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SPTBN1encodes βII-spectrin, the ubiquitously expressed β-spectrin that forms micrometer-scale networks associated with plasma membranes. Mice deficient in neuronal βII-spectrin have defects in cortical organization, developmental delay and behavioral deficiencies. These phenotypes, while less severe, are observed in haploinsufficient animals, suggesting that individuals carrying heterozygous SPTBN1variants may also show measurable compromise of neural development and function. Here we identify heterozygous SPTBN1variants in 29 individuals with developmental, language and motor delays; mild to severe intellectual disability; autistic features; seizures; behavioral and movement abnormalities; hypotonia; and variable dysmorphic facial features. We show that these SPTBN1variants lead to effects that affect βII-spectrin stability, disrupt binding to key molecular partners, and disturb cytoskeleton organization and dynamics. Our studies define SPTBN1variants as the genetic basis of a neurodevelopmental syndrome, expand the set of spectrinopathies affecting the brain and underscore the critical role of βII-spectrin in the central nervous system.

The small GTPase Rap1 and its guanine nucleotide exchange factor, CalDAG‐GEFI (CDGI), are critical for platelet function and hemostatic plug formation. CDGI function is regulated by a calcium binding EF hand regulatory domain and an atypical C1 domain with unknown function.

Cdt1 activity is essential for DNA replication but must be restrained to avoid genotoxic rereplication. Analysis of rare Cdt1 variants associated with Meier–Gorlin dwarfism provides new insight into how Cdt1 normally functions in loading the replicative helicase (MCM) and also reveals a nondegradative role for CDK in avoiding rereplication.

Sro7, a downstream effector of the Sec4 Rab GTPase, acts during Golgi-to-cell surface transport. The binding site for Sec4 occurs within a cleft on Sro7 formed by the junction of two adjacent β-propeller structures. In silico docking and mutational validation experiments point to a precise model for interaction of Sec4-GTP with Sro7.

The DrosophilaMulti Sex Combs (Mxc) protein is necessary for the recruitment of histone mRNA biosynthetic factors to the histone locus body (HLB). Mxc contains multiple domains required for HLB assembly and histone mRNA biosynthesis. Two N-terminal domains of Mxc are essential for promoting HLB assembly via a self-interaction.

Mono-methylation of histone H4 lysine 20 (H4K20me1) is catalyzed by Set8/KMT5A and regulates numerous aspects of genome organization and function. Loss-of-function mutations in Drosophila melanogaster Set8or mammalian KMT5Aprevent H4K20me1 and disrupt development. Set8/KMT5A also has non-histone substrates, making it difficult to determine which developmental functions of Set8/KMT5A are attributable to H4K20me1 and which to other substrates or to non-catalytic roles. Here, we show that human KMT5A can functionally substitute for Set8 during Drosophiladevelopment and that the catalytic SET domains of the two enzymes are fully interchangeable. We also uncovered a role in eye development for the N-terminal domain of Set8 that cannot be complemented by human KMT5A. Whereas Set820/20null mutants are inviable, we found that an R634G mutation in Set8 predicted from in vitroexperiments to ablate catalytic activity resulted in viable adults. Additionally, Set8(R634G) mutants retain significant, albeit reduced, H4K20me1, indicating that the R634G mutation does not eliminate catalytic activity in vivoand is functionally hypomorphic rather than null. Flies engineered to express only unmodifiable H4 histones (H4K20A) can also complete development, but are phenotypically distinct from H4K20R, Set820/20null, and Set8R634Gmutants. Taken together, our results demonstrate functional conservation of KMT5A and Set8 enzymes, as well as distinct roles for Set8 and H4K20me1 in Drosophiladevelopment.Here, Crain et al. demonstrate that mutant animals expressing only unmodifiable histone H4 lysine 20 (H4K20) are developmentally distinct from null mutants of the H4K20 monomethyltransferase, Set8. The authors show that a mutation in the Set8 catalytic domain known to block methyltransferase activity in vitro does not prevent the completion of development or eliminate H4K20 methylation in vivo, indicating that this mutation is not functionally null. Finally, they demonstrate that the human ortholog of Set8, KMT5A, rescues the loss of Set8 despite differing in its N-terminal protein sequence. Overall, this paper highlights the importance of careful in vivo analysis to untangle the diverse functions of histone modifier enzymes and the histone covalent modifications that they make.

