The effect of chronic captopril therapy on adrenergic receptors, plasma noradrenaline and the vascular responses to infused noradrenaline

Adrenergic receptors (alpha 2, beta 2), plasma noradrenaline, heart rate and the pressor responsiveness to infused noradrenaline were examined in ten healthy male volunteers before and after 2 weeks of placebo or captopril therapy in a double blind cross-over study. No significant differences in these measurements were observed between the captopril and placebo treated groups. The study shows that in sodium replete normotensive subjects, long-term angiotensin converting enzyme inhibition does not lead to changes in adrenoceptor density. There is also no alteration in plasma noradrenaline levels nor in the pressor responsiveness to infused noradrenaline. These data suggest that the known interaction between the renin-angiotensin system and the sympathetic nervous system observed in animals is probably of little significance in man.

NO3- transport across the plasma membrane of Arabidopsis thaliana root hairs:Kinetic control by pH and membrane voltage

High-affinity nitrate transport was examined in intact root hair cells of Arabidopsis thaliana using electrophysiological recordings to characterise the response of the plasma membrane to NO₃^-challenge and to quantify transport activity. The NO₃^--associated membrane current was determined using a three-electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in the roots of seedlings grown in the absence of a nitrogen source, but only 4-6 days postgermination. In 6-day-old seedlings, additions of 5-100 μm NO₃^-to the bathing medium resulted in membrane depolarizations of 8-43 mV, and membrane voltage (V_m) recovered on washing NO₃^-from the bath. Voltage clamp measurements carried out immediately before and following NO₃^-additions showed that the NO₃^--evoked depolarizations were the consequence of an inward-directed current that appeared across the entire range of accessible voltages (-300 to +50 mV). Both membrane depolarizations and NO₃^--evoked currents recorded at the free-running voltage displayed quasi-Michaelian kinetics, with apparent values for K_m of 23 ± 6 and 44 ± 11 μm, respectively and, for the current, a maximum of 5.1 ± 0.9 μA cm^-2. The NO₃^-current showed a pronounced voltage sensitivity within the normal physiological range between -250 and -100 mV, as could be demonstrated under voltage clamp, and increasing the bathing pH from 6.1 to 7.4-8.0 reduced the current and the associated membrane depolarizations 3- to 8-fold. Analyses showed a well-defined interaction between the kinetic variables of membrane voltage, pH_o and [NO₃^-]_o. At a constant pH_o of 6.1, depolarization from -250 to -150 mV resulted in an approximate 3-fold reduction in the maximum current but a 10% rise in the apparent affinity for NO₃^-. By contrast, the same depolarization effected an approximate 20% fall in the K_m for transport as a function in [H⁺]_o. These, and additional characteristics of the transport current implicate a carrier cycle in which NO₃^-binding is kinetically isolated from the rate-limiting step of membrane charge transit, and they indicate a charge-coupling stoichiometry of 2(H⁺) per NO₃^-anion transported across the membrane. The results concur with previous studies showing a high-affinity NO₃^-transport system in Arabidopsis that is inducible following a period of nitrogen-limiting growth, but they underline the importance of voltage as a kinetic factor controlling NO₃^-transport at the plant plasma membrane. © 1995 Springer-Verlag New York Inc.

Human mesenchymal stem cells reduce mortality and bacteremia in gram-negative sepsis in mice in part by enhancing the phagocytic activity of blood monocytes

The potential therapeutic value of cell-based therapy with mesenchymal stem cells (MSC) has been reported in mouse models of polymicrobial peritoneal sepsis. However, the mechanisms responsible for the beneficial effects of MSC have not been well defined. Therefore, we tested the therapeutic effect of intravenous bone marrow-derived human MSC in peritoneal sepsis induced by gram-negative bacteria. At 48 h, survival was significantly increased in mice treated with intravenous MSC compared with control mice treated with intravenous fibroblasts (3T3) or intravenous PBS. There were no significant differences in the levels of TNF-a, macrophage inflammatory protein 2, or IL-10 in the plasma. However, there was a marked reduction in the number of bacterial colony-forming units of Pseudomonas aeruginosa in the blood of MSC-treated mice compared with the 3T3 and PBS control groups. In addition, phagocytic activity was increased in blood monocytes isolated from mice treated with MSC compared with the 3T3 and PBS groups. Furthermore, levels of C5a anaphylotoxin were elevated in the blood of mice treated with MSC, a finding that was associated with upregulation of the phagocytosis receptor CD11b on monocytes. The phagocytic activity of neutrophils was not different among the groups. There was also an increase in alternately activated monocytes/macrophages (CD163- and CD206-positive) in the spleen of the MSC-treated mice compared with the two controls. Thus intravenous MSC increased survival from gram-negative peritoneal sepsis, in part by a monocyte-dependent increase in bacterial phagocytosis.

