不可或缺的作用。迄今研究发现,PAK可调节细胞骨架动力学和细胞运动,另外,PAK通过MAPK（mitogen-activated protein kinases）参与转录、细胞凋亡和幸存通路及细胞周期进程;ROCK与肌动蛋白应力纤维介导黏附复合物的形成及与细胞周期进程的调节有关;哺乳动物的PKN与RhoA/B/C相互作用介导细胞骨架调节;MRCK与细胞骨架重排、细胞核转动、微管组织中心再定位、细胞移动和癌细胞侵袭等有关。该文简要介绍Rho小G蛋白下游激酶PAK、ROCK、PKN和MRCK的结构及其在细胞骨架调节中的功能,重点总结它们在真核细胞周期调控中的作用,尤其是在癌细胞周期进程中所发挥的作用,为寻找癌症治疗的新靶点提供理论依据。     

Rho Kinase Enhances Contractions of Rat Mesenteric Collecting Lymphatics

The mechanisms that control phasic and tonic contractions of lymphatic vessels are poorly understood. We hypothesized that rho kinase ROCK, previously shown to increase calcium (Ca²⁺) sensitivity in vascular smooth muscle, enhances lymphatic contractile activity in a similar fashion. Contractions of isolated rat mesenteric lymphatic vessels were observed at a luminal pressure of 2 cm H₂O in a 37°C bath. The expression of ROCK in isolated rat mesenteric lymphatic vessels was assessed by Western blotting and confocal microscopy. The role of ROCK in contractile function was tested using two specific yet structurally distinct inhibitors: H1152 (0.1–10 μM) and Y-27632 (0.5–50 μM). In addition, lymphatics were transfected with constitutively active (ca)-ROCK protein (2 μg/ml) to assess gain of contractile function. Vessel diameter and the concentration of intracellular free Ca²⁺ ([Ca²⁺]_i) were simultaneously measured in a subset of isolated lymphatics loaded with the Ca²⁺-sensing dye fura-2. The results show expression of both the ROCK1 and ROCK2 isoforms in lymphatic vessels. Inhibition of ROCK increased lymphatic end diastolic diameter and end systolic diameter in a concentration-dependent manner. Significant reductions in lymphatic tone and contraction amplitude were observed after treatment 1–10 μM H1152 or 25–50 μM Y-27632. H1152 (10 μM) also significantly reduced contraction frequency. Transient increases in [Ca²⁺]_i preceded each phasic contraction, however this pattern was disrupted by either 10 μM H1152 or 50 μM Y-27632 in the majority of lymphatics studied. The significant decrease in tone caused by H1152 or Y-27632 was not associated with a significant change in the basal [Ca²⁺]_i between transients. Transfection with ca-ROCK protein enhanced lymphatic tone, but was not associated with a significant change in basal [Ca²⁺]_i. Our data suggest that ROCK mediates normal tonic constriction and influences phasic contractions in lymphatics. We propose that ROCK modulates Ca²⁺ sensitivity of contractile proteins in lymphatics.     

A Saccharomyces cerevisiae Model Reveals In Vivo Functional Impairment of the Ogden Syndrome N-Terminal Acetyltransferase NAA10 Ser37Pro Mutant

