Store operated Ca2+ entry dependent contraction of coronary artery smooth muscle: inhibition by peroxide pretreatment

The sarco/endoplasmic reticulum (SER) Ca(2+) pool is refilled by the SER Ca(2+) pump (SERCA) using cytosolic Ca(2+) and/or extracellular Ca(2+) entering the cell. The effects of the SERCA pump inhibitor cyclopiazonic acid (CPA) were studied in pig coronary artery smooth muscle using two protocols. In protocol A, the SERCA pump was inhibited by adding CPA to cells/tissues in Ca(2+)-containing solution, whereas in protocol B, CPA was added to cells/tissues in Ca(2+)-free solution, followed by reintroduction of extracellular Ca(2+). Addition of CPA increased cytosolic Ca(2+) in cultured smooth muscle cells and elicited contraction in de-endothelialized coronary arteries in both protocols. Based on pharmacological experiments, the CPA-induced contraction of de-endothelialized arteries in protocol B resulted from store operated Ca(2+) entry (SOCE). Reactive oxygen species such as peroxides are known to damage the SERCA pump in this tissue. Consistently, CPA-induced contractions were decreased in arteries pre-treated with hydrogen peroxide in protocol A. However, this pretreatment also decreased the force of contraction due to SOCE in protocol B, suggesting that it closed SOCE. We propose that the closure of SOCE triggered by exposure to reactive oxygen species may be a protective mechanism, so that Ca(2+) entry by this pathway is disallowed when SERCA is damaged in pathologies such as ischemia-reperfusion.     

Genetic variation in histidine rich proteins among Indian Plasmodium falciparum population: possible cause of variable sensitivity malaria of rapid diagnostic tests

ABSTRACT: BACKGROUND: Rapid diagnostic tests (RDTs) have revolutionized the diagnosis of malaria. Among the various factors affecting RDTs sensitivity is genetic variation of the antigen used. The genetic variation in PfHRP2 and PfHRP3 proteins was studied among the Indian Plasmodium falciparum isolates. METHODS: One hundred and forty isolates of P. falciparum were collected from six geographical regions of India. Target genes encoding PfHRP2 and PfHRP3 antigens were sequenced to study genetic polymorphism. Minimum detection limit giving a positive rapid diagnostic test was also determined. RESULTS: Extensive variations were observed in amino acid repeat types of PfHRP2 and PfHRP3. PfHRP2 exhibited more polymorphism than PfHRP3. Significant relation was observed between type 2 and type 7 repeats and RDT detection rate as higher number of these repeats showed better sensitivity with RDTs. CONCLUSION: The results provide insights into the genetic diversity of Pfhrp2 and Pfhrp3 genes among Indian P. falciparum population and its relation to RDT sensitivity.     

The solvation interface is a determining factor in peptide conformational preferences

The 21 residue polyalanine-based F(s) peptide was studied using thousands of long, explicit solvent, atomistic molecular dynamics simulations that reached equilibrium at the ensemble level. Peptide conformational preference as a function of hydrophobicity was examined using a spectrum of explicit solvent models, and the peptide length-dependence of the hydrophilic and hydrophobic components of solvent-accessible surface area for several ideal conformational types was considered. Our results demonstrate how the character of the solvation interface induces several conformational preferences, including a decrease in mean helical content with increased hydrophilicity, which occurs predominantly through reduced nucleation tendency and, to a lesser extent, destabilization of helical propagation. Interestingly, an opposing effect occurs through increased propensity for 3(10)-helix conformations, as well as increased polyproline structure. Our observations provide a framework for understanding previous reports of conformational preferences in polyalanine-based peptides including (i) terminal 3(10)-helix prominence, (ii) low pi-helix propensity, (iii) increased polyproline conformations in short and unfolded peptides, and (iv) membrane helix stability in the presence and absence of water. These observations provide physical insight into the role of water in peptide conformational equilibria at the atomic level, and expand our view of the complexity of even the most "simple" of biopolymers. Whereas previous studies have focused predominantly on hydrophobic effects with respect to tertiary structure, this work highlights the need for consideration of such effects at the secondary structural level.     

Genetic diversity in two leading Plasmodium vivax malaria vaccine candidates AMA1 and MSP119 at three sites in India

Plasmodium vivax, a major contributor to the malaria burden in India, has the broadest geographic distribution and shows higher genetic diversity than P. falciparum. Here, we investigated the genetic diversity of two leading P. vivax vaccine candidate antigens, at three geographically diverse malaria-endemic regions in India. Pvama1 and Pvmsp1₁₉ partial coding sequences were generated from one hundred P. vivax isolates in India (Chennai n = 28, Nadiad n = 50 and Rourkela n = 22) and ~1100 published sequences from Asia, South America, North America, and Oceania regions included. These data were used to assess the genetic diversity and potential for vaccine candidacy of both antigens on a global scale. A total of 44 single nucleotide polymorphism (SNPs) were identified among 100 Indian Pvama1 sequences, including 10 synonymous and 34 nonsynonymous mutations. Nucleotide diversity was higher in Rourkela and Nadiad as compared to Chennai. Nucleotide diversity measures showed a strong balancing selection in Indian and global population for domain I of Pvama1, which suggests that it is a dominant target of the protective immune response. In contrast, the Pvmsp1₁₉ region showed highly conserved sequences in India and across the Oceania, South America, North America and Asia, demonstrating low genetic diversity in the global population when compared to Pvama1. Results suggest the possibility of including Pvmsp1₁₉ in a multivalent vaccine formulation against P. vivax infections. However, the high genetic diversity seen in Pvama1 would be more challenging for vaccine development.     