Arabidopsis Rab-E GTPases exhibit a novel interaction with a plasma-membrane phosphatidylinositol-4-phosphate 5-kinase

Rab GTPases of the Arabidopsis Rab-E subclass are related to mammalian Rab8 and are implicated in membrane trafficking from the Golgi to the plasma membrane. Using a yeast two-hybrid assay, Arabidopsis phosphatidylinositol-4-phosphate 5-kinase 2 (PtdIns(4)P 5-kinase 2; also known as PIP5K2), was shown to interact with all five members of the Rab-E subclass but not with other Rab subclasses residing at the Golgi or trans-Golgi network. Interactions in yeast and in vitro were strongest with RAB-E1d[Q74L] and weakest with the RAB-E1d[S29N] suggesting that PIP5K2 interacts with the GTP-bound form. PIP5K2 exhibited kinase activity towards phosphatidylinositol phosphates with a free 5-hydroxyl group, consistent with PtdIns(4)P 5-kinase activity and this activity was stimulated by Rab binding. Rab-E proteins interacted with PIP5K2 via its membrane occupancy and recognition nexus (MORN) domain which is missing from animal and fungal PtdIns(4)P 5-kinases. In plant cells, GFP:PIP5K2 accumulated at the plasma membrane and caused YFP:RAB-E1d to relocate there from its usual position at the Golgi. GFP:PIP5K2 was rapidly turned over by proteasomal activity in planta, and overexpression of YFP:PIP5K2 caused pleiotropic growth abnormalities in transgenic Arabidopsis. We propose that plant cells exhibit a novel interaction in which PIP5K2 binds GTP-bound Rab-E proteins, which may stimulate temporally or spatially localized PtdIns(4,5)P(2) production at the plasma membrane.

Eradication of marine biofilms by atmospheric pressure non-thermal plasma: A potential approach to control biofouling?

Although the antimicrobial activity of atmospheric pressure non-thermal plasmas, including its capacity to eradicate microbial biofilms, has been gaining an ever increasing interest for different medical applications, its potential utilisation in the control of biofouling and biodeterioration has, to date, received no attention. In this study, the ability of atmospheric pressure plasma to eradicate biofilms of four biofouling bacterial species, frequently encountered in marine environments, was investigated. Biofilms were grown on both polystyrene and stainless steel surfaces before being exposed to the plasma source. Viability and biomass of biofilms were evaluated using colony count method and differential Live/Dead fluorescence staining followed by confocal laser scanning microscopy. Rapid and complete eradication of all biofilms under study was achieved after plasma exposures ranging from 60 to 120 s. Confocal microscopy examination showed that plasma treatment has mediated not only cell killing but also varying degrees of physical removal of biofilms. Further investigation and tailored development of atmospheric pressure non-thermal plasma sources for this particular application could provide an additional powerful and effective weapon in the current anti-biofouling armamentarium.

Potential Cellular Targets and Antibacterial Efficacy of Atmospheric Pressure Non-Thermal Plasma

Atmospheric pressure non-thermal plasma (APNTP) has been gaining increasing interest as a new alternative antibacterial approach. Although this approach has demonstrated promising antibacterial activity, its exact mechanism of action remains unclear. Mechanistic elucidation of the antimicrobial activity will facilitate development and rational optimisation of this approach for potential medical applications. In this study, the antibacterial efficacy of an in-house-built APNTP jet was evaluated alongside an investigation of the interactions between APNTP and major cellular components in order to identify the potential cellular targets involved in plasma-mediated bacterial destruction mechanisms. The investigated plasma jet exhibited excellent, rapid antibacterial activity against a selected panel of clinically significant bacterial species including Bacillus cereus, meticillin-resistant Staphylococcus aureus (MRSA), Escherichia coli and Pseudomonas aeruginosa, all of which were completely inactivated within 2 min of plasma exposure. Plasma-mediated damaging effects were observed, to varying degrees, on all of the investigated cellular components including DNA, a model protein enzyme, and lipid membrane integrity and permeability. The antibacterial efficacy of APNTP appears to involve a multiple-target mechanism, which potentially reduces the likelihood of emergence of microbial resistance towards this promising antimicrobial approach. However, cellular membrane damage and resulting permeability perturbation was found to be the most likely rate-determining step in this mechanism. Crown Copyright © 2013.