N-terminal acetylation (Nt-acetylation) occurs on the majority of eukaryotic proteins and is catalyzed by N-terminal acetyltransferases (NATs). Nt-acetylation is increasingly recognized as a vital modification with functional implications ranging from protein degradation to protein localization. Although early genetic studies in yeast demonstrated that NAT-deletion strains displayed a variety of phenotypes, only recently, the first human genetic disorder caused by a mutation in a NAT gene was reported; boys diagnosed with the X-linked Ogden syndrome harbor a p.Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of the NatA complex, and suffer from global developmental delays and lethality during infancy. Here, we describe a Saccharomyces cerevisiae model developed by introducing the human wild-type or mutant NatA complex into yeast lacking NatA (NatA-Δ). The wild-type human NatA complex phenotypically complemented the NatA-Δ strain, whereas only a partial rescue was observed for the Ogden mutant NatA complex suggesting that hNaa10 S37P is only partially functional in vivo. Immunoprecipitation experiments revealed a reduced subunit complexation for the mutant hNatA S37P next to a reduced in vitro catalytic activity. We performed quantitative Nt-acetylome analyses on a control yeast strain (yNatA), a yeast NatA deletion strain (yNatA-Δ), a yeast NatA deletion strain expressing wild-type human NatA (hNatA), and a yeast NatA deletion strain expressing mutant human NatA (hNatA S37P). Interestingly, a generally reduced degree of Nt-acetylation was observed among a large group of NatA substrates in the yeast expressing mutant hNatA as compared with yeast expressing wild-type hNatA. Combined, these data provide strong support for the functional impairment of hNaa10 S37P in vivo and suggest that reduced Nt-acetylation of one or more target substrates contributes to the pathogenesis of the Ogden syndrome. Comparative analysis between human and yeast NatA also provided new insights into the co-evolution of the NatA complexes and their substrates. For instance, (Met-)Ala- N termini are more prevalent in the human proteome as compared with the yeast proteome, and hNatA displays a preference toward these N termini as compared with yNatA.Up to 85% of soluble eukaryotic proteins carry an N-terminal acetyl group at their N terminus, which is the result of a co-translational protein modification referred to as N-terminal protein acetylation (Nt-acetylation) or N^α-acetylation (1). This presumed irreversible protein modification is catalyzed by a specific category of the GCN5-related N-acetyltransferase domain containing superfamily of acetyltransferases; the ribosome associated N-terminal acetyltransferases or NATs¹ (2). NATs catalyze the acetyl transfer from acetyl coenzyme A (Ac-CoA) to a primary α-amine of the first amino acid residue of a nascent protein chain. In eukaryotes, NATs are composed of at least one catalytic subunit and mainly target different substrate N termini based on their N-terminal sequences (3).To date, five human NATs hNatA, hNatB, and hNatC; constituting the major human NAT complexes, and hNatD and hNatF have been identified and their substrate specificity characterized (1, 4–8). In addition, a putative hNatE complex has been described (9–10). Except for NatF, which is only expressed in higher eukaryotes (1), the substrate specificity profiles of the NatA-E complexes seem to be conserved among eukaryotes (5–9, 11–13).Contrary to the original assumption that Nt-acetylation protected proteins from degradation (14), it was more recently demonstrated that this modification creates specific degradation signals (termed Ac/N-degrons) in cellular proteins, thereby diversifying this original view substantially. These degrons target at least some Nt-acetylated proteins for the conditional degradation by a novel branch of the N-end rule pathway, an ubiquitin-dependent proteolytic system (15–16). In addition, numerous reports implicate Nt-acetylation in cellular differentiation, survival, metabolism, and proliferation, thereby linking it to cancer (17–18). As such, Nt-acetylation is now linked to a whole range of molecular implications including protein destabilization and degradation by the Nt-acetylation dependent recruitment of ubiquitin ligases (15–16), protein translocation (19), membrane attachment (20), and protein complex formation (21).Among all characterized NATs, NatA displays the broadest substrate specificity profile and thus represents the primary NAT in terms of substrate N termini as it is responsible for the Nt-acetylation of the methionine aminopeptidase (MetAP) iMet-processed serine, threonine, alanine, glycine, and valine starting N termini (3). The human NatA complex is composed of two essential subunits; the catalytic subunit hNaa10 (hARD1) and the regulatory subunit hNaa15 (NATH/hNAT1) (4). Deregulations of hNaa10 and/or NatA expression have been linked to various signaling molecules including hypoxia inducible factor-1α, DNA methyltransferase1/E-cadherin, β-catenin/cyclin D1, and Bcl-xL, showing its involvement in hypoxia, tumorigenesis, cell cycle progression, and apoptosis (17, 22–26).Recently, the first structures of NATs and a NAT-complex were solved, providing a molecular understanding of the sequence specific Nt-acetylation of protein N termini (27–30). Structural analyses of noncomplexed Naa10 and NatA from Schizosaccharomyces pombe reveal an allosteric modulator function of Naa15 in steering Naa10 specificity and provide a rational for the distinctive substrate specificity profiles observed when assaying non-complexed versus complexed Naa10 (10, 27), with both forms co-existing in cells (10). In particular, three essential catalytic Naa10 residues were found to be incorrectly positioned in non-complexed Naa10, while these shift into the active site in Naa15-complexed Naa10, thereby permitting canonical NatA-mediated Nt-acetylation. Interestingly, noncomplexed Naa10 was shown to efficiently Nt-acetylate glutamate and aspartate starting N termini, whereas poorly acetylating canonical NatA type N termini (10). The study of Liszczak et al. further showed that NatA substrate binding specificity was coupled to the catalytic mechanism being used (27). More specifically, an essential glutamate residue (Glu24 in the protein accession {"type":"entrez-protein","attrs":{"text":"Q9UTI3","term_id":"74625957","term_text":"Q9UTI3"}}Q9UTI3 (Swiss-Prot)) involved in catalysis, precludes methionine from entering the specificity pocket, whereas cognate NatA substrate N-terminal residues can easily be accommodated. Interestingly, and in contrast to NatA, both wild-type Naa10 and Glu24 mutated Naa10 (Naa10 E24A) were still capable of Nt-acetylating acidic amino acid starting N termini, most likely because of the substrate side-chain carboxyl moiety acting as a functional replacement group in the process of catalysis, whereas essentially no activity could be observed when probing a cognate NatA substrate (27).Early yeast studies demonstrated that strains with mutated or deleted NAT genes were viable, but displayed a number of different phenotypes (31). For NatA, the first phenotypes described were defects in sporulation, mating, and entry into stationary phase when NAA10 (ARD1) was mutated (32). Four years later, the overlapping phenotypes of NAA10 and NAA15 (NAT1) mutant strains, revealed, along with other data, that Naa10 and Naa15 are in fact components of the NatA acetyltransferase complex (33–34). As compared with NatA phenotypes, NatB phenotypes are more severe, including slow growth and defects in mitochondrial inheritance (35–36). NatC subunits were initially found to be essential for propagation of the l-A dsRNA virus, and further for growth on nonfermentable carbon sources (37–39). The first reports implicating NAT gene point mutations in human genetic disorders only recently emerged. More specifically, two different point mutations in the X-linked NAA10 gene were both found to cause developmental delays and were linked to the Ogden syndrome (S37P) (40) and intellectual disability (R116W) (41), highlighting the essential importance of NATs and protein Nt-acetylation in biology and disease. Further, in Caenorhabditis elegans (42), Drosophila melanogaster (43), and Trypanosoma brucei (44), Naa10 was proven to be essential and, strengthened by the observed detrimental effects of NAA10 mutations (40–41), the NAA10 gene function is also believed to be essential in human.Ogden syndrome boys harboring the p.Ser37Pro variant in the gene encoding Naa10 are characterized by craniofacial abnormalities, failure to thrive, developmental delay, hypotonia, cardiac arrhythmias, cryptorchidism, and an aged appearance, ultimately resulting in mortality during infancy (40). Although this mutation was shown to significantly impair Naa10 catalytic activity in vitro, we here assessed the influence and functional in vitro and in vivo consequences of this mutation on NatA complex formation and NatA activity in a yeast model. By phenotypic screening in yeast, we show that hNaa10 S37P displays a significantly impaired functionality in vivo. Further, using immunoprecipitation, we show that the human Naa10-Naa15 complex formation is negatively affected by the S37P mutation, and that immunoprecipitated hNatA S37P also displays a reduced in vitro catalytic activity as compared with wild-type hNatA. Finally, quantitative Nt-acetylome analyses suggest that reduced Nt-acetylation of one or more target substrates contributes to the pathogenesis of the Ogden syndrome.     

