Primary structure of the elongation factor G from Escherichia coli. VI. Structure of peptides of cyanogen bromide cleavage of the G-factor molecule

Peptides obtained as a result of cyanogen bromide cleavage of the G-factor have been studied. All 12 peptides embracing the whole structure of fragment T4 have been isolated. For their amino acid sequence determination, cyanogen bromide peptides have been further cleaved with trypsin, chymotrypsin, thermolysin, staphylococcal glutamic protease and BNPS-skatole. The complete primary structure of 9 from 12 cyanogen bromide peptides has been determined.     

Primary structure of the elongation factor G from Escherichia coli. V. Amino acid sequence of the C-terminal domain

The amino acid sequence of the C-terminal domain of the elongation factor G (EF-G) has been studied. The polypeptide chain of the domain consists of 228 amino acid residues, and contains no tryptophan or cysteine residues. To determine its structure, the peptides obtained as a result of the fragment digestion by staphylococcal glutamic protease, cyanogen bromide cleavage, and tryptic hydrolysis of the fragment modified by maleic anhydride have been analyzed, as well as peptides obtained after hydrolyses of cyanogen bromide fragments with chymotrypsin, thermolysin and trypsin.     

Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system

Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.     

Design and formulation of polyplexes based on pluronic-polyethyleneimine conjugates for gene transfer

Previously, we reported the evaluation of several polyplex-based gene delivery systems with respect to their effectiveness, toxicity, and cell type dependence in vitro. One system, P123-g-PEI(2K), a cationic graft block copolymer, is of particular interest as it has been demonstrated to successfully deliver genetic material to murine liver following systemic delivery [Nguyen, H. K., Lemieux, P., Vinogradov, S. V., Gebhart, C. L., Guerin, N., Paradis, G., Bronich, T. K., Alakhov, V. Y., and Kabanov, A. V. (2000) Evaluation of Polyether-Polyethyleneimine Graft Copolymers as Gene Transfer Agents. Gene Ther. 7, 126-138 (1)]. The P123-g-PEI(2K) system requires nonmodified Pluronic P123 as an excipient to stabilize the dispersion. The purpose of the current work was to more closely characterize this system, to assess the role of each component of the system to the overall transfection process. We evaluated particle size, stability, and resistance to nuclease degradation. In addition, cellular uptake and localization of plasmid, as well as transgene expression, were evaluated following in vitro transfection of prostate cancer cells (PC-3) with various individual components of the system. Nonmodified Pluronic alone did not significantly enhance DNA uptake, transgene expression, or DNase protection. Therefore, we conclude that nonmodified Pluronic acted primarily by optimizing the size of the polyplex. Furthermore, though this system displays several characteristics thought desirable of a nonviral gene delivery system, these studies did discriminate a potential limitation of this system for in vivo applications, namely, the insufficient level of protection of plasmid DNA from nuclease degradation. This may limit the effective dose delivered, as well as limiting the effective circulation time. These studies provide vital information that will guide modification of this system to enhance the current in vivo profile.     

The neuroleptic activity of haloperidol increases after its solubilization in surfactant micelles. Micelles as microcontainers for drug targeting

It has been suggested to use surfactant micelles as microcontainers for increasing the efficiency of neuroleptic targeting from blood flow into the brain. The neuroleptic action of haloperidol, intraperitoneally injected into mice in micellar solution of non-ionic block copolymer surfactant (pluronic P-85) in water, increased several-fold if compared with that observed for haloperidol aqueous solution. Incorporation of brain-specific antibodies into haloperidol-containing micelles resulted in additional drastic increase (more than by 2 orders of magnitude) in the drug effect.     

A Catecholic Siderophore Produced by the Thermoresistant Bacillus licheniformis VK21 Strain

Thermophilic and thermoresistant strains of bacilli were screened on a medium containing Chrome Azurol S for the producers of siderophores. It was found that the Bacillus licheniformis VK21 strain dramatically increases secretion of the metabolite, a chelator of Fe³⁺, in response to addition of manganese(II) salts. The growth of the producer on a minimal medium containing MnSO₄ under the conditions of iron deficiency is accompanied by the accumulation of a catecholic product, the content of which reaches maximum at the beginning of the stationary growth phase of culture. In the presence of FeCl₃, the amount of the catecholic product in the medium considerably decreases. The siderophore, called S_VK21, was isolated from the cultural medium and purified by reversed phase HPLC, and its siderophore function was confirmed by the test for the restoration of growth of producer cells in a medium containing EDTA. The UV spectrum of the siderophore has absorption maxima at 248 and 315 nm. According to the amino acid analysis and NMR spectrometry, the metabolite S_VK21 is 2,3-dihydroxybenzoyl-glycyl-threonine.     

Micelles of poly(oxyethylene)-poly(oxypropylene) block copolymer (pluronic) as a tool for low-molecular compound delivery into a cell: phosphorylation of intracellular proteins with micelle incorporated [gamma-32P]ATP.

Pluronic P85 (poly(oxyethylene)-poly(oxypropylene) block copolymer) was used for in vitro delivery of [gamma-32P]ATP into intact Jurkat cells. Negatively charged ATP molecules are not able to penetrate cell plasma membrane. Hence, exogenous [gamma-32P]ATP added to a cell culture does not participate in phosphorylation of intracellular proteins. The addition to cells of [gamma-32P]ATP solubilized in positively charged (containing dodecylamine) pluronic micelles results in considerable increase of protein phosphorylation. In this case the treatment of intact cells with alkaline phosphatase (resulting in dephosphorylation of external proteins) causes no essential decrease of [32P]-incorporation in cell proteins. That gives an evidence of delivery of solubilized ATP into a cell. Under the experimental conditions used, pluronic micelles neither influence the viability of cells nor permeabilize cell plasma membrane.     

Fatty acid acylated antibodies against virus suppress its reproduction in cells

A method for suppression of virus reproduction in cells using fatty acylated antiviral antibodies, which in contrast to non-modified antibodies are capable of intracellular penetration, has been suggested. The addition of stearoylated antiviral antibodies to influenza A/Chili virus-infected cells causes a 100-fold suppression of virus reproduction. Non-modified antibodies do not produce any effect on virus reproduction.     

The cloning and expression of the Staphylococcus aureus enterotoxin A gene in Escherichia coli cells

Gene ent-A has been cloned on phage vector pSL5 with the use of the gene library of S. aureus FR1722(H). It is located within DNA fragment Hind III having 2,500 nucleotide pairs.     

Pluronic micelles as a tool for low-molecular compound vector delivery into a cell: effect of Staphylococcus aureus enterotoxin B on cell loading with micelle incorporated fluorescent dye.

Micelles of pluronic P85 (poly(oxyethylene)-poly(oxypropylene) block copolymer) are used as microcontainers for in vitro delivery of fluorescein into Jurkat and MDCK cells. In order to target the fluorescein containing micelles into the cell, Staphylococcus aureus enterotoxin B (SEB) is covalently conjugated with a pluronic molecule and the conjugate is incorporated into the micelle content. The incorporation of SEB capable of receptor-mediated endocytosis results in a drastic enhancement of the efficiency of cell loading with the fluorescent dye. This effect is not observed under the conditions (4 degrees C) when endocytosis is abolished.