The Sec14-like phosphatidylinositol transfer protein Sfh3 associates with bulk LDs in vegetative cells but targets to a neutral lipid hydrolase-rich LD pool during sporulation. Sfh3 inhibits LD utilization by a PtdIns-4-phosphate–dependent mechanism, and this inhibition prevents prospore membrane biogenesis in sporulating cells.

The G12/13 class of heterotrimeric G proteins, comprising the α-subunits Gα12and Gα13, regulates multiple aspects of cellular behavior, including proliferation and cytoskeletal rearrangements. Although guanine nucleotide exchange factors for the monomeric G protein Rho (RhoGEFs) are well characterized as effectors of this G protein class, a variety of other downstream targets has been reported. To identify Gα12determinants that mediate specific protein interactions, we used a structural and evolutionary comparison between the G12/13, Gs, Gi, and Gq classes to identify “class-distinctive” residues in Gα12and Gα13. Mutation of these residues in Gα12to their deduced ancestral forms revealed a subset necessary for activation of serum response element (SRE)–mediated transcription, a G12/13-stimulated pathway implicated in cell proliferative signaling. Unexpectedly, this subset of Gα12mutants showed impaired binding to heat-shock protein 90 (Hsp90) while retaining binding to RhoGEFs. Corresponding mutants of Gα13exhibited robust SRE activation, suggesting a Gα12-specific mechanism, and inhibition of Hsp90 by geldanamycin or small interfering RNA–mediated lowering of Hsp90 levels resulted in greater downregulation of Gα12than Gα13signaling in SRE activation experiments. Furthermore, the DrosophilaG12/13 homolog Concertina was unable to signal to SRE in mammalian cells, and Gα12:Concertina chimeras revealed Gα12-specific determinants of SRE activation within the switch regions and a C-terminal region. These findings identify Gα12determinants of SRE activation, implicate Gα12:Hsp90 interaction in this signaling mechanism, and illuminate structural features that arose during evolution of Gα12and Gα13to allow bifurcated mechanisms of signaling to a common cell proliferative pathway.

ABSTRACTYersinia pestis, the causative agent of plague, evolved from the gastrointestinal pathogen Yersinia pseudotuberculosis. Both species have numerous type Va autotransporters, most of which appear to be highly conserved. In Y. pestisCO92, the autotransporter genes yapKand yapJshare a high level of sequence identity. By comparing yapKand yapJto three homologous genes in Y. pseudotuberculosisIP32953 (YPTB0365, YPTB3285, and YPTB3286), we show that yapKis conserved in Y. pseudotuberculosis, while yapJis unique to Y. pestis. All of these autotransporters exhibit >96% identity in the C terminus of the protein and identities ranging from 58 to 72% in their N termini. By extending this analysis to include homologous sequences from numerous Y. pestisand Y. pseudotuberculosisstrains, we determined that these autotransporters cluster into a YapK (YPTB3285) class and a YapJ (YPTB3286) class. The YPTB3286-like gene of most Y. pestisstrains appears to be inactivated, perhaps in favor of maintaining yapJ. Since autotransporters are important for virulence in many bacterial pathogens, including Y. pestis, any change in autotransporter content should be considered for its impact on virulence. Using established mouse models of Y. pestisinfection, we demonstrated that despite the high level of sequence identity, yapKis distinct from yapJin its contribution to disseminated Y. pestisinfection. In addition, a mutant lacking both of these genes exhibits an additive attenuation, suggesting nonredundant roles for yapJand yapKin systemic Y. pestisinfection. However, the deletion of the homologous genes in Y. pseudotuberculosisdoes not seem to impact the virulence of this organism in orogastric or systemic infection models.

