Simultaneous determination of metoprolol succinate and amlodipine besylate in human plasma by liquid chromatography-tandem mass spectrometry method and its application in bioequivalence study

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of metoprolol succinate (MPS) and amlodipine besylate (AM) using hydrochlorothiazide (HCTZ) as IS in human plasma. Both the drugs were extracted by simple liquid-liquid extraction with chloroform. The chromatographic separation was performed on a reversed-phase peerless basic C18 column with a mobile phase of methanol-water containing 0.5% formic acid (8:2, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The method was validated over the concentration range of 1-100ng/ml for MPS and 1-15ng/ml AM in human plasma. The MRM transition of m/z 268.10-103.10, m/z 409.10-334.20 and m/z 296.00-205.10 were used to measure MPS, AM and HCTZ (IS), respectively. This method was successfully applied to the pharmacokinetic study of fixed dose combination (FDC) of MPS and AM formulation product after an oral administration to Indian healthy human volunteers.     

Positive feedback and noise activate the stringent response regulator rel in mycobacteria

Phenotypic heterogeneity in an isogenic, microbial population enables a subset of the population to persist under stress. In mycobacteria, stresses like nutrient and oxygen deprivation activate the stress response pathway involving the two-component system MprAB and the sigma factor, SigE. SigE in turn activates the expression of the stringent response regulator, rel. The enzyme polyphosphate kinase 1 (PPK1) regulates this pathway by synthesizing polyphosphate required for the activation of MprB. The precise manner in which only a subpopulation of bacterial cells develops persistence, remains unknown. Rel is required for mycobacterial persistence. Here we show that the distribution of rel expression levels in a growing population of mycobacteria is bimodal with two distinct peaks corresponding to low (L) and high (H) expression states, and further establish that a positive feedback loop involving the mprAB operon along with stochastic gene expression are responsible for the phenotypic heterogeneity. Combining single cell analysis by flow cytometry with theoretical modeling, we observe that during growth, noise-driven transitions take a subpopulation of cells from the L to the H state within a "window of opportunity" in time preceding the stationary phase. It is these cells which adapt to nutrient depletion in the stationary phase via the stringent response. We find evidence of hysteresis in the expression of rel in response to changing concentrations of PPK1. Hysteresis promotes robustness in the maintenance of the induced state. Our results provide, for the first time, evidence that bistability and stochastic gene expression could be important for the development of "heterogeneity with an advantage" in mycobacteria and suggest strategies for tackling tuberculosis like targeting transitions from the low to the high rel expression state.     

Erythropoietin enhancement of rat pancreatic tumor cell proliferation requires the activation of ERK and JNK signals

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor EPOR. Recent studies, however, have shown that the EPOR is additionally present in various cancer cells and EPO induces the proliferation of these cells, suggesting a different function for EPO other than erythropoiesis. Therefore, the purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation and signaling cascades involved in this process, using the rat pancreatic tumor cell line AR42J. Our results showed that AR42J cells expressed EPOR, and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated cells indicated an increased percentage of cells in the S phase, whereas cell numbers in G0/G1 phase were significantly reduced. Phosphorylation of extracellular regulatory kinase 1/2 (ERK1/2) and c-Jun NH₂ terminal kinase 1/2 (JNK1/2) was rapidly stimulated and sustained after EPO addition. Treatment of cells with mitogen-activated protein/ERK kinase (MEK) inhibitor PD98059 or JNK inhibitor SP600125 significantly inhibited EPO-enhanced proliferation and also increased the fraction of cells in G0/G1 phase. Furthermore, the inhibition of JNK using small interference RNA (siRNA) suppressed EPO-enhanced proliferation of AR42J cells. Taken together, our results indicate that AR42J cells express EPOR and that the activation of both ERK1/2 and JNK1/2 by EPO is essential in regulating proliferation and the cell cycle. Thus both appear to play a key role in EPO-enhanced proliferation and suggest that the presence of both is required for EPO-mediated proliferation of AR42J cells. erythropoietin receptor; cell signaling; mitogen-activated protein kinase induction     

Maintaining phase of the crustacean tri-phasic pyloric rhythm

We construct and analyze a model network of the pyloric rhythm of the crustacean stomatogastric ganglion consisting of an oscillator neuron that inhibits two reciprocally inhibitory follower neurons. We derive analytic expressions that determine the phase of firing of the follower neurons with respect to the oscillator. An important aspect of the model is the inclusion of synapses that exhibit short-term synaptic depression. We show that these type of synapses allow there to be a complicated relationship between the intrinsic properties of the neurons and the synapses between them in determining phase relationships. Our analysis reveals the circumstances and ranges of cycle periods under which these properties work in concert with or independently from one another. In particular, we show that phase maintenance over a range of oscillator periods can be enhanced through the interplay of the two follower neurons if the synapses between these neurons are depressing. Since our model represents the core of the oscillatory pyloric network, the results of our analysis can be compared to experimental data and used to make predictions about the biological network.     

Positive and negative aspects of soda/anthraquinone pulping of hardwoods

The positive aspects of the non-sulfur soda/anthraquinone (SAQ) process are mostly tied to improved energy efficiency while lower pulp brightness after bleaching is its most significant drawback. A credible method that quantifies bleachability as well as an approach that solves the problem for SAQ pulps from hardwoods will be described. A straight line correlation (R2=0.904) was obtained between O2 kappa number and final light absorption coefficient (LAC) value after standardized OD0EpD1 bleaching of nine hardwood kraft pulps from three laboratories and one pulp mill. The bleachability of pulps from four different soda processes catalyzed by anthraquinone (AQ) and 2-methylanthraquinone (MAQ) was compared to that of conventional kraft pulps by comparing O2 kappa number decrease and final LAC values. It was observed that a mild hot water pre-hydrolysis improved the bleachability of SAQ pulps to a level equal to that of kraft.     

