Effects of NaCl and iso-osmotic PEG stress on growth,osmolytes accumulation and antioxidant defense in cultured sugarcane cells

In order to discriminate between the ionic and osmotic components of salt stress, sugarcane (Saccharum officinarum L. cv. Co 86032) calli were cultured on media containing NaCl or polyethylene glycol (PEG) 8000 that exerted the same osmotic pressure (−0.7 MPa). PEG stress exposure for 15 days led to significant growth reduction and loss in water content than salt stressed and control tissues. Osmotic adjustment (OA) was observed in callus tissues grown on salt, but was not evident in callus grown on PEG. Oxidative damage to membranes, estimated in terms of accumulation of thiobarbituric acid reactive substances-TBARS and electrolytic leakage was significantly higher in both the stressed calli than the control however, the extent of damage was more in the PEG stressed calli. The stressed callus tissues showed inhibition of ascorbate peroxidase activity, while catalase activity was increased. These results indicate sensitivity of cells to PEG-mediated stress than salt stress and differences in their OA to these two stress conditions. The sensitivity to the osmotic stress indicate that expression of the stress tolerance response requires the coordinated action of different tissues in a plant and hence was not expressed at the cellular level.     

The isolation and immunolocalization of iron-binding compounds produced by Gloeophyllum trabeum

Summary Low molecular weight iron-binding compounds are produced by the brown-rot fungus Gloeophyllum trabeum. These chelators may function in scavenging transition metals for fungal metabolism and extracellular enzyme production. Because of the low molecular mass of the chelate-metal complex (below 1000 Da), and the oxidizing potential of the bound transition metals, certain chelating compounds could also play a role in the early stages of cellulose depolymerization by brown-rot fungi. High-affinity iron-binding compounds were isolated and partially purified from both liquid cultures of the brown-rot Gloeophyllum trabeum and from infected wood. Chelating compounds purified by thin-layer chromatography were used to prepare specific antibodies. These antibodies were shown to detect the chelator in infected wood and liquid fungal cultures by enzyme-linked immunosorbent assay and could be used in immunotransmission electron microscopy to visualize the high-affinity iron-binding compounds in situ. Elucidating the physiological roles of fungal chelate-metal complexes and determining their function in lignocellulose depolymerization will help us to better understand the mechanism of wood biodegradation.Publication no. 1549 Maine Agricultural Experiment Station Offprint requests to: J. Jellison     

Cardiac Specific Expression of Threonine 5 to Alanine Mutant Sarcolipin Results in Structural Remodeling and Diastolic Dysfunction

The functional importance of threonine 5 (T5) in modulating the activity of sarcolipin (SLN), a key regulator of sarco/endoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA) pump was studied using a transgenic mouse model with cardiac specific expression of threonine 5 to alanine mutant SLN (SLNT5A). In these transgenic mice, the SLNT5A protein replaces the endogenous SLN in atria, while maintaining the total SLN content. The cardiac specific expression of SLNT5A results in severe cardiac structural remodeling accompanied by bi-atrial enlargement. Biochemical analyses reveal a selective downregulation of SR Ca²⁺ handling proteins and a reduced SR Ca²⁺ uptake both in atria and in the ventricles. Optical mapping analysis shows slower action potential propagation in the transgenic mice atria. Doppler echocardiography and hemodynamic measurements demonstrate a reduced atrial contractility and an impaired diastolic function. Together, these findings suggest that threonine 5 plays an important role in modulating SLN function in the heart. Furthermore, our studies suggest that alteration in SLN function can cause abnormal Ca²⁺ handling and subsequent cardiac remodeling and dysfunction.     

Rapid and Robust PCR-Based All-Recombinant Cloning Methodology

We report here a PCR-based cloning methodology that requires no post-PCR modifications such as restriction digestion and phosphorylation of the amplified DNA. The advantage of the present method is that it yields only recombinant clones thus eliminating the need for screening. Two DNA amplification reactions by PCR are performed wherein the first reaction amplifies the gene of interest from a source template, and the second reaction fuses it with the designed expression vector fragments. These vector fragments carry the essential elements that are required for the fusion product selection. The entire process can be completed in less than 8 hours. Furthermore, ligation of the amplified DNA by a DNA ligase is not required before transformation, although the procedure yields more number of colonies upon transformation if ligation is carried out. As a proof-of-concept, we show the cloning and expression of GFP, adh, and rho genes. Using GFP production as an example, we further demonstrate that the E. coli T7 express strain can directly be used in our methodology for the protein expression immediately after PCR. The expressed protein is without or with 6xHistidine tag at either terminus, depending upon the chosen vector fragments. We believe that our method will find tremendous use in molecular and structural biology.     

