Identification of specific proteins and glycoproteins associated with membrane fractions isolated from Zea mays L. coleoptiles

The aim of this work was to identify proteins specific for plant cell membranes which could then be used as unique markers. A crude membrane fraction was isolated from corn coleoptiles and separated on non-linear sucrose density gradients. Separation of endoplasmic reticulum (NADH-cytochrome c reductase), mitochondria (cytochrome c oxidase), golgi (inosine diphosphatase), and plasma membranes (N-1-naphthylphthalamic acid-binding) was achieved. The membrane proteins from the gradient fractions were separated using sodium dodecyl sulphate-poly-acrylamide gel electrophoresis and the gels stained with coomassie blue or with concanavalin A/peroxidase to detect glycoproteins. Proteins specific for the various membranes were identified. Five proteins including two glycoproteins were plasma membrane markers. Protoplasts were isolated and iodinated using lactoperoxidase/glucose oxidase covalently attached to beads. Eleven iodinated proteins were found and three of these corresponded to proteins specifically associated with plasma membranes in the density gradients. Two methods for detecting Ca²⁺-binding proteins following sodium dodecylsulphate polyacrylamide gel electrophoresis were employed. The majority of such proteins were found in the endoplasmatic reticulum and one was specific for plasma membranes. In vitro and in vivo phosphorylation of membrane proteins was examined and the majority of proteins phosphorylated were glycoproteins. Two of the phosphorylated proteins (M_r=110,000 and 20,000) were also iodinated on protoplasts and may be part of the plasma membrane ATPases.Abbreviations ER endoplasmic reticulum - IDP inosine diphosphate - NPA N-1-naphthylphthalamic acid     

A system using convertible vectors for screening soluble recombinant proteins produced in Escherichia coli from randomly fragmented cDNAs

Protein insolubility is a major problem when producing recombinant proteins (e.g., to be used as antigens) from large cDNAs in Escherichia coli. Here, we describe a system using three convertible plasmid vectors to screen for soluble proteins produced in E. coli. This system experimentally identified any random cDNA fragments producing soluble protein domains. Shotgun fragments introduced into any of our three plasmids, which contain Gateway recombination sites, fused in-frame to the ORF of the protein tag. These plasmids produced N-terminal GST- and C-terminal three-frame-adaptive FLAG-tagged proteins, kanamycin-resistant gene-tagged proteins (which were pre-selected for in-frame fused cDNAs), or GFP-tagged fusion proteins. The latter is useful as a fluorescence indicator of protein folding. The Gateway recombination sites promote smooth conversion for enrichment of in-frame clones and facilitate both protein solubility assays and final production of proteins without the C-terminal tag. This high-throughput screening method is particularly useful for procedures that require the handling of many cDNAs in parallel.     

Refolding out of guanidine hydrochloride is an effective approach for high-throughput structural studies of small proteins

Low in vivo solubility of recombinant proteins expressed in Escherichia coli can seriously hinder the purification of structural samples for large-scale proteomic NMR and X-ray crystallography studies. Previous results from our laboratory have shown that up to one half of all bacterial and archaeal proteins are insoluble when overexpressed in E. coli. Although a number of strategies may be used to increase in vivo protein solubility, there are no generally applicable methods, and the expression of each insoluble recombinant protein must be individually optimized. For this reason, we have tested a generic denaturation/refolding protein purification procedure to assess the number of structural samples that could be generated by using this methodology. Our results show that a denaturation/refolding protocol is appropriate for many small proteins (相似文献   

Antigenicity of the region encoded by exon8 of the human serine protease, HtrA2/Omi, is associated with its protein solubility

HtrA2/Omi, a mitochondrial serine protease, is pivotal in regulating apoptotic cell death. To determine the location of antigenic determinants in HtrA2/Omi, we expressed a series of the N-terminally truncated HtrA2/Omi as GST fusion proteins in E. coli. We assessed protein solubility and antigenic reactivity of various N-terminally truncated HtrA2/Omi proteins by binding to glutathione beads and immunoblot analyses, respectively. We identified that the region encoded by exon8 of HtrA2/Omi was expressed as a highly soluble form and contains an antigenic determinant specifically recognized by a polyclonal serum against HtrA2/Omi. Our data provide evidence that protein solubility of the specific region in target proteins may contribute to the antigenicity.     

Chemical partitioning of iron,cadmium, nickel and chromium in contaminated soils of south-eastern Nigeria

Abstract

Selected heavy metals Fe, Cd, Ni and Cr were studied in contaminated soil samples collected from south-eastern Nigeria. Geochemical differentiation into different chemical fractions, using Ma and Rao six-step sequential chemical extraction procedure, was carried out to assess the potential mobility and bioavailability of the heavy metals in the soil profiles. The residual fraction was the most important phase for the four heavy metals with the following average percentage values 74.43 for Fe, 37.69 for Cd, 70.11 for Ni and 62.47 for Cr. The carbonate fraction contained an appreciable proportion of Fe, Cd and Ni with the average percentage values of 16.29, 14.86 and 10.47 respectively, while organic fraction was of next importance for Cr with an average percentage value of 27.14. The Fe-Mn oxide fraction also contained 15.86% of Cd. Relatively low amounts of the metals were associated with water soluble and exchangeable fractions. The mobility factors for the metals in all the sites ranged from 8.55 to 40.04 for Fe, 8.66 to 56.58 for Cd, 12.74 to 30.19 for Ni and 0.82 to 7.22 for Cr. The generally low values of mobility factors coupled with significantly high levels of association of the metals with the residual fraction, indicate that the metals do not pose any environmental risk nor hazard.     

