Plant and animal PR1 family members inhibit programmed cell death and suppress bacterial pathogens in plant tissues.

A role for programmed cell death (PCD) has been established as the basis for plant-microbe interactions. A functional plant-based cDNA library screen identified possible anti-PCD genes, including one member of the PR1 family, designated P14a, from tomato. Members of the PR1 family have been subject to extensive research in view of their possible role in resistance against pathogens. The PR1 family is represented in every plant species studied to date and homologues have been found in animals, fungi and insects. However, the biological function of the PR1 protein from plants has remained elusive in spite of extensive research regarding a role in the response of plants to disease. Constitutive expression of P14a in transgenic tomato roots protected the roots against PCD triggered by Fumonisin B1, as did the human orthologue GLIPR1, indicating a kingdom crossing function for PR1. Tobacco plants transformed with a P14a-GFP fusion construct and inoculated with Pseudomonas syringae pv. tabaci revealed that the mRNA was abundant throughout the leaves, but the fusion protein was restricted to the lesion margins, where cell death and bacterial spread were arrested. Vitus vinifera grapes expressing the PR1 homologue P14a as a transgene were protected against the cell death symptoms of Pierce's disease. A pull-down assay identified putative PR1-interacting proteins, including members of the Rac1 immune complex, known to function in innate immunity in rice and animal systems. The findings herein are consistent with a role of PR1 in the suppression of cell death-dependent disease symptoms and a possible mode of action.

FASN regulates cellular response to genotoxic treatments by increasing PARP-1 expression and DNA repair activity via NF-κB and SP1.

Fatty acid synthase (FASN), the sole cytosolic mammalian enzyme for de novo lipid synthesis, is crucial for cancer cell survival and associates with poor prognosis. FASN overexpression has been found to cause resistance to genotoxic insults. Here we tested the hypothesis that FASN regulates DNA repair to facilitate survival against genotoxic insults and found that FASN suppresses NF-κB but increases specificity protein 1 (SP1) expression. NF-κB and SP1 bind to a composite element in the poly(ADP-ribose) polymerase 1 (PARP-1) promoter in a mutually exclusive manner and regulate PARP-1 expression. Up-regulation of PARP-1 by FASN in turn increases Ku protein recruitment and DNA repair. Furthermore, lipid deprivation suppresses SP1 expression, which is able to be rescued by palmitate supplementation. However, lipid deprivation or palmitate supplementation has no effect on NF-κB expression. Thus, FASN may regulate NF-κB and SP1 expression using different mechanisms. Altogether, we conclude that FASN regulates cellular response against genotoxic insults by up-regulating PARP-1 and DNA repair via NF-κB and SP1.

Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras.

Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or, alternatively, it has a different mechanism of substrate binding than other polygalacturonases characterized to date.

Bedside ultrasonography detects significant femoral vessel overlap: implications for central venous cannulation.

OBJECTIVE: Our goal was to gain a better understanding of the femoral vessel anatomy as it relates to central venous cannulation. The primary objective of this study was to use bedside ultrasonography to determine the amount of exposed femoral vein at three sites corresponding to surface anatomy of the landmark-based procedure. METHODS: This cross-sectional study enrolled a random sample of 180 adult patients presenting to a large urban academic emergency department. Subjects underwent standardized ultrasonography to identify and measure the depth and diameter of the femoral vessels and amount of exposed femoral vein at the level of the inguinal ligament (0 cm) 2 cm and 4 cm below. Repeated measures analysis of variance was used to determine significant relationships between vessel measurements and distance from the inguinal ligament. RESULTS: The median age was 44.5 (range 19-90) years; 101 patients were male. The mean (± SD) percentage of exposed vein at the inguinal ligament was 83% (± 21). This decreased significantly (p < 0.01) with increasing distance from the inguinal ligament: 65% (± 25) at 2 cm and 56% (± 30) at 4 cm. At every distance away from the inguinal ligament, there were some subjects with no vein exposed. CONCLUSION: This study demonstrates significant overlap of the femoral vessels at sites where landmark-based femoral vein cannulation is often attempted. Our results suggest that ultrasound guidance would be beneficial as femoral vein cannulation may be difficult or impossible in certain individuals owing to anatomic variations.

Outcome after repair of a sarcoma-related pathologic fracture in dogs: a Veterinary Society of Surgical Oncology Retrospective Study.