Genetic and biochemical evidence is presented that the Exo70 subunit of the exocyst is a direct effector for both Rho3 and Cdc42 GTPases in yeast. Prenylation of these GTPases both promotes the interaction and affects the site of binding within Exo70. Thus, interaction of the Rho GTPases with Exo70 is a key event in spatial regulation of exocytosis.

The Rho3 and Cdc42 members of the Rho GTPase family are important regulators of exocytosis in yeast. However, the precise mechanism by which they regulate this process is controversial. Here, we present evidence that the Exo70 component of the exocyst complex is a direct effector of both Rho3 and Cdc42. We identify gain-of-function mutants in EXO70 that potently suppress mutants in RHO3 and CDC42 defective for exocytic function. We show that Exo70 has the biochemical properties expected of a direct effector for both Rho3 and Cdc42. Surprisingly, we find that C-terminal prenylation of these GTPases both promotes the interaction and influences the sites of binding within Exo70. Finally, we demonstrate that the phenotypes associated with novel loss-of-function mutants in EXO70, are entirely consistent with Exo70 as an effector for both Rho3 and Cdc42 function in secretion. These data suggest that interaction with the Exo70 component of the exocyst is a key event in spatial regulation of exocytosis by Rho GTPases.

ABSTRACTHgbA is the sole TonB-dependent receptor for hemoglobin (Hb) acquisition of Haemophilus ducreyi. Binding of Hb to HgbA is the initial step in heme acquisition from Hb. To better understand this step, we mutagenized hgbAby deletion of each of the 11 putative surface-exposed loops and expressed each of the mutant proteins in transin host strain H. ducreyiFX547 hgbA. All mutant proteins were expressed, exported, and detected on the surface by anti-HgbA immunoglobulin G (IgG). Deletion of sequences in loops 5 and 7 of HgbA abolished Hb binding in two different formats. In contrast, HgbA proteins containing deletions in the other nine loops retained the ability to bind Hb. None of the clones expressing mutant proteins were able to grow on plates containing low concentrations of Hb. Previously we demonstrated in a swine model of chancroid infection that an HgbA vaccine conferred complete protection from a challenge infection. Using anti-HgbA IgG from this study and the above deletion mutants, we show that loops 4, 5, and 7 of HgbA were immunogenic and surface exposed and that IgG directed against loops 4 and 5 blocked Hb binding. Furthermore, loop 6 was cleaved by protease on intact H. ducreyi, suggesting surface exposure. These data implicate a central domain of HgbA (in respect to the primary amino acid sequence) as important in Hb binding and suggest that this region of the molecule might have potential as a subunit vaccine.

HgbA is the sole TonB-dependent receptor for hemoglobin (Hb) acquisition of Haemophilus ducreyi. Binding of Hb to HgbA is the initial step in heme acquisition from Hb. To better understand this step, we mutagenized hgbA by deletion of each of the 11 putative surface-exposed loops and expressed each of the mutant proteins in trans in host strain H. ducreyi FX547 hgbA. All mutant proteins were expressed, exported, and detected on the surface by anti-HgbA immunoglobulin G (IgG). Deletion of sequences in loops 5 and 7 of HgbA abolished Hb binding in two different formats. In contrast, HgbA proteins containing deletions in the other nine loops retained the ability to bind Hb. None of the clones expressing mutant proteins were able to grow on plates containing low concentrations of Hb. Previously we demonstrated in a swine model of chancroid infection that an HgbA vaccine conferred complete protection from a challenge infection. Using anti-HgbA IgG from this study and the above deletion mutants, we show that loops 4, 5, and 7 of HgbA were immunogenic and surface exposed and that IgG directed against loops 4 and 5 blocked Hb binding. Furthermore, loop 6 was cleaved by protease on intact H. ducreyi, suggesting surface exposure. These data implicate a central domain of HgbA (in respect to the primary amino acid sequence) as important in Hb binding and suggest that this region of the molecule might have potential as a subunit vaccine.

