Degradation of Escherichia coli beta-galactosidase fragments in protease-deficient mutants of Salmonella typhimurium.

The degradation rates of several mutationally generated fragments of Escherichia coli beta-galactosidase were determined in wild-type strains of Salmonella typhimurium and in mutant Salmonella strains lacking several proteases and peptidases. Three termination fragments (produced by lacZ545, lacZ521, and lacZX90) and one internal reinitiation (restart) fragment [lacZpi(1)] are degraded in wild-type Salmonella strains at the same rates observed in wild-type Escherichia coli strains. Mutations that lead to loss of peptidases N, A, B, P, and Q or to loss of protease I or II do not affect the decay rates of any of these fragments. In addition, all of these peptidases and proteases are present in E coli mutants carrying deg mutations (deg mutations are known to stabilize beta-galactosidase fragments). Apparently, none of the proteases and peptidases that are currently accessible to direct genetic analysis plays a role in the early steps of the degradation of protein fragments.     

Studies on antigen-induced arthritis in mice. II. Immunologic correlates of arthritis susceptibility in mice.

Antigen-induced arthritis was developed in mice as a model of human rheumatoid arthritis by using methylated bovine serum albumin (mBSA) as antigen. It was found that most strains were susceptible, whereas CBA mice were resistant. We therefore investigated the humoral and cell-mediated immune responses to mBSA in resistant mice (CBA) and susceptible mice (exemplified by C57BL) to determine whether these were associated with susceptibility to arthritis. The resistant strain (CBA) differed from the suceptible strains in the following respects. First, there was a lower humoral immune response to mBSA as measured by passive hemagglutination, but this could be overcome by a larger immunogenic dose. Secondly, there were differences in response to low doses of DNP-mBSA after mBSA carrier preimmunization. Thirdly, there were striking differences in delayed-type hypersensitivity (DTH) to mBSA as determined by a radioisotopic assay in vivo; the response of CBA mice occurred early, at 5 days, declined quickly, and was weaker, whereas that of C57BL mice developed later and was long sustained. Genetic studies of the DTH response with hybrids and backcrosses showed an oligogenic control of immune responsiveness, with one gene being linked to the H-2b allele of the susceptible C57BL mice, and another being independent of the H-2 complex. Our findings indicate that in mice, susceptibility to antigen-induced arthritis with mBSA correlates with a higher responder state to this antigen, and that T cells are the major if not the only determinant of the high responder state.     

Reversible inhibition by diamide of cyclic AMP-dependent protein kinases from bovine thyroid.

The effects of diamide on protein kinases isolated from bovine thyroid were studied. Cyclic AMP-dependent protein kinase activity was directly, rapidly, and reversibly inhibited by diamide. This inhibition was non-competitive with respect to ATP or histone and could be prevented by thiol-reducing agents. However, a cyclic nucleotide-independent thyroid protein kinase was not affected. Our data indicate that diamide specifically inhibits protein kinases which are cyclic AMP-dependent.     

RNA-DNA hybridization analyses of tRNA-Val-3b in Drosophila melanogaster

Summary Transfer RNA was extracted from 50–300 mg of adult flies and specifically labeled in vitro. The level of individual isoacceptors was quantitated by efficient annealing to Drosphila tRNA genes carried on recombinant DNA plasmids immobilized on nitrocellulose filters. The level of tRNA _3b ^Val in the tRNA isolated from flies deficient in the major tRNA _3b ^Val loci has been examined. The results show that deletion of the major tRNA _3b ^Val loci resulted in a reduction of approximately 50% in the level of tRNA _3b ^Val but did not produce the Minute phenotype; furthermore the effects of deficiencies at two loci were approximately additive.     

Repertoires of T cells directed against a large protein antigen, beta-galactosidase. I. Helper cells have a more restricted specificity repertoire than proliferative cells

The proliferative and helper T cell repertoires were compared in the CBA/J mouse for the response to the large protein antigen, tetrameric beta-galactosidase (GZ = 1021 a.a/monomer). The systems assessed the ability of cyanogen bromide (CB) peptides of GZ to: 1) prime for a T cell proliferative response to GZ; or 2) generate T cell help, measured by the production of anti-FITC PFC in the in vitro response to GZ-FITC. Priming for in vitro proliferation was attempted with 11 CB peptides comprising 70% of the GZ molecule. Strong priming was found with five peptides and intermediate priming was found with four other peptides; two peptides were without effect (CB-20 = a.a 767-862, and CB-4 = a.a. 188-202). Despite this indication of generally dispersed recognition of GZ epitopes, only two CB peptides, CB-2 (a.a. 3-92) and CB-10 (a.a. 378-418) were able to induce a T helper cell response. The surprising dearth of helper T cell-inducing epitopes may be peculiar to the limited fluorescein (FITC) substitution on GZ-FITC (17-25 FITC residues per tetramer) or it may reflect the constraints involved in T cell recognition required for T-B collaboration. Also considered was the possibility that the helper T cell repertoire might be distinct from the proliferative repertoire, the latter reflecting DNA synthesis and recruitment by other functional T cell subpopulations.     

Effects of pH and ionic strength on the structure of collagen fibrils

The roles of pH and ionic strength on the structure and stability of collagen fibrils have been investigated by means of x-ray and neutron diffraction techniques. High-angle x-ray diffraction shows that a salt concentration of 0.5M KCl is sufficient to reduce the osmotic swelling and related disordering in the pH range 1–3. The relative intensities of the low-angle meridional x-ray and neutron diffraction Bragg reflections vary with pH. Difference Fourier syntheses between pH 7 and 1.6 data indicate, for both x-ray and neutron diffraction, a reduced scattering contribution from the telopeptides at low pH. Lyotropic relaxation is a crucial step in the appearance at low pH of a doubling of the 668-Å axial periodicity (D) of collagen fibrils. These results suggest that electrostatic interactions are essential for the structural stability of the telopeptide regions and of the 1D and 3D intermolecular staggers between collagen molecules.