Normalization of nitric oxide flux improves physiological parameters of porcine kidneys maintained on pulsatile perfusion.

Early endothelial damage and resultant reduction in the beneficial production of nitric oxide (NO) derived from the endothelial NO synthase (eNOS) are phenomena associated with the functional degradation of transplanted kidneys. In contrast, the inflammation characteristic of kidney preservation leads to the later, detrimental expression of the inducible NO synthase (iNOS). We reasoned that provision of low-level NO (to compensate for lack of eNOS) using the chemical NO donor S-nitrosoglutathione (GSNO), along with an iNOS inhibitor (N-omega iminoethyl-L-lysine; L-NIL), might "normalize" NO levels and therefore be beneficial in maintenance of flow. Non-heartbeating donor porcine kidneys were subjected to 30-45 min warm ischemic time and stored from 3 to 30 h, simulating the time required for national sharing. The kidneys were then machine preserved with Belzer MPS (BMP) at a set systolic pressure of 40 mmHg. Eight kidneys were perfused for 5h with BMP only (Group 1 control), 8 kidneys with BMP+GSNO only (Group 2), and 8 kidneys with BMP+GSNO+L-NIL (Group 3). Lower vascular resistance (VR) is a predictor of improved end-organ function. Both Group 2 and 3 kidneys demonstrated statistically significant reduction in VR as compared to Group 1 kidneys, with Group 3 kidneys demonstrating a greater drop in VR than Group 2. Reduced oxygen saturation suggests a higher metabolic rate. Only Group 3 had lower oxygen saturation as compared to Group 1. Increased Ca2+ concentration in the perfusate is a predictor of worse end-organ function. Group 2, but not Group 3, had a higher perfusate Ca2+ concentration than Group 1. The combination of suppression of harmful amounts of NO, while supplying a constant low-level amount of NO, may improve pulsatile kidney preservation.     

Monitoring photodynamic therapy of solid tumors online by BOLD-contrast MRI

Antivascular photodynamic therapy (PDT) of tumors with palladium-bacteriopheophorbide (TOOKAD) relies on in situ photosensitization of the circulating drug by local generation of cytotoxic reactive oxygen species, which leads to rapid vascular occlusion, stasis, necrosis and tumor eradication. Intravascular production of reactive oxygen species is associated with photoconsumption of O(2) and consequent evolution of paramagnetic deoxyhemoglobin. In this study we evaluate the use of blood oxygenation level-dependent (BOLD) contrast magnetic resonance imaging (MRI) for real-time monitoring of PDT efficacy. Using a solid tumor model, we show that TOOKAD-PDT generates appreciable attenuation (25-40%) of the magnetic resonance signal, solely at the illuminated tumor site. This phenomenon is independent of, though augmented by, ensuing changes in blood flow. These results were validated by immunohistochemistry and intravital microscopy. The concept of photosensitized BOLD-contrast MRI may have intraoperative applications in interactive guidance and monitoring of antivascular cancer therapy, PDT treatment of macular degeneration, interventional cardiology and possibly other biomedical disciplines.     

Onset of DNA aggregation in presence of monovalent and multivalent counterions

We address theoretically aggregation of DNA segments by multivalent polyamines such as spermine and spermidine. In experiments, the aggregation occurs above a certain threshold concentration of multivalent ions. We demonstrate that the dependence of this threshold on the concentration of DNA has a simple form. When the DNA concentration c(DNA) is smaller than the monovalent salt concentration, the threshold multivalent ion concentration depends linearly on c(DNA), having the form alphac(DNA) + beta. The coefficients alpha and beta are related to the density profile of multivalent counterions around isolated DNA chains, at the onset of their aggregation. This analysis agrees extremely well with recent detailed measurements on DNA aggregation in the presence of spermine. From the fit to the experimental data, the number of condensed multivalent counterions per DNA chain can be deduced. A few other conclusions can then be reached: 1), the number of condensed spermine ions at the onset of aggregation decreases with the addition of monovalent salt; 2), the Poisson-Boltzmann theory overestimates the number of condensed multivalent ions at high monovalent salt concentrations; and 3), our analysis of the data indicates that the DNA charge is not overcompensated by spermine at the onset of aggregation.     

Replicative senescence enhances apoptosis induced by pemphigus autoimmune antibodies in human keratinocytes

We have recently shown that skin lesions of the autoimmune disease pemphigus vulgaris are associated with Fas-mediated apoptosis. Here, we describe the induction of the Fas-dependent apoptosis pathway in cultured keratinocytes by pemphigus vulgaris autoantibodies (PV-IgG), as seen from a variety of cellular, morphological and biochemical parameters. All apoptotic characters appear stronger and faster in aged cultures than in young, showing increased susceptibility of senescent keratinocytes to PV-IgG-mediated apoptotic death and culture lesions. Together with immunosenescence, this phenomenon may explain the late onset of pemphigus disease.     