A STUDY OF THE EFFECTS OF STATIC AND EXTREMELY LOW FREQUENCY MAGNETIC FIELDS ON LIPID PEROXIDATION PRODUCTS IN SUBCELLULAR FIBROBLAST FRACTIONS

Cultured fibroblasts isolated from murine livers by tissue trypsinization were exposed to a static magnetic field (0.490 T) and to extremely low frequency (ELF) magnetic field (50 Hz, 0.020 T). The cultures were exposed to magnetic fields on four consecutive days for exposure times of 2, 4, 8, 16, 32, and 64 min. After such exposures and obtaining of fibroblast subcellular fractions, lipid peroxidation product—malondialdehyde (MDA) was measured. Increased peroxidation of fibroblasts' membrane structures exposed to an ELF magnetic field was observed in subcellular fractions—microsomal, mitochondrial, and nuclear. No changes in peroxidation of membrane structures were found in fibroblasts exposed to a static magnetic field.     

黑河下游湿地土壤有机氮组分剖面的分布特征

结合野外调查,用Bremner法研究了黑河下游湿地不同土壤类型的有机氮组分,结果表明:在0—50 cm土层,5种土壤有机氮均以酸解性氮为主,占全氮的71.04%—81.79%。泥炭土、沼泽土、草甸土、亚高山草甸土所含的酸解氮、非酸解氮和酸解氮组分氨态氮、氨基酸态氮、氨基糖态氮含量的剖面分布总体上均随土层深度的增加而呈降低趋势,而风沙土却相反,上述有机氮组分呈升高趋势。5种土壤酸解氮及其组分氨态氮、氨基酸态氮、氨基糖态氮占全氮比例的剖面分布总体上均随土层深度的增加而呈降低趋势,而非酸解氮却呈升高趋势。5种土壤酸解未知态氮含量及占全氮比例均在剖面分布上无明显特征。在0—30 cm各相同土层内,5种土壤酸解氮各组分含量及占全氮比例的大小顺序均为氨基酸态氮氨态氮未知态氮氨基糖态氮;而在30—50 cm土层,5种土壤酸解氮各组分含量及占全氮比例的大小顺序均无明显特征。此外,黑河下游湿地土壤干化、沙化过程中,表层0—10 cm土壤有机氮组分含量变化明显,其中土壤氨态氮对生态环境变化最为敏感。     

Chitosan-based delivery systems for curcumin: A review of pharmacodynamic and pharmacokinetic aspects

Effective drug delivery is one of the most important issues associated with the administration of therapeutic agents that have low oral bioavailability. Curcumin is an active ingredient in the turmeric plant, which has low oral bioavailability due to its poor aqueous solubility. One strategy that has been considered for enhancing the aqueous solubility, and, thus, its oral bioavailability, is the use of chitosan as a carrier for curcumin. Chitosan is a biodegradable and biocompatible polymer that is relatively water-soluble. Therefore, various studies have sought to improve the aqueous solubility of chitosan. The use of different pharmaceutical excipients and formulation strategies has the potential to improve aqueous solubility, formulation processing, and the overall delivery of hydrophobic drugs. This review focuses on various methods utilized for chitosan-based delivery of curcumin.     

An automated high-throughput screening method for the identification of high-yield, soluble protein variants using cell-free expression and systematic truncation

A highly automated method for rapidly identifying soluble protein variants with good expression yields has been developed. This method is based on a commercially available in vitro protein expression system. It consists of two polymerase chain reactions (PCR) followed by in vitro protein expression and protein quantification by dot blot. The PCR protocols have been improved and optimized to allow automation using commercial fluid handling devices. A PCR primer design program has also been implemented to streamline protein variant design. This automated protocol is highly reliable and has tremendously improved the throughput of expression screening as compared to conventional cell-based methods and manual in vitro methods. We have applied this method to 32 problematic targets from the TB Structural Genomics Consortium. Experimental results of these studies are reported.     

Precipitation of porcine insulin with carbon dioxide

Recent works have pointed to the use of volatile electrolytes such as carbon dioxide (CO₂) dissolved in aqueous solutions as a promising alternative to the precipitating agents conventionally used for protein recovery in the food and pharmaceutical industries. In this work we investigated experimental and theoretical aspects of the precipitation of porcine insulin, a biomolecule of pharmaceutical interest, using CO₂ as an acid‐precipitating agent. The solubility of porcine insulin in NaHCO₃ solutions in pressurized CO₂ was determined as a function of temperature and pressure, with a minimum being observed close to the protein isoelectric point. A thermodynamic model was developed and successfully utilized to correlate the experimental data. Insulin was considered a polyelectrolyte in the model and its self‐association reactions were also taken into account. The biological activity of insulin was maintained after precipitation with CO₂, although some activity can be lost if foam is formed in the depressurization step. Biotechnol. Bioeng. 2009;103: 909–919. © 2009 Wiley Periodicals, Inc.