Humanization of yeast to produce complex terminally sialylated glycoproteins

Yeast is a widely used recombinant protein expression system. We expanded its utility by engineering the yeast Pichia pastoris to secrete human glycoproteins with fully complex terminally sialylated N-glycans. After the knockout of four genes to eliminate yeast-specific glycosylation, we introduced 14 heterologous genes, allowing us to replicate the sequential steps of human glycosylation. The reported cell lines produce complex glycoproteins with greater than 90% terminal sialylation. Finally, to demonstrate the utility of these yeast strains, functional recombinant erythropoietin was produced.     

Characterization of storage proteins in wild (Glycine soja) and cultivated (Glycine max) soybean seeds using proteomic analysis

A combined proteomic approach was applied for the separation, identification, and comparison of two major storage proteins, beta-conglycinin and glycinin, in wild (Glycine soja) and cultivated (Glycine max) soybean seeds. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with three different immobilized pH gradient (IPG) strips was an effective method to separate a large number of abundant and less-abundant storage proteins. Most of the subunits of beta-conglycinin were well-separated in the pH range 3.0-10.0, while acidic and basic glycinin polypeptides were well-separated in pH ranges 4.0-7.0 and 6.0-11.0, respectively. Although the overall distribution pattern of the protein spots was similar in both genotypes using pH 3.0-10.0, variations in number and intensity of protein spots were better resolved using a combination of pH 4.0-7.0 and pH 6.0-11.0. The total number of storage protein spots detected in wild and cultivated genotypes was approximately 44 and 34, respectively. This is the first study reporting the comparison of protein profiles of wild and cultivated genotypes of soybean seeds using proteomic tools.     

Assessment of indigenous Nepalese soybean as a potential germplasm resource for improvement of protein in North American cultivars

Soybeans contain approximately 40% protein and 20% oil and represents an important source of protein in animal rations and human diets. Attempts are being made to increase further the overall protein content of soybeans by utilization of exotic germplasms. In this study, soybean cultivars from Nepal have been characterized and their potential as a germplasm resource for improvement of the protein content and quality of North American cultivars assessed. Soybean cultivars 'Sathia', 'Seti', 'Kavre', and 'Soida Chiny', indigenous to various regions of Nepal, contained 42-45% protein, which is significantly higher in comparison to that of the North American cultivar 'Williams 82' (39%). Fractionation of seed protein by high-resolution two-dimensional gel electrophoresis revealed differences in the protein profiles of these cultivars. Various isoelectric forms of glycinin and beta-conglycinin were identified by comparing the matrix-assisted laser desorption ionization time-of-flight mass fingerprinting data against the National Center for Biotechnology Information nonredundant database. Nepalese cultivar Sathia was distinct, lacking some isoelectric forms of acidic and basic glycinin subunits while expressing other unique forms. The contribution of these unique protein spots present in either Sathia or Williams 82 to the total protein content was quantified using scanning laser densitometry. Distinct restriction fragment length polymorphisms (RFLP) for group 1 glycinin genes were observed among the tested Nepalese genotypes, indicating sequence variation among the cultivars. Conversely, evaluation of RFLP for the genes encoding group 2 glycinins, beta-conglycinin, and Bowman-Birk proteinase inhibitors indicated a high degree of conservation in these genes. Determination of amino acid composition, a reflection of protein quality, indicated that the arginine content of the Nepalese soybeans ranged from 7.7 to 8.1%, which was 5-10% higher than the 7.4% expressed in Williams 82. Additionally, Karve and Seti contained significantly more cysteine than Williams 82. Nepalese high-protein soybeans having a desirable amino acid composition hold potential to increase the protein quality and diversity of North American cultivars.     

Surface sites for engineering allosteric control in proteins

Statistical analyses of protein families reveal networks of coevolving amino acids that functionally link distantly positioned functional surfaces. Such linkages suggest a concept for engineering allosteric control into proteins: The intramolecular networks of two proteins could be joined across their surface sites such that the activity of one protein might control the activity of the other. We tested this idea by creating PAS-DHFR, a designed chimeric protein that connects a light-sensing signaling domain from a plant member of the Per/Arnt/Sim (PAS) family of proteins with Escherichia coli dihydrofolate reductase (DHFR). With no optimization, PAS-DHFR exhibited light-dependent catalytic activity that depended on the site of connection and on known signaling mechanisms in both proteins. PAS-DHFR serves as a proof of concept for engineering regulatory activities into proteins through interface design at conserved allosteric sites.     

