Differences in the transient kinetics of the binding of D-ADP and its mirror image L-ADP to human 3-phosphoglycerate kinase revealed by the presence of 3-phosphoglycerate

L-Nucleosides comprise a new class of antiviral and anticancer agents that are converted in vivo by a cascade of kinases to pharmacologically active nucleoside triphosphates. The last step of the cascade may be catalyzed by 3-phosphoglycerate kinase (PGK), an enzyme that has low specificity for nucleoside diphosphate (NDP): NDP + 1,3-bisphosphoglycerate <--> NTP + 3-phosphoglycerate. Here we compared the kinetics of the formation of the complexes of human PGK with d- and its mirror image l-ADP and the effect of 3-phosphoglycerate (PG) on these by exploiting the fluorescence signal of PGK that occurs upon its interaction with nucleotide substrate. Two types of experiment were carried out: equilibrium (estimation of dissociation constants) and stopped-flow (transient kinetics of the interactions). We show that under our experimental conditions (buffer containing 30% methanol, 4 degrees C) PGK binds d- and l-ADP with similar kinetics. However, whereas PG increased the dissociation rate constant for d-ADP by a factor of 8-which is a kinetic explanation for "substrate antagonism"-PG had little effect on this constant for l-ADP. We explain this difference by a molecular modeling study that showed that the beta-phosphates of d- and l-ADP have different orientations when bound to the active site of human PGK. The difference is unexpected because l-ADP is almost as catalytically competent as d-ADP [ Varga, A. et al. (2008) Biochem. Biophys. Res. Commun. 366, 994-1000].     

Development of an Automated and Sensitive Microfluidic Device for Capturing and Characterizing Circulating Tumor Cells (CTCs) from Clinical Blood Samples

Current analysis of circulating tumor cells (CTCs) is hindered by sub-optimal sensitivity and specificity of devices or assays as well as lack of capability of characterization of CTCs with clinical biomarkers. Here, we validate a novel technology to enrich and characterize CTCs from blood samples of patients with metastatic breast, prostate and colorectal cancers using a microfluidic chip which is processed by using an automated staining and scanning system from sample preparation to image processing. The Celsee system allowed for the detection of CTCs with apparent high sensitivity and specificity (94% sensitivity and 100% specificity). Moreover, the system facilitated rapid capture of CTCs from blood samples and also allowed for downstream characterization of the captured cells by immunohistochemistry, DNA and mRNA fluorescence in-situ hybridization (FISH). In a subset of patients with prostate cancer we compared the technology with a FDA-approved CTC device, CellSearch and found a higher degree of sensitivity with the Celsee instrument. In conclusion, the integrated Celsee system represents a promising CTC technology for enumeration and molecular characterization.     

Biofilm inhibition and anti-Candida activity of a cationic lipo-benzamide molecule with twin-nonyl chain

A series of cationic lipo-benzamide compounds with varying lengths of hydrocarbon chains (C2M–C18M) were evaluated for anti-Candida activity. Four compounds harbouring 8–11 hydrocarbon chains demonstrated concentration-dependent inhibition of fungal cell growth with Minimum Inhibitory Concentration (MIC) of ≤6.2?µg?ml^?1. The most active compound (C9M) inhibited growth of both Candida albicans and non-albicans strains and is equally active against pairs of azole sensitive and resistant clinical isolates of C. albicans. Compound C9M also inhibited different stages of Candida biofilms. Scanning Electron Microscopy (SEM) of Candida cells after C9M treatment was also done and no significant cell lysis was observed. Hemolysis assay was performed and only 2.5% haemolysis was observed at MIC concentration.     

In vitro leishmanicidal effects of the anti-fungal drug natamycin are mediated through disruption of calcium homeostasis and mitochondrial dysfunction

Natamycin, a Food and Drug Administration approved anti-fungal drug, and also used as a food additive was evaluated for anti-leishmanial activity since it is known to specifically bind to ergosterol, which is essential to these parasites but absent in mammals. Promising anti-proliferative activity was observed in both promastigote and amastigote forms of the parasite with IC₅₀ values of 15 and 8 µM respectively and a selective index of 12.5. The ultrastructural effects of natamycin on both forms of the parasite and physiological effects on promastigotes were studied in detail for the first time. Electron microscopic observations in treated cells revealed sub-cellular changes like plasma membrane alterations, accumulation of vesicles in the flagellar pocket and extensive mitochondrial damage. Natamycin treatment in promastigotes resulted in elevation of cytosolic calcium (Ca²⁺) levels which caused irreversible loss of mitochondrial membrane potential. This resulted in depletion of cellular ATP levels along with ROS generation finally leading to apoptosis-like and necrotic cell death. In view of our observations along with the safety profile of an existing anti-fungal drug, natamycin may be further investigated for repurposing it as a promising drug candidate against Leishmaniasis.     

Fractal fluctuations in human respiration.

The present study was designed to characterize respiratory fluctuations in awake, healthy adult humans under resting conditions. For this purpose, we recorded respiratory movements with a strain-gauge pneumograph in 20 subjects. We then used Allan factor, Fano factor, and dispersional analysis to test whether the fluctuations in the number of breaths, respiratory period, and breath amplitude were fractal (i.e., time-scale-invariant) or random in occurrence. Specifically, we measured the slopes of the power laws in the Allan factor, Fano factor, and dispersional analysis curves for original time series and compared these with the slopes of the curves for surrogates (randomized data sets). In addition, the Hurst exponent was calculated from the slope of the power law in the Allan factor curve to determine whether the long-range correlations among the fluctuations in breath number were positively or negatively correlated. The results can be summarized as follows. Fluctuations in all three parameters were fractal in nine subjects. There were four subjects in whom only the fluctuations in number of breaths and breath amplitude were fractal, three subjects in whom only the fluctuations in number of breaths were fractal, and two subjects in whom only fluctuations in breath number and respiratory period were fractal. Time-scale-invariant behavior was absent in the two remaining subjects. The results indicate that, in most cases, apparently random fluctuations in respiratory pattern are, in fact, correlated over more than one time scale. Moreover, the data suggest that fractal fluctuations in breath number, respiratory period, and breath amplitude are controlled by separate processes.     

Medullary lateral tegmental field neurons influence the timing and pattern of phrenic nerve activity in cats.

In an effort to characterize the role of the medullary lateral tegmental field (LTF) in regulating respiration, we tested the effects of selective blockade of excitatory (EAA) and inhibitory amino acid (IAA) receptors in this region on phrenic nerve activity (PNA) of vagus-intact and vagotomized cats anesthetized with dial-urethane. We found distinct patterns of changes in central respiratory rate, duration of inspiratory and expiratory phases of PNA (Ti and Te, respectively), and I-burst amplitude after selective blockade of EAA and IAA receptors in the LTF. First, blockade of N-methyl-D-aspartate (NMDA) receptors significantly (P < 0.05) decreased central respiratory rate primarily by increasing Ti but did not alter I-burst amplitude. Second, blockade of non-NMDA receptors significantly reduced I-burst amplitude without affecting central respiratory rate. Third, blockade of GABAA receptors significantly decreased central respiratory rate by increasing Te and significantly reduced I-burst amplitude. Fourth, blockade of glycine receptors significantly decreased central respiratory rate by causing proportional increases in Ti and Te and significantly reduced I-burst amplitude. These changes in PNA were markedly different from those produced by blockade of EAA or IAA receptors in the pre-B?tzinger complex. We propose that a proper balance of excitatory and inhibitory inputs to several functionally distinct pools of LTF neurons is essential for maintaining the normal pattern of PNA in anesthetized cats.