509 research outputs found
Detection of capillovirus-like particles in a grapevine affected with rugose wood
Research Not
Grapevine virus C and grapevine leaf roll associated virus 2 are serologically related and appear to be the same virus
Protein extracted from grapevines infected with GLRaV-2 virus was subjected to electrophoresis, followed by Western blots. A protein band of about 23 kDa was detected in all infected plants. When GVC antibodies were used on blots obtained from the same infected plants, a similar protein band was detected in all infected plants. To address the possibility of the presence of another virus with the same molecular weight, the gene coding for the coat protein of GLRaV-2 was cloned and expressed in E. coli. The expressed protein reacted positively to both GLRaV- 2 and GVC antibodies. Using Immunosorbent Electron Microscopy (ISEM), polyclonal antibodies prepared against either GVC or GLRaV-2 trapped and decorated GLRaV-2 particles. The cDNA from GVC-infected grapevines and Nicotiana benthamiana were cloned and sequenced. All of the clones that were sequenced had the same sequence as GLRaV-2. Based on the data obtained, we concluded that GVC is the same virus as GLRaV-2. Keywords
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A role for ceramide accumulation in age-related cardiac mitochondrial dysfunction
Ceramides are a group of lipids in the sphingolipid family that are potent cell signaling molecules. Elevations in ceramide levels above the norm generally lead to apoptosis. Evidence from in vitro studies suggests that accumulation of ceramides within mitochondria leads to dysfunction of the mitochondria. This includes inhibition of the electron transport chain and release of reactive oxygen species (ROS). Large elevations of mitochondrial ceramide may also lead to death of the cell as they promote membrane permeability transition, which allows the release of cytochrome c and other apoptogenic factors. With age, cardiac mitochondria show a similar dysfunctional phenotype to that found in conditions of acute ceramide elevation. We therefore hypothesize that ceramides may be accumulating in cardiac mitochondria from aged animals.
To elucidate the characteristics of the sphingolipidome, we developed both mitochondrial isolation techniques and tandem mass spectrometry assays to specifically and sensitively monitor mitochondrial sphingolipids. Using these techniques, it was found that mitochondria contain six distinct ceramide species with highly saturated lipid moieties. Using young (3-6 months old; which corresponds to a post-adolescent human) and old (24 to 28 months old; which corresponds to an elderly human) Fischer 344 rat hearts, we found that aging leads to a significant increase in total mitochondrial ceramides (32%, p < 0.03), with C₁₆-, C₁₈-, and C₂₄:₁-ceramides showing the largest percent increases (72.3%, 73.4%, and 77.7%, respectively, p < 0.05). Furthermore, the age-associated elevation in ceramide levels correlated to a 28% decrease in the activity of complex IV of the electron transport chain (p < 0.05), which could be replicated in vitro by inducing a ceramide accumulation in mitochondria isolated from young animals.
Mitochondria do not contain enzymes for de novo ceramide biosynthesis, rather, these organelles have sphingomyelinases, a family of enzymes that cleave the phosphorylcholine headgroup from nascent pools of sphingomyelin. Specifically, mitochondria contain the magnesium-requiring isoform of sphingomyelinase with a neutral pH optima (nSMase). Recent work has shown that nSMase activity is inversely regulated by glutathione status. Because cardiac mitochondrial glutathione (mGSH) declines by up to 60% with age, we hypothesized that the loss in mGSH leads to an increase in ceramides through the upregulation of nSMase.
To determine whether loss of mGSH plays a role in the regulation of mitochondrial nSMase activity, mGSH levels were depleted by treating freshly isolated hepatocytes with 3-hydroxy-4-pentenoate (3HP). It was found that 3HP rapidly depleted mGSH in a concentration-dependent manner (EC₅₀ = 232 μM, p < 0.05). Moreover, this depletion led to an increase in nSMase activity (24 ± 3% at 250 μM 3HP, p < 0.05), and an increase in total ceramide levels (27%, p < 0.05). These findings suggest that mGSH status plays a critical role in the maintenance of ceramide levels within mitochondria. Furthermore, because nSMase activity is regulated by mGSH levels, we hypothesized that any agent promoting an increase in mGSH would reverse the ceramide accumulation seen in cardiac mitochondria from aged animals.
