Mammalian ribosomal protein: analysis by electrophoresis on polyacrylamide gel.

Ribosomal protein from five mammalian tissues when analyzed by discontinuous electrophoresis on polyacrylamide gel at pH 4.5 yielded 24 bands. Densitometric tracings indicated that the patterns of the basic ribosomal proteins from the several tissues were qualitatively similar. Protein from Escherichia coli ribosomes analyzed at pH 4.5 gave 29 bands, and the pattern was different from that of mammalian ribosomal protein. No distinct band was found when mammalian ribosomal protein was analyzed at pH 8.3 (acidic proteins). Ribosomal protein from Escherichia coli gave eight bands at pH 8.3. Thus, the structure of the genes responsible for synthesis of ribosomal protein in several mammalian tissues is the same, and different genes direct synthesis of ribosomal protein in bacteria.

Effect of polymyxin B on liposomal membranes derived from Escherichia coli lipids.

The specificity of the action of polymyxin B was studied using liposomes as a model membrane system. Liposomes prepared from total lipids of Gram-negative bacteria Escherichia coli, a mixture of purified E. coli phosphatidylethanolamine and cardiolipin and a mixture of phosphatidylethanolamine and phosphatidylglycerol, were extemely sensitive to polymyxin while those prepared from lipids of Gram-positive bacteria Streptococcus sanguis, lipids of sheep erythrocyte membranes, mixtures of egg lecithin and negatively charged amphiphatic molecules, were less sensitive to the action of the antibiotic. Cholesterol was shown to suppress the polymyxin-induced response in liposomes.

Use of a fluorescein derivative of phosphatidylethanolamine as a pH probe at water/lipid interfaces.

A fluorescent indicator for the determination of pH in the vicinity of water/lipid interfaces was produced by the covalent linkage of fluoresceinisothiocyanate to Escherichia coli phosphatidylethanolamine. When embedded in monolayers spread on an air/water interface, its apparent pK was shown to be only slightly affected by the nature of the polar headgroups or the packing density of the host phospholipids. Its properties were not affected by the ion content of the subphase. For small unilamellar vesicles, its properties were only affected when localized in the inner layer. This probe could therefore be of value in the study of proton fluxes along biological membranes.

Solubilization and reconstitution of proline carrier in Escherichia coli; quantitative analysis and optimal conditions.

Proline carrier of Escherichia coli was extracted from the carrier-overproducing membranes with dodecylmaltoside in the presence of phospholipid. The solubilized carrier showed the same proline binding activity as that in normal membranes. As judged from determinations of the binding activity in the micellar state as a marker of active carrier and the radioactivity of N-[ethyl-2-3H]ethylmaleimide-labeled carrier as a marker of carrier polypeptide, 80% of the carrier molecules in the membranes were extracted. Optimal conditions for reconstitution of the solubilized carrier were established. By a combination of freeze-thawing, sonication and dilution procedures, 70% of the solubilized carrier molecules were incorporated into proteoliposomes and the restored active transport of proline showed an apparent Kt of 1 microM and turnover number of 0.6 s-1. The transport of proline was driven by a membrane potential in a Na+ (or Li+)-dependent manner.

Giant liposomes as model membranes for immunological studies: spontaneous insertion of purified K1-antigen (poly-alpha-2,8-NeuAc) of Escherichia coli.

A flow chamber has been constructed to use giant liposomes (diameter 5-50 microns) as model membranes for immunological studies and other experiments involving the interaction with water-soluble compounds. As an example of immunological importance, the insertion of purified K-antigen from Escherichia coli K1 has been studied. Despite its large hydrophilic part (poly-alpha-2,8-NeuAc), which is capped at its potential reducing end with phosphatidic acid acting as a lipid anchor group, this water-soluble material is readily incorporated into liposomal membranes of dimyristoylphosphatidylcholine (DMPC). The incorporation has been proven by immunofluorescence using a FITC-labeled monoclonal anti-K1-IgG. Without the lipid residue, however, no binding of poly-alpha-2,8-NeuAc to the liposomes has been observed. This could be shown by using colominic acid, an oligomeric form of alpha-2,8-NeuAc with free reducing ends instead of purified K1-antigen. The possibility for further manipulation of this model system has been shown by using a poly-alpha-2,8-NeuAc cleaving enzyme (endoneuraminidase). The function of the endoneuraminidase has been proven by showing no binding of the antibody after enzyme treatment of K1-bearing liposomes as well as by rapid loss of fluorescence of a previously bound FITC-antibody.