Plasma IL-6 levels correlate with clinical and ultrasound measures of arthritis in patients with systemic lupus erythematosus

ObjectiveThe objective of this paper is to elucidate the role of specific cytokines in lupus (SLE) arthritis.MethodsFifty SLE and 40 RA patients had an ultrasound (US) scan of their hand as per standardized protocols. US scores were expressed per joint and as a total 'US activity' score, (sum of power Doppler (PD) and grey-scale synovial hypertrophy scores in all joints) and a total erosion score. SLE disease activity was assessed (BILAG and SELENA-SLEDAI). Plasma levels of IL-6, TNF-alpha and BLyS were measured using sandwich ELISA kits (Quantikine kits, R & D).ResultsOn the basis of the US results SLE patients were divided into three groups: erosive arthritis (n?=?20), non-erosive arthritis (n?=?18) and those with a normal US scan (n?=?12). Across the SLE groups plasma IL-6 levels correlated with CRP (p?

Ambient Temperature Hydrocarbon Selective Catalytic Reduction of NOx Using Atmospheric Pressure Nonthermal Plasma Activation of a Ag/Al2O3 Catalyst

Atmospheric pressure nonthermal-plasma-activated catalysis for the removal of NOx using hydrocarbon selective catalytic reduction has been studied utilizing toluene and n-octane as the hydrocarbon reductant. When the plasma was combined with a Ag/Al2O3 catalyst, a strong enhancement in activity was observed when compared with conventional thermal activation with high conversions of both. NOx and hydrocarbons obtained at temperature at temperature ≤250 °C, where the silver catalyst is normally inactive. Importantly, even in the absence of an external heat source, significant activity was obtained. This low temperature activity provides the basis for applying nonthermal plasmas to activate emission control catalysts during cold start conditions, which remains an important issue for mobile and stationary applications.

A novel RCE1 isoform is required for H-Ras plasma membrane localization and is regulated by USP17

Processing of the 'CaaX' motif found on the C-termini of many proteins, including the proto-oncogene Ras, requires the ER (endoplasmic reticulum)-resident protease RCE1 (Ras-converting enzyme 1) and is necessary for the proper localization and function of many of these 'CaaX' proteins. In the present paper, we report that several mammalian species have a novel isoform (isoform 2) of RCE1 resulting from an alternate splice site and producing an N-terminally truncated protein. We demonstrate that both RCE1 isoform 1 and the newly identified isoform 2 are required to reinstate proper H-Ras processing and thus plasma membrane localization in RCE1-null cells. In addition, we show that the deubiquitinating enzyme USP17 (ubiquitin-specific protease 17), previously shown to modulate RCE1 activity, can regulate the abundance and localization of isoform 2. Furthermore, we show that isoform 2 is ubiquitinated on Lys43 and deubiquitinated by USP17. Collectively, the findings of the present study indicate that RCE1 isoform 2 is required for proper 'CaaX' processing and that USP17 can regulate this via its modulation of RCE1 isoform 2 ubiquitination.

Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens

The emergence of multidrug-resistant pathogens within the clinical environment is presenting a mounting problem in hospitals worldwide. The 'ESKAPE' pathogens (Enterococcusfaecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) have been highlighted as a group of causative organisms in a majority of nosocomial infections, presenting a serious health risk due to widespread antimicrobial resistance. The stagnating pipeline of new antibiotics requires alternative approaches to the control and treatment of nosocomial infections. Atmospheric pressure nonthermal plasma (APNTP) is attracting growing interest as an alternative infection control approach within the clinical setting. This study presents a comprehensive bactericidal assessment of an in-house-designed APNTP jet both against biofilms and planktonic bacteria of the ESKAPE pathogens. Standard plate counts and the XTT metabolic assay were used to evaluate the antibacterial effect of APNTP, with both methods demonstrating comparable eradication times. APNTP exhibited rapid antimicrobial activity against all of the ESKAPE pathogens in the planktonic mode of growth and provided efficient and complete eradication of ESKAPE pathogens in the biofilm mode of growth within 360 s, with the exception of A. baumannii where a >4log reduction in biofilm viability was observed. This demonstrates its effectiveness as a bactericidal treatment against these pathogens and further highlights its potential application in the clinical environment for the control of highly antimicrobial-resistant pathogens.

