Identification of proteins potentially involved in proximal-distal pattern formation in the regenerating forelimb of the newt.

We have used high resolution two-dimensional gel electrophoresis to identify and characterize proteins that may represent products of genes involved in establishing positional information along the proximal-distal axis of the regenerating forelimb of the newt Notophthalmus viridescens. At least 24 proteins have been found whose synthesis and (or) abundance is increased in proximal (midstylopodial) regenerates relative to midzeugopodial (distal) regenerates at either of two regeneration stages, the early dedifferentiation and moderate bud stages. Four of these same proteins show an axial asymmetry at both stages. Ten distal-specific proteins were also identified, although only one was common to both stages. More significantly, 6 of these 34 proteins (molecular masses of 73, 73, 51.5, 44.0, 19.5, and 16.5 kilodaltons and isoelectric points of 6.70, 6.74, 6.0, 6.05, 5.9, and 6.98, respectively) are regulated to proximal levels by treatment of distal regenerates with retinoic acid (RA) at both stages. An additional five are proximalized by RA at only one regeneration stage. Since the effect of RA is to proximalize positional information in blastema cells, these 11 proteins represent gene products that could be involved in a biochemical cascade leading to the establishment of positional information in the regenerating limb along this axis.     

A rapid method for capture and identification of immunogenic proteins in Bordetella pertussis enriched membranes fractions: a fast-track strategy applicable to other microorganisms

Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (> 95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins.     

Small Angle X-Ray Scattering from Lipid-Bound Myelin Basic Protein in Solution

The structure of myelin basic protein (MBP), purified from the myelin sheath in both lipid-free (LF-MBP) and lipid-bound (LB-MBP) forms, was investigated in solution by small angle x-ray scattering. The water-soluble LF-MBP, extracted at pH < 3.0 from defatted brain, is the classical preparation of MBP, commonly regarded as an intrinsically unfolded protein. LB-MBP is a lipoprotein-detergent complex extracted from myelin with its native lipidic environment at pH > 7.0. Under all conditions, the scattering from the two protein forms was different, indicating different molecular shapes. For the LB-MBP, well-defined scattering curves were obtained, suggesting that the protein had a unique, compact (but not globular) structure. Furthermore, these data were compatible with earlier results from molecular modeling calculations on the MBP structure which have been refined by us. In contrast, the LF-MBP data were in accordance with the expected open-coil conformation. The results represent the first direct structural information from x-ray scattering measurements on MBP in its native lipidic environment in solution.     

Numerical analysis of normalized whole-cell protein profiles after sodium dodecyl sulphate-polyacrylamide gel electrophoresis

A technique is described for mathematically normalizing whole-cell protein profiles after sodium dodecyl sulphate-polyacrylamide gel electrophoresis to obtain standardized absolute migration distances using two internal Mr standards. A soft laser scanning densitometer was used to measure protein band migration distances in wet, silver-stained gels. The normalized values were superior to the unnormalized migration distances and common RF values in reducing the inter- and intragel variability of the protein band positions. A procedure is described for clustering normalized bacterial protein profiles using a sample data set obtained from the type strains of four Legionella species.     

Sequence analysis of the 17-kilodalton-antigen gene from Rickettsia rickettsii.

DNA obtained from the Sheila Smith strain of Rickettsia rickettsii was digested to completion with the restriction endonucleases BamHI and SalI and ligated with the plasmid vector pUC19. The ligation mixture was used to transform Escherichia coli. A total of 465 bacterial clones were screened for antigen production with hyperimmune rabbit serum. One of the reactive clones, containing a recombinant plasmid designated pSS124, was solubilized and subjected to immunoblot analysis and revealed expression of a 17-kilodalton protein reactive with anti-R. rickettsii serum that comigrated with an antigen from R. rickettsii. A 1.6-kilobase PstI-BamHI fragment from pSS124 was subcloned and continued to direct synthesis of the 17-kilodalton antigen. The nucleotide sequence was determined for this 1.6-kilobase subclone, which encompassed the gene encoding the polypeptide as well as flanking regions containing potential regulatory sequences. The open reading frame consisted of 477 nucleotides that specified a 159-amino-acid protein with a calculated molecular weight of 16,840. The deduced amino acid sequence contained a hydrophobic sequence near the amino terminus that resembled signal peptides described for E. coli. The carboxy terminus was hydrophilic in nature and probably contained the exposed epitopes.     

Comparative effects of roxithromycin and erythromycin on cellular immune functions in vitro. 2. Chemotaxis and phagocytosis of 3H-Staphylococcus aureus by human macrophages

The in vitro effects of roxithromycin on human macrophage activity were compared with those of erythromycin. Half the MIC of roxithromycin significantly enhanced the phagocytosis of 3H-labelled Staphylococcus aureus by human macrophages. Bacterial phagocytosis was also increased after pre-incubation of staphylococci with roxithromycin. Erythromycin displayed a less stimulating effect. Pre-incubation of macrophages with the drugs caused a depression of staphylococcal uptake by phagocytes. Macrophage chemotaxis was unchanged in the presence of both macrolides.