OBJECTIVE: To report outcome in dogs after internal fixation of a sarcoma-related pathologic fracture of the appendicular skeleton. STUDY DESIGN: Multi-institutional case series. ANIMALS: Dogs (n=16). METHODS: Medical records of participating VSSO members were reviewed for dogs with pathologic fracture associated with a confirmed bone sarcoma of the appendicular skeleton repaired by external or internal fixation. Dogs were included if they had a histological diagnosis of osteosarcoma or sarcoma and excluded if they had radiation before fracture. Data collected were analyzed for signalment, fracture location, staging performed, method of fracture fixation, histopathology, adjunctive treatment and outcome. RESULTS: Signalment and fracture location of 16 dogs that met the inclusion criteria was similar to dogs with appendicular OSA without fracture. One of 14 dogs had pulmonary metastasis and 3 of 5 dogs had bone metastasis. Bone plate or interlocking nail were used for repair in 12 dogs. Limb use immediately after surgery in 13 dogs was good (4), weight-bearing but lame (7) and non-weight bearing (2). Adjunctive therapy was administered in 5 dogs (chemotherapy, 3; radiation, 4; pamidronate, 3). Survival time ranged from 18 to 897 days; median survival was 166 days. CONCLUSIONS: Repair of pathologic fracture can result in palliation and prolonged survival.

Surgical treatment of mineralized and nonmineralized supraspinatus tendinopathy in twenty-four dogs.

OBJECTIVE: To report and compare the clinical diagnosis, surgical treatment, histopathologic changes, and outcomes of dogs with mineralized and nonmineralized supraspinatus tendinopathy (ST). STUDY DESIGN: Case series. ANIMALS: Dogs (n=24) with ST. METHODS: Medical records (1995-2006) of dogs with ST that had surgical treatment were reviewed. Results of clinical examination, diagnostic imaging, surgery, histopathology of resected tendon tissue, and outcome were compared between dogs with mineralized and nonmineralized ST. RESULTS: There were 15 dogs with mineralized ST and 9 with nonmineralized ST. Chronic, unilateral, intermittent or waxing-waning lameness, and pain elicited on palpation of the cranial aspect of the shoulder were the most consistent findings. On ultrasonographic or magnetic resonance imaging (MRI) of 35 shoulders, enlargement of the supraspinatus tendon (54%), increased fluid content (63%), and medial displacement of the biceps tendon (60%) were observed. Eleven of 12 dogs with bilateral abnormalities only had unilateral lameness. Surgery was performed in 30 shoulders. Resected tendon specimens had myxomatous degeneration and/or cartilaginous metaplasia in 11 of 13 dogs in the mineralized group and all 9 dogs in the nonmineralized group. Functional outcome after surgery was poor in 3 dogs and good-to-excellent in 16. CONCLUSIONS: Mineralized and nonmineralized ST have many similarities. Although lameness is usually unilateral, the supraspinatus tendon may be affected bilaterally. CLINICAL RELEVANCE: Ultrasonography and MRI are good imaging techniques for detection of ST especially the nonmineralized form. Surgical treatment results in good recovery of limb function. Nonmineralized ST is a recently described disorder in dogs and evaluation of more cases is necessary to determine outcome after surgical or medical treatment.

Leptosphaeria maculans elicits apoptosis coincident with leaf lesion formation and hyphal advance in Brassica napus.

Programmed cell death, with many of the morphological markers of apoptosis, is increasingly recognized as an important process in plant disease. We have investigated the involvement and potential role of apoptosis during the formation of leaf lesions by the fungus Leptosphaeria maculans on susceptible Brassica napus cv. Westar. There were no signs of host cell damage until 7 to 8 days postinoculation (dpi), when trypan-blue-stained leaf mesophyll cells were first detected. Hyphae were visible in the intercellular spaces of the inoculated area from 5 dpi and were associated with trypan-blue-stained cells at 8 to 9 dpi. Hallmarks of apoptosis, observed coincident with or immediately prior to the formation of leaf lesions at 8 to 10 dpi, included membrane shrinkage of the mesophyll cell cytoplasm, loss of cell to cell contact in mesophyll cells, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of nuclei in apparently "healthy" tissue immediately adjacent to dead areas. Hyphae were highly branched and prolific in the "healthy" tissue immediately adjacent to dead areas 9 to 10 dpi, and formed pycnidia inside dead areas 11 to 12 dpi. Coinfiltration of the tetrapeptide caspase inhibitor Ac-DEVD-CHO with spores of the pathogen significantly suppressed development of leaf lesions but did not affect fungus viability. We hypothesize that L. maculans elicits apoptosis as a dependent component of pathogenesis in susceptible B. napus, and that the fungus uses apoptotic cells as a source of nutrition for reproduction and further growth.

Programmed cell death suppression in transformed plant tissue by tomato cDNAs identified from an Agrobacterium rhizogenes-based functional screen.

