Enzyme interactions of 2,10-ethanoandrostene-3,17-dione: aromatase and 17 beta-hydroxysteroid dehydrogenase

An analogue of androstenedione containing an ethano bridge between carbons 2 and 10 of the A ring of the steroid, 1, has been evaluated as an inhibitor and a possible substrate of human placental aromatase. This compound was found to be a competitive inhibitor versus androstenedione (Kis = 25 +/- 2 nM) of the aromatase activity. Analyses of the incubation mixtures of 1 with human placental microsomes and NADPH by GC-MS indicated the formation of a new compound having an increase in molecular weight of 2 mass units (300 m.u.) from that of the parent steroid (298 m.u.). Subsequent analyses of incubations of 1 with an isolated 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) from Pseudomonas testosteronii in the presence of NADPH resulted in the formation of a new compound having the same retention time and molecular mass as that found for the product from the placental microsome incubation. Consequently, steroid 1 is both an inhibitor of human placental aromatase and a substrate for 17 beta-HSD.     

Heritable lymphocyte function-associated antigen-1 deficiency: abnormalities of cytotoxicity and proliferation associated with abnormal expression of LFA-1

The effect of heritable LFA-1 deficiency on T lymphocyte function was measured. After primary mixed lymphocyte stimulation, all six patients studied showed diminished allospecific T lymphocyte cytolytic and NK activity as compared with kindred and normal controls. MLR and mitogen-induced proliferative responses were consistently depressed. LFA-1-deficient, EBV-transformed B cell lines were poor stimulators of T cell responses. Primary cytolytic responses by lymphocytes from severely LFA-1-deficient patients (less than 0.2% of normal surface expression) were consistently more profoundly depressed than those by lymphocytes from moderately deficient patients (about 5% of normal surface expression). These results demonstrate the importance of LFA-1 in lymphocyte function. After repeated MLR restimulation, proliferative and cytolytic capacity improved and CTL lines could be established from all patients. Cytolysis by lines from one but not a second severe patient, and by four of four moderate patients, was inhibited by anti-LFA-1 MAb, and at 10-fold lower concentrations than required for inhibition of killing by control CTL lines. The locus of inhibition was on the target cell for the severely deficient CTL line, and on both the target and effector cells for moderately deficient CTL lines. In contrast, the locus of inhibition for normal CTL is on the effector cell. These findings show that LFA-1 can participate bidirectionally in cell interactions. The in vitro results are discussed in terms of the clinical findings in patients.     

Modification of hemoglobin A with dimethyl adipimidate. Contribution of individual reacted subunits to changes in oxygen affinity.

The effect of dimethyl adipimidate, a bifunctional imidoester, on the oxygen affinity of hemoglobin A has been studied. Treatment of human oxyhemoglobin with 5 mM dimethyl adipimidate at pH 8.5, room temperature is accompanied by an increase in oxygen affinity in the presence and absence of 2,3-diphosphoglyceric acid. Circular dichroism measurements in the ultraviolet region indicate that dimethyl adipimidate-treated hemoglobin exhibits a reduced conformational change upon deoxygenation. In order to study the contribution of reacted individual subunits, alpha and beta subunits of dimethyl adipimidate-treated and untreated hemoglobin have been separated and reconstituted to form hybrid tetramers containing either the alpha-treated (alpha t beta c) or the beta-treated subunits (alpha c beta t). Electrophoresis on sodium dodecyl sulfate polyacrylamide gels of isolated alpha and beta globin subunits as well as hybrid tetramers from dimethyl adipimidate-treated hemoglobin reveals that 20% of the globin subunits are cross-linked. In the absence of 2,3-diphosphoglyceric acid, modification of alpha subunits increases the oxygen affinity and reduces the conformational change of the tetramer upon deoxygenation whereas modification of beta subunits has no effect. However, treatment of beta subunits decreases the effect of 2,3-diphosphoglyceric acid on the oxygen affinity of the hybrids and reduces the 2,3-diphosphoglyceric acid-induced spectral changes in oxyhemoglobin. Therefore the interaction of dimethyl adipimidate with both the alpha and beta subunits contributes to regulating the oxygen affinity of human hemoglobin.     

Complex temporal decay: a complete analysis

Relaxation dynamics is universal in science and engineering; its study serves to parameterize a system's response and to help identify a microscopic model of the processes involved. When measured data for a phenomenon cannot be fitted using one exponential, the choice of an alternative function to describe the decay becomes nontrivial. Here, we contrast two different, but fundamentally related approaches to fitting nontrivial decay curves; exponential decomposition and the gamma probability density function. Copyright © 2013 John Wiley & Sons, Ltd.     