Response to lysophosphatidic acid in Xenopus oocytes and its rapid desensitization: the role of Gq and Go G-protein families

Native Xenopus oocytes exhibit dose‐dependent depolarizing current responses to lysophosphatidic acid (LPA), with EC50 = 0.18 μM. Responses to LPA were subject to pronounced rapid desensitization. When oocytes were challenged with 5 nM LPA, the response was <10% of the maximal. Subsequent addition of 0.5 μM LPA resulted in 50–70% desensitization, when compared to naïve controls. Injection of antisense oligodeoxyoligonucleotides (ASODNs) targeted at either of the two endogenous LPA receptors inhibited the LPA response by approximately 50%, but did not alter the degree of rapid desensitization. To study the involvement of G‐proteins in rapid homologous desensitization of responses to LPA, we selectively depleted native G‐proteins by injection of specific ASDONs. Injection of ASDONs targeted at Gαq family mRNAs (mainly Gα11) reduced the response to 0.5 μM LPA by 50%. ASDONs targeted at either Gαo or Gαo1 caused a large decrease in the amount of their cognate mRNAs and the Gαo family proteins, while the response to LPA was inhibited by up to 30%. Injection of ASDONs targeted at Gαo1 mRNA decreased rapid desensitization from 69 to 23%, while pertussis toxin (PTX) completely abolished it. Expression of two dominant negative mutants of the human Gαo family homologs either decreased or virtually abolished rapid desensitization. Microinjection of CaCl₂ demonstrated that 50% of rapid desensitization could be attributed to inhibition of Ca²⁺ activation of chloride channels. We propose that the apparent degenerate coupling of different G‐proteins to LPA receptors in Xenopus oocytes actually serves both the generation of the response (by Gq and Go G‐protein families) and its desensitization (mostly by Go G‐protein family). © 2004 Wiley‐Liss, Inc.     

Expression of streptavidin in tomato resulted in abnormal plant development that could be restored by biotin application

Biotin is an essential cofactor for a variety of carboxylase and decarboxylase reactions and is involved in diverse metabolic pathways of all organisms. In the present study we tested the hypothesis that controlling biotin availability by the expression of Streptomyces avidinii streptavidin, would impede plant development. Transient expression of streptavidin fused to plant signal peptide, bacterial signal peptide or both, in tomato (Lycopersicon esculentum cv. VF36) plants resulted in various levels of tissue impairment, exhibited as lesion development on 1-week-old tomato seedlings. The least toxic construct was introduced to tomato (stable transformation) under the constitutive CaMV 35S promoter, and lesions appeared on stems, flower morphologies were modified and numbers and sizes of fruits were altered. Furthermore, tissue-specific expression of the streptavidin, by means of the beta-phaseolin or TobRB7 promoters, resulted in localised effects, i.e., impaired seed formation or seedless fruits, respectively, with no alteration in the morphology of the other plant organs. External application of biotin on streptavidin-expressing tomato plants prevented the degeneration symptoms and facilitated normal plant development. It can be concluded that expression of streptavidin in the plant cell can lead to local and temporal deficiencies in biotin availability, impairing developmental processes while biotin application restores plant growth cycle.     

Inflammatory modulation of hepatocyte apoptosis by nitric oxide: in vivo, in vitro, and in silico studies

Nitric oxide (NO*) and its reaction products are key players in the physiology and pathophysiology of inflammatory settings such as sepsis and shock. The consequences of the expression of inducible NO* synthase (iNOS, NOS-2) can be either protective or damaging to the liver. We have delineated two distinct hepatoprotective actions of NO*: the stimulation of cyclic guanosine monophosphate and the inhibition of caspases by S-nitrosation. In contrast, iNOS/NO* promotes hepatocyte death under conditions of severe redox stress, such as hemorrhagic shock or ischemia/reperfusion. Redox stress activates an unknown molecular switch that transforms NO*, which is hepatoprotective under resting conditions, into an agent that induces hepatocyte death. We hypothesize that the magnitude of the redox stress is a major determinant for the effects of NO* on cell survival by controlling the chemical fate of NO*. To address this hypothesis, we have carried out studies in relevant in vivo and in vitro settings. Moreover, we have constructed an initial mathematical model of caspase activation and coupled it to a model describing some of the reactions of NO* in hepatocytes. Our studies suggest that modulation of iron, oxygen, and superoxide may dictate whether NO* is hepatoprotective or hepatotoxic.     

INDI: a computational framework for inferring drug interactions and their associated recommendations

Inferring drug–drug interactions (DDIs) is an essential step in drug development and drug administration. Most computational inference methods focus on modeling drug pharmacokinetics, aiming at interactions that result from a common metabolizing enzyme (CYP). Here, we introduce a novel prediction method, INDI (INferring Drug Interactions), allowing the inference of both pharmacokinetic, CYP‐related DDIs (along with their associated CYPs) and pharmacodynamic, non‐CYP associated ones. On cross validation, it obtains high specificity and sensitivity levels (AUC (area under the receiver‐operating characteristic curve)?0.93). In application to the FDA adverse event reporting system, 53% of the drug events could potentially be connected to known (41%) or predicted (12%) DDIs. Additionally, INDI predicts the severity level of each DDI upon co‐administration of the involved drugs, suggesting that severe interactions are abundant in the clinical practice. Examining regularly taken medications by hospitalized patients, 18% of the patients receive known or predicted severely interacting drugs and are hospitalized more frequently. Access to INDI and its predictions is provided via a web tool at http://www.cs.tau.ac.il/～bnet/software/INDI , facilitating the inference and exploration of drug interactions and providing important leads for physicians and pharmaceutical companies alike.