Biochemical and Physiological Responses of Cucumis sativus Cultivars to Different Combinations of Low-Temperature and High Humidity

Low-temperature and high humidity are typical environmental factors in the plastic tunnel and solar greenhouse during the cold season that restricts plant growth and development. Herein, we investigated the impact of different combinations of low-temperature and high humidity (day/night: T1 15/10 °C?+?95%, T2 12/8 °C?+?95%, and T3 9/5 °C?+?95%) along with a control (CK 25/18 °C?+?80%) on cucumber cultivars viz: Zhongnong37 (ZN37: resistant) and Shuyanbailv (SYB: sensitive). The low-temperature and high humidity stresses increased electrolyte leakage (EL), malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and intercellular concentration of carbon dioxide (Ci), and reduced morphological indices, relative water content (RWC), net photosynthesis rate (Pn), stomatal conductance (Gs), transpiration rate (E) and leaf pigments in both cultivars as compared to control (CK). Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) were decreased in cv. SYB under stress conditions as compared to cv. ZN37. Low-temperature and high humidity treatments showed an increase in proline and soluble protein content in cv. ZN37 as compared to cv. SYB. Abscisic acid (ABA) and jasmonic acid (JA) were augmented while indole-3-acetic acid (IAA), zeatin (ZT), zeatin riboside (ZR), and gibberellic acid (GA) were decreased in both cultivars. Under T3 (9/5 °C?+?95%), Pn, protoporphyrin, and ZT were extremely decreased by 71.3%, 74.3%, and 82.4%, respectively, in cv. SYB compared to control. Moreover, principal component analysis (PCA) based on physiochemical traits confirmed that cv. ZN37 had the strongest correlation with antioxidant enzymes, proline, and soluble protein content than cv. SYB under low-temperature and high humidity treatments. Our results suggest that a stress-tolerant cultivar mitigates stress damage in cucumber transplants by regulating photosynthetic efficiency, antioxidant capacity and hormonal profile when compared to a stress-sensitive cultivar.

     

Topographic regulation of neuronal intermediate filaments by phosphorylation, role of peptidyl-prolyl isomerase 1: significance in neurodegeneration

The neuronal cytoskeleton is tightly regulated by phosphorylation and dephosphorylation reactions mediated by numerous associated kinases, phosphatases and their regulators. Defects in the relative kinase and phosphatase activities and/or deregulation of compartment-specific phosphorylation result in neurodegenerative disorders. The largest family of cytoskeletal proteins in mammalian cells is the superfamily of intermediate filaments (IFs). The neurofilament (NF) proteins are the major IFs. Aggregated forms of hyperphosphorylated tau and phosphorylated NFs are found in pathological cell body accumulations in the central nervous system of patients suffering from Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis. The precise mechanisms for this compartment-specific phosphorylation of cytoskeletal proteins are not completely understood. In this review, we focus on the mechanisms of neurofilament phosphorylation in normal physiology and neurodegenerative diseases. We also address the recent breakthroughs in our understanding the role of different kinases and phosphatases involved in regulating the phosphorylation status of the NFs. In addition, special emphasis has been given to describe the role of phosphatases and Pin1 in phosphorylation of NFs.     

Human NOC3 is essential for DNA replication licensing in human cells

Noc3p (Nucleolar Complex-associated protein) is an essential protein in budding yeast DNA replication licensing. Noc3p mediates the loading of Cdc6p and MCM proteins onto replication origins during the M-to-G₁ transition by interacting with ORC (Origin Recognition Complex) and MCM (Minichromosome Maintenance) proteins. FAD24 (Factor for Adipocyte Differentiation, clone number 24), the human homolog of Noc3p (hNOC3), was previously reported to play roles in the regulation of DNA replication and proliferation in human cells. However, the role of hNOC3 in replication licensing was unclear. Here we report that hNOC3 physically interacts with multiple human pre-replicative complex (pre-RC) proteins and associates with known replication origins throughout the cell cycle. Moreover, knockdown of hNOC3 in HeLa cells abrogates the chromatin association of other pre-RC proteins including hCDC6 and hMCM, leading to DNA replication defects and eventual apoptosis in an abortive S-phase. In comparison, specific inhibition of the ribosome biogenesis pathway by preventing pre-rRNA synthesis, does not lead to any cell cycle or DNA replication defect or apoptosis in the same timeframe as the hNOC3 knockdown experiments. Our findings strongly suggest that hNOC3 plays an essential role in pre-RC formation and the initiation of DNA replication independent of its potential role in ribosome biogenesis in human cells.     

