Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.     

Novel drug designing approach for dual inhibitors as anti-inflammatory agents: implication of pyridine template

Compounds incorporating thiophene moiety, a pi excess five membered heterocycle, have attracted a great deal of research interest, owing to the therapeutic utility of the template as useful drug molecular scaffolding. We report the synthesis and pharmacological evaluation of thiophenes substituted with 4-methanesulfonyl benzoyl moiety at the fifth position of the ring, as possible anti-inflammatory lead candidates. The aryl sulfonyl methyl thiophene analogs AP29, AP82, and AP37, when screened for anti-inflammatory activity in carrageenin induced rat paw edema, an acute in vivo model, exhibited moderate to good activity at a dose level of 100 mg/kg body weight P.o compared to Ibuprofen. In a five day formalin induced rat paw edema, a chronic in vivo anti-inflammatory model, candidates AP29, AP82, and AP37 inhibited the disease progression by 53%, 34%, and 65%, respectively on the fifth day, at a dose level of 100 mg/kg body weight P.o compared to Rofecoxib, Ibuprofen, and Dexamethasone at therapeutic doses which gave a protection of 53.8%, 81.5%, and 81.5%, respectively. The replacement of the 4-methanesulfonyl benzoyl moiety in AP82 with the pyridine template, 3,5-dimethyl-4-methoxy-2-pyridyl function, gave rise to AP84, which was less active in the acute model, but gave 54% and 75% protection both during the first day and fifth day, respectively, in the chronic model. A dual mechanism of action is proposed for AP84, a non-steroidal drug which has exhibited remarkable activity when compared to the steroid dexamethasone. These results open up new avenues in designing novel anti-inflammatory drugs as dual inhibitors with the incorporation of a pyridine template as part of the pharmacophore.     

Effects of an antiprogestin onapristone on the endometrium of bonnet monkeys: morphometric and ultrastructural studies

Our previous studies demonstrated the ability of low doses of antiprogestin ZK 98.299 (onapristone) to inhibit fertility in bonnet monkeys. In the present study cumulative effects of low doses of ZK 98.299 on the endometrial cytoarchitecture of bonnet monkeys were analyzed. Treatment with either the vehicle (n = 3) or onapristone at 2.5 mg (n = 4) or 5.0 mg (n = 3) was initiated on Day 5 of the first menstrual cycle and thereafter repeated every third day for four to seven consecutive cycles. The last treatment cycles were anovulatory in two animals treated with 2.5 mg and all animals treated with 5.0 mg. Endometrial biopsies were collected on Day 8 after the midcycle estradiol peak in ovulatory menstrual cycles and on Day 20 in anovulatory menstrual cycles during the last treatment cycle. Ultrathin sections of the fixed endometrium were stained with toluidine blue for morphometric analysis and uranyl acetate and lead citrate for ultrastructural analysis. The ZK 98.299-treated animals showed a dose-dependent endometrial atrophy as evident by a decrease in the height and diameter of the glands and early signs of compaction in the stroma. Ultrastructural analysis also revealed dose-dependent degenerative changes in the subcellular organelles such as the nucleus, mitochondria, endoplasmic reticulum, lysosomes, and Golgi apparatus. This suggests that long-term treatment with low doses of ZK 98.299 leads to the suppression of estrogen-dependent endometrial proliferation. However, this blockade operates independent of estradiol receptor (ER) and progesterone receptor (PR) concentrations as the expressions of these steroid receptors did not show any significant changes even after prolonged treatment. The study demonstrated an antiestrogenic effect of ZK 98.299 on endometrium after prolonged treatment in bonnet monkeys.     

