Bacterial Expression,Purification and Characterization of a Rice Voltage-Dependent,Anion-Selective Channel Isoform,OsVDAC4

The voltage-dependent anion-selective channel (VDAC) is the most abundant protein in the mitochondrial outer membrane and forms the major conduit for metabolite transport across this membrane. VDACs from different sources show varied primary sequence but conserved functional properties. Here, we report on the characterization of a rice channel, OsVDAC4, which complements a VDAC1 deficiency in yeast. We present a consensus secondary structure prediction of an N-terminal α-helix and 19 β-strands. Bacterially expressed OsVDAC4 was purified from inclusion bodies into detergent-containing solution, where it is largely helical. Detergent-solubilized OsVDAC4 inserts spontaneously into artificial membranes of two topologies—spherical liposomes and planar bilayers. Insertion into liposomes results in an increase in β-structure. Transport of polyethylene glycols was used to estimate a pore diameter of ~2.6 nm in liposomes. Channels formed in planar bilayers exhibit large conductance (4.6 ± 0.3 nS in 1 M KCl), strong voltage dependence and weak anion selectivity. The open state of the channel is shown to be permeable to ATP. These data are consistent with a large β-barrel pore formed by OsVDAC4 on inserting into membranes. This study forms a platform to carry out studies of the interaction of OsVDAC4 with putative modulators.     

Inhibition of autophagy stimulate molecular iodine-induced apoptosis in hormone independent breast tumors

Estrogen receptor negative (ER^−ve) and p53 mutant breast tumors are highly aggressive and have fewer treatment options. Previously, we showed that molecular Iodine (I₂) induces apoptosis in hormone responsive MCF-7 breast cancer cells, and non-apoptotic cell death in ER^−ve–p53 mutant MDA-MB231 cells (Shrivastava, 2006). Here we show that I₂ (3 μM) treatment enhanced the features of autophagy in MDA-MB231 cells. Since autophagy is a cell survival response to most anti-cancer therapies, we used both in vitro and in vivo systems to determine whether ER^−ve mammary tumors could be sensitized to I₂-induced apoptosis by inhibiting autophagy. Autophagy inhibition with chloroquine (CQ) and inhibitors for PI3K (3MA, LY294002) and H+/ATPase (baflomycin) resulted in enhanced cell death in I₂ treated MDA-MB231 cells. Further, CQ (20 μM) in combination with I₂, showed apoptotic features such as increased sub-G1 fraction (∼5-fold), expression of cleaved caspase-9 and -3 compared to I₂ treatment alone. Flowcytometry of I₂ and CQ co-treated cells revealed increase in mitochondrial membrane permeability (p < 0.01) and translocation of cathepsin D activity to cytosol relative to I₂ treatment. For in vivo studies ICRC mice were transplanted subcutaneously with MMTV-induced mammary tumors. A significant reduction in tumor volumes, as measured by MRI, was found in I₂ and CQ co-treated mice relative to I₂ or vehicle treated mice. These data indicate that inhibition of autophagy renders ER^−ve breast tumor cells more sensitive to I₂ induced apoptosis. Thus, I₂ together with autophagy inhibitor could have a potential tumorostatic role in ER^−ve aggressive breast tumors that may be evaluated in future studies.     

Reexamining hydroxamate inhibitors of botulinum neurotoxin serotype A: extending towards the β-exosite

Botulinum neurotoxins (BoNTs) are the most toxic proteins known to man, exposure to which results in flaccid paralysis. Given their extreme potency, these proteins have become studied as possible weapons of bioterrorism; however, effective treatments that function after intoxication have not progressed to the clinic. Here, we have reexamined one of the most effective inhibitors, 2,4-dichlorocinnamyl hydroxamate, in the context of the known plasticity of the BoNT/A light chain metalloprotease. Our studies have shown that modifications of this compound are tolerated and result in improved inhibitors, with the best compound having an IC(50) of 0.23 μM. Given the inconsistency of structure-activity relationship trends observed across similar compounds, this data argues for caution in extrapolating across structural series.     

Two-Arm Randomized Pilot Intervention Trial to Decrease Sitting Time and Increase Sit-To-Stand Transitions in Working and Non-Working Older Adults

Background

Excessive sitting has been linked to poor health. It is unknown whether reducing total sitting time or increasing brief sit-to-stand transitions is more beneficial. We conducted a randomized pilot study to assess whether it is feasible for working and non-working older adults to reduce these two different behavioral targets.