A Functional Calvin Cycle Is Not Indispensable for the Light Activation of C4 Phosphoenolpyruvate Carboxylase Kinase and Its Target Enzyme in the Maize Mutant bundle sheath defective2-mutable1

We used a pale-green maize (Zea mays L.) mutant that fails to accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to test the working hypothesis that the regulatory phosphorylation of C₄ phosphoenolpyruvate carboxylase (PEPC) by its Ca²⁺-insensitive protein-serine/threonine kinase (PEPC kinase) in the C₄ mesophyll cytosol depends on cross-talk with a functional Calvin cycle in the bundle sheath. Wild-type (W22) and bundle sheath defective2-mutable1 (bsd2-m1) seeds were grown in a controlled environment chamber at 100 to 130 μmol m⁻² s⁻¹ photosynthetic photon flux density, and leaf tissue was harvested 11 d after sowing, following exposure to various light intensities. Immunoblot analysis showed no major difference in the amount of polypeptide present for several mesophyll- and bundle-sheath-specific photosynthetic enzymes apart from Rubisco, which was either completely absent or very much reduced in the mutant. Similarly, leaf net CO₂-exchange analysis and in vitro radiometric Rubisco assays showed that no appreciable carbon fixation was occurring in the mutant. In contrast, the sensitivity of PEPC to malate inhibition in bsd2-m1 leaves decreased significantly with an increase in light intensity, and there was a concomitant increase in PEPC kinase activity, similar to that seen in wild-type leaf tissue. Thus, although bsd2-m1 mutant plants lack an operative Calvin cycle, light activation of PEPC kinase and its target enzyme are not grossly perturbed.     

