Impaired Autophagy Flux is Associated with Proinflammatory Microglia Activation Following Japanese Encephalitis Virus Infection

Neurochemical Research - Role of autophagy in Japanese encephalitis viral (JEV) infection is not well known. In the present study, we reported the role of autophagy flux in microglia activation,...     

Tetraplex Formation of d(GGGGGTTTTT): 1H NMR Study in Solution

Abstract

The oligonucleotide d(G₅T₅) can in principle form a fully matched duplex with G · T pairing and/or a tetraplex. Non-denaturing gel electrophoresis, circular dichroism and NMR experiments show that the tetraplex is exclusively formed by this oligomer in solution. In the presence of its complementary strand d(A₅C₅) at low temperature, d(G₅T₅) forms the tetraplex over the normally expected Watson-Crick duplex. However, when d(G₅T₅) and d(A₅C₅) are mixed together in equimolar amounts and heated for several minutes at 85°C, and then allowed to cool, the product was essentially the Watson-Crick duplex. The lack of resolution in the 500 MHz ¹H NMR spectra and the presence of extensive spin diffusion do not allow us to derive a quantitative structure for the tetraplex from the NMR data. However, we find good qualitative agreement between the NOESY and MINSY data and a theoretically derived stereochemically sound structure in which the G's and T's are part of a parallel tetraplex.     

Structure and Dynamics of Netropsin-Poly(dA-dT)· Poly(dA-dT) Complex: 500 MHz 1H NMR Studies

Abstract

Antibiotic netropsin is known to bind specifically to A and T regions in DNA; the mode of binding being non-intercalative. Obviously, H-bonding between the proton donors of netropsin and acceptors N3 of A and 02 of T comes as a strong possibility which might render this specificity. In netropsin there could be 8 proton donors: four terminal amino groups and four internal imino groups. However, methylation of the terminal amino groups does not alter the binding affinity of netropsin to DNA—but the modification of the internal imino groups significantly lowers the binding affinity. Hence, the logical conclusion is that netropsin may specifically interact with A and T through H-bonding and in order to do so, it should approach the helix from the minor groove. The present paper provides experimental data which verify the conclusion mentioned above.

Using poly(dA-dT)? poly(dA-dT) as a model system it was observed following a thorough theoretical stereochemical analysis that netropsin could bind to -(T-A-T) sequence of the polymer in the B-form through the minor groove by forming specific B-bonding. Models could be either right or left-handed B-DNA with a mono or dinucleotide repeat.

By monitoring the ³¹P signals of free poly(dA-dT) ? poly(dA-dT) and netropsin-poly(dA-dT)? poly(dA-dT) complex we show that the drug changes the DNA structure from essentially a mononucleotide repeat to that of very dominant dinucleotide repeat; however the base- pairing in the DNA-drug complex remain to be Watson-Crick. Whether H-bonding is the specific mode of interaction was judged by monitoring the imino protons of netropsin in the presence of poly(dA-dT) ? poly(dA-dT). This experiment was conducted in 90% H₂O + 10% D₂O Using the time-shared long pulse. It was found that exchangeable imino protons of netropsin appear in the drug-DNA complex and disappear upon increasing the D₂O content; thus confirming that H-bonding is indeed the specific mode of interaction. From these and several NOE measurements, we propose a structure for poly(dA-dT)? poly(dA-dT(-netropsin complex.

In summary, experimental data indicate that netropsin binds to poly(dA-dT)? poly(dA-dT) by forming specific hydrogen bonds and that the binding interaction causes the structure to adopt a Watson-Crick paired dinucleotide repeat motif. The proposed hydrogen bonds can form only if the drug approaches the DNA from the minor groove. Within the NMR time scale the interaction between the ligand and DNA is a fast one. From the NOE experimental data, it appears that poly(dA-dT)? poly(dA-dT) in presence of netropsin exists as an equilibrium mixture of right- and left-handed B-DNA duplexes with a dinucleotide repeat—with a predominance of the left-handed form. The last conclusion is a soft one because it was very difficult to make sure the absence of spin diffusion. In a 400 base pairs long DNA duplex- drug complex (as used in this study), equilibrium between right and left-handed helices can also mean the existence of both helical domains in the same molecule with fast interchange between these domains or/and unhindered motion/propagation of these domains along the helix axis.     