Control of Aeromonas hydrophila Infection in Spotted Snakehead, Channa punctatus, by Solanum nigrum L., a Medicinal Plant

The present study describes the potential of black nightshade, Solanum nigrum , to control Aeromonas hydrophila infection in spotted snakehead, Channa punctatus . Similar sizes of Ch.   punctatus were grouped into three categories as follows: control (injected with sterilized water), Ae.   hydrophila infected   +   untreated, and Ae.   hydrophila infected   +   treated with S.   nigrum leaf extract. Hematological parameters (white blood cell [WBC], red blood cell [RBC], hemoglobin [Hb], and packed cell volume [PCV]) were analyzed in these fishes once every 5   d from Days 5 to 30 postexposure. Preliminary antibacterial assay reported that leaf extracted with ethyl acetate had the maximum inhibition compared to other parts of the plant (i.e., berries and whole plant) or when extracted with other solvent extracts (chloroform and ethanol). Except for WBC count, all other parameters showed a declining trend from Day 5 onward and reaching to a minimum on Day 30 in Ae.   hydrophila -infected fish, whereas the WBC count increased. The infected fish treated with S.   nigrum leaf extract reported a significant reversion to normal RBC, WBC, Hb, and PCV levels and the skin ulceration recovered within a short period. The antiulcer potential of the medicinal plant as described in humans may be extended to the fish population.     

Seed Invigoration by Hydration–Dehydration for Improvement of Seed Quality in Safflower (Carthamus tinctorius L.)

Three midstorage hydration-dehydration treatments viz., soaking, moisture equilibration and moist-sand conditioning followed by drying, each at varying durations for preservation of vigour and viability during storage in safflower var. K.1, were evaluated for their efficacy. Soaking seeds for 3 h followed by dehydration proved to be the best. Maintenance of membrane integrity and counteraction of lipid peroxidation may be the possible reasons for such advantage as evidenced by lower electrical conductance and peroxide values in the best treatment.     

Preliminary evaluation of anti-pyretic and anti-ulcerogenic activities of Sida cordifolia methanolic extract

The anti-pyretic and anti-ulcerogenic properties of methanolic extract of Sida cordifolia aerial parts (MESC) were investigated in rats. Oral dose of 500 mg/kg MESC significantly reduced pyrexia induced by TAB vaccine. MESC exhibited significant anti-ulcerogenic effect against aspirin and ethanol induced damage. Both these properties were comparable to the reference drugs.     

Assessment of impact of aquaculture on Kolleru Lake (India) using remote sensing and Geographical Information System

Aquaculture is one of the fastest growing food industries and the rapid growth of aquaculture worldwide has resulted in growing concerns about its impact on important ecosystems. Kolleru Lake, India's largest fresh water body, and Ramsar site have undergone tremendous changes due to the development of aquaculture. To assess the impact of aquaculture on Kolleru, satellite data were found appropriate because of the synoptic‐detailed overview and accuracy. Satellite data of IRS 1D, LISS III from 2004 and Survey of India topographic maps from 1967 were processed using image processing techniques in erdas imagine and analysed in Geographical Information System (GIS) such as arc gis 9.1. Land use map prepared from the satellite data was verified in the field using Global Positioning System to check the land and water use classes and its areal extent. It was estimated from the topographic maps of 1967 that the total lake boundary area was 180.38 km², in which 70.70 km² had water throughout the year and 100.97 km² had water during the rainy season. The digital image processing of 2004 satellite data revealed that Kolleru was no longer a lake and the lake area of 62.65 km² (34.73%) only remained in a degraded state, extensively colonized by macrophytes. The total loss of lake area was 109.02 km² between 1967 and 2004, in which aquaculture was developed in 99.74 km², which represented 55.3% of the 1967 lake area. The maximum conversion to aquaculture occurred from the lake liable to be inundated during the 1967 rainy season. The area under agriculture was 16.62 km² in 2004, the increase in lake area for agriculture between 1967 and 2004 being 8.22 km² (4.55% of lake). If human induced degradation is allowed to continue, the lake will very soon disappear. Now, the Government of Andhra Pradesh, India, has initiated schemes and laws to restore the lake to its pre‐development state. Lessons learnt from the Kolleru Lake cautioned that there is a need for regular monitoring of important water resources throughout the world to protect the biodiversity of the earth.