Herp Promotes Degradation of Mutant Huntingtin: Involvement of the Proteasome and Molecular Chaperones

In neurodegenerative diseases, pathogenic proteins tend to misfold and form aggregates that are difficult to remove and able to induce excessive endoplasmic reticulum (ER) stress, leading to neuronal injury and apoptosis. Homocysteine-induced endoplasmic reticulum protein (Herp), an E3 ubiquitin ligase, is an important early marker of ER stress and is involved in the ubiquitination and degradation of many neurodegenerative proteins. However, in Huntington’s disease (HD), a typical polyglutamine disease, whether Herp is also involved in the metabolism and degradation of the pathogenic protein, mutant huntingtin, has not been reported. Therefore, we studied the relationship between Herp and N-terminal fragments of huntingtin (HttN-20Q and HttN-160Q). We found that Herp was able to bind to the overexpressed Htt N-terminal, and this interaction was enhanced by expansion of the polyQ fragment. Confocal microscopy demonstrated that Herp was co-localized with the HttN-160Q aggregates in the cytoplasm and tightly surrounded the aggregates. Overexpression of Herp significantly decreased the amount of soluble and insoluble HttN-160Q, promoted its ubiquitination, and inhibited its cytotoxicity. In contrast, knockdown of Herp resulted in more HttN-160Q protein, less ubiquitination, and stronger cytotoxicity. Inhibition of the autophagy-lysosomal pathway (ALP) had no effect on the function of Herp. However, blocking the ubiquitin-proteasome pathway (UPP) inhibited the reduction in soluble HttN-160Q caused by Herp. Interestingly, blocking the UPP did not weaken the ability of Herp to reduce HttN-160Q aggregates. Deletions of the N-terminal of Herp weakened its ability to inhibit HttN-160Q aggregation but did not result in a significant increase in its soluble form. However, loss of the C-terminal led to a significant increase in soluble HttN-160Q, but Herp still maintained the ability to inhibit aggregate formation. We further found that the expression level of Herp was significantly increased in HD animal and cell models. Our findings suggest that Herp is a newly identified huntingtin-interacting protein that is able to reduce the cytotoxicity of mutant huntingtin by inhibiting its aggregation and promoting its degradation. The N-terminal of Herp serves as the molecular chaperone to inhibit protein aggregation, while its C-terminal functions as an E3 ubiquitin ligase to promote the degradation of misfolded proteins through the UPP. Increased expression of Herp in HD models may be a pro-survival mechanism under stress.     

Isoalantolactone Enhances the Radiosensitivity of UMSCC-10A Cells via Specific Inhibition of Erk1/2 Phosphorylation

Background

Although radiotherapy is one of the mainstream approaches for the treatment of head and neck squamous cell carcinoma (HNSCC), this cancer is always associated with resistance to radiation. In this study, the mechanism of action of isoalantolactone as well as its radiosensitizing effect was investigated in UMSCC-10A cells.

Methods

The radiosensitization of UMSCC-10A cells treated with isoalantolactone was analyzed by colony formation assay. The radiosensitization effects of isoalantolactone on cell proliferation, cell cycle and apoptosis regulation were examined by BrdU incorporation assay, DNA content assay and flow cytometry, respectively. Western blotting was performed to determine the effects of isoalantolactone combined with radiation on the protein expression of Mek, extracellular signal-regulated kinase (Erk1/2) as well as phosphorylated Mek and Erk1/2. Erk1/2 knockdown by siRNA was used to confirm that isoalantolactone specifically inhibited the activation of Erk1/2 signaling pathway in UMSCC-10A cells.

Results

Isoalantolactone enhanced the radiosensitivity of UMSCC-10A cells; the sensitivity enhanced ratios (SERs) were 1.44 and 1.63, respectively, for 2.5 and 5 μM. Moreover, isoalantolactone enhanced radiation-induced cell proliferation and apoptosis and cell cycle arrested at G2/M phase. Furthermore, no marked changes were observed in the expression of total Erk1/2 and Mek protein after radiation treatment. However, isoalantolactone was significantly reduced radiation-induced the phosphorylation of Erk1/2, whereas it altered the phosphorylation of Mek to a lesser extent. In addition, the radiosensitivity of UMSCC-10A cells with Erk1/2 knockdown was increased. Isoalantolactone cannot further prevent the proliferation of UMSCC-10A cells with Erk1/2 knockdown which other mechanism regulated cell proliferation.

Conclusion

Our results suggested that isoalantolactone enhanced radiation-induced apoptosis, cell cycle arrested and reduced the cell proliferation of UMSCC-10A cells via specifically inhibited the phosphorylation of Erk1/2. Thus a low concentration of isoalantolactone may be used to overcome the resistance of UMSCC-10A cells to radiation and may be a promising radiosensitizer in cancer therapy.     