A simple method for the preparation of large quantities of pure plasmid DNA.

Polyethylene glycol quantitatively precipitates plasmid DNA of molecular weight 6-123-10-6, from cleared lysates of plasmid-carrying bacterial strains, After resuspension and density-gradient centrifugation of the precipitated DNA, it is unchanged in length and in transformation efficiency for Escherichia coli K12. Plasmid DNA can be easily prepared in large quantities by including a polyethylene glycol precipitation step in standard plasmid isolation procedures.

Characterisation of channels induced in planar bilayer membranes by detergent solubilised Escherichia coli porins.

Purified OmpF, OmpC, NmpC, PhoE and Lc (Protein 2) porins from the Escherichia coli outer membrane were incorporated into planar phospholipid bilayer membranes and the permeability properties of the pores studied. Triton X-100 solubilised porin samples showed large and reproducible increases in membrane conductivity composed of discreet single-channel events. The magnitude of the cation selectivity found for the porins was in the order OmpC greater than OmpF greater than NmpC = Lc; PhoE was anion selective. For the cation selective porins the cation/anion permeability ratios in a variety of solutes ranged from 6 to 35. Further information on the internal structure of the porins was obtained by examination of the single-channel conductance and this was used to interpret macroscopic observations and to estimate single-channel diameters. The same porins solubilised in SDS exhibited slight conductance increase with no observable single-channel activity. Use of on-line microcomputer techniques confirmed the ohmic current vs. voltage behaviour for all the single porin channels examined.

Induction of elongation factor Tu-GDP crystal polymorphism by polyethylene glycol contaminants.

Trypsin-modified elongation factor (EF-)Tu-GDP from Escherichia coli is known to crystallize in several different unit cells under apparently identical conditions. The crystal polymorphism was investigated and found to be correlated with the source of polyethylene glycol used in the crystallization procedure. The use of highly purified polyethylene glycol promoted the growth of a new crystal form belonging to space group P2(1)2(1)2(1) with cell dimensions of a = 71.9 A, b = 74.7 A, c = 170.9 A and two molecules per asymmetric unit. In extensive crystallization trials, substances that typically contaminate commercial preparations of polyethylene glycol were screened. The final results show that the presence of the divalent anions, HPO4(2-) or SO4(2-), at different concentrations induce the growth of two known crystal forms belonging to space groups C222(1) and P4(3)2(1)2. The relevancy of the findings is discussed.

Escherichia coli hemolysin permeabilizes small unilamellar vesicles loaded with calcein by a single-hit mechanism.

Escherichia coli hemolysin produces small unilamellar lipid vesicles permeable to the fluorescent dye calcein by forming pores through their membrane. The process of permeabilization proceeds as a pseudo first-order reaction, indicating that the toxin is active as a monomer; consistently no evidence for cooperativity has been found in a dose-response titration. The rate of interaction increases on lowering the pH of the solution and by introducing negatively charged lipids into the vesicles. The overall pore formation mechanism resembles that of other toxins of bacterial origin such as colicins, diphtheria, tetanus and botulinum toxin.

Studies on interaction of 5 S RNA with ribosomal proteins.

Proteins of the large ribosomal subunit of rat liver (TP 60) were immobilized by diffusion transfer onto nitrocellulose after two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Incubation of the TP 60 blots with 32P-labeled 5 S RNA under defined ionic conditions (300 mM KCl, 20 mM MgCl2) resulted in specific binding to a limited set of ribosomal proteins consisting of proteins L3, L4, L6, L13/15 and--to a lesser extent--L7 and L19. Under identical conditions, blots with proteins of the small ribosomal subunit (TP 40) did not bind 5 S RNA.

An amplification technology for improving sensitivity when measuring components in biological samples.