The genetic regulation of programmed cell death (PCD) is well characterized in animal systems, but largely unresolved in plants. This research was designed to identify plant genes that can suppress PCD triggered in plants by Fumonisin B1 (FB1). Agrobacterium rhizogenes was used to transform individual members of a cDNA library into tomato roots, which were then screened for resistance to FB1. Cellular changes elicited during FB1-induced PCD include chromatin condensation, fragmentation into pycnotic DNA bodies, TUNEL positive reactions, ROS accumulation, and eventual loss of membrane integrity. Several cDNA library members collectively overexpressed in a transformed root population revealed PCD suppressive action and were recovered by PCR. One of the FB1 suppressive genes was homologous to metallothionein, and shared sequence homology to the animal ortholog reported to suppress PCD through interference with formation or activity of reactive oxygen species (ROS). The metallothionein recovered in this screen suppressed ROS accumulation in FB1-treated roots and prevented symptoms of PCD. Anti-PCD genes recovered by this screen represent potential sources of resistance to PCD-dependent plant diseases, while the screen should be useful to identify genes capable of suppressing PCD triggered by other effectors, including those expressed by root pathogens during infection.

Treatment of ununited anconeal process in 8 dogs by osteotomy and dynamic distraction of the proximal part of the ulna

A surgical technique for the treatment of ununited anconeal process in dogs treated by osteotomy and dynamic distraction of the proximal part of the ulna using a linear external skeletal fixator was evaluated. In all cases the osteotomy was distracted 1mm each day after the surgery until desired distraction had been achieved. Eight dogs and 9 joints diagnosed with ununited anconeal process were treated. The success of the procedure was determined by comparing clinical signs of lameness and degree of arthrosis at the time of diagnosis to 6 months after the surgical intervention. Radiographic union occurred in 88.9 percent of the affected joints between 21 and 42 days after the surgical procedure. Clinically, six elbows were classified as good, two as satisfactory and one as unsatisfactory. Six months after surgery two elbows had no arthrosis, one had Grade 1, two Grade 2 and one Grade 3. It is concluded the combination of ulnar osteotomy and dynamic distraction of the olecranon by a linear external skeletal fixator is a feasible procedure for the treatment of ununited anconeal process in dogs.

Foi avaliada nova técnica de tratamento da não união do processo ancôneo em cães através da osteotomia e distração dinâmicada porção proximal da ulna utilizando fixador externo. Em todos os casos a taxa de distração foi de 1mm a cada dia após a cirurgia até que a distração desejada fosse adquirida. Oito cães com nove articulações afetadas foram tratados. O sucesso do tratamento foi determinado pela comparação dos sinais clínicos de claudica-ção e grau de artrose no tempo do diagnóstico, até 6 meses após a intervenção cirúrgica. Clinicamente, seis cotovelos foram classificados como bons, dois como satisfatório e um como insatisfatório. Seis meses após a cirurgia, dois cotovelos não apresentavam artrose, um apresentava Grau 1, dois Grau 2 e um Grau 3. Conclui-se que a combinação de osteotomia da ulna com distração dinâmica do olécrano por fixador externo linear é procedimento factível no tratamento da não união do processo ancôneo em cães.

Augmentation of intertransverse process lumbar fusion.

Spinal fusion surgery for alleviation of intractable lower back pain in humans is currently a primary therapeutic technique, with failure rates averaging between 5 to 35%. Implanted and external source-based electrical stimulation devices have been investigated in an attempt to increase osteogenesis at the fusion site in an attempt to reduce spinal fusion failure rates. The purpose of our study was to evaluate the efficacy of two co-processor systems and an additional system with an SIS generation field at 15.8 mA (rms) using biomechanical, dual-energy X-ray absorptiometry (DXA), and histomorphometric analyses, in rabbits following dorsolateral (= posteriolateral [in humans]) spinal fusion. Fifty-six male New Zealand White underwent bilateral lumbar spinal fusion by performing decortication of the transverse processes of lumbar vertebrae four and five with placement of autogenic cancellous bone graft harvested from the ilial wings. Four study groups were designated based on the type of IES device used for stimulation or as a control. Eight weeks after surgery all subjects were sacrificed and the quality and strength of the fusion masses were compared using radiographic, biomechanical, histomorphometry, and qualitative histological evaluation. While some variation existed within and between groups, Group 2 showed a significant improvement in all parameters measured as compared to the control group (P < 0.05). The use of adjunct non-invasive surface IES for improving bony fusion rates for patients undergoing lumbar spinal fusion is supported by this study.

Fas signaling induces raft coalescence that is blocked by cholesterol depletion in human RPE cells undergoing apoptosis.