Antioxidant associated chemoprevention by selenomethionine in murine tumor model

Effectiveness of selenium in different forms like sodium selenite, selenocysteine and selenomethionine has been compared in four different doses, namely 4, 6, 8 and 10 ppm of each, in terms of their bioavailability and prolongation of survival of Dalton's lymphorna (DL) bearing mice. Selenomethionine, at a dose of 8 ppm, was found to be the most bioavailable and least cytotoxic form that was capable of increasing the life span of the tumour bearing hosts maximally (almost two-fold). Benef iciality of selenomethionine has also been studied by observing continuous changes brought about by this compound on the glutathione (GSH) level, glutathione peroxidase (GPx) activity and extent of lipid peroxidation in the hepatic tissue of the tumour bearing hosts, which are indispensable for a cell to function normally and are found to exhibit significantly altered behaviour in neoplastic cells. Selenomethionine caused the maintenance of high steady state GSH level and a normal GPx activity during the fist phase of tumour growth. It also controlled lipid peroxidation during the first 15-20 days following tumour transplantation. These conditions helped in the maintenance of intracellular redox balance, cellular integrity and metabolic rhythms of cells in DL bearing mice receiving selenomethionine.Affiliation     

Rotaviral Enterotoxin Nonstructural Protein 4 Targets Mitochondria for Activation of Apoptosis during Infection

Viruses have evolved to encode multifunctional proteins to control the intricate cellular signaling pathways by using very few viral proteins. Rotavirus is known to express six nonstructural and six structural proteins. Among them, NSP4 is the enterotoxin, known to disrupt cellular Ca²⁺ homeostasis by translocating to endoplasmic reticulum. In this study, we have observed translocation of NSP4 to mitochondria resulting in dissipation of mitochondrial membrane potential during virus infection and NSP4 overexpression. Furthermore, transfection of the N- and C-terminal truncated NSP4 mutants followed by analyzing NSP4 localization by immunofluorescence microscopy identified the 61–83-amino acid region as the shortest mitochondrial targeting signal. NSP4 exerts its proapoptotic effect by interacting with mitochondrial proteins adenine nucleotide translocator and voltage-dependent anion channel, resulting in dissipation of mitochondrial potential, release of cytochrome c from mitochondria, and caspase activation. During early infection, apoptosis activation by NSP4 was inhibited by the activation of cellular survival pathways (PI3K/AKT), because PI3K inhibitor results in early induction of apoptosis. However, in the presence of both PI3K inhibitor and NSP4 siRNA, apoptosis was delayed suggesting that the early apoptotic signal is initiated by NSP4 expression. This proapoptotic function of NSP4 is balanced by another virus-encoded protein, NSP1, which is implicated in PI3K/AKT activation because overexpression of both NSP4 and NSP1 in cells resulted in reduced apoptosis compared with only NSP4-expressing cells. Overall, this study reports on the mechanism by which enterotoxin NSP4 exerts cytotoxicity and the mechanism by which virus counteracts it at the early stage for efficient infection.     

Monosodium glutamate impairs the contraction of uterine visceral smooth muscle ex vivo of rat through augmentation of acetylcholine and nitric oxide signaling pathways

The aim of the study was to examine the toxic effects of Monosodium glutamate (MSG), an extensively used food additive, on the contraction of uterine visceral smooth muscle (UVSM) in rat and to elucidate the probable neurocrine mechanism involved in it. MSG produced significant potentiation of the force and inhibition of frequency of uterus recorded ex vivo in chronic MSG exposure and in single dose acute experiments. MSG also produced significant potentiation of force of acetylcholine induced contraction and no alterations in atropine induced contraction of uterus. Further, MSG produced significant increase in force and frequency of contraction of neostigmine incubated uterus. We have found significant potentiation of the post pause force of contraction of uterus when MSG was applied in adrenaline incubated uterus. MSG also produced significant decrease in frequency of contraction of sodium nitroprusside incubated uterus; increase in frequency of N-ω-Nitro-l-Arginine Methyl Ester incubated uterus and no significant changes in frequency of contraction of methylene blue incubated uterus. These results indicate that MSG potentiates the force of contraction of UVSM predominantly by augmenting the activity of cholinergic intrinsic efferents and inhibits the frequency of contraction probably by augmenting the activity of nitrergic efferents. In conclusion, MSG potentiates the force and inhibits the frequency of contraction of UVSM, and the MSG induced effect is probably mediated through the augmentation of acetylcholine and nitric oxide signaling pathways.     

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.