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Endonuclease VII is an enzyme from bacteriophage T4 capable of resolving four-arm Holliday junction intermediates in recombination. Since natural Holliday junctions have homologous (2-fold) sequence symmetry, they can branch migrate, creating a population of substrates that have the branch point at different sites. We have explored the substrate requirements of endonuclease VII by using immobile analogs of Holliday junctions that lack this homology, thereby situating the branch point at a fixed site in the molecule. We have found that immobile junctions whose double-helical arms contain fewer than nine nucleotide pairs do not serve as substrates for resolution by endonuclease VII. Scission of substrates with 2-fold symmetrically elongated arms produces resolution products that are a function of the particular arms that are lengthened. We have confirmed that the scission products are those of resolution, rather than nicking of individual strands, by using shamrock junction molecules formed from a single oligonucleotide strand. A combination of end-labeled and internally labeled shamrock molecules has been used to demonstrate that all of the scission is due to coordinated cleavage of DNA on opposite sides of the junction, 3' to the branch point. Endonuclease VII is known to cleave the crossover strands of Holliday junctions in this fashion. The relationship of the long arms to the cleavage direction suggests that the portion of the enzyme which requires the minimum arm length interacts with the pair of arms containing the 3' portion of the crossover strands on the bound surface of the antiparallel junction.

Three heterochromatin-like domains have been identified in Saccharomyces cerevisiae that are refractory to transcription by Pol II, the silent mating-type loci, telomeres and the ribosomal DNA. Previous work has shown that chromatin remodelers can regulate silent chromatin. Here, we report the findings of an investigation into the role of ISW2 in transcriptional silencing at the rDNA. We show that the levels of retrotransposition and mRNA from a genetically marked Ty1 element located in the rDNA were increased significantly in isw2Delta cells, while transcript levels from Ty1 elements outside of the rDNA were not increased in cells lacking ISW2. Additionally, we show that Isw2 is not required for silencing at a telomere. Our findings demonstrate that Isw2 is required for transcriptional silencing at the rDNA and emphasize the differences in the regulation of transcriptional silencing at silent loci in S. cerevisiae.

A 3-year field study was conducted at Blackville, S.C., to evaluate the potential of using resistant pepper (Capsicum annuum L.) cultivars as a rotation crop for managing the southern root-knot nematode [Meloidogyne incognita (Kofoid and White) Chitwood]. The experiment was a split-plot with main plots arranged in a randomized complete-block design. In 1993, the entire experimental site was infested with M. incognita by inoculating a planting of susceptible PA-136 cayenne pepper with eggs of M. incognita race 3. In 1994, the main plots were planted to either highly resistant `Carolina Cayenne' or its susceptible sibling line PA-136. In 1995, `Carolina Cayenne' and the susceptible bell cultivars California Wonder and Keystone Resistant Giant were grown as subplots in each of the original main plots. `Carolina Cayenne' plants were unaffected by the previous crop. Previous cropping history, however, had a significant impact on the performance of the bell cultivars; the mean galling response was less (P < 0.01) and the yield was 2.8 times greater (P < 0.01) in the main plots previously cropped with `Carolina Cayenne' than in those previously cropped with PA-136. These results suggest that resistant pepper cultivars have considerable merit as a rotation crop for managing M. incognita infestations in soils used for growing high-value vegetables.

The Set1-containing complex, COMPASS, methylates histone H3 on lysine 4 (K4) in Saccharomyces cerevisiae. Despite the preferential association of K4-trimethylated H3 with regions of the genome that are transcribed by RNA polymerase II, transcriptional silencing is one of the few cases in S. cerevisiae where histone-methylation defects have a clear effect on gene expression. To better understand the role of COMPASS in transcriptional silencing, we have determined which members of COMPASS are required for silencing at the ribosomal DNA locus (rDNA), a telomere, and the silent mating loci (HM) using Northern analyses. Our findings indicate that most members of COMPASS are required for silencing at the rDNA and telomere, while none are required for silencing of endogenous genes at the HM loci. To complement gene-expression analysis, quantitative Western blot experiments were performed to determine the members of COMPASS that are required for methylation of histone H3. While most are required for trimethylation, cells lacking certain COMPASS proteins maintain reduced levels of K4 mono- and dimethylated H3, suggesting that some COMPASS members have redundant function. Finally, we show Paf1 is required for silencing and K4-methylated H3 at the rDNA, suggesting a possible direct role for K4-methylated H3 in gene silencing.