Kraft black liquor for improving the productivity of Spirulina biomass

Summary Mass cultivation of Spirulina for commercial application suffers from poor productivity when measured against laboratory results or theoretical projections. Wider applications of algal products require that this gap be reduced. Addition of eucalyptus kraft black liquor at a maximum of 0.1% to Spirulina cultures enhanced biomass productivity by at least 40%. The factors enhancing Spirulina biomass productivity were insoluble at low pH, of low molecular mass and stable to high temperature. Single addition of kraft black liquor in outdoor continuous cultures afforded sustained enhancement in biomass productivity for at least eight weeks.     

Chemodiversity and α-Glucosidase Activity of Eucalyptus Species from Northwestern Himalaya,India

The aim of current work was to determine essential oils (EOs) composition from three Eucalyptus species, including E. citriodora, E. camaldulensis and E. globulus and assess their α-glucosidase inhibitory activity. The EOs were collected using the hydrodistillation technique and characterized by GC/MS, GC-FID and NMR. The isolated EOs from leaves parts of Eucalyptus species varied from 0.56 to 1.0 % on fresh weight basis. The content of the EOs was distinct according to the species. The most abundant metabolites were identified as citronellal (0–83.0 %), 1,8-cineole (0.2–44.8 %), spathulenol (0.4–16.1 %) α-pinene (0.4–15.9 %), p-cymene (3.7–11.9 %), citronellol (0–8.6 %), β-eudesmol (5.3–8.6 %) and β-pinene (0–7.1 %). The EOs obtained from targeted samples exhibited strong α-glucosidase inhibitory activity. These results are encouraging and underline that the EOs of Eucalyptus species may be a promising alternative source of natural antidiabetic.     

Tricyclic 4,4-dimethyl-3,4-dihydrochromeno[3,4-d]imidazole derivatives as microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors: SAR and in vivo efficacy in hyperalgesia pain model

A series of substituted tricyclic 4,4-dimethyl-3,4-dihydrochromeno[3,4-d]imidazole derivatives have been synthesized and their mPGES-1 biological activity has been disclosed in detail. Structure-activity relationship (SAR) optimization provided inhibitors with excellent mPGES-1 potency and low to moderate PGE₂ release A549 cell potency. Among the mPGES-1 inhibitors studied, 7, 9 and 11l provided excellent selectivity over COX-2 (>200-fold) and >70-fold selectivity for COX-1 except 11l, which exhibited dual mPGES-1/COX-1 activity. Furthermore, the above tested mPGES-1 inhibitors demonstrated good metabolic stability in liver microsomes, high plasma protein binding (PPB) and no significant inhibition observed in clinically relevant CYP isoforms. Besides, selected mPGES-1 tool compounds 9 and 11l provided good in vivo pharmacokinetic profile and oral bioavailability (%F = 33 and 85). Additionally, the representative mPGES-1 tool compounds 9 and 11l revealed moderate in vivo efficacy in the LPS-induced thermal hyperalgesia guinea pig pain model.     

Identification and characterization of candidates involved in production of OMEGAs in microalgae: a gene mining and phylogenomic approach

Abstract

Optimizing the production of the high-value renewables such as OMEGAs through pathway engineering requires an in-depth understanding of the structure–function relationship of genes involved in the OMEGA biosynthetic pathways. In this preliminary study, our rationale is to identify and characterize the ～221 putative genes involved in production of OMEGAs using bioinformatic analysis from the Streptophyte (plants), Chlorophyte (green algae), Rhodophyta (red algae), and Bacillariophyta (diatoms) lineages based on their phylogenomic profiling, conserved motif/domain organization and physico-chemical properties. The MEME suite predicted 12 distinct protein domains, which are conserved among these putative genes. The phylogenomic analysis of the putative candidate genes [such as FAD2 (delta-12 desaturase); ECR (enoyl-CoA reductase); FAD2 (delta-12 desaturase); ACOT (acyl CoA thioesterase); ECH (enoyl-CoA hydratase); and ACAT (acetyl-CoA acyltransferase)] with similar domains and motif patterns were remarkably well conserved. Furthermore, the subcellular network prediction of OMEGA biosynthetic pathway genes revealed a unique interaction between the light-dependent chlorophyll biosynthesis and glycerol-3-phosphate dehydrogenase, which predicts a major cross-talk between the key essential pathways. Such bioinformatic analysis will provide insights in finding the key regulatory genes to optimize the productivity of OMEGAs in microalgal cell factories.