Interplay between Cytoskeletal Stresses and Cell Adaptation under Chronic Flow

Using stress sensitive FRET sensors we have measured cytoskeletal stresses in α-actinin and the associated reorganization of the actin cytoskeleton in cells subjected to chronic shear stress. We show that long-term shear stress reduces the average actinin stress and this effect is reversible with removal of flow. The flow-induced changes in cytoskeletal stresses are found to be dynamic, involving a transient decrease in stress (phase-I), a short-term increase (3–6 min) (Phase-II), followed by a longer-term decrease that reaches a minimum in ∼20 min (Phase-III), before saturating. These changes are accompanied by reorganization of the actin cytoskeleton from parallel F-actin bundles to peripheral bundles. Blocking mechanosensitive ion channels (MSCs) with Gd³⁺ and GsMTx4 (a specific inhibitor) eliminated the changes in cytoskeletal stress and the corresponding actin reorganization, indicating that Ca²⁺ permeable MSCs participate in the signaling cascades. This study shows that shear stress induced cell adaptation is mediated via MSCs.     

Loss of CMD2‐mediated resistance to cassava mosaic disease in plants regenerated through somatic embryogenesis

Cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) are the two most important viral diseases affecting cassava production in Africa. Three sources of resistance are employed to combat CMD: polygenic recessive resistance, termed CMD1, the dominant monogenic type, named CMD2, and the recently characterized CMD3. The farmer‐preferred cultivar TME 204 carries inherent resistance to CMD mediated by CMD2, but is highly susceptible to CBSD. Selected plants of TME 204 produced for RNA interference (RNAi)‐mediated resistance to CBSD were regenerated via somatic embryogenesis and tested in confined field trials in East Africa. Although micropropagated, wild‐type TME 204 plants exhibited the expected levels of resistance, all plants regenerated via somatic embryogenesis were found to be highly susceptible to CMD. Glasshouse studies using infectious clones of East African cassava mosaic virus conclusively demonstrated that the process of somatic embryogenesis used to regenerate cassava caused the resulting plants to become susceptible to CMD. This phenomenon could be replicated in the two additional CMD2‐type varieties TME 3 and TME 7, but the CMD1‐type cultivar TMS 30572 and the CMD3‐type cultivar TMS 98/0505 maintained resistance to CMD after passage through somatic embryogenesis. Data are presented to define the specific tissue culture step at which the loss of CMD resistance occurs and to show that the loss of CMD2‐mediated resistance is maintained across vegetative generations. These findings reveal new aspects of the widely used technique of somatic embryogenesis, and the stability of field‐level resistance in CMD2‐type cultivars presently grown by farmers in East Africa, where CMD pressure is high.     

Candidate gene based association analysis of salt tolerance in traditional and improved varieties of rice (Oryza sativa L.)

Journal of Plant Biochemistry and Biotechnology - Rice is a staple food for more than half of the world population but the productivity of rice plant is attenuated in salt-stressed...     

A Structure-Guided Mutational Analysis of Simian Virus 40 Large T Antigen: Identification of Surface Residues Required for Viral Replication and Transformation

Simian virus 40 large T antigen (TAg) transforms cells in culture and induces tumors in rodents. Genetic studies suggest that TAg interaction with the chaperone hsp70 and tumor suppressors pRb and p53 may not be sufficient to elicit complete transformation of cells. In order to identify additional cellular factors important for transformation, we designed mutations on the solvent-exposed surface of TAg. We hypothesized that surface residues would interact directly with cellular targets and that the mutation of these residues might disrupt this interaction without perturbing TAg''s global structure. Using structural data, we identified 61 amino acids on the surface of TAg. Each surface amino acid was changed to an alanine. Furthermore, five patches containing clusters of charged amino acids on the surface of TAg were identified. Within these patches, we selectively mutated three to four charged amino acids and thus generated five mutants (patch mutants 1 to 5). We observed that while patch mutants 3 and 4 induced foci in REF52 cells, patch mutants 1 and 2 were deficient in focus formation. We determined that the patch 1 mutant is defective in p53 binding, thus explaining its defect in transformation. The patch 2 mutant can interact with the Rb family members and p53 like wild-type TAg but is unable to transform cells, suggesting that it is defective for action on an unknown cellular target essential for transformation. Our results suggest that the histone acetyltransferase CBP/p300 is one of the potential targets affected by the mutations in patch 2.Simian virus 40 (SV40) large T antigen is a multifunctional protein that is essential for productive viral infection and for cellular transformation (26). T antigen possesses several biochemical activities, some of which map to discrete domains that can act independently and/or coordinately. To effect replication and transformation, T antigen binds to several cellular targets via different domains/regions. For example, during replication, T antigen associates with components of the cellular replication apparatus such as DNA polymerase α, replication protein A, and topoisomerase I (11, 14, 24, 31, 39). Three regions of T antigen are essential to elicit cellular transformation (1, 2, 36). The LXCXE motif mediates binding to the members of the Rb family (pRb, p107, and p130) and in conjunction with the J domain results in the inactivation of the Rb family function. While these domains reside in the N terminus of T antigen, a third transforming function in the C terminus of T antigen is essential for inactivation of the tumor suppressor p53. Genetic studies suggest that inactivation of pRb and p53 is not always sufficient to induce T-antigen-mediated transformation (7, 30, 38), thus indicating the presence of additional targets of T antigen contributing to transformation. In the past few years, several additional targets of T antigen, including CBP/p300, Bub1, Cul7, Fbw7, and IRS-1, have been discovered (8, 9, 12, 17, 29, 40, 42); however, their roles in T-antigen-mediated transformation are not clear. T antigen also targets the DNA-damage-sensing and -processing complex Mre11-Rad50-Nbs1 and may induce genetic instability that contributes to transformation (10, 42). The issue is complicated further by the observation that T antigen has redundant functions, that is, it can act on critical targets via multiple mechanisms (7, 37).One of the key strategies to delineate functions of T antigen required for transformation is the use of amino acid substitution and truncation mutants. However, a caveat to this approach is the production of mutants that are defective in transformation due to a loss of integrity of the secondary, or even local, structure. In this study, we combined available sequence data with structural information to design mutants. Sequence alignments allow the identification of conserved amino acid residues, while structural data provide information about amino acid residues on the surface of the molecule. This approach allows us to combine structural elements and target binding sites. In addition, identification of residues conserved across species, followed by mutation of these conserved residues, will likely yield better insights into common biological pathways. Using this method, we have generated four mutants, of which two are defective in transformation and, thus, of great interest for the identification of novel cellular pathways regulating cell growth and proliferation.     