MEK kinase 2 and the adaptor protein Lad regulate extracellular signal-regulated kinase 5 activation by epidermal growth factor via Src

Lad is an SH2 domain-containing adaptor protein that binds MEK kinase 2 (MEKK2), a mitogen-activated protein kinase (MAPK) kinase kinase for the extracellular signal-regulated kinase 5 (ERK5) and JNK pathways. Lad and MEKK2 are in a complex in resting cells. Antisense knockdown of Lad expression and targeted gene disruption of MEKK2 expression results in loss of epidermal growth factor (EGF) and stress stimuli-induced activation of ERK5. Activation of MEKK2 and the ERK5 pathway by EGF and stress stimuli is dependent on Src kinase activity. The Lad-binding motif is encoded within amino acids 228 to 282 in the N terminus of MEKK2, and expression of this motif blocks Lad-MEKK2 interaction, resulting in inhibition of Src-dependent activation of MEKK2 and ERK5. JNK activation by EGF is similarly inhibited by loss of Lad or MEKK2 expression and by blocking the interaction of MEKK2 and Lad. Our studies demonstrate that Src kinase activity is required for ERK5 activation in response to EGF, MEKK2 expression is required for ERK5 activation by Src, Lad and MEKK2 association is required for Src activation of ERK5, and EGF and Src stimulation of ERK5-regulated MEF2-dependent promoter activity requires a functional Lad-MEKK2 signaling complex.     

UDP-galactose 4-epimerase from Escherichia coli: equilibrium unfolding studies

UDP-galactose 4-epimerase from Escherichia coli is a homodimer of 39 kDa subunit with non-covalently bound NAD acting as cofactor. The enzyme can be reversibly reactivated after denaturation and dissociation using 8 M urea at pH 7.0. There is a strong affinity between the cofactor and the refolded molecule as no extraneous NAD is required for its reactivation. Results from equilibrium denaturation using parameters like catalytic activity, circular-dichroism, fluorescence emission (both intrinsic and with extraneous fluorophore 1-aniline 8-naphthalene sulphonic acid), 'reductive inhibition' (associated with orientation of NAD on the native enzyme surface), elution profile from size-exclusion HPLC and light scattering have been compiled here. These show that inactivation, integrity of secondary, tertiary and quaternary structures have different transition mid-points suggestive of non-cooperative transition. The unfolding process may be broadly resolved into three parts: an active dimeric holoenzyme with 50% of its original secondary structure at 2.5 M urea; an active monomeric holoenzyme at 3 M urea with only 40% of secondary structure and finally further denaturation by 6 M urea leads to an inactive equilibrium unfolded state with only 20% of residual secondary structure. Thermodynamical parameters associated with some transitions have been quantitated. The results have been discussed with the X-ray crystallographic structure of the enzyme.     

Salicylic Acid Induces Changes in Mango Fruit that Affect Oviposition Behavior and Development of the Oriental Fruit Fly,Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis (Hendel) is an important quarantine pest around the globe. Although measures for its control are implemented worldwide through IPM and male annihilation, there is little effect on their population. Hence, there is a need for new strategies to control this minacious pest. A strategy that has received negligible attention is the induction of ‘natural plant defenses’ by phytohormones. In this study, we investigated the effect of salicylic acid (SA) treatment of mango fruit (cv. Totapuri) on oviposition and larval development of B. dorsalis. In oviposition choice assays, gravid females laid significantly less eggs in SA treated compared to untreated fruit. Headspace volatiles collected from SA treated fruit were less attractive to gravid females compared to volatiles from untreated fruit. GC-MS analysis of the headspace volatiles from SA treated and untreated fruit showed noticeable changes in their chemical compositions. Cis-ocimene and 3-carene (attractants to B. dorsalis) were reduced in the headspace volatiles of treated fruit. Further, reduced pupae formation and adult emergence was observed in treated fruit compared to control. Increased phenol and flavonoid content was recorded in treated fruit. We also observed differential expression of anti-oxidative enzymes namely catalase (CAT), polyphenoloxidase (PPO) and peroxidase (POD). In summary, the results indicate that SA treatment reduced oviposition, larval development and adult emergence of B. dorsalis and suggest a role of SA in enhancing mango tolerance to B. dorsalis.     