Rhizoctonia foliar blight of soybean is found in tropical and subtropical areas worldwide. In the US, it is found mainly in the states of the Gulf Coast region. On 13 September, 2005, the Clemson University Plant Problem Clinic received a soybean sample from Florence County, SC showing severe foliar blight. The cultivars Northrup King 7325 and 76-L9 were affected. Hyphae typical of Rhizoctonia species were observed on the foliar lesions, and the genus was confirmed by microscopic examination. Accepted for publication 7 August 2006. Published 30 October 2006.

In This Chapter Learn How to Install Web Matrix Learn How to Install the Microsoft Mobile Internet Toolkit Discover the Web Matrix IDE Elements Learn How to Use FTP Workspaces Create Pseudo-Project-Oriented Workspaces Create Projectless Workspaces Discover the Online Component Gallery Learn About the Web Matrix File Types

In This Chapter Learn Methods to Improve Unmanaged Component Performance Discover Downloadable Controls That Improve User Performance Learn How to Use Output Caching Learn How to Work with the Performance Console CCreate a Vary By None Page Create a Vary Cache By Browser Page reate a Vary Cache By Headers Page Create a Vary Cache By Parameters Page

In This Chapter Use the XML Support That Web Matrix Provides Use Microsoft XML Notepad to View XML-Formatted Files Create Applications with the XMLEditGrid Control Create Applications with the XML File Page Create Applications with the XSL Transform Page Create Applications with the XML Schema Page Design an XML Data Display Application

In This Chapter Create a Mobile Device Emulation Environment Learn to Design with Mobile Support in Mind Create an Application with the Simple Mobile Page Design a Simple Mobile Solution Create an Application with the Simple Mobile User Control Design a Mobile Solution with User Controls Design a Secure Mobile Application Learn to Create Applications for a Specific Device

In This Chapter Learn How to Configure MSDE Using MSDE Query Learn How to Use the Code Builders Build Your Own SQL Scripts Create an Application Using a Simple Stored Procedure Create an Application Using the DataList and Repeater Controls Develop a Simple Data Display Application Discover How to Use Data Grids Discover How to Use Data Reporting Develop a Complete Database Application Learn About the Limitations of the Web Matrix Approach

In This Chapter Learn How to Access Files Using Web Matrix Create an HTML Page Create a Text File Create a Style Sheet Develop a Simple HTML Example Learn to Use the ASP.NET Page Discover the Differences Between Code Behind and Inline Code Create a WEB.CONFIG Page Create a GLOBAL.ASAX Page Learn About the ASP.NET HTTP Handler Page

In This Chapter Understand How Web Services Affect You Develop Applications Using XML Web Services Learn to Use the Simple Page Learn to Use the SOAP Headers Page Explore Techniques for Developing Custom Web Service Pages Create a Simple Web Service Application Learn to Use the Custom Class Create a Custom Class Learn to Use Output Caching Design with Enhanced Performance in Mind

In This Chapter Discover What Web Matrix Can Do for You Learn HowYou Can Use Web Matrix in Your Next Project Learn About Web Matrix Limitations Obtain an Overview of the Web Matrix Features Create a Development Server Setup Create a Development Client Setup

In This Chapter Learn to Use the Component and Control Tools Provided with the .NET Framework Develop Applications Using Existing Components Customize the Toolbox Tabs in Web Matrix to Meet Your Needs Develop Applications Using Existing Controls Learn to Use the Class Page Create an Application That Implements a Component Learn to Use the ASP.NET User Control Create an Application That Uses an ASP.NET User Control

In This Chapter Understand Web Site Security Requirements Create a Project with the Login Page Create a Project with the Logout Page Create a Project with the Config File Page Learn Methods for Creating a Better Password Scheme Design a General Security Example Use Windows-Validated Security