Immunochemical characterization of antisera reactivities toN-acetyl-d-glucosamine oligosaccharides with theβ(1–4)-glycosidic linkage

The (1–4)-linked oligosaccharides ofN-acetyl-d-glucosamine (GlcNAc) isolated from chitin were used to prepare synthetic immunogens and antigens by reductive amination of (GicNAc)_n to bovine serum albumin (BSA). The rabbit antisera produced to the (GlcNAc)_n-BSA conjugates were characterized using an enzyme-linked immunosorbent assay (ELISA) system under conditions that, only the antibodies with carbohydrate specificity were reactive with the solid-phase adsorbed (GlcNAc)_n-BSA antigens. Inhibition assays using the (GlcNAc)_n-BSA, (GlcNAc)_n oligosaccharides, and the reduced oligosaccharides showed a relative specificity of the antisera for the chain length of the (GlcNAc)_n sequences. For example, the anti-(GlcNAc)₅-and anti-(GlcNAc)₄-sera were inhibited best by the longer chain (GlcNAc)_n ologosaccharides with the antibody combining sites directed mainly to the cyclic GlcNAc residues of the (GlcNAc)_n-BSA conjugates. The antibody combining sites were in part directed to the acyclic moiety of the reducing end of the oligosaccharides as shown by the increased inhibitory activities of the reduced (GlcNAc)_n oligosaccharides particularly, with the anti-(GlcNAc)₂-and anti-(GlcNAc)₃-sera. The best hapten inhibitors for the anti-(GlcNAc)₂-BSA and anti-(GlcNAc)₁-BSA sera were theN-butylamine derivatives of (GlcNAc)₂ and (GlcNAc)₁, respectively, indicating that the antibodies were also reactive with the secondary amine formed between the reducing end of the oligosaccharides and the -amino groups of lysine.Abbreviations ELISA enzyme-linked immunosorbent assay - BSA bovine serum albumin - GicNAc N-acetyl-d-glucosamine - (GlcNAc)_n Oligosaccharides containing GlcNAc in 1–4 linkages - (GlcNAc)₂ DGlcNAc(1–4)-d-GlcNAc - (GlcNAc)₃ (GlcNAc)₄ and (GlcNAc)₅, the homologous oligosaccharides of (GlcNAc)₂ - PBS phosphate buffered saline (0.01 M sodium phosphate, pH73 containing 0.15%M (NaCl) - PBSA PBS containing 1% BSA and 0.1% Tween-20 - ONPG o-nitrophenyl--d-galactopyranoside     

Strain improvement of Aspergillus sp. and Penicillium sp. by induced mutation for biotransformation of alpha-pinene to verbenol

Variants of Aspergillus sp. and Penicillium sp. obtained after treatment with colchicine, ethyl methanesulphonate (EMS), or ultraviolet (UV) irradiation indicated varying levels of significant increases in their efficiency to transform alpha-pinene to verbenol. In case of Aspergillus sp. the UV-induced variant was the best performer with a 15-fold increase in biotransformation efficiency compared to the wild type. In case of colchicine and EMS-induced variants the biotransformation increases were 2- and 8-fold, respectively. The UV-induced variant of Penicillium sp. was capable of eight fold increase in efficiency while the colchicine- and EMS-induced variants were 1.5- and 2-fold, respectively. The variants were characterised with respect to changes in colony morphology, spore dimension, DNA content, and products formed, viz. verbenol and verbenone.     

