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Minimally invasive exodontia is among the long-sought-for development aims of safe dental medicine. In this paper, we aim, for the first time, to examine whether the enzymatic disruption of the periodontal ligament fibers reduces the force required for tooth extraction. To this end, recombinantly expressed clostridial collagenase G variant purified from Escherichia coli was injected into the periodontal ligament of mesial and distal roots of the first and second split porcine mandibular premolars. The vehicle solution was injected into the corresponding roots on the contralateral side. Following sixteen hours, the treated mandibles were mounted on a loading machine to measure the extraction force. In addition, the effect of the enzyme on the viability of different cell types was evaluated. An average reduction of 20% in the applied force (albeit with a large variability of 50 to 370 newton) was observed for the enzymatically treated roots, reaching up to 50% reduction in some cases. Importantly, the enzyme showed only a minor and transient effect on cellular viability, without any signs of toxicity. Using an innovative model enabling the analytical measurement of extraction forces, we show, for the first time, that the enzymatic disruption of periodontal ligament fibers substantially reduces the force required for tooth extraction. This novel technique brings us closer to atraumatic exodontia, potentially reducing intra- and post-operative complications and facilitating subsequent implant placement. The development of novel enzymes with enhanced activity may further simplify the tooth extraction process and present additional clinical relevance for the broad range of implications in the oral cavity.

Objective : To assess the prevalence of posterior crossbite and/or deep bite among patients diagnosed with temporomandibular disorders (TMD) compared to a non-TMD population. Methods : Four hundred ninety-four patients were enrolled: 345 with TMD according to the diagnostic criteria for TMD and 149 without TMD (control group). The chi-square test was used for statistical analysis. Results : A statistically significant association was found between sleep and awake bruxism and painful TMDs ( p < .001 for both), but not with disc displacement. There was no association between posterior crossbite and/or deep bite with either painful TMD or disc displacement. Conclusion : Within the study limitations, sleep bruxism and awake bruxism may be related to pain in the TMD, and the severity of a deep overbite and presence of a unilateral or bilateral posterior crossbite should not be considered risk factors or as having any effect on the pain associated with TMD and/or disc displacement.

Populations of Eruca sativa (Brassicaceae) from desert and Mediterranean (Med) habitats in Israel differ in their defense against larvae of the generalist Spodoptera littoralis but not the specialist Pieris brassicae. Larvae of the generalist insect feeding on plants of the Med population gained significantly less weight than those feeding on the desert plants, and exogenous application of methyl jasmonate (MJ) on leaves of the Med plants significantly reduced the level of damage created by the generalist larvae. However, MJ treatment significantly induced resistance in plants of the desert population, whereas the generalist larvae caused similar damage to MJ-induced and noninduced plants. Analyses of glucosinolates and expression of genes in their synthesis pathway indicated that defense in plants of the Med population against the generalist insect is governed by the accumulation of glucosinolates. In plants of the desert population, trypsin proteinase inhibitor activity was highly induced in response to herbivory by S. littoralis. Analysis of genes in the defense-regulating signaling pathways suggested that in response to herbivory, differences between populations in the induced levels of jasmonic acid, ethylene, and salicylic acid mediate the differential defenses against the insect. In addition, expression analysis of myrosinase-associated protein NSP2 suggested that in plants of the desert population, glucosinolates breakdown products were primarily directed to nitrile production. We suggest that proteinase inhibitors provide an effective defense in the desert plants, in which glucosinolate production is directed to the less toxic nitriles. The ecological role of nitrile production in preventing infestation by specialists is discussed.