Lipoic acid (LA) is a naturally occurring dithiol compound used for many years as an anti-inflammatory agent. LA-supplementation has been shown to increase cellular and mGSH by increasing cellular cysteine levels in the aging heart. In order to determine whether LA reverses the age-associated ceramidosis in cardiac mitochondria, young and old F344 rats were pair-fed LA [0.2% (w/w) in the diet] against controls for two weeks and cardiac mitochondria were subsequently isolated and analyzed. It was found that LA-treatment reversed the age-associated decline in mGSH levels [decreased 43% with age (p < 0.05)], and reduced nSMase activity [increased 103% with age (p < 0.05)]. Ceramide levels were reduced [elevated 32% with age (p < 0.03)] so that they were no longer different from young controls and complex IV activity restored t youthful levels [declined 28% with age (p < 0.05)].
In conclusion, this dissertation provides evidence to support a new mechanism that explains, at least in part, the progression of mitochondrial dysfunction in the aging heart, and may also contribute to understanding the age-related loss of cardiomyocytes. It also provides mechanistic insights into the overall health benefits of LA supplementation and supports its use as a safe, natural, and "age-essential" micronutrient
Federal Trust Responsibility and Conflicts of Interest: Environmental Protection or Natural Resource Development
Comparing symptoms and emotion recognition in African American and White samples with schizophrenia
Racial status has an important role in schizophrenia, with African American samples being rated lower than White participants on a range of constructs. In many studies, however, demographic factors are not accounted for. In the present study, African American (n = 106) and White participants (n = 81) were compared on symptom severity and emotion recognition scales while controlling for other demographic factors. Contrary to our hypothesis, there were no differences in symptoms between racial groups. However, White participants performed better on an emotion recognition measure than African Americans. These differences were most prominent in response to negatively-valenced stimuli. This study replicated previous findings of racial differences in emotion recognition but not symptom severity. Future research should assess the role of racial identity on symptom severity. In addition, further research is needed to assess if utilising multi-ethnic stimuli improves performance by racial minorities on emotion recognition measures
Androgen Deprivation Therapy Potentiates the Efficacy of Vascular Targeted Photodynamic Therapy of Prostate Cancer Xenografts
WST11 vascular targeted photodynamic therapy (VTP) is a local ablation approach relying upon rapid, free radical-mediated destruction of tumor vasculature. A phase III trial showed that VTP significantly reduced disease progression when compared with active surveillance in patients with low-risk prostate cancer. The aim of this study was to identify a druggable pathway that could be combined with VTP to improve its efficacy and applicability to higher risk prostate cancer tumors. Transcriptome analysis of VTP-treated tumors (LNCaP-AR xenografts) was used to identify a candidate pathway for combination therapy. The efficacy of the combination therapy was assessed in mice bearing LNCaP-AR or VCaP tumors. Gene set enrichment analysis identifies the enrichment of androgen-responsive gene sets within hours after VTP treatment, suggesting that the androgen receptor (AR) may be a viable target in combination with VTP. We tested this hypothesis in mice bearing LNCaP-AR xenograft tumors by using androgen deprivation therapy (ADT), degarelix, in combination with VTP. Compared with either ADT or VTP alone, a single dose of degarelix in concert with VTP significantly inhibited tumor growth. A sharp decline in serum prostate-specific antigen (PSA) confirmed AR inhibition in this group. Tumors treated by VTP and degarelix displayed intense terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining 7 days after treatment, supporting an increased apoptotic frequency underlying the effect on tumor inhibition. Improvement of local tumor control following androgen deprivation combined with VTP provides the rationale and preliminary protocol parameters for clinical trials in patients presented with locally advanced prostate cancer.