GTPase activity of dynamin and resulting conformation change are essential for endocytosis

Dynamin is a large GTPase with a relative molecular mass of 96,000 (Mr 96K) that is involved in clathrin-mediated endocytosis and other vesicular trafficking processes. Although its function is apparently essential for scission of newly formed vesicles from the plasma membrane, the nature of dynamin's role in the scission process is still unclear. It has been proposed that dynamin is a regulator (similar to classical G proteins) of downstream effectors. Here we report the analysis of several point mutants of dynamin's GTPase effector (GED) and GTPase domains. We show that oligomerization and GTP binding alone, by dynamin, are not sufficient for endocytosis in vivo. Rather, efficient GTP hydrolysis and an associated conformational change are also required. These data argue that dynamin has a mechanochemical function in vesicle scission.

Atmospheric cold plasma process for vegetable leaf decontamination: A feasibility study on radicchio (red chicory, Cichorium intybus L.)

Cold plasma is an emerging non-thermal processing technology that could be used for large scale leaf decontamination as an alternative to chlorine washing. In this study the effect of an atmospheric cold plasma apparatus (air DBD, 15 kV) on the safety, antioxidant activity and quality of radicchio (red chicory, Cichorium intybus L.) was investigated after 15 and 30 min of treatment (in afterglow at 70 mm from the discharge, at 22 °C and 60% of RH) and during storage. Escherichia coli O157:H7 inoculated on radicchio leaves was significantly reduced after 15 min cold plasma treatment (-1.35 log MPN/cm²). However, a 30 min plasma treatment was necessary to achieve a significant reduction of Listeria monocytogenes counts (-2.2 log CFU/cm²). Immediately after cold plasma treatment, no significant effects emerged in terms of antioxidant activity assessed by the ABTS and ORAC assay and external appearance of the radicchio leaves. Significant changes between treated and untreated radicchio leaves are quality defects based on the cold plasma treatment. Atmospheric cold plasma appears to be a promising processing technology for the decontamination of leafy vegetables although some criticalities, that emerged during storage, need to be considered in future studies.

The Activity of Breast Milk and Monoclonal HIV-specific Antibodies in Mother-to-Child Transmission

Mother-to-child transmission of HIV is a unique setting that allows us to explore both the correlates of protective immunity and the characteristics of transmitted variants. This thesis first describes the levels and functional capacity of breast milk HIV-specific antibodies in 19 women with high plasma viral loads. Neutralizing antibodies (Nabs) were detected in breast milk supernatant (BMS) of 4 of 19 women examined, were of low potency and were not associated with infant infection. The low NAb activity in BMS was reflected in binding antibody levels with HIV envelope specific IgG titers being 2.2 log10 lower in BMS versus plasma. In contrast, non- neutralizing antibodies (nNAbs) capable of antibody dependent cell-mediated cytotoxicity (ADCC) were detected in the BMS from all 19 women. BMS ADCC activity was associated with envelope-specific IgG titers (p = 0.014) and was inversely associated with infant infection risk (p = 0.039). Our data indicate that BMS has limited HIV neutralizing activity, however, BMS ADCC activity is a correlate of transmission that may impact infant infection risk. In the second part of this thesis the neutralization sensitivity of 111 variants of diverse subtypes obtained from mothers and infants was determined against 7 HIVspecific broadly neutralizing monoclonal antibodies (mAbs) (NIH45-46w, VRC01, PGT128, PGT121, PG9 PGT145 and b12). Maternal and infant variants did not differ in their neutralization sensitivity to these mAbs and neither did variants from transmitting versus those from non-transmitting women. However, subtype A viruses were iii significantly more sensitive to neutralization by NIH45-46w and VRC01 (p= 0.0001 in both cases) and PGT145 (p=0.03) compared to non-subtype A viruses. Together, NIH45- 46w and PGT128 neutralization profiles resulted in 100% coverage of the variants tested. These data suggest that the epitopes targeted by these mAbs are present and accessible in both circulating and transmitted variants and that a combination of antibodies would provide maximum coverage against diverse subtypes commonly found in HIV endemic regions. Overall, this data suggest that an antibody based HIV vaccine capable of eliciting antibodies of multiple specificities that can mediate ADCC and/or neutralizing activity can provide protection and conquer the genetic diversity displayed by HIV.