In today's rapidly changing technological world, it is increasingly important that web and desktop applications be accessible to everyone, including those of us with special needs. However, the legal requirements for accessibility are often convoluted and diffucult to understand, making it impossible for most developers to comply with the laws. In the first book on the market to address the topic, Author John Paul Mueller leads you through the maze of Section 508the congressional law ensuring that all Americans have access to information technologyand offers comprehensive guidance on how to quickly and easily update your applications to conform to Section 508 requirements. To begin, Mueller outlines the Section 508 requirements that are relevant to web and desktop application developers, and explains the laws in language you can understand. Using extensive and varied program examples, Mueller continues on to teach you to write web and desktop applications that not only conform to Section 508 standards, but look forward to ensure long-term universal accessibility for all types of computer users. Specific topics include writing accessible web applications and desktop applications using Visual Basic .NET and Visual C# .NET, developing special application capabilities, using Microsoft and other third-party tools to develop accessible features, developing accessible scripting solutions, and more. The book even includes a directory of helpful organizations, agencies, and resources to help you with any further accessibility concerns you may have.

A new equisetin derivative, CJ-21,058 (I) was isolated from the fermentation broth of an unidentified fungus CL47745. It shows antibacterial activity against Gram-positive multi-drug resistant bacteria by inhibiting ATP-dependent translocation of precursor proteins across a bacterial cell membrane.

Equipment was developed and tested for injection and broadcast application of municipal solid waste (MSW) compost at selected rates to agricultural land for cotton production. Replicated tests were conducted to determine the effects of injected versus broadcast applied compost on soil parameters (organic matter, soil compaction, and soil fertility) and plant growth. All broadcast application rates of compost significantly reduced hardpan formation in the top 15 cm of soil compared to no compost application. In addition, all rates of injected material significantly reduced soil compaction in the E– and B–horizons (15 to 45 cm). Broadcast application of compost significantly increased soil organic matter content 6 and 12 weeks after planting proportional to the compost application rate. In addition, soil nitrogen content was significantly higher in the broadcast application plots 6 weeks after planting. MSW compost (broadcast or injected) significantly increased plant N, P, and K contents compared to no compost application. Increases in plant nitrogen were proportional to application rates. In addition, injected application increased plant sulfur compared to no compost application. All rates of compost (injected or broadcast applications) significantly increased cotton lint yield compared to no compost application. Yield increase was proportional to application rates. For the 26.9–Mg/ha injected application treatment, yield increases were 23, 24, and 44% in 1997, 1998, and 1999, respectively, compared to no compost application. Vitazyme increased plant N, P, and K contents with no effects on Ca, Mg, and S. Vitazyme increased cotton lint yield 35 kg/ha or 3%. In addition, soil nitrogen content 6 weeks after planting in plots treated with Vitazyme was 12% higher than no–Vitazyme plots.

A model study was performed to evaluate a new in-stream grade-control structure. A rock chute, a loose riprap structure, was modified to include three riffle-pool sequences. The structure is designed as an in-stream grade-stabilization measure that allows fish passage and biological enhancement. This paper summarizes the results of a three phase model study that examined the seepage characteristics, rock stability, flow velocities, and overall hydraulic performance of this structure. The models suggest that the structure will perform well and provide a viable new treatment technique for in- stream grade stabilization.

Recent studies have suggested that soluble forms of B7-1 and B7-2 may exist, but transcripts that code for these molecules have not been previously described. In this study, we report the cloning and characterization of an alternatively spliced soluble form of porcine B7-1 (sB7-1) that lacks exons coding for both the transmembrane and cytoplasmic domains. Northern blot analysis of RNA from alveolar macrophages revealed an approximate 3:1 ratio of the transmembrane form of B7-1 mRNA relative to sB7-1 mRNA. Porcine B7-1 was present on the surface of both B and T cells following stimulation with PMA/ionomycin. A histidine-tagged form of porcine sB7-1 (sB7-1-His) interacted with both CD28 and CTLA-4, and effectively blocked IL-2 production from human responder cells stimulated with PHA and either porcine or human stimulator cells. In addition, sB7–1-His inhibited human T cell proliferation in response to porcine or human peripheral blood leukocytes. This study is the first report of an alternatively spliced form of B7 that codes for a soluble protein. Furthermore, these data demonstrate that porcine B7-1 interacts with the human receptors CD28 and CTLA-4, suggesting a potential role for this molecule in pig to human xenotransplantation. Possible physiological functions for the soluble form of B7-1 are discussed.