The viral V2 protein is one of the key factors that Tomato yellow leaf curl geminivirus (TYLCV), a major tomato pathogen worldwide, utilizes to combat the host defense. Besides suppressing the plant RNA silencing defense by targeting the host SGS3 component of the silencing machinery, V2 also interacts with the host CYP1 protein, a papain-like cysteine protease likely involved in hypersensitive response reactions. The biological effects of the V2-CYP1 interaction, however, remain unknown. We addressed this question by demonstrating that V2 inhibits the enzymatic activity of CYP1, but does not interfere with post-translational maturation of this protein., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Tomato yellow leaf curl virus (TYLCV), a major tomato pathogen causing extensive crop losses, is a whitefly-transmitted geminivirus. V2 mutants of TYLCV-Is and related viruses tend to induce symptomless infection with attenuated viral DNA levels, while accumulating close to wild-type DNA levels in protoplasts, suggesting V2 as a movement protein. The discovery of plant-silencing mechanisms and viral silencing suppressors, V2 included, led us to reconsider V2׳s involvement in viral movement. We studied two mutant versions of the virus, one impaired in V2 silencing-suppression activity, and another carrying a non-translatable V2. While both mutant viruses spread in the infected plant to newly emerged leaves at the same rate as the wild-type virus, their DNA-accumulation levels were tenfold lower than in the wild-type virus. Thus, we suggest that the setback in virus proliferation, previously ascribed to a movement impediment, is due to lack of silencing-suppression activity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

One the most intriguing, yet least studied, aspects of the bacterium-host plant interaction is the role of the host ubiquitin/proteasome system (UPS) in the infection process. Increasing evidence indicates that pathogenic bacteria subvert the host UPS to facilitate infection. Although both mammalian and plant bacterial pathogens are known to use the host UPS, the first prokaryotic F-box protein, an essential component of UPS, was identified in Agrobacterium. During its infection, which culminates in genetic modification of the host cell, Agrobacterium transfers its T-DNA--as a complex (T-complex) with the bacterial VirE2 and host VIP1 proteins--into the host cell nucleus. There the T-DNA is uncoated from its protein components before undergoing integration into the host genome. It has been suggested that the host UPS mediates this uncoating process, but there is no evidence indicating that this activity can unmask the T-DNA molecule. Here we provide support for the idea that the plant UPS uncoats synthetic T-complexes via the Skp1/Cullin/F-box protein VBF pathway and exposes the T-DNA molecule to external enzymatic activity.

The V2 protein of Tomato yellow leaf curl geminivirus (TYLCV) is an RNA-silencing suppressor that counteracts the innate immune response of the host plant. However, this anti-host defense function of V2 may include targeting of other defensive mechanisms of the plant. Specifically, we show that V2 recognizes and directly binds the tomato CYP1 protein, a member of the family of papain-like cysteine proteases which are involved in plant defense against diverse pathogens. This binding occurred both in vitro and in vivo, within living plant cells. The V2 binding site within mCYP1 was identified in the direct proximity to the papain-like cysteine protease active site.

Bhendi yellow vein mosaic disease is caused by a complex consisting of a monopartite begomovirus associated with a β-satellite. The C2 protein of bhendi yellow vein mosaic virus (BYVMV) is a suppressor of post-transcriptional gene silencing and also functions as a transcriptional activator. To explore the molecular mechanisms of its nuclear trafficking and self-interaction, fusion proteins of fluorescent proteins with wild-type or mutated constructs of BYVMV C2 were expressed in tobacco protoplasts. Analyses revealed that the BYVMV C2 nuclear localization signal (NLS) was located in the N terminus of the protein, comprising aa 17-31 of C2. NLSs are recognized by a class of soluble transport receptors termed karyopherins α and β. The BYVMV C2 NLS was found to be necessary for this protein's interaction with its nuclear import mediator, karyopherin α, ensuring its nuclear localization. Nevertheless, when deleted, C2 was found in both the cytoplasm and the nucleus, suggesting NLS-independent nuclear import of this protein. Homotypic interaction of BYVMV C2 was also found, which correlates with the nuclear localization needed for efficient activation of transcription.