In Saskatoon in 2002, as one of the key strategies for the in motionhealth promotion strategy, the Forever… in motionprogram was developed with the general goal of increasing opportunities for physical activity among older adults living in congregate housing. The three components of the program were a low-intensity exercise program, informal socialization and educational sessions. The objective of the present study was to examine whether participation in this program positively influenced participants’ physical, emotional, psychological and social well-being. A quasi-experimental, pretest/post-test design was employed to examine the impact of the program on various aspects of participant well-being. Thirty-six program participants and a comparison group of 22 non-participants from two congregate housing facilities took part in the study. The pretest was administered to the study and comparison groups before or shortly after the 12-week session commenced, and the post-test was administered after the 12-week session had concluded. Pretest and post-test assessment consisted of self-report measures of 1) vitality, 2) self-rated health, 3) mental health, 4) social functioning, 5) role limitations due to emotional problems, 6) physical activity-related knowledge, and 7) self-efficacy for exercise. A multivariate analysis of covariance (MANCOVA) was conducted using the seven post-test scores as dependent variables and the pretest scores as covariates. After adjusting for differences in baseline characteristics, the findings revealed statistically significant improvements in self-reported health and self-efficacy for exercise in the program participant group as compared with non-participants. The results of this study suggest that a relatively low-cost, low-intensity exercise program such as the Forever…in motionprogram may positively influence the well-being of older adults living in congregate housing. However, additional research with a larger number of participants and a more rigorous study design is needed to further elucidate the health benefits of the Forever… in motionprogram. Le programme Forever… in motionétait l’un des éléments clés de la stratégie de promotion de la santé in motionimplantée en 2002 à Saskatoon. Globalement, le programme visait à accroître les possibilités d’activité physique offertes aux personnes âgées vivant dans des habitations collectives. Il comportait trois volets: un programme de conditionnement à faible impact, une socialisation informelle et des séances de sensibilisation. Nous avons voulu déterminer si la participation à ce programme a eu une influence positive sur le bien-être physique, affectif, psychologique et social des participants. Nous avons employé une méthode pré-test/post-test quasi expérimentale pour analyser l’incidence du programme sur divers aspects du bien-être. Trente-six participants au programme et un groupe témoin de 22 non-participants ont été recrutés dans deux habitations collectives. Le prétest a été administré au groupe des participants et au groupe témoin avant ou peu après le début de la session de 12 semaines. Le post-test a été administré après la fin des 12 semaines. L’évaluation pré- et post-test comportait des mesures autodéclarées: 1) de la vitalité, 2) de l’état de santé auto-évalué, 3) de la santé mentale, 4) des relations sociales, 5) des contraintes dues à des troubles affectifs, 6) des connaissances sur l’activité physique et 7) de l’auto-efficacité par rapport au conditionnement physique. Nous avons effectué une analyse multivariée de la covariance (mancova) en utilisant les sept scores au post-test comme variables dépendantes et les scores au pré-test comme covariables. Après les rajustements nécessaires pour tenir compte des écarts dans les caractéristiques de base, nous avons noté des améliorations significatives de l’état de santé auto-évalué et de l’auto-efficacité par rapport au conditionnement physique dans le groupe des participants au programme, comparés aux non-participants. Un programme d’activité physique à faible intensité et relativement peu coûteux, comme Forever… in motion, pourrait positivement influencer le bien-être des personnes âgées vivant dans des habitations collectives. Pour déterminer avec précision les avantages pour la santé de Forever... in motion, il faudrait pousser la recherche en recrutant un plus grand nombre de participants et en resserrant la méthode de l’étude.