Implications from predictions of HLA-DRB1 binding peptides in the membrane proteins of Corynebacterium diphtheriae

The aerobic gram positive bacterium Corynebacterium diphtheriae causes diphtheria, a respiratory tract illness characterized by symptoms such as sore throat, low fever, and an adherent membrane on the tonsils, pharynx, and/or nasal cavity. Therefore, it is important to develop preventive vaccines for diphtheria. The availability of the 2,488,635 bp long complete sequence for the C. diphtheriae genome provides an opportunity to understand cell mediated immune response using Computational Biology tools from the bacterial proteome sequence data. We selected 355 membrane proteins from the C. diphtheriae proteome using annotation data to identify potential HLA-DRB1 binding short peptide using modeling, simulations and predictions. This exercise identified 30 short peptides in membrane proteins showing binding capability to HLA-DRB1 alleles. These peptides serve as outline for the understanding of cell mediated immune response to C. diphtheriae. It should be noted that the predicted data to be verified using binding assays for further consideration.     

Perillyl alcohol as a radio-/chemosensitizer in malignant glioma

The prognosis for patients with malignant glioma has not significantly changed in two decades, despite advances in surgery, radiation, and chemotherapy, emphasizing the growing need for novel approaches to glioma therapy. Perillyl alcohol (POH) is a naturally occurring monoterpene that has been shown to possess chemotherapeutic as well as chemopreventive activity in animal tumor models and is currently in Phase I and Phase II clinical trials. In the present study, we have demonstrated that POH is an effective radiosensitizer at clinically relevant doses of radiation using established glioma cell lines. POH caused a transient arrest in the G2/M phase of the cell cycle and induced apoptosis in glioma cells. POH treatment sensitized glioma cells to Fas-mediated apoptosis, which was further augmented in the presence of ionizing radiation and abrogated in the presence of antagonistic antibody. POH-induced radiosensitization was partially inhibited in glioma cells expressing dominant negative Fas-associated death domain and completely inhibited in glioma cells overexpressing the cytokine response modifier A. In addition, POH treatment resulted in a dose-dependent sensitization to cisplatin and doxorubicin induced cytotoxicity in glioma cells, highlighting its usefulness as a potent radio/chemosensitizer in the treatment of malignant glioma.     

On the role of anesthesia on the body/brain temperature differential in rats

Different anesthetics often produce distinctly different effects on blood flow, oxygen consumption and other physiological parameters in animal studies. We investigated the influence of two common anesthetics—alpha-chloralose and chloral hydrate—on the body-core/brain temperature differential in rats. The results indicate a remarkable difference of 4.34±0.64 °C (mean±SD) in the body-core/brain-cortical temperature differential observed under alpha-chloralose anesthesia compared to a relatively smaller differential of 2.73±0.40 °C under chloral hydrate anesthesia in rats. Temperature gradients within the brain are around 0.3 °C/mm in both cases. Thus, the anesthetic utilized has the potential to markedly influence brain temperature and therefore other important physiological parameters in the brain.     

S-Glutathionylation of p47phox sustains superoxide generation in activated neutrophils

Post-translational modifications (PTMs) induced conformational changes of proteins can cause their activation or inactivation. Neutrophils clear pathogen through phagocytosis and oxidative burst generation, while participate in inflammation through sustained and uncontrolled generation of ROS. In activated PMNs, cytosolic NOX-2 subunit p47phox following phosphorylation interacts with p67phox, p40phox and along with Rac2 translocate to the membrane. Phosphorylation of p47phox subunit occurs in both short spurts as well as sustained ROS generation, suggesting towards the unidentified molecular mechanism(s) driving these two diverse outcomes by various stimuli. The present study demonstrates that in PMA or NO treated neutrophils a subunit of NOX2, p47phox gets glutathionylated to sustain ROS generation along with a decrease in catalase, Grx-1 activity and change in GSH/GSSG ratio. Surprisingly, fMLP treated cells neither showed sustained ROS production nor glutathionylation of p47phox. S-Glutathionylation was always secondary to phosphorylation of p47phox and inhibition of glutathionylation did not alter phosphorylation but specifically impaired sustained ROS production. Interestingly, forced S-glutathionylation of p47phox converted the fMLP induced ROS generation into sustained release of ROS. We then identified the glutathionylation susceptible cysteine residues of p47phox by LC-MS/MS with IAM switch mapping. Site-directed mutagenesis of cysteine residues further mitigated p47phox S-glutathionylation. Thus, we demonstrate that p47phox S-glutathionylation plays an essential key role in the sustained ROS generation by human neutrophils.