The capsid protein (CP) of Tomato yellow leaf curl virus-Israel (TYLCV-IL), encoded by the v1 gene, is the only known component of the viral capsid. Three point mutations introduced into the conserved NLS region of the CP were investigated. One mutant, in which the Arg at position 19 was converted to Leu, had the most significant effect on the CP-CP homotypic interaction as well as on CP's interaction with its nuclear receptor karyopherin α1 and with the protein GroEL. The latter has been suggested to protect the virions in the insect vector hemolymph. These effects were first observed by yeast two-hybrid assay and then confirmed in tobacco protoplasts by measuring fluorescence resonance energy transfer (FRET) between YFP- and CFP-tagged proteins. Most importantly, when the point mutation converting Arg 19 to Leu was introduced into the full-length TYLCV genome, it disrupted its ability to cause symptoms., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Macromolecules may transfer between the cytoplasm and the nucleus only through specific gates - the nuclear pore complexes (NPCs). Translocation of nucleic acids and large proteins requires the presence of a nuclear localization signal (NLS) within the transported molecule. This NLS is recognized by a class of soluble transport receptors termed karyopherins α and beta. We previously characterized the expression pattern of the tomato karyopherin α 1 (LeKAPα1) promoter in transformed tobacco plants. Expression of LeKAPα1 was mainly observed in growing tissues where cell division and extension is rapid. The expression pattern of LeKAPα1 resembled that of auxin-responsive genes. This led us to suggest that auxin participates in the regulation of LeKAPα1 expression. Here we characterized the correlation between auxin level and the activity of the LeKAPα1 promoter. To this end, transgenic tobacco plants carrying the GUS reporter gene under the control of the LeKAPα1 promoter were treated with various levels of exogenous auxin. We also studied transgenic plants in which we increased the endogenous levels of auxin. For this, we expressed in plants both the LeKAPα1 promoter-GUS reporter and the Agrobacterium tumefaciens iaaM gene, which increases the endogenous levels of auxin. The results indicate that the auxin indole-3-acetic acid (IAA) can induce LeKAPα1 expression. We also identified that the sites and levels of LeKAPα1 expression correlated with the endogenous pathways of polar auxin transport.

Introduction: Identification of the dental arch form of the orthodontic patient is a key aspect for achieving a stable, functional, and esthetic dentition. Failure to customize preformed archwires with the patient's arch form might increase the probability of relapse and lead to an unnatural smile. The primary objective of this study was to identify the arch forms of Israeli subjects with dental normocclusion and malocclusions. The secondary objective was to clarify the morphologic differences between Israeli and North American white subjects with various malocclusions., Methods: The sample included 134 Israeli (40 Class I, 61 Class II, and 33 Class III) and 160 North American (60 Class I, 50 Class II, and 50 Class III) subjects. The most facial portion of 13 proximal contact areas was digitized from photocopied images of the mandibular dental arches. Clinical bracket points were calculated for each tooth based on mandibular tooth thickness data. Four linear and 2 proportional measurements were made. The dental arches were classified as square, ovoid, and tapered forms to determine and compare the frequency distributions between the 2 ethnic groups., Results: The most frequent mandibular arch form of the Israeli group was found to be ovoid as opposed to tapered in the North American white group. The white population had statistically significant decreased arch widths and increased arch depths compared with the Israeli population. The analysis of the Israeli sample indicated that, as the malocclusion shifts from Class III through Class I to Class II, the weight of the arch form tends to shift from square and ovoid to ovoid and tapered., Conclusions: Our results suggest that, when treating Israeli patients, one should expect to use the preformed ovoid arch form orthodontic wires in a significant percentage of patients., (Copyright © 2011 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.)

The highly regenerative capacity of the human adult oral mucosa suggests the existence of a robust stem cell (SC) population in its lamina propria (OMLP). The purpose of this study was to characterize the availability, growth, immunophenotype, and potency of this presumable SC population. Cells positive for the embryonic stem cell transcription factors Oct4 and Sox2 and for p75 formed distinct cord-like structure in the OMLP. Regardless of donor age, trillions of cells, termed human oral mucosa stem cells (hOMSC), 95% of which express mesenchymal stromal cell markers, were simply, and reproducibly produced from a biopsy of 3-4 x 2 x 1 mm(3). A total of 40-60% of these cells was positive for Oct4, Sox2, and Nanog and 60-80% expressed constitutively neural and neural crest SC markers. hOMSC differentiated in culture into mesodermal (osteoblastic, chondroblastic, and adipocytic), definitive endoderm and ectodermal (neuronal) lineages. Unexpectedly, hOMSC treated with dexamethasone formed tumors consisting of two germ layer-derived tissues when transplanted in severe combined immune deficiency mice. The tumors consisted of tissues produced by neural crest cells during embryogenesis-cartilage, bone, fat, striated muscle, and neural tissue. These results show that the adult OMLP harbors a primitive SC population with a distinct primitive neural-crest like phenotype and identifies the in vivo localization of putative ancestors for this population. This is the first report on ectodermal- and mesodermal-derived mixed tumors formation by a SC population derived from a nonmalignant somatic adult human tissue.