Molecular dynamics simulations coupled with functional analyses of the major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p identify structural elements involved in regulating the ability of Sec14p to execute phospholipid exchange. The molecular dynamics simulations suggest large rigid body motions within the Sec14p molecule accompany closing and opening of an A10/T4/A11helical gate, and that "state-of-closure" of this helical gate determines access to the Sec14p phospholipid binding cavity. The data also project that conformational dynamics of the helical gate are controlled by a hinge unit (residues F212, Y213, K239, I240, and I242) that links to the N- and C-terminal ends of the helical gate, and by a novel gating module (composed of the B1LB2and A12LT5substructures) through which conformational information is transduced to the hinge. The 114TDKDGR119motif of B1LB2plays an important role in that transduction process. These simulations offer new mechanistic possibilities for an important half-reaction of the Sec14p phospholipid exchange cycle that occurs on membrane surfaces after Sec14p has ejected bound ligand, and is reloading with another phospholipid molecule. These conformational transitions further suggest structural rationales for known disease missense mutations that functionally compromise mammalian members of the Sec14-protein superfamily.

Molecular dynamics simulations coupled with functional analyses of the major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p identify structural elements involved in regulating the ability of Sec14p to execute phospholipid exchange. The molecular dynamics simulations suggest large rigid body motions within the Sec14p molecule accompany closing and opening of an A10/T4/A11helical gate, and that “state-of-closure” of this helical gate determines access to the Sec14p phospholipid binding cavity. The data also project that conformational dynamics of the helical gate are controlled by a hinge unit (residues F212, Y213, K239, I240, and I242) that links to the N- and C-terminal ends of the helical gate, and by a novel gating module (composed of the B1LB2and A12LT5substructures) through which conformational information is transduced to the hinge. The 114TDKDGR119motif of B1LB2plays an important role in that transduction process. These simulations offer new mechanistic possibilities for an important half-reaction of the Sec14p phospholipid exchange cycle that occurs on membrane surfaces after Sec14p has ejected bound ligand, and is reloading with another phospholipid molecule. These conformational transitions further suggest structural rationales for known disease missense mutations that functionally compromise mammalian members of the Sec14-protein superfamily.

Artificial transcription factors (ATFs) are proteins designed to specifically bind and regulate genes. Because of their DNA-binding selectivity and modular organization, arrays of zinc finger (ZF) domains have traditionally been used to build the ATF's DNA-binding domains. ATFs have been designed and constructed to regulate a variety of therapeutic targets. Recently, novel combinatorial technologies have been developed to induce expression of any gene of interest or to modify cellular phenotypes. Large repertoires of ATFs have been generated by recombination of all available sequence-specific ZF lexicons. These libraries comprise millions of ATFs with unique DNA-binding specificities. The ATFs are produced by combinatorial assembly of three- and six-ZF building blocks and are linked to activator or repressor domains. Upon delivery into a cell population, any gene in the human genome can potentially be regulated. ATF library members generate genome-wide, experimental perturbations of gene expression, resulting in a phenotypically diverse population, or cellular library. A variety of phenotypic screenings can be applied to select for cells exhibiting a phenotype of interest. The ATFs are then used as genetic probes to identify the targeted genes responsible for the phenotypic switch. In this review we will summarize several applications of ATF library screenings in gene discovery, biotechnology, and disease therapeutics.

Set2 methylation of histone H3 at lysine 36 (K36) has recently been shown to be associated with RNA polymerase II (Pol II) elongation in Saccharomyces cerevisiae. However, whether this modification is conserved and associated with transcription elongation in other organisms is not known. Here we report the identification and characterization of the Set2 ortholog responsible for K36 methylation in the fission yeast Schizosaccharomyces pombe. We find that similar to the budding yeast enzyme, S. pombe Set2 is also a robust nucleosome-selective H3 methyltransferase that is specific for K36. Deletion of the S. pombe set2+ gene results in complete abolishment of K36 methylation as well as a slow-growth phenotype on plates containing synthetic medium. These results indicate that Set2 is the sole enzyme responsible for this modification in fission yeast and is important for cell growth under stressed conditions. Using the chromatin immunoprecipitation assay, we demonstrate that K36 methylation in S. pombe is associated with the transcribed regions of Pol II-regulated genes and is devoid in regions that are not transcribed by Pol II. Consistent with a role for Set2 in transcription elongation, we find that S. pombe Set2 associates with the hyperphosphorylated form of Pol II and can fully rescue K36 methylation and Pol II interaction in budding yeast cells deleted for Set2. These results, along with our finding that K36 methylation is highly conserved among eukaryotes, imply a conserved role for this modification in the transcription elongation process.