Pin1-dependent prolyl isomerization modulates the stress-induced phosphorylation of high molecular weight neurofilament protein

Aberrant phosphorylation of neuronal cytoskeletal proteins is a key pathological event in neurodegenerative disorders such as Alzheimer disease (AD) and amyotrophic lateral sclerosis, but the underlying mechanisms are still unclear. Previous studies have shown that Pin1, a peptidylprolyl cis/trans-isomerase, may be actively involved in the regulation of Tau hyperphosphorylation in AD. Here, we show that Pin1 modulates oxidative stress-induced NF-H phosphorylation. In an in vitro kinase assay, the addition of Pin1 substantially increased phosphorylation of NF-H KSP repeats by proline-directed kinases, Erk1/2, Cdk5/p35, and JNK3 in a concentration-dependent manner. In vivo, dominant-negative (DN) Pin1 and Pin1 small interfering RNA inhibited epidermal growth factor-induced NF-H phosphorylation. Because oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, we studied the role of Pin1 in stressed cortical neurons and HEK293 cells. Both hydrogen peroxide (H(2)O(2)) and heat stresses induce phosphorylation of NF-H in transfected HEK293 cells and primary cortical cultures. Knockdown of Pin1 by transfected Pin1 short interference RNA and DN-Pin1 rescues the effect of stress-induced NF-H phosphorylation. The H(2)O(2) and heat shock induced perikaryal phospho-NF-H accumulations, and neuronal apoptosis was rescued by inhibition of Pin1 in cortical neurons. JNK3, a brain-specific JNK isoform, is activated under oxidative and heat stresses, and inhibition of Pin1 by Pin1 short interference RNA and DN-Pin1 inhibits this pathway. These results implicate Pin1 as a possible modulator of stress-induced NF-H phosphorylation as seen in neurodegenerative disorders like AD and amyotrophic lateral sclerosis. Thus, Pin1 may be a potential therapeutic target for these diseases.     

Multiplexed Integrating Plasmids for Engineering of the Erythromycin Gene Cluster for Expression in Streptomyces spp. and Combinatorial Biosynthesis

Bacteria in the genus Streptomyces and its close relatives are prolific producers of secondary metabolites with antibiotic activity. Genome sequencing of these bacteria has revealed a rich source of potentially new antibiotic pathways, whose products have never been observed. Moreover, these new pathways can provide novel genes that could be used in combinatorial biosynthesis approaches to generate unnatural analogues of existing antibiotics. We explore here the use of multiple orthologous integrating plasmid systems, based on the int/attP loci from phages TG1, SV1, and ϕBT1, to express the polyketide synthase (PKS) for erythromycin in a heterologous Streptomyces host. Streptomyces strains containing the three polyketide synthase genes eryAI, eryAII, and eryAIII expressed from three different integrated plasmids produced the aglycone intermediate, 6-deoxyerythronolide B (6-dEB). A further pair of integrating plasmids, both derived from the ϕC31 int/attP locus, were constructed carrying a gene cassette for glycosylation of the aglycone intermediates, with or without the tailoring gene, eryF, required for the synthesis of erythronolide B (EB). Liquid chromatography-mass spectrometry of the metabolites indicated the production of angolosaminyl-6-dEB and angolosaminyl-EB. The advantages of using multiplexed integrating plasmids for engineering expression and for combinatorial biosynthesis were demonstrated.     

Methods to produce marker-free transgenic plants

Selectable marker genes (SMGs) have been extraordinarily useful in enabling plant transformation because of the low efficiency of transgene integration. The most used SMGs encode proteins resistant to antibiotics or herbicides and use negative selection, i.e., by killing nontransgenic tissue. However, there are perceived risks in wide-scale deployment of SMG-transgenic plants, and therefore research has recently been performed to develop marker-free systems. In this review, transformation using markers not based on antibiotic or herbicide resistance genes, as well as different systems of marker gene deletion, are discussed.     

