Redox-regulation of Erk1/2-directed phosphatase by reactive oxygen species: role in signaling TPA-induced growth arrest in ML-1 cells

Extracellular signal-regulated kinase (Erk)1/2 activity signals myeloid cell differentiation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Previously, we reported that Erk1/2 activation (phosphorylation) induced by TPA required reactive oxygen species (ROS) as a second messenger. Here, we hypothesized that ROS generated in response to TPA inhibit Erk1/2-directed phosphatase activity, which leads to an increase phosphorylation of Erk1/2 to signal p21(WAF1/Cip1)-mediated growth arrest in ML-1 cells. Incubation of ML-1 cells with TPA resulted in a marked accumulation of phosphorylated Erk1/2, and is subsequent to H2O2 generation. Interestingly, post-TPA-treatment with N-acetylcysteine (NAC) stimulated a marked and a rapid dephosphorylation of Erk1/2, suggesting a regeneration of Erk1/2-directed phospahatase activity by NAC. ROS generation in ML-1 cells induced by TPA was suggested to occur in the mitochondrial electron transport chain (METC) based on the following observations: (i) undifferentiated ML-1 cells not only lack p67-phox and but also express a low level of p47-phox key components required for NADPH oxidase enzymatic activity, (ii) pretreatment with DPI, an inhibitor of NADH- and NADPH-dependent enzymes, or rhein, an inhibitor of complex I, blocked the ROS generation, and (iii) examination of the microarray analysis data and Western blot analysis data revealed an induction of MnSOD expression at both mRNA and protein levels in response to TPA. MnSOD is a key member of the mitochondrial defense system against mitochondrial-derived superoxide. Together, this study suggested that TPA stimulated ROS generation as a second messenger to activate Erk1/2 via a redox-mediated inhibition of Erk1/2-directed phosphatase in ML-1 cells.     

Transgenic switchgrass (Panicum virgatum L.) targeted for reduced recalcitrance to bioconversion: a 2‐year comparative analysis of field‐grown lines modified for target gene or genetic element expression

Transgenic Panicum virgatum L. silencing (KD) or overexpressing (OE) specific genes or a small RNA (GAUT4‐KD, miRNA156‐OE, MYB4‐OE, COMT‐KD and FPGS‐KD) was grown in the field and aerial tissue analysed for biofuel production traits. Clones representing independent transgenic lines were established and senesced tissue was sampled after year 1 and 2 growth cycles. Biomass was analysed for wall sugars, recalcitrance to enzymatic digestibility and biofuel production using separate hydrolysis and fermentation. No correlation was found between plant carbohydrate content and biofuel production pointing to overriding structural and compositional elements that influence recalcitrance. Biomass yields were greater for all lines in the second year as plants establish in the field and standard amounts of biomass analysed from each line had more glucan, xylan and less ethanol (g/g basis) in the second‐ versus the first‐year samples, pointing to a broad increase in tissue recalcitrance after regrowth from the perennial root. However, biomass from second‐year growth of transgenics targeted for wall modification, GAUT4‐KD, MYB4‐OE, COMT‐KD and FPGS‐KD, had increased carbohydrate and ethanol yields (up to 12% and 21%, respectively) compared with control samples. The parental plant lines were found to have a significant impact on recalcitrance which can be exploited in future strategies. This summarizes progress towards generating next‐generation bio‐feedstocks with improved properties for microbial and enzymatic deconstruction, while providing a comprehensive quantitative analysis for the bioconversion of multiple plant lines in five transgenic strategies.     

Characterisation of carboxymethyl cellulose and polyacrylamide graft copolymer

The synthesis of carboxymethyl cellulose-g-polyacrylamide was carried out by a ceric ion induced solution polymerization technique. By varying the amount of catalyst and monomer, six different grades of graft copolymers were synthesized. These graft copolymers were characterized by elemental analysis, infrared spectroscopy, rheological studies, scanning electron microscopy, thermal analysis, viscosity measurement and X-ray diffractometry. They exhibit distinguished flocculation characteristics in various suspensions and effluents. Their flocculation and viscosifying characteristics are drastically enhanced on their hydrolysis.     