Combined Inhibition of IGF-1R/IR and Src Family Kinases Enhances Antitumor Effects in Prostate Cancer by Decreasing Activated Survival Pathways

Background

Treatment of metastatic prostate cancer (PCa) with single agents has shown only modest efficacy. We hypothesized dual inhibition of different pathways in PCa results in improved tumor inhibition. The Src family kinases (SFK) and insulin-like growth factor-1 (IGF-1) signaling axes are aberrantly activated in both primary PCa and bone metastases and regulate distinct and overlapping functions in PCa progression. We examined the antitumor effects of combined inhibition of these pathways.

Materials and Methods

Src andIGF-1 receptor (IGF-1R) inhibition was achieved in vitro by short hairpin (sh)RNA and in vitro and in vivo by small molecule inhibitors (dasatinib and BMS-754807, against SFK and IGF-1R/Insulin Receptor(IR), respectively).

Results

In vitro, inhibition of IGF-1 signaling affected cell survival and proliferation. SFK blockade alone had modest effects on proliferation, but significantly enhanced the IGF-1R blockade. These findings correlated with a robust inhibition of IGF-1-induced Akt1 phophorylation by dasatinib, whereas Akt2 phosphorylation was SFK independent and only inhibited by BMS-754807. Thus, complete inhibition of both Akt genes, not seen by either drug alone, is likely a major mechanism for the decreased survival of PCa cells. Furthermore, dasatinib and BMS-754807 inhibited in vivo growth of the primary human xenograft MDA PCa 133, with corresponding inhibition of Akt in tumors. Also, both orthotopic and intratibial tumor growth of PC-3 cells were more potently inhibited by dual SFK and IGF-1R/IR blockade compared to either pathway alone, with a corresponding decrease in bone turnover markers.

Conclusions

Dual IGF-1R/IR and SFK inhibition may be a rational therapeutic approach in PCa by blocking both independent and complementary processes critical to tumor growth.     

Detection of Alpha-Rod Protein Repeats Using a Neural Network and Application to Huntingtin

A growing number of solved protein structures display an elongated structural domain, denoted here as alpha-rod, composed of stacked pairs of anti-parallel alpha-helices. Alpha-rods are flexible and expose a large surface, which makes them suitable for protein interaction. Although most likely originating by tandem duplication of a two-helix unit, their detection using sequence similarity between repeats is poor. Here, we show that alpha-rod repeats can be detected using a neural network. The network detects more repeats than are identified by domain databases using multiple profiles, with a low level of false positives (<10%). We identify alpha-rod repeats in approximately 0.4% of proteins in eukaryotic genomes. We then investigate the results for all human proteins, identifying alpha-rod repeats for the first time in six protein families, including proteins STAG1-3, SERAC1, and PSMD1-2 & 5. We also characterize a short version of these repeats in eight protein families of Archaeal, Bacterial, and Fungal species. Finally, we demonstrate the utility of these predictions in directing experimental work to demarcate three alpha-rods in huntingtin, a protein mutated in Huntington''s disease. Using yeast two hybrid analysis and an immunoprecipitation technique, we show that the huntingtin fragments containing alpha-rods associate with each other. This is the first definition of domains in huntingtin and the first validation of predicted interactions between fragments of huntingtin, which sets up directions toward functional characterization of this protein. An implementation of the repeat detection algorithm is available as a Web server with a simple graphical output: http://www.ogic.ca/projects/ard. This can be further visualized using BiasViz, a graphic tool for representation of multiple sequence alignments.     

Wolbachia Enhances West Nile Virus (WNV) Infection in the Mosquito Culex tarsalis

Novel strategies are required to control mosquitoes and the pathogens they transmit. One attractive approach involves maternally inherited endosymbiotic Wolbachia bacteria. After artificial infection with Wolbachia, many mosquitoes become refractory to infection and transmission of diverse pathogens. We evaluated the effects of Wolbachia (wAlbB strain) on infection, dissemination and transmission of West Nile virus (WNV) in the naturally uninfected mosquito Culex tarsalis, which is an important WNV vector in North America. After inoculation into adult female mosquitoes, Wolbachia reached high titers and disseminated widely to numerous tissues including the head, thoracic flight muscles, fat body and ovarian follicles. Contrary to other systems, Wolbachia did not inhibit WNV in this mosquito. Rather, WNV infection rate was significantly higher in Wolbachia-infected mosquitoes compared to controls. Quantitative PCR of selected innate immune genes indicated that REL1 (the activator of the antiviral Toll immune pathway) was down regulated in Wolbachia-infected relative to control mosquitoes. This is the first observation of Wolbachia-induced enhancement of a human pathogen in mosquitoes, suggesting that caution should be applied before releasing Wolbachia-infected insects as part of a vector-borne disease control program.