Chromatographic separation and sensitive determination of teriflunomide,an active metabolite of leflunomide in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive, selective and high throughput liquid chromatography tandem mass spectrometry (LC–ESI-MS/MS) method has been developed for the determination of teriflunomide, an active metabolite of leflunomide in human plasma. Plasma samples were prepared by liquid–liquid extraction of teriflunomide and valsartan as internal standard (IS) in ethyl acetate from 200 μL human plasma. The chromatographic separation was achieved on an Inertsil ODS-3 C18 (50 mm × 4.6 mm, 3 μm) analytical column using isocratic mobile phase, consisting of 20 mM ammonium acetate–methanol (25:75, v/v), at a flow-rate of 0.8 mL/min. The precursor → product ion transition for teriflunomide (m/z 269.0 → 82.0) and IS (m/z 434.1 → 350.3) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and negative ion mode. The method was validated over a wide dynamic concentration range of 10.1–4001 ng/mL. Matrix effect was assessed by post-column infusion experiment and the mean process efficiency were 91.7% and 88.2% for teriflunomide and IS respectively. The method was rugged and rapid with a total run time of 2.0 min and is applied to a bioequivalence study of 20 mg leflunomide (test and reference) tablet formulation in 12 healthy Indian male subjects under fasting condition.     

Modulations in the intestinal disaccharide hydrolases and membrane dynamics: Effect of non-steroidal anti-inflammatory drugs aspirin and nimesulide

The present study was designed to evaluate the influence of two commonly prescribed non-steroidal anti-inflammatory drugs (NSAIDs), aspirin and nimesulide on the biochemical composition and membrane dynamics of rat intestine. Female Wistar rats were divided into three different groups viz: Group I (Control), Group II (aspirin-treated, 50 mg/kg body weight) and Group III (nimesulide-treated, 10 mg/kg body weight). After 28 days, biochemical estimations in both drug treated groups showed an increase in sucrase, lactase, maltase and alkaline phosphatase as compared to the control. Alterations in the intestinal membrane dynamics by fluidity studies and Fourier Transform Infra Red (FTIR) spectroscopy also showed considerable changes. The alterations in the histoarchitecture of the intestine were also seen, which correlated well with the changes in structure and composition of the intestine. The use of NSAIDs like aspirin and nimesulide may cause the gastrointestinal side effects due to initial changes in the enzyme activities and membrane dynamics.     

The CBL–CIPK signaling module in plants: a mechanistic perspective

In a given environment, plants are constantly exposed to multitudes of stimuli. These stimuli are sensed and transduced to generate a diverse array of responses by several signal transduction pathways. Calcium (Ca²⁺) signaling is one such important pathway involved in transducing a large number of stimuli or signals in both animals and plants. Ca²⁺ engages a plethora of decoders to mediate signaling in plants. Among these groups of decoders, the sensor responder complex of calcineurin B‐like protein (CBL) and CBL‐interacting protein kinases (CIPKs) play a very significant role in transducing these signals. The signal transduction mechanism in most cases is phosphorylation events, but some structural role for the pair has also come to light recently. In this review, we discuss the structural nature of the sensor‐responder duo; their mechanism of substrate phosphorylation and also their structural role in modulating targets. Moreover, the mechanism of complex formation and mechanistic role of protein phosphatases with CBL–CIPK module has been mentioned. A comparison of CBL–CIPK with other decoders of Ca²⁺ signaling in plants also signifies the relatedness and diversity in signaling pathways. Further an attempt has been made to compare this aspect of Ca²⁺ signaling pathways in different plant species to develop a holistic understanding of conservation of stimulus–response‐coupling mediated by this Ca²⁺–CBL–CIPK module.     

A Novel Link between Fic (Filamentation Induced by cAMP)-mediated Adenylylation/AMPylation and the Unfolded Protein Response