Statins enhance the efficacy of HER2-targeting radioligand therapy in drug-resistant gastric cancers
Human epidermal growth factor receptor 2 (HER2) is overexpressed in various cancer types. HER2-targeting trastuzumab plus chemotherapy is used as first-line therapy for HER2-positive recurrent or primary metastatic gastric cancer, but intrinsic and acquired trastuzumab resistance inevitably develop over time. To overcome gastric cancer resistance to HER2-targeted therapies, we have conjugated trastuzumab with a beta-emitting therapeutic isotope, lutetium-177, to deliver radiation locally to gastric tumors with minimal toxicity. Because trastuzumab-based targeted radioligand therapy (RLT) requires only the extramembrane domain binding of membrane-bound HER2 receptors, HER2-targeting RLT can bypass any resistance mechanisms that occur downstream of HER2 binding. Leveraging our previous discoveries that statins, a class of cholesterol-lowering drugs, can enhance the cell surface-bound HER2 to achieve effective drug delivery in tumors, we proposed that the combination of statins and
Positron Emission Tomography/Computed Tomography with Gallium-68-labeled Prostate-specific Membrane Antigen Detects Relapse After Vascular-targeted Photodynamic Therapy in a Prostate Cancer Model
BACKGROUND: Evaluating the efficacy of focal therapy for prostate cancer is limited by current approaches and may be improved with biological imaging techniques.
OBJECTIVE: We assessed whether positron emission tomography/computed tomography with gallium-68-labeled prostate-specific membrane antigen (Ga-PSMA PET/CT) can be used to predict relapse after vascular-targeted photodynamic therapy (VTP).
DESIGN, SETTING, AND PARTICIPANTS: A total of 1×10 LNCaP cells were grafted subcutaneously in the flanks of 6-8-wk-old SCID mice. Of 24 mice with measurable tumors 6 wk after tumor implantation, 20 were treated with VTP (150mW/cm) to ablate the tumors. Blood prostate-specific antigen (PSA) levels were assessed, and ⁶⁸Ga-PSMA PET/CT images were performed 1 d before VTP and 1 and 4 wk after.
OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Local tumor relapse was evaluated by histology, and tumors were analyzed by prostate-specific membrane antigen (PSMA) and PSA immunohistochemistry. T tests and Kruskal-Wallis tests were used to determine significance.
RESULTS AND LIMITATIONS: Four weeks after VTP, 11 (65%) mice had complete responses and six (35%) had tumor relapses confirmed by histology (hematoxylin and eosin, and PSMA immunohistochemistry). All mice with local relapse had positive Ga-PSMA PET/CT findings 4 wk after VTP; all complete responders did not. One week after VTP, the relapse detection sensitivity of Ga-PSMA PET/CT was 75%, whereas the sensitivity of PSA was only 33%. Compared with controls, relapsed tumors had a three-fold reduction in the number of cells with strong PSA staining by immunohistochemistry (1.5% vs 4.5%; p=0.01).
CONCLUSIONS: In a preclinical prostate cancer model, we show that Ga-PSMA PET/CT can identify and predict relapse earlier than blood PSA level. These findings support further testing in clinical trials.
PATIENT SUMMARY: Positron emission tomography/computed tomography with gallium-68-labeled prostate-specific membrane antigen may be used to follow and evaluate treatment outcomes in men who receive focal therapy for prostate cancer
Development of extinction imagers for the determination of atmospheric optical extinction: final report
The primary goals of this project for JTO and ONR (Grant N00014-07-1-1060) were to further develop Extinction Imagers for use in the ocean environment, and to extend the capabilities into the Short Wave IR (SWIR). Extinction Imaging is a method for determining the effective extinction coefficient over an extended path using a sensor at one end of the path. It uses calibrated imagers to acquire the relative radiance of a dark target near the other the end of the path and the horizon sky in the direction of the dark target. It is completely passive and thus covert, and the hardware is robust and relatively inexpensive. It uses rigorous equations, which determine the extinction coefficient from the measured apparent contrast of the radiance of the dark target with respect to the horizon sky.
The project was very successful. We found that the ocean surface could readily be used as a dark target in red and SWIR wavelengths. Both the red and the SWIR measurement results were excellent for daytime. Comparisons with standard instruments, as well as uncertainty analysis, indicated that extinction imagers provide better measurements of the atmospheric extinction losses over extended paths than other methods of which we are aware.
Our secondary goals were to address the night regime, and to address slanted paths above the horizontal. Regarding night, we found that the visible sensor acquired excellent data, but the ocean surface was not a good dark target in our wavelengths. Recommendations on the handling of night are given in the report. Regarding the lines of sight above the horizon, we developed a slant path algorithm that determines beam transmittance. It performed very well. Recommendations are made regarding integration of these techniques for military applications.Joint Technology Office via Office of Naval ResearchGrant N00014-07-1-106
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