The properties that make DNA such an effective molecule for its biological role as genetic material also make it a superb molecule for nanoconstruction. One key to using DNA for this purpose is to produce stable complex motifs, such as branched molecules. Combining branched species by sticky ended interactions, leads to N- connected stick figures whose edges consist of double helical DNA. Zero node removal or reciprocal crossover, leads to complex fused motifs, such as rigid multi-crossover molecules and paranemic crossover molecules. Multi-crossover molecules have been used to produce 2D arrays and a nanomechanical device. Algorithmic assembly and the use of complex complementarities for joining units are goals in progress that are likely to produce new capabilities for DNA nanotechnology.

Discordant xenografts surviving the initial hyperacute rejection phase may be subject to cellular rejection processes mediated by infiltrating leukocytes including T cells, NK cells and monocytes. The stable adhesion of these cell types to endothelial cells is due to the molecular interaction of the integrins VLA-4 and LFA-1 with their ligands vascular cell adhesion molecule (VCAM) and ICAM-1 present on the endothelial cells. Human VLA-4 binds to porcine VCAM, and blocking mAbs specific for porcine VCAM have been developed. We have localized the epitope of the anti-porcine VCAM blocking mAbs 2A2 and 3F4 to domains 1 and 2, respectively. Humanized antibodies (IgG4 isotype) were constructed from these anti-porcine VCAM antibodies and demonstrated to inhibit adhesion of Ramos, Jurkat and YT cells, as well as purified resting and activated human T cells, to porcine aortic endothelial cells (PAEC). These cell types express both LFA-1 as well as VLA-4, suggesting blockade of human VLA-4 interaction with porcine VCAM may alone be sufficient to significantly impair adhesion of human leukocytes to porcine endothelial cells. The chimeric anti-porcine VCAM (pVCAM) HuG4 antibodies promoted increased adhesion of Fc receptor (FcR) positive cells such as U937 monocytic cells to PAEC. In contrast, chimeric anti-porcine VCAM antibodies created using the CH1 and hinge region from human IgG2 and the CH2 and CH3 regions from human IgG4 (HuG2/G4 antibodies) inhibited binding of FcR positive cells to PAEC. These chimeric anti-pVCAM antibodies should allow delineation of the in vivo role of VLA-4/VCAM interaction in porcine-to-primate xenotransplants. Further, the design of the HuG2/G4 antibodies should render them efficacious in multiple settings requiring elimination of FcR binding.

It has been shown that peripheral T cell tolerance can be induced by systemic antigen administration. We have been interested in using this phenomenon to develop antigen-specific immunotherapies for T cell-mediated autoimmune diseases. In patients with the demyelinating disease multiple sclerosis (MS), multiple potentially autoantigenic epitopes have been identified on the two major proteins of the myelin sheath, myelin basic protein (MBP) and proteolipid protein (PLP). To generate a tolerogenic protein for the therapy of patients with MS, we have produced a protein fusion between the 21.5-kD isoform of MBP (MBP21.5) and a genetically engineered form of PLP (deltaPLP4). In this report, we describe the effects of treatment with this agent (MP4) on clinical disease in a murine model of demyelinating disease, experimental autoimmune encephalomyelitis (EAE). Treatment of SJL/J mice with MP4 after induction of EAE either by active immunization or by adoptive transfer of activated T cells completely prevented subsequent clinical paralysis. Importantly, the administration of MP4 completely suppressed the development of EAE initiated by the cotransfer of both MBP- and PLP-activated T cells. Prevention of clinical disease after the intravenous injection of MP4 was paralleled by the formation of long-lived functional peptide-MHC complexes in vivo, as well as by a significant reduction in both MBP- and PLP-specific T cell proliferative responses. Mice treated with MP4 were resistant to disease when rechallenged with an encephalitogenic PLP peptide emulsified in CFA, indicating that MP4 administration had a prolonged effect in vivo. Administration of MP4 was also found to markedly ameliorate the course of established clinical disease. Finally, MP4 therapy was equally efficacious in mice defective in Fas expression. These results support the conclusion that MP4 protein is highly effective in suppressing disease caused by multiple neuroantigen epitopes in experimentally induced demyelinating disease.