The ubiquitin/26S proteasome pathway is a basic biological mechanism involved in the regulation of a multitude of cellular processes. Increasing evidence indicates that plants utilize the ubiquitin/26S proteasome pathway in their immune response to pathogen invasion, emphasizing the role of this pathway during plant-pathogen interactions. The specific functions of proteasomal degradation in plant-pathogen interactions are diverse, and do not always benefit the host plant. Although in some cases, proteasomal degradation serves as an effective barrier to help plants ward off pathogens, in others, it is used by the pathogen to enhance the infection process. This review discusses the different roles of the ubiquitin/26S proteasome pathway during interactions of plants with pathogenic viruses, bacteria, and fungi.

The human body displays central circadian rhythms of activity. Recent findings suggest that peripheral tissues, such as bone, possess their own circadian clocks. Studies have shown that osteocalcin protein levels oscillate over a 24-hour period, yet the specific skeletal sites involved and its transcriptional profile remain unknown. The current study aimed to test the hypothesis that peripheral circadian mechanisms regulate transcription driven by the osteocalcin promoter. Transgenic mice harboring the human osteocalcin promoter linked to a luciferase reporter gene were used. Mice of both genders and various ages were analyzed non-invasively at sequential times throughout 24-hour periods. Statistical analyses of luminescent signal intensity of osteogenic activity from multiple skeletal sites indicated a periodicity of ~ 24 hrs. The maxillomandibular complex displayed the most robust oscillatory pattern. These findings have implications for dental treatments in orthodontics and maxillofacial surgery, as well as for the mechanisms underlying bone remodeling in the maxillomandibular complex.

The application of novel assays for basic cell research is tightly linked to the development of easy-to-use and versatile tools and protocols for implementing such technologies for a wide range of applications and model species. The bimolecular fluorescence complementation (BiFC) assay is one such novel method for which tools and protocols for its application in plant cell research are still being developed. BiFC is a powerful tool which enables not only detection, but also visualization and subcellular localization of protein-protein interactions in living cells. Here we describe the application of BiFC in plant cells while focusing on the use of our versatile set of vectors which were specifically designed to facilitate the transformation, expression and imaging of protein-protein interactions in various plant species. We discuss the considerations of using our system in various plant model systems, the use of single versus multiple expression cassettes, the application of our vectors using various transformation methods and the use of internal fluorescent markers which can assist in signal localization and easy data acquisition in living cells.

Fluorescence molecular tomography (FMT) is a novel tomographic near-infrared (NIR) imaging modality that enables 3D quantitative determination of fluorochrome distribution in tissues of live small animals at any depth. This study demonstrates a noninvasive, quantitative method of monitoring engineered bone remodeling via FMT. Murine mesenchymal stem cells overexpressing the osteogenic gene BMP2 (mMSCs-BMP2) were implanted into the thigh muscle and into a radial nonunion bone defect model in C3H/HeN mice. Real-time imaging of bone formation was performed following systemic administration of the fluorescent bisphosphonate imaging agent OsteoSense, an hydroxyapatite-directed bone-imaging probe. The mice underwent imaging on days 7, 14, and 21 postimplantation. New bone formation at the implantation sites was quantified using micro-computed tomography (micro-CT) imaging. A higher fluorescent signal occurred at the site of the mMSC-BMP2 implants than that found in controls. Micro-CT imaging revealed a mass of mature bone formed in the implantation sites on day 21, a finding also confirmed by histology. These findings highlight the effectiveness of FMT as a functional platform for molecular imaging in the field of bone regeneration and tissue engineering., ((c) 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

The V2 protein of tomato yellow leaf curl geminivirus (TYLCV) functions as an RNA-silencing suppressor that counteracts the innate immune response of the host plant. The host-cell target of V2, however, remains unknown. Here we show that V2 interacts directly with SlSGS3, the tomato homolog of the Arabidopsis SGS3 protein (AtSGS3), which is known to be involved in the RNA-silencing pathway. SlSGS3 genetically complemented an AtSGS3 mutation and restored RNA silencing, indicating that SlSGS3 is indeed a functional homolog of AtSGS3. A point mutant of V2 that is unable to bind SlSGS3 also lost its ability to suppress RNA silencing, suggesting a correlation between the V2-SlSGS3 interaction in planta and the suppressor activity of V2.