Cortical potential imaging using L-curve and GCV method to choose the regularisation parameter

Background

The electroencephalography (EEG) is an attractive and a simple technique to measure the brain activity. It is attractive due its excellent temporal resolution and simple due to its non-invasiveness and sensor design. However, the spatial resolution of EEG is reduced due to the low conducting skull. In this paper, we compute the potential distribution over the closed surface covering the brain (cortex) from the EEG scalp potential. We compare two methods – L-curve and generalised cross validation (GCV) used to obtain the regularisation parameter and also investigate the feasibility in applying such techniques to N170 component of the visually evoked potential (VEP) data.

Methods

Using the image data set of the visible human man (VHM), a finite difference method (FDM) model of the head was constructed. The EEG dataset (256-channel) used was the N170 component of the VEP. A forward transfer matrix relating the cortical potential to the scalp potential was obtained. Using Tikhonov regularisation, the potential distribution over the cortex was obtained.

Results

The cortical potential distribution for three subjects was solved using both L-curve and GCV method. A total of 18 cortical potential distributions were obtained (3 subjects with three stimuli each – fearful face, neutral face, control objects).

Conclusions

The GCV method is a more robust method compared to L-curve to find the optimal regularisation parameter. Cortical potential imaging is a reliable method to obtain the potential distribution over cortex for VEP data.

     

Symbiotic influence of endophytic Bacillus pumilus on growth promotion and probiotic potential of the medicinal plant Ocimum sanctum

Bacillus pumilus was isolated from surface-sterilized tissues of the medicinal plant Ocimum sanctum. Scanning electron microscopic (SEM) imaging confirmed the presence of a rod shaped bacterium within the plant tissues. The bacterium was identified as B. pumilus by biochemical analyses and 16S rRNA gene sequencing. In vitro analyses indicate that the isolated strain of B. pumilus was endowed with multiple plant growth promotion (PGP) traits such as phosphate solubilization and the production of indole acetic acid (IAA), siderophore and hydrogen cyanide (HCN). Phosphate solubilization (37.3 μg ml^?1) and IAA production (36.7 μg ml^?1) by the isolate was found to reach a maximum after 60 h of incubation. Siderophore mediated iron sequestration by B. pumilus may confer a competitive advantage to the host with respect to pathogen inhibition. Siderophore produced by the isolate was found to be of a trihydroxamate type with hexadentate nature. The B. pumilus isolate also exhibited cellulolytic, proteolytic and chitinolytic activity. Cell free supernatant, culture filtrates of the isolate were found to suppress the growth of fungal phytopathogens. The culture filtrate retained its antifungal activity even after exposure to heat. In addition to PGP, the isolate exhibited probiotic properties such as acid tolerance (pH2), bile salt tolerance (2 %), auto-aggregation, antibiotic resistance and the absence of haemolytic activity. These finding suggest the possibility of utilizing this endophytic strain of B. pumilus as a bioinoculant to enhance plant growth and also as a probiotic.     

A Preliminary Report on a Combined Gram-Pappen-Heim Stain for Formalin Fixed Tissues

Although the color of indigo is strongly dependent on its environment, it is blue in most commonly encountered situations. Indigo's absorption at such long wavelengths for such a small molecule is unique, and I provide here an overview of the concepts advanced to account for this feature. A traditional valence–bond approach may be used to provide a reasonable qualitative explanation. A more rigorous, quantitative explanation is provided by molecular orbital methods of varying degrees of sophistication and several explanations have been proposed based on these models. Commonly, it is suggested that the important structural unit in determining color is based on the cross-conjugated “H-chromophore” concept. A second closely related explanation describes it as two symmetrically coupled merocyanine chains. Another proposal suggests that the basic chromophore may be interpreted as the aza analogue of two coupled anti aromatic-cyclopentadienyl ions. PiSYSTEM, a commercially available quantum mechanics program, has been used to provide a successful quantitative account of the colors of indigo and indirubin, a red isomer.     

Acute induction of autophagy as a novel strategy for cardioprotection: getting to the heart of the matter