Regenerated silica-based RNA purification columns to address the short supply of RNA purification kits for COVID-19 diagnosis

Molecular Biology Reports - RT-qPCR technique is the current world-wide method used for the early detection of SARS-CoV2 RNA in the suspected clinical samples. Viral RNA extraction is the key...     

Ageing of chloroplasts in vitro I. Quantitative analysis of the degradation of pigments, proteins and nucleic acids

Patterns of the degradation of various photosynthetic pigments,proteins and nucleic acids have been studied during the ageingof isolated chloroplasts in the light and dark. Ageing causesdegradation of chlorophylls and carotenoids, but the rate ofdegradation of both pigments was faster during light than duringdark ageing. Carotenoids are degraded much faster than chlorophyllsboth in the light and dark. The relatively greater degradationof carotenoids than chlorophylls in the dark suggests the involvementof some mechanism other than the photodestruction of carotenoidsduring ageing. The rate of decline for the DNA content is appreciablyslower than that of RNA and chloroplast-protein, but the degradationof latter two macromolecules is less than that of the chlorophyllsand carotenoids. (Received October 30, 1978; )     

The Mystery of Oxygen Evolution: Analysis of Structure and Function of Photosystem II,the Water-Plastoquinone Oxido-Reductase

Photosystem II (PS II) of thylakoid membrane of photosynthetic organisms has drawn attention of researchers over the years because it is the only system on Earth that provides us with oxygen that we breathe. In the recent past, structure of PS II has been the focus of research in plant science. The report of X-ray crystallographic structure of PS II complex by the research groups of James Barber and So Iwata in UK (K.N. Ferreira et al. Science 303: 1831–38, 2004) is a milestone in the area of research in photosynthesis. It follows the pioneering and elegant work from the laboratories of Horst Witt and W. Saenger in Germany (A. Zouni et al. Nature 409: 739–743, 2001), and J. Shen in Japan (N. Kamiya and J. Shen, Proc Natl Acad Sci USA 100: 98–103, 2003). It is time to analyze the historic events during the long journey made by the researchers to arrive at this point. This review makes an attempt to critically review the growth of the advancement of concepts and knowledge on the photosystem in the background of technological development. We conclude the review with perspectives on research and technology that should reveal the complete story of PS II of thylakoid in the future.     

Global expression profiles from C57BL/6J and DBA/2J mouse lungs to determine aging-related genes.

This study identified gene expression profiles that provided evidence for genomic mechanisms underlying the pathophysiology of aging lung. Aging lungs from C57BL/6 (B6) and DBA/2 (D2) mouse strains differ in physiology and morphometry. Lungs were harvested from B6 mice at 2, 18, and 26 mo and from D2 mice at 2 and 18 mo of age. Purified RNA was subjected to oligonucleotide microarray analyses, and differential expression analyses were performed for comparison of various data sets. A significant majority of differentially expressed genes were upregulated with aging in both strains. Aging D2 lungs uniquely exhibited upregulation in stress-response genes including xenobiotic detoxification cascades. In contrast, aging B6 lungs showed downregulation of heat shock-response genes. Age-dependent downregulation of genes common to both B6 and D2 strains included several collagen genes (e.g., Col1a1 and Col3a1). There was a greater elastin gene (Eln) expression in D2 mice at 2 mo, and Eln was uniquely downregulated with age in this strain. The matrix metalloproteinase 14 gene (Mmp14), critical to alveolar structural integrity, was also downregulated with aging in D2 mice only. Several polymorphisms in the regulatory and untranslated regions of Mmp14 were identified between strains, suggesting that variation in Mmp14 gene regulation contributes to accelerated aging of lungs in D2 mice. In summary, lungs of B6 and D2 mice age with variable rates at the gene expression level, and these quantifiable genomic differences provide a template for understanding the variability in age-dependent changes in lung structure and function.     

An efficient method for medium throughput screening of cuticular wax composition in different plant species

Introduction

Most aerial plant organs are covered by a cuticle, which largely consists of cutin and wax. Cuticular waxes are mixtures of dozens of compounds, mostly very-long-chain aliphatics that are easily extracted by solvents. Over the last four decades, diverse cuticular wax analysis protocols have been developed, most of which are complex and time-consuming, and need to be adapted for each plant species or organ. Plant genomics and breeding programs often require mid-throughput metabolic phenotyping approaches to screen large numbers of individuals and obtain relevant biological information.