Removal of the pro-domain does not affect the conformation of the procaspase-3 dimer.

We have investigated the oligomeric properties of procaspase-3 and a mutant that lacks the pro-domain (called pro-less variant). In addition, we have examined the interactions of the 28 amino acid pro-peptide when added in trans to the pro-less variant. By sedimentation equilibrium studies, we have found that procapase-3 is a stable dimer in solution at 25 degrees C and pH 7.2, and we estimate an upper limit for the equilibrium dissociation constant of approximately 50 nM. Considering the expression levels of caspase-3 in Jurkat cells, we predict that procaspase-3 exists as a dimer in vivo. The pro-less variant is also a dimer, with little apparent change in the equilibrium dissociation constant. Thus, in contrast with the long pro-domain caspases, the pro-peptide of caspase-3 does not appear to be involved in dimerization. Results from circular dichroism, fluorescence anisotropy, and FTIR studies demonstrate that the pro-domain interacts weakly with the pro-less variant. The data suggest that the pro-peptide adopts a beta-structure when in contact with the protein, but it is a random coil when free in solution. In addition, when added in trans, the pro-peptide does not inhibit the activity of the mature caspase-3 heterotetramer. On the other hand, the active caspase-3 does not efficiently hydrolyze the pro-domain at the NSVD(9) sequence as occurs when the pro-peptide is in cis to the protease domain. Based on these results, we propose a model for maturation of the procaspase-3 dimer.     

Dimeric procaspase-3 unfolds via a four-state equilibrium process.

We have examined the folding and assembly of a catalytically inactive mutant of procaspase-3, a homodimeric protein that belongs to the caspase family of proteases. The caspase family, and especially caspase-3, is integral to apoptosis. The equilibrium unfolding data demonstrate a plateau between 3 and 5 M urea, consistent with an apparent three-state unfolding process. However, the midpoint of the second transition as well as the amplitude of the plateau are dependent on the protein concentration. Overall, the data are well described by a four-state equilibrium model in which the native dimer undergoes an isomeration to a dimeric intermediate, and the dimeric intermediate dissociates to a monomeric intermediate, which then unfolds. By fitting the four-state model to the experimental data, we have determined the free energy change for the first step of unfolding to be 8.3 +/- 1.3 kcal/mol. The free energy change for the dissociation of the dimeric folding intermediate to two monomeric intermediates is 10.5 +/- 1 kcal/mol. The third step in the unfolding mechanism represents the complete unfolding of the monomeric intermediate, with a free energy change of 7.0 +/- 0.5 kcal/mol. These results show two important points. First, dimerization of procaspase-3 occurs as a result of the association of two monomeric folding intermediates, demonstrating that procaspase-3 dimerization is a folding event. Second, the stability of the dimer contributes significantly to the conformational free energy of the protein (18.8 of 25.8 kcal/mol).     

Purple membrane lipid control of bacteriorhodopsin conformational flexibility and photocycle activity.

Specific lipids of the purple membrane of Halobacteria are required for normal bacteriorhodopsin structure, function, and photocycle kinetics [Hendler, R.W. & Dracheva, S. (2001) Biochemistry (Moscow)66, 1623-1627]. The decay of the M-fast intermediate through a path including the O intermediate requires the presence of a hydrophobic environment near four charged aspartic acid residues within the cytoplasmic loop region of the protein (R. W. Hendler & S. Bose, unpublished results). On the basis of the unique ability of squalene, the most hydrophobic purple membrane lipid, to induce recovery of M-fast activity in Triton-treated purple membrane, we proposed that this uncharged lipid modulates an electrostatic repulsion between the membrane surface of the inner trimer space and the nearby charged aspartic acids of the cytoplasmic loop region to promote transmembrane alpha-helical mobility with a concomitant increase in the speed of the photocycle. We examined Triton-treated purple membranes in various stages of reconstitution with native lipid suspensions using infrared spectroscopic techniques. We demonstrate a correlation between the vibrational half-width parameter of the protein alpha-helical amide I mode at 1660 cm-1, reflecting the motional characteristics of the transmembrane helices, and the lipid-induced recovery of native bacteriorhodopsin properties in terms of the visible absorbance maxima of ground state bacteriorhodopsin and the mean decay times of the photocycle M-state intermediates.     

Superior Appetite Hormone Profile After Equivalent Weight Loss by Gastric Bypass Compared to Gastric Banding

The goal of this study was to understand the mechanisms of greater weight loss by gastric bypass (GBP) compared to gastric banding (GB) surgery. Obese weight‐ and age‐matched subjects were studied before (T0), after a 12 kg weight loss (T1) by GBP (n = 11) or GB (n = 9), and at 1 year after surgery (T2). peptide YY_3–36 (PYY_3–36), ghrelin, glucagon‐like peptide‐1 (GLP‐1), leptin, and amylin were measured after an oral glucose challenge. At T1, glucose‐stimulated GLP‐1 and PYY levels increased significantly after GBP but not GB. Ghrelin levels did not change significantly after either surgery. In spite of equivalent weight loss, leptin and amylin decreased after GBP, but not after GB. At T2, weight loss was greater after GBP than GB (P = 0.003). GLP‐1, PYY, and amylin levels did not significantly change from T1 to T2; leptin levels continued to decrease after GBP, but not after GB at T2. Surprisingly, ghrelin area under the curve (AUC) increased 1 year after GBP (P = 0.03). These data show that, at equivalent weight loss, favorable GLP‐1 and PYY changes occur after GBP, but not GB, and could explain the difference in weight loss at 1 year. Mechanisms other than weight loss may explain changes of leptin and amylin after GBP.