Long-term citalopram administration reduces responsiveness of HPA axis in patients with major depression: relationship with S-citalopram concentrations in plasma and cerebrospinal fluid (CSF) and clinical response.

RATIONALE: A dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is a well-documented neurobiological finding in major depression. Moreover, clinically effective therapy with antidepressant drugs may normalize the HPA axis activity. OBJECTIVE: The aim of this study was to test whether citalopram (R/S-CIT) affects the function of the HPA axis in patients with major depression (DSM IV). METHODS: Twenty depressed patients (11 women and 9 men) were challenged with a combined dexamethasone (DEX) suppression and corticotropin-releasing hormone (CRH) stimulation test (DEX/CRH test) following a placebo week and after 2, 4, and 16 weeks of 40 mg/day R/S-CIT treatment. RESULTS: The results show a time-dependent reduction of adrenocorticotrophic hormone (ACTH) and cortisol response during the DEX/CRH test both in treatment responders and nonresponders within 16 weeks. There was a significant relationship between post-DEX baseline cortisol levels (measured before administration of CRH) and severity of depression at pretreatment baseline. Multiple linear regression analyses were performed to identify the impact of psychopathology and hormonal stress responsiveness and R/S-CIT concentrations in plasma and cerebrospinal fluid (CSF). The magnitude of decrease in cortisol responsivity from pretreatment baseline to week 4 on drug [delta-area under the curve (AUC) cortisol] was a significant predictor (p<0.0001) of the degree of symptom improvement following 16 weeks on drug (i.e., decrease in HAM-D21 total score). The model demonstrated that the interaction of CSF S-CIT concentrations and clinical improvement was the most powerful predictor of AUC cortisol responsiveness. CONCLUSION: The present study shows that decreased AUC cortisol was highly associated with S-CIT concentrations in plasma and CSF. Therefore, our data suggest that the CSF or plasma S-CIT concentrations rather than the R/S-CIT dose should be considered as an indicator of the selective serotonergic reuptake inhibitors (SSRIs) effect on HPA axis responsiveness as measured by AUC cortisol response.

A novel anti-CD19 monoclonal antibody (GBR 401) with high killing activity against B cell malignancies.

BACKGROUND: CD19 is a B cell lineage specific surface receptor whose broad expression, from pro-B cells to early plasma cells, makes it an attractive target for the immunotherapy of B cell malignancies. In this study we present the generation of a novel humanized anti-CD19 monoclonal antibody (mAb), GBR 401, and investigate its therapeutic potential on human B cell malignancies. METHODS: GBR 401 was partially defucosylated in order to enhance its cytotoxic function. We analyzed the in vitro depleting effects of GBR 401 against B cell lines and primary malignant B cells from patients in the presence or in absence of purified NK cells isolated from healthy donors. In vivo, the antibody dependent cellular cytotoxicity (ADCC) efficacy of GBR 401 was assessed in a B cell depletion model consisting of SCID mice injected with healthy human donor PBMC, and a malignant B cell depletion model where SCID mice are xenografted with both primary human B-CLL tumors and heterologous human NK cells. Furthermore, the anti-tumor activity of GBR 401 was also evaluated in a xenochimeric mouse model of human Burkitt lymphoma using mice xenografted intravenously with Raji cells. Pharmacological inhibition tests were used to characterize the mechanism of the cell death induced by GBR 401. RESULTS: GBR 401 exerts a potent in vitro and in vivo cytotoxic activity against primary samples from patients representing various B-cell malignancies. GBR 401 elicits a markedly higher level of ADCC on primary malignant B cells when compared to fucosylated similar mAb and to Rituximab, the current anti-CD20 mAb standard immunotherapeutic treatment for B cell malignancies, showing killing at 500 times lower concentrations. Of interest, GBR 401 also exhibits a potent direct killing effect in different malignant B cell lines that involves homotypic aggregation mediated by actin relocalization. CONCLUSION: These results contribute to consolidate clinical interest in developing GBR 401 for treatment of hematopoietic B cell malignancies, particularly for patients refractory to anti-CD20 mAb therapies.