Objectives

To generate a fast, simple and user-friendly methodology able to capture most wax complexity independently of the plant, cultivar and organ.

Methods

Here we present a simple GC–MS method for screening relatively small wax amounts, sampled by short extraction with a versatile, uniform solvent. The method will be tested and validated in leaves and fruits from three different crop species: tomato (Solanum lycopersicum), apple (Malus domestica) and hybrid aspen (Populus tremula × tremuloides).

Results

Consistent results were obtained in tomato cultivar M82 across three consecutive years (2010–2012), two organs (leaf and fruit), and also in two different tomato (M82 and MicroTom) and apple (Golden Delicious and Granny Smith) cultivars. Our results on tomato wax composition match those reported previously, while our apple and hybrid aspen analyses provide the first comprehensive cuticular wax profile of these species.

Conclusion

This protocol allows standardized identification and quantification of most cuticular wax components in a range of species.

     

Mechanical insights of oxythiamine compound as potent inhibitor for human transketolase-like protein 1 (TKTL1 protein)

Transketolase is a connecting link between glycolytic and pentose phosphate pathway, which is considered as the rate-limiting step due to synthesis of large number of ATP molecule and it can be proposed as a plausible target facilitating the growth of cancerous cells suggesting its potential role in cancer. Oxythiamine, an antimetabolite has been proved to be an efficient anticancerous compound in vitro, but its structural elucidation of the inhibitory mechanism has not yet been done against the human transketolase-like 1 protein (TKTL1). The three-dimensional (3D) structure of TKTL1 protein was modeled and subjected for refinement, stability and validation. Based on the reported homologs of transketolase (TKT), the active site residues His46, Ser49, Ser52, Ser53, Ile56, Leu82, Lys84, Leu123, Ser125, Glu128, Asp154, His160, Thr216 and Lys218 were identified and considered for molecular-modeling studies. Docking studies reveal the H-bond interactions with residues Ser49 and Lys218 that could play a major role in the activity of TKTL1. Molecular dynamics (MD) simulation study was performed to reveal the comparative stability of both native and complex forms of TKTL1. MD trajectory at 30?ns, confirm the role of active site residues Ser49, Lys84, Glu128, His160 and Lys218 in suppressing the activity of TKTL1. Glu128 is observed to be the most important residue for deprotonation state of the aminopyrimidine moiety and preferred to be the site of inhibitory action. Thus, the proposed mechanism of inhibition through in silico studies would pave the way for structure-oriented drug designing against cancer.     

Formulation of Sustained-Release Dosage Form of Verapamil Hydrochloride by Solid Dispersion Technique Using Eudragit RLPO or Kollidon®SR

The release of verapamil hydrochloride from tablets with Eudragit RLPO or Kollidon®SR with different drug-to-polymer ratios were investigated with a view to develop twice-daily sustained-release dosage form by solid dispersion (SD) technique. The SDs containing Eudragit RLPO or Kollidon®SR at drug-polymer ratios of 1:1, 1:2, and 1:3 with verapamil hydrochloride were developed using solvent evaporation technique. The physical mixtures of drug and both polymers were prepared by using simple mixing technique at the same ratio as solid dispersion. The physicochemical properties of solid dispersion were evaluated by using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and differential scanning calorimetry (DSC). The study of DSC, XRD, and FTIR could not show significant interaction between verapamil HCl and Kollidon®SR or Eudragit RLPO. The solid dispersions or physical mixtures were compressed to tablets. The tablets were prepared with solid dispersions containing Eudragit RLPO or Kollidon®SR, with all the official requirements of tablet dosage forms fulfilled. Tablets prepared were evaluated for the release of verapamil hydrochloride over a period of 12 h in pH 6.8 phosphate buffer using US Pharmacopoeia type II dissolution apparatus. The in vitro drug release study revealed that the tablet containing Eudragit has extended the release rate for 12 h whereas the tablet containing Kollidon®SR at the same concentration has extended the release rate up to 8 h. The in vitro release profile and the mathematical models indicate that release of verapamil hydrochloride can be effectively controlled from a tablet containing solid dispersions of Eudragit RLPO. The reduction of size fraction of the SD system from 200–250 to 75–125 μm had a great effect on the drug release.