Altered lipid composition and enhanced lipid production in green microalga by introduction of brassica diacylglycerol acyltransferase 2

Higher lipid biosynthesis and accumulation are important to achieve economic viability of biofuel production via microalgae. To enhance lipid content, Chlamydomonas reinhardtii was genetically engineered with a key enzyme diacylglycerol acyltransferase (BnDGAT2) from Brassica napus, responsible for neutral lipid biosynthesis. The transformed colonies harbouring aph7 gene, screened on hygromycin‐supplemented medium, achieved transformation frequency of ~120 ± 10 colonies/1 × 10⁶ cells. Transgene integration and expression were confirmed by PCR, Southern blots, staining lipid droplets, proteins and spectro‐fluorometric analysis of Nile red‐stained cells. The neutral lipid is a major class (over 80% of total lipids) and most significant requirement for biodiesel production; this was remarkably higher in the transformed alga than the untransformed control. The levels of saturated fatty acids in the transformed alga decreased to about 7% while unsaturated fatty acids increased proportionately when compared to wild type cells. Polyunsaturated fatty acids, especially α‐linolenic acid, an essential omega‐3 fatty acid, were enhanced up to 12% in the transformed line. Nile red staining confirmed formation of a large number of lipid globules in the transformed alga. Evaluation of long‐term stability and vitality of the transgenic alga revealed that cryopreservation produced significantly higher quantity of lipid than those maintained continuously over 128 generations on solid medium. The overexpression of BnDGAT2 significantly altered the fatty acids profile in the transformed alga. Results of this study offer a valuable strategy of genetic manipulation for enhancing polyunsaturated fatty acids and neutral lipids for biofuel production in algae.     

The BLOC-1 complex promotes endosomal maturation by recruiting the Rab5 GTPase-activating protein Msb3

Membrane microcompartments of the early endosomes serve as a sorting and signaling platform, where receptors are either recycled back to the plasma membrane or forwarded to the lysosome for destruction. In metazoan cells, three complexes, termed BLOC-1 to -3, mediate protein sorting from the early endosome to lysosomes and lysosome-related organelles. We now demonstrate that BLOC-1 is an endosomal Rab-GAP (GTPase-activating protein) adapter complex in yeast. The yeast BLOC-1 consisted of six subunits, which localized interdependently to the endosomes in a Rab5/Vps21-dependent manner. In the absence of BLOC-1 subunits, the balance between recycling and degradation of selected cargoes was impaired. Additionally, our data show that BLOC-1 is both a Vps21 effector and an adapter for its GAP Msb3. BLOC-1 and Msb3 interacted in vivo, and both mutants resulted in a redistribution of active Vps21 to the vacuole surface. We thus conclude that BLOC-1 controls the lifetime of active Rab5/Vps21 and thus endosomal maturation along the endocytic pathway.     

Development of cloning vectors and transformation methods for<Emphasis Type="Italic"> Amycolatopsis</Emphasis>

The genus Amycolatopsis is of industrial importance, as its species are known to produce commercial antibiotics. It belongs to the family Pseudonocardiaceae and has an eventful taxonomic history. Initially strains were identified as Streptomyces, then later as Nocardia. However, based on biochemical, morphological and molecular features, the genus Amycolatopsis, containing seventeen species, was created. The development of molecular genetic techniques for this group has been slow. The scarcity of molecular genetic tools including stable plasmids, antibiotic resistance markers, transposons, reporter genes, cloning vectors, and high efficiency transformation protocols has made progress slow, but efforts in the past decade have led to the development of cloning vectors and transformation methods for these organisms. Some of the cloning vectors have broad host range (pRL series) whereas others have limited host range (pMEA300 and pMEA100). The cloning vector pMEA300 has been completely sequenced, while only the minimal replicon (pA-rep) has been sequenced from pRL plasmids. Direct transformation of mycelia and electroporation are the most widely applicable methods for transforming species of Amycolatopsis. Conjugational transfer from Escherichia coli has been reported only in the species A. japonicum, and gene disruption and replacements using homologous recombination are now possible in some strains. Electronic Publication     

A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy.

Methods

Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR.

Results

In vitro binding experiments of ¹²⁵I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney.

Conclusions

These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.     

Growth of tomato and an ABA-deficient mutant (sitiens) under saline conditions

To determine whether ABA accumulation inhibits or promotes shoot growth under stress, an ABA-deficient mutant tomato, sitiens, and its wild-type, the cultivar Rheinlands Rhum, were exposed to moderate salinity stress. Plants were grown at 75 m M NaCl for 2 weeks under conditions of moderate or high relative humidity (70% and 95% RH, respectively). At 70% RH, shoot DW and relative growth rate were reduced more in sitiens than in the cultivar, but the major difference between genotypes was in the degree of injury suffered by older leaves. Most leaves of sitiens died after 2 weeks, but those of the cultivar remained alive. When plants were grown at 95% RH, to maximize the leaf water status of both genotypes, there was no significant effect of salt on shoot DW of either genotype. However, there was still considerable leaf death in sitiens whereas no visible injury appeared in the cultivar. Cl^– accumulated to higher levels in leaf tissues than Na⁺, but to similar concentrations in both genotypes, and so could not explain the injury in the sitiens leaves. The results indicate that ABA maintains rather than inhibits new growth under stress, and has a major effect on preservation of older leaves.     