PURPOSE: To investigate whether the signaling events occurring in Fas-mediated apoptosis alter raft membrane formation in human RPE cells. METHODS: Formation of lipid rafts in cultured human retinal pigment epithelial cells (ARPE-19) was studied by confocal microscopy, with fluorescein-labeled cholera toxin subunit B binding protein (BODIPY)-labeled ganglioside GM1 lipid after Fas-L induction of apoptosis. Apoptosis was assessed by fluorescein-labeled annexin V detection of phosphatidylserine externalization and quadrant analysis with flow cytometry. Membrane rafts were localized into membrane vesicles by passing BODIPY-labeled GM1 RPE cells through a 2-microm-pore polycarbonate membrane using an extruder device. The labeled fractions, containing vesicles enriched in GM1, were detected by flow cytometry and then analyzed for the presence of Fas protein. RESULTS: Differential punctate staining of membrane rafts was demonstrated in normal and FasL-induced apoptotic human ARPE-19 cells in culture by confocal microscopy, using cholera toxin B and GM1 labeling of extruded vesicles. The lipid raft-associated vesicles were derived by plasma membrane dissociation, via a newly developed whole-cell extrusion technique that produced 2-microm vesicles with both GM1 lipid and Fas protein abundance enriched in a subpopulation of the membrane-derived vesicles. The amount of Fas protein in the vesicles containing raft domains markedly increased in FasL-treated cells. Treatment of human ARPE 19 cells with methyl beta-cyclodextrin after FasL induction of apoptosis resulted in cellular cholesterol depletion and markedly reduced the incidence of Fas-receptor localization in GM1 rafts. CONCLUSIONS: Human ARPE-19 cells in culture contain membrane rafts with apoptotic signaling effectors uniformly distributed in the native state. The cells stimulated to undergo apoptosis appear to use membrane rafts in the death-signaling process by mobilization of rafts to localized regions of the membrane that are now enriched with apoptotic signaling effectors. Fas signaling induces apoptotic raft formation that results in polar condensation, or capping, of the rafts in the late stages of apoptosis. A novel extrusion technique is described that allows localization and enrichment of rafts into membrane vesicles, which can be assayed by flow cytometry. Cholesterol depletion, after Fas ligand activation of apoptosis, reduced raft formation in cells induced to undergo apoptosis. Therapeutic implications for the treatment of retinal disorders are discussed.

Prevalence of cranial cruciate ligament rupture in a population of dogs with lameness previously attributed to hip dysplasia: 369 cases (1994-2003).

OBJECTIVE: To determine the prevalence of cranial cruciate ligament rupture (CCLR) in dogs with lameness previously attributed to canine hip dysplasia (CHD). DESIGN: Retrospective study. ANIMALS: 369 client-owned dogs. PROCEDURES: Hospital medical records from 1994 to 2003 were reviewed for dogs in which the referring veterinarian had diagnosed hip dysplasia or hip pain. Dogs were designated as having hind limb lameness because of partial or complete CCLR or CHD. RESULTS: 8% of dogs were sexually intact females, 43% were spayed females, 14% were sexually intact males, and 35% were castrated males. Mean age was 3.8 years (range, 3 months to 15 years). The most common breeds were the Labrador Retriever (21%), German Shepherd Dog (13%), and Golden Retriever (11%). The prevalence of CCLR as the cause of hind limb lameness was 32% (95% confidence interval, 27.2% to 36.8%). The distribution of CCLR among hind limbs was left (29%), right (28%), and bilateral (43%). Of 119 dogs with CCLR, 94% had concurrent radiographic signs of CHD, 92% had stifle joint effusion, and 81% had a cranial drawer sign. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of the high prevalence of CCLR in dogs referred for lameness because of CHD, it is important to exclude other sources of stifle joint disease before making recommendations for treatment of CHD.

Expression of the antiapoptotic baculovirus p35 gene in tomato blocks programmed cell death and provides broad-spectrum resistance to disease.

The sphinganine analog mycotoxin, AAL-toxin, induces a death process in plant and animal cells that shows apoptotic morphology. In nature, the AAL-toxin is the primary determinant of the Alternaria stem canker disease of tomato, thus linking apoptosis to this disease caused by Alternaria alternata f. sp. lycopersici. The product of the baculovirus p35 gene is a specific inhibitor of a class of cysteine proteases termed caspases, and naturally functions in infected insects. Transgenic tomato plants bearing the p35 gene were protected against AAL-toxin-induced death and pathogen infection. Resistance to the toxin and pathogen co-segregated with the expression of the p35 gene through the T3 generation, as did resistance to A. alternata, Colletotrichum coccodes, and Pseudomonas syringae pv. tomato. The p35 gene, stably transformed into tomato roots by Agrobacterium rhizogenes, protected roots against a 30-fold greater concentration of AAL-toxin than control roots tolerated. Transgenic expression of a p35 binding site mutant (DQMD to DRIL), inactive against animal caspases-3, did not protect against AAL-toxin. These results indicate that plants possess a protease with substrate-site specificity that is functionally equivalent to certain animal caspases. A biological conclusion is that diverse plant pathogens co-opt apoptosis during infection, and that transgenic modification of pathways regulating programmed cell death in plants is a potential strategy for engineering broad-spectrum disease resistance in plants.