A new technology for improving the sensitivity in measuring components in biological samples is described. The method is based on the use of spherical microbeads (detection beads) which contain a large amount of immobilized enzyme and a reagent with biospecific affinity for the component to be detected. These microbeads have been used in a 'sandwich reaction' for visualization of P-fimbriated Escherichia coli which has a known receptor structure (Gal(alpha 1-4)Gal(beta)). In the initial step the bacteria were enriched on a solid support (e.g., a plastic film or beads (greater than 150 microns)) to which the receptor structure had been covalently bound. In the next step detection beads coupled with enzyme and receptor structure were added and finally a chromogenic substrate for the enzyme was used for visualization. A sensitivity of 10(5) bacteria/ml was reached. The detection beads are of general utility and might be useful for the detection of lectins on other pathogens.

In situ enzyme immunodetection of surface or intracellular bacterial antigens using nitrocellulose sheets.

We describe an immunological method which allows the in situ colorimetric detection of translated DNA fragments in bacteria. In the absence of lysis only cell surface proteins are detected. For cytoplasmic proteins, lysis is required. The procedure comprises the following steps: bacteria are lysed, the proteins are transferred onto a disc of nitrocellulose sheet, the remaining protein sites are blocked, the disc is successively soaked in a solution of antibodies specific for the protein to be detected and in a solution of peroxidase-labelled anti-IgG antibody solution. Finally, the immune complexes are made visible by enzyme substrate incubation. We describe the application of this method to the detection of the LamB protein, the LacZ protein, and a LamB-polio VP1 chimera translated from cloned DNA fragment in E. coli.

Chemical composition of the washed cells of Streptococcus sanguis (804) and Streptococcus mutans (B-14).

Washed cells of Streptococcus saguis (804) and Streptococcus mutans (B14) were analyzed for water, ash, protein, carbohydrate, lipid, and nucleic acid content. Lipids were analyzed by gas chromatography. Ash was subjected to elemental analysis. These data were compared with the published values for the composition of dental plaque.

Some studies on the preservation of indometacin suspensions intended for ophthalmic use.

Suspensions of indometacin (1% w/v), buffered to pH 5.6, may be satisfactorily preserved by 0.002% w/v phenylmercuric nitrate in the presence of hydroxypropylmethylcellulose (0.5% w/v), despite 90% of the preservative being adsorbed to the indometacin powder. Polyvinyl alcohol (1.4%) could be used as an alternative suspending agent.

[Role of the N-terminal sequence (1-26) in the dimerization of protein L7 in solution]. / Rol' N-kontsevoi posledovatel'nosti (1-26) dlia dimerizatsii belka L7 v rastvore.

Native protein L7 from E. coli ribosomes, oxidized protein and a fragment of protein L7 containing the sequence 27--120 obtained by cleavage of the native protein by cyanogen bromide were investigated by sedimentation analysis. It was found that protein L7 exists in solution in a dimer form while the protein oxidized by hydrogen peroxide and the fragment 27--120 do not form a dimer. On the basis of these investigations a conclusion is made that the ability for dimerization is stimulated by N-terminal regions of the protein L7 molecule.

[A study of the reaction between antibodies and DNA by the method of immune complex formation on the membranes of nitrocellulose filters]. / Izuchenie reaktsii mezhdu antitelami i DNK metodom fiksatsii immunykh kompleksov na membrannykh nitrotselliuloznykh fil'trakh

The conditions were developed to measure antibodies to denatured and native DNA. It was shown that membrane filters treated with alkali adsorbed denatured DNA complexed with antibodies. Utilizing this method it was shown that antibodies to denatured DNA react with pyrimidines (probably thymine). This assay provides proof of the wide existence antibodies to native DNA in the sera of patients with systemic lupus erythematosus. DNA-binding of sera was significantly depended on reaction condition.

[Adsorption of spin-labeled flocculants on E. coli cells]. / Izuchenie adsorbtsii spin-mechenykh flokuliantov na kletkakh E. coli.

The interaction of intact E. coli cells with polymeric flocculants polyethylenimine and dextran was being studied by spin-labelling. The nitroxyl groups of spin-labelled polymers are reduced during formation of the adsorption layer on the cell surface. Some peculiarities of redox reactions between polymer macroradicals and units of the bacterial electron-transport chain were studied depending of temperature and flocculation regime. The course of the reduction of spin-labelled polymer radicals and characteristics of EPR spectra of the flocculant give evidence on the formation of a loose polymeric layer on the surface of E. coli cells.