Efficient high‐throughput biological process characterization: Definitive screening design with the Ambr250 bioreactor system

The burgeoning pipeline for new biologic drugs has increased the need for high‐throughput process characterization to efficiently use process development resources. Breakthroughs in highly automated and parallelized upstream process development have led to technologies such as the 250‐mL automated mini bioreactor (ambr250?) system. Furthermore, developments in modern design of experiments (DoE) have promoted the use of definitive screening design (DSD) as an efficient method to combine factor screening and characterization. Here we utilize the 24‐bioreactor ambr250? system with 10‐factor DSD to demonstrate a systematic experimental workflow to efficiently characterize an Escherichia coli (E. coli) fermentation process for recombinant protein production. The generated process model is further validated by laboratory‐scale experiments and shows how the strategy is useful for quality by design (QbD) approaches to control strategies for late‐stage characterization. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1388–1395, 2015     

Dasatinib Attenuates Pressure Overload Induced Cardiac Fibrosis in a Murine Transverse Aortic Constriction Model

Reactive cardiac fibrosis resulting from chronic pressure overload (PO) compromises ventricular function and contributes to congestive heart failure. We explored whether nonreceptor tyrosine kinases (NTKs) play a key role in fibrosis by activating cardiac fibroblasts (CFb), and could potentially serve as a target to reduce PO-induced cardiac fibrosis. Our studies were carried out in PO mouse myocardium induced by transverse aortic constriction (TAC). Administration of a tyrosine kinase inhibitor, dasatinib, via an intraperitoneally implanted mini-osmotic pump at 0.44 mg/kg/day reduced PO-induced accumulation of extracellular matrix (ECM) proteins and improved left ventricular geometry and function. Furthermore, dasatinib treatment inhibited NTK activation (primarily Pyk2 and Fak) and reduced the level of FSP1 positive cells in the PO myocardium. In vitro studies using cultured mouse CFb showed that dasatinib treatment at 50 nM reduced: (i) extracellular accumulation of both collagen and fibronectin, (ii) both basal and PDGF-stimulated activation of Pyk2, (iii) nuclear accumulation of Ki67, SKP2 and histone-H2B and (iv) PDGF-stimulated CFb proliferation and migration. However, dasatinib did not affect cardiomyocyte morphologies in either the ventricular tissue after in vivo administration or in isolated cells after in vitro treatment. Mass spectrometric quantification of dasatinib in cultured cells indicated that the uptake of dasatinib by CFb was greater that that taken up by cardiomyocytes. Dasatinib treatment primarily suppressed PDGF but not insulin-stimulated signaling (Erk versus Akt activation) in both CFb and cardiomyocytes. These data indicate that dasatinib treatment at lower doses than that used in chemotherapy has the capacity to reduce hypertrophy-associated fibrosis and improve ventricular function.     

Colorimetric estimation of human glucose level using γ-Fe2O3 nanoparticles: An easily recoverable effective mimic peroxidase

This article reports simple, green and efficient synthesis of γ-Fe₂O₃ nanoparticles (NPs) (maghemite) through single-source precursor approach for colorimetric estimation of human glucose level. The γ-Fe₂O₃ NPs, having cubic morphology with an average particle size of 30 nm, exhibited effective peroxidase-like activity through the catalytic oxidation of peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H₂O₂ producing a blue-colored solution. On the basis of this colored-reaction, we have developed a simple, cheap, highly sensitive and selective colorimetric method for estimation of glucose using γ-Fe₂O₃/TMB/glucose–glucose oxidase (GOx) system in the linear range from 1 to 80 μM with detection limit of 0.21 μM. The proposed glucose sensor displays faster response, good stability, reproducibility and anti-interference ability. Based on this simple reaction process, human blood and urine glucose level can be monitored conveniently.