An in vitro approach to study chromosomal DNA damage

In this study a simple electrophoresis approach has been proposed for assessing DNA damage per chromosome in vitro. Novel procedures of gel casting, sample loading, electrophoresis and quantification of damage have been suggested. Sets of Saccharomyces cerevisiae chromosomes subjected to DNA damage by Bleomycin, Co⁶⁰--radiation alone and in combination with Hoechst were studied in detail. Statistical analyses showed that damage induced by Bleomycin bore linear positive correlation with %GA (r=0.97) and %GT (r=0.61) contents of chromosomes. Samples pre-treated with Hoechst showed much less damage by Co⁶⁰--irradiation as compared to samples not treated with Hoechst but exposed to Co⁶⁰--irradiation. The `protective effect of Hoechst' bore linear positive correlation (r=0.8) with %TAT content of chromosomes.     

Mitochondrial VDAC and hexokinase together modulate plant programmed cell death

The voltage-dependent anion channel (VDAC) and mitochondrially located hexokinase have been implicated both in pathways leading to cell death on the one hand, and immortalization in tumor formation on the other. While both proteins have also been implicated in death processes in plants, their interaction has not been explored. We have examined cell death following heterologous expression of a rice VDAC in the tobacco cell line BY2 and in leaves of tobacco plants and show that it is ameliorated by co-expression of hexokinase. Hexokinase also abrogates death induced by H₂O₂. We conclude that the ratio of expression of the two proteins and their interaction play a major role in modulating death pathways in plants.     

Phosphorylation status of the NR2B subunit of NMDA receptor regulates its interaction with calcium/calmodulin-dependent protein kinase II

Ca²⁺ influx through NMDA-type glutamate receptor at excitatory synapses causes activation of post-synaptic Ca²⁺/calmodulin-dependent protein kinase type II (CaMKII) and its translocation to the NR2B subunit of NMDA receptor. The major binding site for CaMKII on NR2B undergoes phosphorylation at Ser1303, in vivo . Even though some regulatory effects of this phosphorylation are known, the mode of dephosphorylation of NR2B-Ser1303 is still unclear. We show that phosphorylation status at Ser1303 enables NR2B to distinguish between the Ca²⁺/calmodulin activated form and the autonomously active Thr286-autophosphorylated form of CaMKII. Green fluorescent protein–α-CaMKII co-expressed with NR2B sequence in human embryonic kidney 293 cells was used to study intracellular binding between the two proteins. Binding in vitro was studied by glutathione- S -transferase pull-down assay. Thr286-autophosphorylated α-CaMKII or the autophosphorylation mimicking mutant, T286D-α-CaMKII, binds NR2B sequence independent of Ca²⁺/calmodulin unlike native wild-type α-CaMKII. We show enhancement of this binding by Ca²⁺/calmodulin. Phosphorylation or a phosphorylation mimicking mutation on NR2B (NR2B-S1303D) abolishes the Ca²⁺/calmodulin-independent binding whereas it allows the Ca²⁺/calmodulin-dependent binding of α-CaMKII in vitro . Similarly, the autonomously active mutants, T286D-α-CaMKII and F293E/N294D-α-CaMKII, exhibited Ca²⁺-independent binding to non-phosphorylatable mutant of NR2B under intracellular conditions. We also show for the first time that phosphatases in the brain such as protein phosphatase 1 and protein phosphatase 2A dephosphorylate phospho-Ser1303 on NR2B.     

Dengue virus infection induces broadly cross-reactive human IgM antibodies that recognize intact virions in humanized BLT-NSG mice

The development of small animal models that elicit human immune responses to dengue virus (DENV) is important since prior immunity is a major risk factor for developing severe dengue disease. This study evaluated anti-DENV human antibody (hAb) responses generated from immortalized B cells after DENV-2 infection in NOD-scid IL2rγ^null mice that were co-transplanted with human fetal thymus and liver tissues (BLT-NSG mice). DENV-specific human antibodies predominantly of the IgM isotype were isolated during acute infection and in convalescence. We found that while a few hAbs recognized the envelope protein produced as a soluble recombinant, a number of hAbs only recognized epitopes on intact virions. The majority of the hAbs isolated during acute infection and in immune mice were serotype-cross-reactive and poorly neutralizing. Viral titers in immune BLT-NSG mice were significantly decreased after challenge with a clinical strain of dengue. DENV-specific hAbs generated in BLT-NSG mice share some of the characteristics of Abs isolated in humans with natural infection. Humanized BLT-NSG mice provide an attractive preclinical platform to assess the immunogenicity of candidate dengue vaccines.     

Detection of glucose using immobilized bienzyme on cyclic bisureas–gold nanoparticle conjugate

A highly sensitive electrochemical glucose sensor has been developed by the co-immobilization of glucose oxidase (GOx) and horseradish peroxidase (HRP) onto a gold electrode modified with biocompatible cyclic bisureas–gold nanoparticle conjugate (CBU–AuNP). A self-assembled monolayer of mercaptopropionic acid (MPA) and CBU–AuNP was formed on the gold electrode through a layer-by-layer assembly. This modified electrode was used for immobilization of the enzymes GOx and HRP. Both the HRP and GOx retained their catalytic activity for an extended time, as indicated by the low value of Michaelis–Menten constant. Analytical performance of the sensor was examined in terms of sensitivity, selectivity, reproducibility, lower detection limit, and stability. The developed sensor surface exhibited a limit of detection of 100 nM with a linear range of 100 nM to 1 mM. A high sensitivity of 217.5 μA mM⁻¹ cm⁻² at a low potential of −0.3 V was obtained in this sensor design. Various kinetic parameters were calculated. The sensor was examined for its practical clinical application by estimating glucose in human blood sample.