ABSTRACTSet2 methylation of histone H3 at lysine 36 (K36) has recently been shown to be associated with RNA polymerase II (Pol II) elongation in Saccharomyces cerevisiae. However, whether this modification is conserved and associated with transcription elongation in other organisms is not known. Here we report the identification and characterization of the Set2 ortholog responsible for K36 methylation in the fission yeast Schizosaccharomyces pombe. We find that similar to the budding yeast enzyme, S. pombeSet2 is also a robust nucleosome-selective H3 methyltransferase that is specific for K36. Deletion of the S. pombe set2+gene results in complete abolishment of K36 methylation as well as a slow-growth phenotype on plates containing synthetic medium. These results indicate that Set2 is the sole enzyme responsible for this modification in fission yeast and is important for cell growth under stressed conditions. Using the chromatin immunoprecipitation assay, we demonstrate that K36 methylation in S. pombeis associated with the transcribed regions of Pol II-regulated genes and is devoid in regions that are not transcribed by Pol II. Consistent with a role for Set2 in transcription elongation, we find that S. pombeSet2 associates with the hyperphosphorylated form of Pol II and can fully rescue K36 methylation and Pol II interaction in budding yeast cells deleted for Set2. These results, along with our finding that K36 methylation is highly conserved among eukaryotes, imply a conserved role for this modification in the transcription elongation process.

A survey of kcat/Km values supports the view that most enzymes combine with substrates at rates that approach the limits imposed by diffusional encounter. The closeness of that approach precludes the obligate participation of any rare species of the substrate, or any rare species of the enzyme, in productive binding. Proceeding from the ground state through the transition state, the enzyme–substrate complex is assumed to remain in a state of quasi-equilibrium with the free enzyme and the unbound substrate in solution. Because the forces of attraction between an enzyme and substrate approach a maximum in the transition state for substrate transformation, the enzyme and substrate are also likely to approach a maximal state of distortion from their native ground-state structures at that stage in the reaction, furnishing a natural explanation of induced fit. There is now abundant evidence, from mutations of enzymes and substrates, that interactions between the enzyme and substrate, using ordinary chemical forces of attraction, are so strongly synergistic that they may suffice to explain the very high affinities that are achieved in the transition state. Copyright © 2004 John Wiley & Sons, Ltd.

A survey of kcat/Kmvalues supports the view that most enzymes combine with substrates at rates that approach the limits imposed by diffusional encounter. The closeness of that approach precludes the obligate participation of any rare species of the substrate, or any rare species of the enzyme, in productive binding. Proceeding from the ground state through the transition state, the enzyme–substrate complex is assumed to remain in a state of quasi‐equilibrium with the free enzyme and the unbound substrate in solution. Because the forces of attraction between an enzyme and substrate approach a maximum in the transition state for substrate transformation, the enzyme and substrate are also likely to approach a maximal state of distortion from their native ground‐state structures at that stage in the reaction, furnishing a natural explanation of induced fit. There is now abundant evidence, from mutations of enzymes and substrates, that interactions between the enzyme and substrate, using ordinary chemical forces of attraction, are so strongly synergistic that they may suffice to explain the very high affinities that are achieved in the transition state. Copyright © 2004 John Wiley & Sons, Ltd.

Calcium-binding domains in the α-subunit of integrins contain a central loop structure. To examine the importance of the loop structure, a series of αIIb mutants containing changes to the calcium-liganding amino acids have been constructed. Significantly, none of the mutant αIIbβ3 complexes was detected on the surface of transfected cells, but mutant pro-αIIb was detected in cell lysates in complex with β3. To study the importance of the regions flanking the second calcium-binding domain for ligand-binding and ligand-binding specificity, three αIIb/α5 chimaeras containing α5 sequences flanking or flanking and including the second calcium-binding domain were constructed. The chimaera containing both α5-flanking regions was not expressed on the cell surface, but FR1 and FR2, substituting either the first or second flanking region, were expressed. FR1β3-transfected cells lost the ability to adhere to fibrinogen and to support aggregation and had minimal fibrinogen-binding ability. The heterodimer complex was less stable than the wild-type. FR2β3-transfected cells adhered to fibrinogen and bound soluble fibrinogen with higher affinity when compared with wild-type. In addition, the heterodimer complex was more stable than wild-type. These results indicate that the conformation of the second calcium-binding domain is critical for maturation of the αIIbβ3 complex and expression on the cell surface and that the surrounding sequences are critical for αIIbβ3 function.