Insights into structure, dynamics and hydration of locked nucleic acid (LNA) strand-based duplexes from molecular dynamics simulations

Locked nucleic acid (LNA) is a chemically modified nucleic acid with its sugar ring locked in an RNA-like (C3′-endo) conformation. LNAs show extraordinary thermal stabilities when hybridized with DNA, RNA or LNA itself. We performed molecular dynamics simulations on five isosequential duplexes (LNA–DNA, LNA–LNA, LNA–RNA, RNA–DNA and RNA–RNA) in order to characterize their structure, dynamics and hydration. Structurally, the LNA–DNA and LNA–RNA duplexes are found to be similar to regular RNA–DNA and RNA–RNA duplexes, whereas the LNA–LNA duplex is found to have its helix partly unwound and does not resemble RNA–RNA duplex in a number of properties. Duplexes with an LNA strand have on average longer interstrand phosphate distances compared to RNA–DNA and RNA–RNA duplexes. Furthermore, intrastrand phosphate distances in LNA strands are found to be shorter than in DNA and slightly shorter than in RNA. In case of induced sugar puckering, LNA is found to tune the sugar puckers in partner DNA strand toward C3′-endo conformations more efficiently than RNA. The LNA–LNA duplex has lesser backbone flexibility compared to the RNA–RNA duplex. Finally, LNA is less hydrated compared to DNA or RNA but is found to have a well-organized water structure.     

Non‐invasive optical assessment of viscosity of middle ear effusions in otitis media

Eustachian tube dysfunction can cause fluid to collect within the middle ear cavity and form a middle ear effusion (MEE). MEEs can persist for weeks or months and cause hearing loss as well as speech and learning delays in young children. The ability of a physician to accurately identify and characterize the middle ear for signs of fluid and/or infection is crucial to provide the most appropriate treatment for the patient. Currently, middle ear infections are assessed with otoscopy, which provides limited and only qualitative diagnostic information. In this study, we propose a method utilizing cross‐sectional depth‐resolved optical coherence tomography to noninvasively measure the diffusion coefficient and viscosity of colloid suspensions, such as a MEE. Experimental validation of the proposed technique on simulated MEE phantoms with varying viscosity and particulate characteristics is presented, along with some preliminary results from in vivo and ex vivo samples of human MEEs.

In vivo Optical Coherence Tomography (OCT) image of a human tympanic membrane and Middle Ear Effusion (MEE) (top), with a CCD image of the tympanic membrane surface (inset). Below is the corresponding time‐lapse M‐mode OCT data acquired along the white dotted line over time, which can be analyzed to determine the Stokes–Einstein diffusion coefficient of the effusion.     

Sequence-specific delivery of a quinone methide intermediate to the major groove of DNA.

Silyl-protected phenol derivatives serve as convenient precursors for generating highly electrophilic quinone methide intermediates under biological conditions. Reaction is initiated by addition of fluoride and has previously exhibited proficiency in DNA alkylation and cross-linking. This approach has now been extended to the modification of duplex DNA through triplex recognition and fluoride-dependent quinone methide induction. Both oligonucleotides of a model duplex were alkylated in a sequence specific manner by an oligonucleotide conjugate that is consistent with triplex association. Optimum reaction required the presence of the two complementary target sequences and a pH of below 6.5. In addition, one guanine in each strand adjacent to the triplex region was the predominant site of alkylation. The yield of modification varied from approximately 20% for the purine-rich strand to only 4% for the pyrimidine-rich strand. This surprising difference indicates that the linker between the recognition and reactive elements may limit productive interaction between the quinone methide and the reactive nucleophiles of DNA. Restricted orientation of this intermediate may also be responsible for the lack of target cross-linking at detectable levels.     

Agrobacterium-mediated Transfer of Arginine Decarboxylase and Ornithine Decarboxylase Genes to Datura innoxia Enhances Shoot Regeneration and Hyoscyamine Biosynthesis

The genes (adc and odc) for two enzymes, arginine decarboxylase and ornithine decarboxylase involved in polyamine biosynthesis, were introduced into anther-derived calli of Datura innoxia through Agrobacterium tumefaciens. The transformed calli exhibited increased regeneration frequency as compared to control. Transgenic lines showed higher polyamine levels, mainly in the putrescine titre, and such lines also yielded a high level of the alkaloid, hyoscyamine. The results suggest that polyamines can modulate in vitro morphogenesis and polyamine biosynthetic pathway can be exploited for enhancement of polyamine-derived alkaloids of pharmaceutical importance.     

Studies on a Doubleheaded Protease Inhibitor from Phaseolus mungo

A doubleheaded protease inhibitor showing inhibition of bovine pancreatic trypsin and α-chymotrypsin was isolated and purified from the seeds of Phaseolus mungo. The molecular weight of the protease inhibitor was found to be 14.2 kD by SDS-PAGE analysis and gel filtration. The native inhibitor inhibited trypsin and α-chymotrypsin stoichiometrically at the molar ratio 1:1 and 2:1 respectively. The Ki app for trypsin was found to be 0.35 nM and for α-chymotrypsin to be 2.4 nM. Bovine pepsin was not inhibited by the inhibitor. However, the pepsin treated inhibitor was still able to inhibit trypsin and α-chymotrypsin. The inhibitor was stable in 8M urea. Addition of 0.2 M mercaptoethanol resulted in significant loss of inhibitory activity. The inhibitor was extremely heat stable with only 50% loss of inhibitory activity after heating for 100°C for 20 min. Thus, the Phaseolus mungo trypsin/chymotrypsin inhibitor resembles other Bowman-Birk protease inhibitors.