There is no question that necrosis and apoptosis contribute to cardiomyocyte death in the setting of myocardial ischemia-reperfusion. Indeed, considerable effort and resources have been invested in the development of novel therapies aimed at attenuating necrotic and apoptotic cell death, with the ultimate goal of applying these strategies to reduce infarct size and improve outcome in patients suffering acute myocardial infarction (MI) or ‘heart attack’. However, an issue that remains controversial is the role of autophagy in determining the fate of ischemic-reperfused cardiomyocytes: i.e., is induction of autophagy detrimental or protective? Recent data from our group obtained in the clinically relevant, in vivo swine model of acute MI provide novel evidence of a positive association between pharmacological upregulation of autophagy (achieved by administration of chloramphenicol succinate (CAPS)) and increased resistance to myocardial ischemia-reperfusion injury.Key words: myocardial ischemia, myocardial infarction, ischemia-reperfusion injury, autophagy, chloramphenicolOngoing controversy concerning the role of autophagy in myocardial ischemia-reperfusion injury (pro-survival versus pro-death) may be an example of the ‘Goldilocks Principle’: uncontrolled ‘pathophysiological’ induction of autophagy in response to an extreme or prolonged stress reportedly contributes to cardiac cell death, whereas a modest ‘physiological’ upregulation of autophagy may be beneficial. Indeed, in support of this latter concept, a growing body of evidence obtained in isolated cardiomyocytes and rodent models has revealed that acute, pre-ischemic induction of autophagy can confer a cardioprotective phenotype. The objective of our recent publication was to extend this paradigm to a clinically relevant, large animal (swine) model and establish whether pharmacological upregulation of autophagy would render the heart resistant to lethal ischemia-reperfusion injury and thus limit myocardial infarct size.To test this concept, anesthetized pigs were assigned to receive our candidate drug, chloramphenicol succinate (CAPS) or placebo and, at 10 min after treatment, underwent 45 min of coronary artery occlusion followed by 3 h of reperfusion. Administration of CAPS results in a rapid and robust upregulation in molecular markers of autophagy: at 10 min post-treatment (the time corresponding to the onset of the sustained ischemic insult), we observed a 2.4- and 6.2-fold increase in expression of Beclin 1 and LC3-II, respectively, versus baseline. However, most notably, CAPS-treated pigs displayed a profound, ~50% reduction in infarct size when compared with placebo-controls. To investigate whether the favorable effect of CAPS was retained when administered in a more clinically relevant manner, an additional cohort of pigs received CAPS at 15 min before the onset of reperfusion. Efficacy was maintained (albeit attenuated) with delayed treatment, with mean infarct size reduced by ~27% versus controls.The novel aspect of our study is the unequivocal documentation of a significant infarct-sparing effect of CAPS in a well-established pre-clinical model of ischemia-reperfusion injury, thereby bringing the concept of cardioprotection via pharmacological upregulation of autophagy one step closer to future clinical evaluation. Nonetheless, our use of the swine model has an inherent weakness: although we have shown compelling evidence of an association between induction of autophagy and reduction of infarct size, the pig is not amenable to the application of genetic and molecular tools that would yield definitive documentation of cause-and-effect.As acknowledged in our recent publication, an issue of particular relevance in establishing the mechanism by which CAPS confers cardioprotection is the tight and complex interaction between autophagy and the PtdIns3K-Akt-mTOR signaling pathway. Specifically, class III PtdIns3K is an activator of autophagy and, via its interaction with Beclin 1, plays a pivotal role in initiating autophagosome formation, whereas class I PtdIns3K purportedly suppresses autophagy. Interaction at the level of mTOR is multifaceted, bi-directional and has been reported to exert both positive and negative feedback; i.e., while activation of mTOR is associated with inhibition of autophagy, there is evidence of self-regulation of autophagy by autophagy-induced inhibition of mTOR and, in at least one model, co-activation of autophagy and mTOR. PtdIns3K-Akt-mTOR are also components of the ‘Reperfusion Injury Salvage Kinase’ or RISK pathway, a canonical cardioprotective signal transduction pathway that, when activated, has been shown in multiple models to attenuate lethal ischemia-reperfusion injury. Akt signaling is upregulated by a host of protective strategies including ischemic preconditioning (considered the ‘gold standard’ of cardioprotection) and pharmacological preconditioning-mimetic agents. It is therefore perhaps not surprising that administration of CAPS is accompanied by an increase in expression of phospho-Akt.If CAPS treatment is associated with both an induction of autophagy (as documented in our study) and, as with many cardioprotective strategies, upregulation of Akt signaling, this raises two intriguing and interrelated possibilities. First, autophagy and PtdIns3K-Akt-mTOR signaling may yield additive benefit. Second, we speculate that co-activation of the Akt signaling pathway may, by these complex bi-directional interactions, assist in establishing an appropriate balance and maintaining autophagy in a favorable, pro-survival ‘Goldilocks’ state. Our data clearly demonstrate that CAPS is cardioprotective, and may, via induction of autophagy, provide a novel and clinically relevant therapy to attenuate myocardial ischemia-reperfusion injury. However, detailed molecular investigation will be required to ‘get to the heart’ of the mechanisms underlying the reduction of infarct size seen with CAPS treatment.     

Impaired mitophagy at the heart of injury

Recent publications link mitophagy mediated by PINK1 and Parkin with cardioprotection and attenuation of inflammation and cell death. The field is in need of methods to monitor mitochondrial turnover in vivo to support the development of new therapies targeting mitochondrial turnover.