ABSTRACTThe adeno-associated virus type 2 (AAV) replication (Rep) proteins Rep78 and 68 (Rep78/68) exhibit a number of biochemical activities required for AAV replication, including specific binding to a 22-bp region of the terminal repeat, site-specific endonuclease activity, and helicase activity. Individual and clusters of charged amino acids were converted to alanines in an effort to generate a collection of conditionally defective Rep78/68 proteins. Rep78 variants were expressed in human 293 cells and analyzed for their ability to mediate replication of recombinant AAV vectors at various temperatures. The biochemical activities of Rep variants were further characterized in vitro by using Rep68 His-tagged proteins purified from bacteria. The results of these analyses identified a temperature-sensitive (ts) Rep protein (D40,42,44A-78) that exhibited a delayed replication phenotype at 32°C, which exceeded wild-type activity by 48 h. Replication activity was reduced by more than threefold at 37°C and was undetectable at 39°C. Stability of the Rep78 protein paralleled replication levels at each temperature, further supporting atsphenotype. Replication differences resulted in a 3-log-unit difference in virus yields between the permissive and nonpermissive temperatures (2.2 × 106and 3 × 103, respectively), demonstrating that this is a relatively tight mutant. In addition to the tsRep mutant, we identified a nonconditional mutant with a reduced ability to support viral replication in vivo. Additional characterization of this mutant demonstrated an Mg2+-dependent phenotype that was specific to Rep endonuclease activity and did not affect helicase activity. The two mutants described here are unique, in that Rep tsmutants have not previously been described and the D412A Rep mutant represents the first mutant in which the helicase and endonuclease functions can be distinguished biochemically. Further understanding of these mutants should facilitate our understanding of AAV replication and integration, as well as provide novel strategies for production of viral vectors.

Fogo selvagem (FS) is a blistering skin disease caused by pathogenic IgG4 autoantibodies to desmoglein 1 (DSG1). Preclinical FS and leishmaniasis are endemic to certain regions of Brazil and exhibit nonpathogenic anti-DSG1 antibodies. Recurring bites from Lutzomyia longipalpis, the sand fly vector of leishmaniasis, immunize individuals with L. longipalpissalivary antigens LJM17 and LJM11. We measured the antibody responses to LJM17, LJM11, and DSG1 in normal settlers and patients with FS from an endemic focus of FS and nonendemic control populations. We also immunized mice with these antigens and assessed the IgG response. Healthy individuals and patients with FS from endemic areas had significantly higher values of IgG4 anti-LJM17 antibodies than nonendemic controls (P< 0.001 for both). The levels of IgG anti-DSG1 and IgG4 anti-LJM17 and anti-LJM11 antibodies correlated positively in normal settlers and patients with FS. Mice immunized with recombinant LJM17 produced IgG1 antibodies (human IgG4 homolog) that strongly cross-reacted with recombinant DSG1; these IgG1 antibodies were inhibited by LJM17, LJM11, and DSG1 in a dose-dependent manner. However, they did not bind human or mouse epidermis by indirect immunofluorescence. Lastly, we identified short-sequence homologies of surface-exposed residues within the human DSG1 ectodomain and LJM17. Inoculation by LJM17 from L. longipalpis-elicited DSG1–cross-reactive IgG4 antibodies may lead to FS in genetically predisposed individuals.

The ArabidopsisG protein α subunit exhibits properties necessary and sufficient for receptor-independent activation.