Monitoring gene expression in vitro and in vivo, is crucial when analyzing osteogenesis and developing effective bone gene therapy protocols. Until recently, molecular analytical tools were only able to detect protein expression either in vitro or in vivo. These systems include histology and immunohistochemistry, fluorescent imaging, PET (micro-PET), CT (micro-CT), and bioluminescent imaging. The last is the only system to date that can enable efficient quantitative monitoring of gene expression both in vitro and in vivo. Effective bioluminescent imaging in bone can be achieved by using transgenic mice harboring the luciferase reporter gene, downstream of an osteogenesis specific promoter. The aim of this chapter is to comprehensively describe the various protocols needed for the detection of bioluminescence in bone development and repair.

Nuclear proteins are involved in many critical biological processes within plant cells and, therefore, are in the focus of studies that usually begin with demonstrating that the protein of interest indeed exhibits nuclear localization. Thus, studies of plant nuclear proteins would be facilitated by a convenient experimental system for identification of proteins that are actively imported into the cell nucleus and visualization of their nuclear accumulation in vivo. To this end, we developed a system of vectors that allows screening for cDNAs coding for nuclear proteins in a simple genetic assay in yeast cells, and verification of nuclear accumulation in planta following one-step transfer and autofluorescent tagging of the identified clones into a multiple cloning site-compatible and reading frame-compatible plant expression vector. In a recommended third experimental step, the plant expression cassette containing the identified clone can be transferred, also by a one-step cloning, into a binary multigene expression vector for transient or stable coexpression with any other proteins.

The coat protein (CP) of Tomato yellow leaf curl virus (TYLCV), encoded by the v1 gene, is the only known component of the viral capsid. In addition, the CP plays a role in the virus transport into the host cell nucleus where viral genes are replicated and transcribed. In this study, we analyzed the effect of small interfering double-stranded RNAs (siRNAs), derived from an intron-hairpin RNA (ihpRNA) construct and targeting the v1 gene product, on CP accumulation. Transient assays involving agroinfiltration of the CP-silencing construct followed by infiltration of a fused GFP-CP (green fluorescent protein-coat protein) gene showed down-regulation of GFP expression in Nicotiana benthamiana. Some of the transgenic tomato plants (cv. Micro-Tom), expressing the siRNA targeted against the TYLCV CP gene, did not show disease symptoms 7 weeks post-inoculation with the virus, while non-transgenic control plants were infected within 2 weeks post inoculation. The present study demonstrates, for the first time, that siRNA targeted against the CP of TYLCV can confer resistance to the virus in transgenic tomato plants, thereby enabling flowering and fruit production.

The Israeli isolate of Tomato yellow leaf curl geminivirus (TYLCV-Is) is a major tomato pathogen, causing extensive crop losses both in the New and Old World. Surprisingly, however, little is known about the molecular mechanisms of TYLCV-Is interactions with tomato cells. Here, we have identified a TYLCV-Is protein, V2, which acts as a suppressor of RNA silencing and which is unrelated to presently known viral suppressors. Specifically, V2, but not other proteins of TYLCV-Is, inhibited RNA silencing of a reporter transgene, GFP. This inhibition elevated the cellular levels of the GFP transcript and the GFP protein, but it had no apparent effect on the accumulation of GFP-specific short interfering RNAs (siRNAs), suggesting that TYLCV-Is V2 targets a step in the RNA silencing pathway which is subsequent to the Dicer-mediated cleavage of dsRNA. Visualization of the sub-cellular localization of TYLCV-Is V2 in plant protoplasts and tissues showed that this protein is associated with cytoplasmic strands and inclusion bodies in the cortical regions of the cell.