Numerous group I introns in both prokaryotes and eukaryotes behave as mobile genetic elements. The functional requirements for intron mobility were determined in the T4 phage system using an in vivo assay to measure intron homing with wild-type and mutant derivatives. Thus, it was demonstrated that intron mobility occurs in the context of phage recombination-dependent replication, a pathway that uses overlapping subsets of replication and recombination functions. The functional requirements for intron homing and the nature of recombinant products are only partially consistent with the accepted double-strand-break repair (DSBR) model for intron inheritance, and implicate additional homing pathways. Whereas ambiguities in resolvase requirements and underrepresentation of crossover recombination products are difficult to rationalize strictly by DSBR, these properties are most readily consistent with a synthesis-dependent strand annealing (SDSA) pathway. These pathways share common features in the strand invasion steps, but differ in subsequent repair synthesis and resolution steps, influencing the genetic consequences of the intron transfer event.

I-TevI, a double-strand DNA endonuclease involved in the mobility of the td intron of phage T4, is highly unusual in that it binds and cleaves intronless td alleles (td homing sites) in a site-specific but sequence-tolerant manner. The endonuclease binds to sequences flanking the intron insertion site and near the remote cleavage site, located 23 and 25 nucleotides away on the top and bottom strands, respectively. Mapping studies indicate that I-TevI has both sequence and distance sensors that function during cut-site selection. Although I-TevI cleavage of many insertion and deletion variants of the homing site is impaired, double-strand breaks are generated at positions that collectively span two turns of the helix, indicating that the interaction is extraordinarily flexible. However, the endonuclease does exhibit spacing preferences between its binding domains, and sequence preferences near the cleavage site, with the G:C pair at -23 implicated as a cleavage determinant. Furthermore, I-TevI appears to function through interactions across the minor groove at the cleavage site, as it does at the intron insertion site, and to be capable of cleaving sequentially, first on the bottom and then on the top strand. These properties of I-TevI are incorporated in a model wherein the endonuclease effects distant cleavage via a flexible hinge.

Exposure of mature T cells to their specific antigen normally results in proliferation, but in the presence of high and repeated doses of antigen we have found that these same T cells undergo programmed cell death. The conditions required for antigen to induce the death of mature T cells include: an initial mitogenic stimulus that induces T cell responsiveness to growth lymphokines such as interleukin 2 (IL-2) or interleukin 4 (IL-4), T cell progression through the cell cycle, and finally strong T cell receptor (TCR) restimulation while still under the influence of growth lymphokine. Our data suggest that antigen-induced T cell death is part of a feedback regulatory loop which senses and controls the magnitude of the T cell response to antigen, that we have termed, “Propriocidal regulation.” Propriocidal regulation provides an explanation for the phenomenon of high dose suppression, and a mechanism for the peripheral deletion of T cells resulting in antigen-specific immunological tolerance. In this review, we will discuss data demonstrating the effeciveness of an immunotherapy based on the propriocidal mechanism to delete autoantigen-reactive T cells in experimental allergic encephalomyelitis (EAE), a mouse model for multiple sclerosis (MS).

The control of structure on the nanoscale relies on intermolecular interactions whose specificity and geometry can be treated on a predictive basis. DNA fulfills this criterion, and provides an extremely convenient construction medium: The sticky-ended association of DNA molecules occurs with high specificity, and it results in the formation of double helical DNA, whose structure is well known. The use of stable branched DNA molecules permits one to make stick-figures. We have used this strategy to construct in solution a covalently closed DNA molecule whose helix axes have the connectivity of a cube: The molecule has twelve double helical edges; every edge is two helical turns in length, resulting in a hexacatenane, each of whose strands corresponds to a face of the object. We have developed a solid-support-based synthetic methodology that is more effective than solution synthesis. The key features of the technique are control over the formation of each edge of the object, and the topological closure of each intermediate. The isolation of individual objects on the surface of the support eliminates cross-reactions between growing products. The solid-support-based methodology has been used to construct a molecule whose helix axes have the connectivity of a truncated octahedron. This figure has 14 faces, of which six are square and eight are hexagonal; this Archimedean polyhedron contains 24 vertices and 36 edges, and is built from a 14-catenane of DNA. Knotted molecules appear to be the route for cloning DNA objects. It is possible to construct three knotted topologies, as well as a simple cyclic molecule from a single precursor, by control of solution conditions. Control of both branching and braiding topology is strong in this system, but control of 3-D structure remains elusive. Our key aim is the formation of prespecified 2-D and 3-D periodic structures for use in diffraction experiments. Another application envisioned is scaffolding for the assembly of molecular electronic devices.