Purification and characterization of a novel thermo-active amidase from Geobacillus subterraneus RL-2a

A thermostable amidase produced by Geobacillus subterraneus RL-2a was purified to homogeneity, with a yield of 9.54 % and a specific activity of 48.66 U mg^?1. The molecular weight of the native enzyme was estimated to be 111 kDa. The amidase of G. subterraneus RL-2a is constitutive in nature, active at a broad range of pH (4.5–11.5) and temperature (40–90 °C) and has a half-life of 5 h and 54 min at 70 °C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co²⁺, Hg²⁺, Cu²⁺, Ni²⁺, and thiol reagents. The presence of mid-chain aliphatic and amino acid amides enhances the enzymatic activity. The acyl transferase activity was detected with propionamide, butyramide and nicotinamide. The enzyme showed moderate stability toward toluene, carbon tetrachloride, benzene, ethylene glycol except acetone, ethanol, butanol, propanol and dimethyl sulfoxide. The K _m and V _max of the purified amidase with nicotinamide were 6.02 ± 0.56 mM and 132.6 ± 4.4 μmol min^?1 mg^?1 protein by analyzing Michaelis–Menten kinetics. The results of MALDI-TOF analysis indicated that this amidase has homology with the amidase of Geobacillus sp. C56-T3 (gi|297530427). It is the first reported wide-spectrum thermostable amidase from a thermophilic G. subterraneus.     

CHPVDB ‐ a sequence annotation database for Chandipura Virus

Databases containing proteomic information have become indispensable for virology studies. As the gap between the amount of sequence information and functional characterization widens, increasing efforts are being directed to the development of databases. For virologist, it is therefore desirable to have a single data collection point which integrates research related data from different domains. CHPVDB is our effort to provide virologist such a one‐step information center. We describe herein the creation of CHPVDB, a new database that integrates information of different proteins in to a single resource. For basic curation of protein information, the database relies on features from other selected databases, servers and published reports. This database facilitates significant relationship between molecular analysis, cleavage sites, possible protein functional families assigned to different proteins of Chandipura virus (CHPV) by SVMProt and related tools.     

Autonomic Cardiovascular Responses in Acclimatized Lowlanders on Prolonged Stay at High Altitude: A Longitudinal Follow Up Study

Acute exposure to hypobaric hypoxia at high altitude is reported to cause sympathetic dominance that may contribute to the pathophysiology of high altitude illnesses. The effect of prolonged stay at high altitude on autonomic functions, however, remains to be explored. Thus, the present study aimed at investigating the effect of high altitude on autonomic neural control of cardiovascular responses by monitoring heart rate variability (HRV) during chronic hypobaric hypoxia. Baseline electrocardiography (ECG) data was acquired from the volunteers at mean sea level (MSL) (<250 m) in Rajasthan. Following induction of the study population to high altitude (4500–4800 m) in Ladakh region, ECG data was acquired from the volunteers after 6 months (ALL 6) and 18 months of induction (ALL 18). Out of 159 volunteers who underwent complete investigation during acquisition of baseline data, we have only included the data of 104 volunteers who constantly stayed at high altitude for 18 months to complete the final follow up after 18 months. HRV parameters, physiological indices and biochemical changes in serum were investigated. Our results show sympathetic hyperactivation along with compromise in parasympathetic activity in ALL 6 and ALL 18 when compared to baseline data. Reduction of sympathetic activity and increased parasympathetic response was however observed in ALL 18 when compared to ALL 6. Our findings suggest that autonomic response is regulated by two distinct mechanisms in the ALL 6 and ALL 18. While the autonomic alterations in the ALL 6 group could be attributed to increased sympathetic activity resulting from increased plasma catecholamine concentration, the sympathetic activity in ALL 18 group is associated with increased concentration of serum coronary risk factors and elevated homocysteine. These findings have important clinical implications in assessment of susceptibility to cardio-vascular risks in acclimatized lowlanders staying for prolonged duration at high altitude.     

Evaluation of Lateral-Flow Clostridium botulinum Neurotoxin Detection Kits for Food Analysis

The suitability and sensitivity of two in vitro lateral-flow assays for detecting Clostridium botulinum neurotoxins (BoNTs) in an assortment of foods were evaluated. Toxin extraction and preparation methods for various liquid, solid, and high-fat-content foods were developed. The lateral-flow assays, one developed by the Naval Medical Research Center (Silver Spring, MD) and the other by Alexeter Technologies (Gaithersburg, MD), are based on the immunodetection of BoNT types A, B, and E. The assays were found to be rapid and easy to perform with minimum requirements for laboratory equipment or skills. They can readily detect 10 ng/ml of BoNT types A and B and 20 ng/ml of BoNT type E. Compared to other in vitro detection methods, these assays are less sensitive, and the assessment of a result is strictly qualitative. However, the assay was found to be simple to use and to require minimal training. The assays successfully detected BoNT types A, B, and E in a wide variety of foods, suggesting their potential usefulness as a preliminary screening system for triaging food samples with elevated BoNT levels in the event of a C. botulinum contamination event.