Bhendi yellow vein mosaic disease (BYVMD) is caused by a complex consisting of a monopartite begomovirus BYVMV and a satellite DNA beta component. BYVMV represents a new member of the emerging group of monopartite begomoviruses requiring a satellite component for symptom induction. Here we report the results of the transient expression of green fluorescent protein (GFP) fused with the betaC1 and coat protein (CP) coding regions, in the epidermal cells of Nicotiana benthamiana. GFPCP was found to be targeted into the nucleus whereas GFPbetaC1 was localized towards the periphery of the cell. The sub-cellular localization of the betaC1 protein has been compared with that of the CP in yeast cells using a genetic system for detection of protein nuclear import and export. Expression of betaC1 ORF in transgenic N. benthamiana under the control of the Cauliflower mosaic virus 35S promoter produced severe developmental abnormalities in the plant, like distorted stem, leaves and stunting of the plant. We also present the results on the interaction of CP and betaC1 proteins using yeast two hybrid analysis, suggesting a collaborative role in the inter- and intracellular dynamics of BYVMD.

Angiogenesis is mandatory for reperfusion of viable tissues, and lack of vascularization may cause ischemia. The increasing disparity between the demand and availability of adequate substitutes for small-diameter human blood vessels has prompted an intensive search for artificial materials or biological allograft tissues, both of which usually fail in the long term. The objective of this study was to pioneer a novel model for in vivo guided angiogenesis based on a specific design process of a filamentous polymeric scaffold with endothelial cells in a 3-dimensional culture system. To our knowledge, this is the first report of an in vivo guided angiogenesis approach based on a 2-step model, composed of endothelial cells and a filamentous polymeric scaffold framework. Endothelial cells that had been cultured on a specifically designed filamentous polymeric scaffold within a regulated dynamic tissue culture system were shown in vivo to induce guided angiogenesis. Cells seeded on a biodegradable polymeric scaffold were implanted into mice. On day 28 after implantation, analysis revealed a guided angiogenic process along the path of the implanted polymeric scaffold as well as initial evidence for early maturation of engineered vessels, allowing red blood cells to flow through the forming lumina of new vessels as the polymer degraded. The authors conclude that in vivo guided angiogenesis can be achieved by combining endothelial cells with biodegradable filamentous polymeric scaffolds and that this model can lay the cornerstone for vascular engineering and future development of clinically available protocols aimed to treat life-threatening cardiovascular conditions.

Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing protein-protein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-terminal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multi-gene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific protein-protein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types.

The plant-infecting geminiviruses deliver their genome and viral proteins into the host cell nucleus. Members of the family Geminiviridae possess either a bipartite genome composed of two approximately 2.6 kb DNAs or a monopartite genome of approximately 3.0 kb DNA. The bipartite genome of Bean dwarf mosaic virus (BDMV) encodes several karyophilic proteins, among them the capsid protein (CP) and BV1 (nuclear shuttle protein). A CP is also encoded by the monopartite genome of Tomato yellow leaf curl virus (TYLCV). Here, an in vitro assay system was used for direct demonstration of nuclear import of BDMV BV1 and TYLCV CP, as well as synthetic peptides containing their putative nuclear localization signals (NLSs). Full-length recombinant BDMV BV1 and TYLCV CP mediated import of conjugated fluorescently labelled BSA molecules into nuclei of permeabilized mammalian cells. Fluorescently labelled and biotinylated BSA conjugates bearing the synthetic peptides containing aa 3-20 of TYLCV CP (CP-NLS) or aa 84-106 of BDMV BV1 (BV1-NLS) were also imported into the nuclei of permeabilized cells. This import was blocked by the addition of unlabelled BSA-NLS peptide conjugates or excess unlabelled free NLS peptides. The CP- and BV1-NLS peptides also mediated nuclear import of fluorescently labelled BSA molecules into the nuclei of microinjected mesophyll cells of Nicotiana benthamiana leaves, demonstrating their biological function in intact plant tissue. BV1-NLS and CP-NLS were shown to mediate specific binding to importin alpha, both in vitro and in vivo. These results are consistent with a common nuclear-import pathway for CP and BV1, probably via importin alpha.

SUMMARY Nuclear import and export are crucial processes for any eukaryotic cell, as they govern substrate exchange between the nucleus and the cytoplasm. Proteins involved in the nuclear transport network are generally conserved among eukaryotes, from yeast and fungi to animals and plants. Various pathogens, including some plant viruses, need to enter the host nucleus to gain access to its replication machinery or to integrate their DNA into the host genome; the newly replicated viral genomes then need to exit the nucleus to spread between host cells. To gain the ability to enter and exit the nucleus, these pathogens encode proteins that recognize cellular nuclear transport receptors and utilize the host's nuclear import and export pathways. Here, we review and discuss our current knowledge about the molecular mechanisms by which plant viruses find their way into and out of the host cell nucleus.