The maintenance of endoplasmic reticulum (ER) homeostasis is a critical aspect of determining cell fate and requires a properly functioning unfolded protein response (UPR). We have discovered a previously unknown role of a post-translational modification termed adenylylation/AMPylation in regulating signal transduction events during UPR induction. A family of enzymes, defined by the presence of a Fic (filamentation induced by cAMP) domain, catalyzes this adenylylation reaction. The human genome encodes a single Fic protein, called HYPE (Huntingtin yeast interacting protein E), with adenylyltransferase activity but unknown physiological target(s). Here, we demonstrate that HYPE localizes to the lumen of the endoplasmic reticulum via its hydrophobic N terminus and adenylylates the ER molecular chaperone, BiP, at Ser-365 and Thr-366. BiP functions as a sentinel for protein misfolding and maintains ER homeostasis. We found that adenylylation enhances BiP''s ATPase activity, which is required for refolding misfolded proteins while coping with ER stress. Accordingly, HYPE expression levels increase upon stress. Furthermore, siRNA-mediated knockdown of HYPE prevents the induction of an unfolded protein response. Thus, we identify HYPE as a new UPR regulator and provide the first functional data for Fic-mediated adenylylation in mammalian signaling.     

CaMtw1, a member of the evolutionarily conserved Mis12 kinetochore protein family, is required for efficient inner kinetochore assembly in the pathogenic yeast Candida albicans

Proper assembly of the kinetochore, a multi-protein complex that mediates attachment of centromere DNA to spindle microtubules on each chromosome, is required for faithful chromosome segregation. Each previously characterized member of the Mis12/Mtw1 protein family is part of an essential subcomplex in the kinetochore. In this work, we identify and characterize CaMTW1, which encodes the homologue of the human Mis12 protein in the pathogenic budding yeast Candida albicans. Subcellular localization and chromatin immunoprecipitation assays confirmed CaMtw1 is a kinetochore protein. CaMtw1 is essential for viability. CaMtw1-depleted cells and cells in which CaMtw1 was inactivated with a temperature-sensitive mutation had reduced viability, accumulated at the G2/M stage of the cell cycle, and exhibited increased chromosome missegregation. CaMtw1 depletion also affected spindle length and alignment. Interestingly, in C. albicans, CaMtw1 and the centromeric histone, CaCse4, influence each other for kinetochore localization. In addition, CaMtw1 is required for efficient kinetochore recruitment of another inner kinetochore protein, the CENP-C homologue, CaMif2. Mis12/Mtw1 proteins have well-established roles in the recruitment and maintenance of outer kinetochore proteins. We propose that Mis12/Mtw1 proteins also have important co-dependent interactions with inner kinetochore proteins and that these interactions may increase the fidelity of kinetochore formation.     

Functional characterization of the Saccharomyces cerevisiae protein Chl1 reveals the role of sister chromatid cohesion in the maintenance of spindle length during S-phase arrest

Background

Reconstructing the evolutionary history of a species is challenging. It often depends not only on the past biogeographic and climatic events but also the contemporary and ecological factors, such as current connectivity and habitat heterogeneity. In fact, these factors might interact with each other and shape the current species distribution. However, to what extent the current population genetic structure reflects the past and the contemporary factors is largely unknown. Here we investigated spatio-temporal genetic structures of Nile tilapia (Oreochromis niloticus) populations, across their natural distribution in Africa. While its large biogeographic distribution can cause genetic differentiation at the paleo-biogeographic scales, its restricted dispersal capacity might induce a strong genetic structure at micro-geographic scales.

Results

Using nine microsatellite loci and 350 samples from ten natural populations, we found the highest genetic differentiation among the three ichthyofaunal provinces and regions (Ethiopian, Nilotic and Sudano-Sahelian) (R _ST = 0.38 - 0.69). This result suggests the predominant effect of paleo-geographic events at macro-geographic scale. In addition, intermediate divergences were found between rivers and lakes within the regions, presumably reflecting relatively recent interruptions of gene flow between hydrographic basins (R _ST = 0.24 - 0.32). The lowest differentiations were observed among connected populations within a basin (R _ST = 0.015 in the Volta basin). Comparison of temporal sample series revealed subtle changes in the gene pools in a few generations (F = 0 - 0.053). The estimated effective population sizes were 23 - 143 and the estimated migration rate was moderate (m ~ 0.094 - 0.097) in the Volta populations.

Conclusions

This study revealed clear hierarchical patterns of the population genetic structuring of O. niloticus in Africa. The effects of paleo-geographic and climatic events were predominant at macro-geographic scale, and the significant effect of geographic connectivity was detected at micro-geographic scale. The estimated effective population size, the moderate level of dispersal and the rapid temporal change in genetic composition might reflect a potential effect of life history strategy on population dynamics. This hypothesis deserves further investigation. The dynamic pattern revealed at micro-geographic and temporal scales appears important from a genetic resource management as well as from a biodiversity conservation point of view.