Serum organochlorine pesticides residues and risk of cancer: A case-control study

Organochlorine pesticides (OCPs) are frequently used worldwide as insecticides, fungicides, herbicides and termiticides and have been associated with a variety of cancers in animal and human studies. In the present study, we examined residues of fourteen OCPs in the serum samples of diagnosed cancer patients and healthy residents of Karachi, Pakistan. A random collection of fasting blood samples was carried out from the donors with informed consent. Serum was separated within 2?h of blood collection and was then subjected to extraction with organic solvents followed by purification with florisil column. The final organic extract of each serum sample was processed with Gas Chromatograph coupled with Electron Capture Detector (GC-ECD). OCPs were detected in 97.59% of the cancer cases and 93.75% of the healthy subjects. Mean concentrations of total OCPs (ΣOCPs) was found elevated in the cancer group (0.606?mg/kg) compared with the control group (0.322?mg/kg). Endosulfan was the highest prevalent OCP with a mean concentration of 0.214?mg/kg in the cancer group and 0.166?mg/kg in the control group. The second most prevalent OCP was 4,4-DDE with a mean concentration of 0.131?mg/kg in the cancer group and 0.019?mg/kg in the control group. Highest level of ΣOCPs was detected in the breast cancer cases (20.411?mg/kg) with a mean level of (2.041?mg/kg). In light of the obtained results and available literature on the subject, it has been concluded that OCPs are positively associated with the risk of various cancers in humans.     

Differential protein expression profiles of gastric epithelial cells following Helicobacter pylori infection using ProteinChips

Helicobacter pylori infects approximately half of the world's population and the bacterium is associated with gastric cancer and peptic and duodenal ulcers. In this study, Surface Enhanced Laser Desorption /Ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was used to identify the biomarkers from H. pylori infected gastric epithelial cells (GEC) to understand key mechanisms associated with pathogenesis. Using different chip surfaces, differential protein expression profile of GEC was obtained and several upregulated or downregulated biomarkers were detected on GEC, following H. pylori infection. Four different H. pylori infected GECs were compared based on their expression of MHC class II, a receptor reported to trigger apoptosis. One biomarker was identified in H. pylori infected GEC as Annexin A2 (Annexin II) from the flow through of the anion-exchange resin. The increased expression of Annexin II in GEC following H. pylori infection was further confirmed by Western Blot analyses and indicates its involvement in H. pylori pathogenesis.     

Tetracycline up-regulates COX-2 expression and prostaglandin E2 production independent of its effect on nitric oxide

Tetracyclines (doxycycline and minocycline) augmented (one- to twofold) the PGE2 production in human osteoarthritis-affected cartilage (in the presence or absence of cytokines and endotoxin) in ex vivo conditions. Similarly, bovine chondrocytes stimulated with LPS showed (one- to fivefold) an increase in PGE2 accumulation in the presence of doxycycline. This effect was observed at drug concentrations that did not affect nitric oxide (NO) production. In murine macrophages (RAW 264.7) stimulated with LPS, tetracyclines inhibited NO release and increased PGE2 production. Tetracycline(s) and L-N-monomethylarginine (L-NMMA) (NO synthase inhibitor) showed an additive effect on inhibition of NO and PGE2 accumulation, thereby uncoupling the effects of tetracyclines on NO and PGE2 production. The enhancement of PGE2 production in RAW 264.7 cells by tetracyclines was accompanied by the accumulation of both cyclooxygenase (COX)-2 mRNA and cytosolic COX-2 protein. In contrast to tetracyclines, L-NMMA at low concentrations (< or = 100 microM) inhibited the spontaneous release of No in osteoarthritis-affected explants and LPS-stimulated macrophages but had no significant effect on the PGE2 production. At higher concentrations, L-NMMA (500 microM) inhibited NO release but augmented PGE2 production. This study indicates a novel mechanism of action of tetracyclines to augment the expression of COX-2 and PGE2 production, an effect that is independent of endogenous concentration of NO.