Agrobacterium most likely can transform virtually all known plant species, and experimental protocols for Agrobacterium-mediated genetic transformation of yet more plant species, ecotypes, and cultivars are published almost on a daily basis. Interestingly, the Agrobacterium host range is not limited to the plant kingdom, and it has been shown to transform many species of fungi and even prokaryotes. The ability of Agrobacterium to genetically transform HeLa cells further widens the range of potential hosts of Agrobacterium to include humans and perhaps other animal species. Furthermore, because mammalian cells significantly differ from plant cells, they provide a useful experimental system for identification and functional characterization of plant-specific factors involved in the transformation process. Here, we present basic procedures for transfection and Agrobacterium-mediated genetic transformation of mammalian cells. We also demonstrate the use of mammalian cells for studies of the cellular components of the genetic transformation pathway.

Cell-to-cell tobacco mosaic virus movement protein (TMV MP) mediates viral spread between the host cells through plasmodesmata. Although several host factors have been shown to interact with TMV MP, none of them coresides with TMV MP within plasmodesmata. We used affinity purification to isolate a tobacco protein that binds TMV MP and identified it as calreticulin. The interaction between TMV MP and calreticulin was confirmed in vivo and in vitro, and both proteins were shown to share a similar pattern of subcellular localization to plasmodesmata. Elevation of the intracellular levels of calreticulin severely interfered with plasmodesmal targeting of TMV MP, which, instead, was redirected to the microtubular network. Furthermore, in TMV-infected plant tissues overexpressing calreticulin, the inability of TMV MP to reach plasmodesmata substantially impaired cell-to-cell movement of the virus. Collectively, these observations suggest a functional relationship between calreticulin, TMV MP, and viral cell-to-cell movement.

Rhizobium rhizogenes, a soil bacterium, is the causative agent of the neoplastic disease hairy root. Upon incubation of Rhizobium rhizogenes A4 with coniferyl alcohol, a lignin precursor, bacterial virulence on cotton cotyledon slices was stimulated. This was observed both in numbers of root hairs produced and in their length. Stimulation was maximized after exposure of bacteria to 150 microg/mL of coniferyl alcohol for 4 h. This was shown to be at the early log phase of bacterial growth.

The karyopherin alpha1 (LeKAPalpha 1) gene of tomato (Lycopersicon esculentum) encodes a receptor involved in nuclear import. To analyze the expression pattern of this gene, a genomic clone containing its upstream region was isolated and sequenced. To study the promoter functionality, a 2170 bp fragment (LM1), was fused to glucuronidase (GUS) and introduced into petunia cells by particle bombardment. For further characterization of the promoter, one inverse and three deletion constructs were studied in cell suspension. To follow its expression in tobacco leaves, transgenic plants expressing GUS under the control of the LM1 promoter were made. Expression of LM1-GUS was largely restricted to actively growing leaf regions, suggesting possible involvement of active cell division and plant growth regulators in LeKAPalpha 1 expression.

The results of the present work demonstrate that core histones are able to penetrate the plasma membrane of plant cells. Confocal microscopy has revealed that incubation of petunia protoplasts with fluorescently labeled core histones resulted in cell penetration and nuclear import of the externally added histones. Intracellular accumulation was also confirmed by an ELISA-based quantitative method using biotin-labeled histones. Penetration into petunia protoplasts and cultured cells was found to be non-saturable, occurred at room temperature and at 4 degrees C and was not inhibited by Nocodazole. Furthermore, penetration of the biotinylated histone was neither blocked by the addition of an excess of free biotin molecules, nor by non-biotinylated histone molecules. All these results clearly indicate that the observed uptake is due to direct translocation through the cell plasma membrane and does not occur via endocytosis. Our results also show that the histones H2A and H4 were able to mediate penetration of covalently attached BSA molecules demonstrating the potential of the histones as carriers for the delivery of macromolecules into plant cells. To the best of our knowledge, the findings of the present paper demonstrate, for the first time, the activity of cell penetrating proteins (CPPs) in plant cells.