Activation kinetics of a Bacillus subtilis menaquinone biosynthetic gene promoter (the menCD promoter) were measured during growth and sporulation, with the aid of a menCD'-lacZ translational gene fusion. Transient maximal activation was seen shortly after the end of exponential growth in unbuffered complex medium containing a low glucose concentration. These activation kinetics were correlated with transient acidification of the medium under conditions permitting TCA cycle function during the post-exponential period, while mutations that blocked TCA cycle function (cit mutants) were associated with sustained acidification and promoter activation during this period. In cit+ strains, buffering of the medium to pH 5.7 caused sustained maximal activation, while buffering to pH 7.2 prevented enhancement of activation. The menCD promoter appears to be responsive to extracellular acidic pH.

Target spot of soybean (Glycine max (L.) Merr.) caused by Corynespora cassiicola (Berk. & Curt.), although found in most soybean-growing countries, is considered to be a disease of limited importance (1) and has never been reported to cause soybean yield loss in the southeastern United States (2,3). Soybean plants submitted to the North Carolina Plant Disease and Insect Clinic (NCPDIC) in August 2004 from Beaufort, Robeson, Wilson, and Johnston counties, NC had symptoms consistent with target spot. Symptoms consisted of roughly circular, necrotic leaf lesions from minute to 11 mm in diameter, though typically approximately 4 to 5 mm in diameter, and with a yellow margin. Large lesions occasionally exhibited a zonate pattern often associated with this disease. Microscopic examination of the lesions revealed the presence of spores (conidia) typical of C. cassiicola (1). Conidia were mostly three to five septate with a central hilum at the base and ranged in size from 7 to 22 wide × 39 to 520 μm long. Three commercial soybean fields near Blackville, SC (Barnwell County) were severely affected by this disease and it caused premature defoliation. Nineteen of twenty-seven maturity group VII and VIII genotypes in the 2004 Clemson University soybean variety trial near Blackville, SC had visible symptoms of target spot. Heavy rainfall associated with hurricanes during September 2004 probably enhanced the incidence of this disease, and yield suppression due to target spot was estimated at 20 to 40% in some fields. In 2005, 20 of 161 soybean samples submitted to the NCPDIC or collected in surveys from 16 counties were positive for target spot on the basis of microscopic examination. Target spot also was diagnosed in six counties (Baldwin, DeKalb, Elmore, Fayette, Macon, and Pickens) in Alabama and in four additional counties (Bamberg, Hampton, Orange-burg, and Calhoun) in South Carolina in 2005. Records from the NCPDIC indicate that target spot had not been diagnosed on soybean in North Carolina since 1981. The large increase in incidence of target spot in the southeast may be related to changes in weather patterns, changes in pathogen virulence, and/or the introduction of more susceptible host genotypes. References: (1) J. B. Sinclair. Target spot. Page 27 in: Compendium of Soybean Diseases. G. L. Hartman et al. eds. The American Phytopathological Society, St. Paul, MN, 1999. (2) J. A. Wrather et al. Plant Dis. 79:1076. 1995. (3) J. A. Wrather et al. On-line publication. doi:10.1094/PHP-2003-0325-01-RV. Plant Health Progress, 2003.

A root-knot nematode (Meloldogyne incognita) project was initiated in a field of infested sandy loam (EREC) in 1991 and continued. There were ten sweetpotato entries consisting of six cultivars (Beauregard. Excel, Georgia Jet, Jewel, Red Jewel, and Sumor), three advanced lines (W-270, W-274, and W-279) and PI 399161 which were selected for their diversity in disease reactions and other traits. Each entry was planted in the same plots each year to monitor effects of continuous cropping, disease reactions, yield and population shifts of the pathogen. Marketable yields were reduced each year for Georgia Jet and Red Jewel, but not for Beauregard. Internal necrosis in the storage roots was most severe for Beauregard. Several of the highly resistant entries, especially Sumor and W-279, performed well each year, including high yields, good quality. and little or no nematode reproduction. This study demonstrates the considerable economic benefits of a high level of durable resistance to root knot in sweetpotato.