Methods

Thirty adults (15 workers and 15 non-workers) age 50–70 years were randomized to one of two conditions (a 2-hour reduction in daily sitting or accumulating 30 additional brief sit-to-stand transitions per day). Sitting time, standing time, sit-to-stand transitions and stepping were assessed by a thigh worn inclinometer (activPAL). Participants were assessed for 7 days at baseline and followed while the intervention was delivered (2 weeks). Mixed effects regression analyses adjusted for days within participants, device wear time, and employment status. Time by condition interactions were investigated.

Results

Recruitment, assessments, and intervention delivery were feasible. The ‘reduce sitting’ group reduced their sitting by two hours, the ‘increase sit-to-stand’ group had no change in sitting time (p < .001). The sit-to-stand transition group increased their sit-to-stand transitions, the sitting group did not (p < .001).

Conclusions

This study was the first to demonstrate the feasibility and preliminary efficacy of specific sedentary behavioral goals.

Trial Registration

clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02544867","term_id":"NCT02544867"}}NCT02544867     

Studies on a drought resistant legume: The moth bean,Vigna aconitifolia (Jacq) marechal. I. Protoplast culture and organogenesis

High yields of viable protoplasts were obtained from callus cultures derived from shoot apices of Vigna aconitifolia (JACQ) Marechal. The protoplasts divided and formed cell clusters on modified MS medium. The protoplast-derived callus formed multiple shoot buds on MS and B5 basal media without supplements, on MS medium containing supplements and on B5 medium containing charcoal (0.25%). Shoot formation occurred.Abbreviations MS Murashige and Skoog (1962) - B5 B5 basal medium (Gamborg et al 1968) - CM coconut milk - NAA 1-napthaleneacetic acid - BA 6-benzyladenine - IAA indole-3-acetic acid - 2,4 D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - CB Cellulase from Penicillium funiculosum - C Cellulase from Trichoderma reesei - CRIO Cellulase R10 - MR10 Macerozyme R10 - PDS Potassium dextran sulphate NCL communication No. 3376     

Preparation of lactose free milk by fermentation using immobilizedSaccharomyces fragilis

Summary Saccharomyces fragilis cells (40% w/v) were immobilized in 2% Ca-alginate and were used in a batch process for the removal of lactose from milk by fermentation. Immobilized cells (10 g) could completely desugarate 100 mL of milk in 3.5 h. The immobilized preparation was used repeatedly in 15 batches without decrease in the activity.     

Late-onset adrenal hyperplasia in north Indian hirsute women

The occurrence of late-onset congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency was studied in 60 consecutive hirsute women by means of adrenocorticotrophin (ACTH)-stimulated serum 17-hydroxyprogesterone (17-OHP) levels. Five (8.3%) women had an exaggerated response (ACTH-stimulated 17-OHP 3,160 +/- 560 ng/dl). All of them had regular periods and 3 were virilized. The other 2 were indistinguishable from those with idiopathic hirsutism or polycystic ovarian disease.     

GMP production and characterization of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1) for phase I/II clinical trials

The bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(UCHT1), was developed for treatment of T-cell leukemia, autoimmune diseases and tolerance induction for transplantation. To obtain clinical grade bivalent anti-T cell immunotoxin for phase I/II clinical trials, a single batch of 120 L bioreactor culture was performed using the Pichia pastoris mutEF2JC307-8(2) strain expressing the bivalent anti-T cell immunotoxin. After 162 h induction of the culture by methanol, the culture medium was harvested by a 0.1 microm hollow-fiber microfiltration step. The recombinant protein was purified by a 3-step purification procedure (Butyl 650 M capturing step, borate anion exchange step and final Poros anion exchange step). The final material was filter sterilized, aseptically vialed, and stored at -80 degrees C. Expression level was 207 mg/L of culture supernatant and the final production yield was 69.6% or 144.2mg/L of culture supernatant. The final product was characterized by multiple assays. Vialed product was sterile. The drug concentration was 0.8 mg/mL in 150 mM NaCl, 5% glycerol, 1mM EDTA, and 5mM Tris (pH 8.0). Purity by SDS-PAGE was 98%. Aggregates by Superdex 200 HPLC were <1%. Potency revealed a 20 h IC(50) of 17f M on Jurkat cells. Endotoxin level was 0.02 U/mg. Chemical and biologic assays confirmed the purity, composition, and functional activities of the molecule. The drug did not react with tested frozen human tissue sections except for T cells. LD(10) in mice was between 500 and 75 0microg/kg. There was no evidence of loss of solubility, proteolysis, aggregation, or loss of potency over 1.5 year at -80 degrees C. The scalable synthesis of this protein drug should be useful for production for phase I/II clinical trials and can be applicable for other diphtheria toxin fusion drugs for clinical development.