Viral delivery of the therapeutic gene bone morphogenetic protein-2 (BMP-2) is a promising approach for bone regeneration. The human parvovirus adeno-associated virus (AAV) type 2 is considered one of the most encouraging viral vector systems because of its high transduction rates and biosafety ratings. Bone morphogenetic protein-2 is a highly potent osteoinductive protein, which induces bone formation in vivo and osteogenic differentiation in vitro. The exogenous regulation of BMP-2 expression in bone-regenerating sites is required to control BMP-2 protein secretion, thus promoting safe and controlled bone formation and regeneration. We have therefore constructed a dual-construct vector for the recombinant AAV (rAAV)-based recombinant human BMP-2 (rhBMP-2) gene delivery system, which is regulated by the tetracycline-sensitive promoter (TetON). Each vector was encapsidated separately, yielding two recombinant viruses. We evaluated the efficiency of rAAV-hBMP-2 to induce bone formation in ectopic and orthotopic sites. Doxycycline (Dox), an analogue of tetracycline, was orally administered to mice via their drinking water to induce rhBMP-2 expression. Bone formation was measured using quantitative imaging-microcomputerized tomography and cooled charge-coupled device imaging-to detect osteogenic activity at the cellular level, detecting osteocalcin expression. The rAAV-hBMP-2-treated mice that were given Dox demonstrated bone formation in both in vivo models compared to none in mice prevented from receiving Dox. Thus, the Tet-regulated rAAV-hBMP-2 vector is an effective means of induction and regulation of bone regeneration and repair.

Adult stem cells reside in adult tissues and serve as the source for their specialized cells. In response to specific factors and signals, adult stem cells can differentiate and give rise to functional tissue specialized cells. Adult mesenchymal stem cells (MSCs) have the potential to differentiate into various mesenchymal lineages such as muscle, bone, cartilage, fat, tendon and ligaments. Adult MSCs can be relatively easily isolated from different tissues such as bone marrow, fat and muscle. Adult MSCs are also easy to manipulate and expand in vitro. It is these properties of adult MSCs that have made them the focus of cell-mediated gene therapy for skeletal tissue regeneration. Adult MSCs engineered to express various factors not only deliver them in vivo, but also respond to these factors and differentiate into skeletal specialized cells. This allows them to actively participate in the tissue regeneration process. In this review, we examine the recent achievements and developments in stem-cell-based gene therapy approaches and their applications to bone, cartilage, tendon and ligament tissues that are the current focus of orthopedic medicine.

Statement of Problem: Controversy exists concerning the preferred type of final restoration of endodontically treated molars regarding their resistance to fracture under occlusal load., Purpose: The aim of this study was to assess the resistance to fracture of endodontically treated molars with various degrees of tooth structure loss restored with amalgam under simulated occlusal load., Material and Methods: . The study teeth consisted of 90 noncarious, nonrestored molars stored in physiological saline solution. The teeth were endodontically treated and randomly divided into 9 experimental groups of 10 specimens each with various degrees of tooth loss, ranging from a conservative endodontic access to removal of all cusps. All teeth were restored with a standardized technique to their original contour with amalgam. Each specimen was mounted onto a specialized jig for loading at the central fossa at a 30-degree angle to the long tooth axis. The resistance to fracture, under continuous compressive force at a cross-head speed of 2 mm/minute, was applied by a universal testing machine and was recorded. One-way analysis of variance with Scheffé contrast was used to statistically compare the differences between the groups at significance level P<.05., Results: One-way analysis of variance showed that the group with a conservative endodontic access (1137.6 +/- 311.6 N) and the group with removal of all cusps (1261.4 +/- 195.1 N) presented a significantly higher resistance to fracture when compared with the other groups (P<.05). There was no significant difference in resistance to fracture under the simulated load between the other 7 groups, which ranged from 655.8 +/- 229.4 to 906.3 +/- 168.1 N (P>.05)., Conclusion: Within the limitations of this study, the endodontically treated molars with a conservative endodontic access or after removal of all cusps that were restored to their original contour with amalgam presented the highest resistance to fracture under a simulated occlusal load.