Searching for autolysin functions. Characterization of a pneumococcal mutant deleted in the lytA gene

The first mutant of Streptococcus pneumoniae showing a complete deletion in the lytA gene coding for the N-acetylmuramyl-L-alanine amidase has been isolated and characterized. This amidase was previously the only autolysin detected in this species. This mutant shows a normal growth rate and can be transformed using either chromosomal or plasmid DNA. The most remarkable biological consequences of the absence of the amidase are the formation of small chains (six to eight cells) and the absence of lysis in the stationary phase of growth. In addition, this mutant exhibits a tolerant response against the beta-lactam antibiotics.     

Cellular interactions of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)-specific suppressor factors. I. Inhibition of the activity of GAT-specific helper T cell clones by monoclonal GAT-specific suppressor T cell factors

Considerable information concerning the serology and biochemistry of antigen-specific, T cell-derived suppressor factors has been obtained with the use of T cell hybridomas as a source of homogeneous material. Similarly, knowledge of helper T cell products and receptors is accumulating from studies of helper T cell clones and hybridomas. Our strategy for studying the mechanisms by which suppressor factors inhibit responses was to determine whether monoclonal suppressor factors could inhibit antibody responses specific for L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) in cultures containing unprimed splenic B cells, macrophages, and GAT-specific T cell clones as a source of helper activity. The MHC-restricted, two chain suppressor factors, GAT-TsF2, inhibited these responses if the helper T cell clones and suppressor factor were derived from H-2-compatible mice. Furthermore, responses were inhibited by briefly pulsing T cell clones with GAT-TsF2 in the presence of GAT, indicating that suppressor factors need not be present continuously. In addition, helper T cell clones adsorbed syngeneic, but not allogeneic, GAT-TsF2 in the presence of GAT. Adsorption also requires a shared antigenic specificity between the H-2b-derived helper T cells and TsF2 factor. Thus, helper T cells can serve as the cellular target of antigen-specific, MHC-restricted GAT-TsF2, and cloned helper T cells can be used as a homogeneous target population for analysis of the molecular mechanisms of T cell suppression.     

B cell subsets in spleens of BALB/c mice: identification and isolation of MMTV-expressing and MMTV-responding subpopulations

The DNA of BALB/c mice contains two genomic- and one subgenomic-size MMTV proviruses that appear to be preferentially expressed in their spleen cells, although intact MMTV virions cannot be detected in the tissues of these mice. This retrovirus antigen expression is restricted to a subpopulation of B lymphocytes, as determined by double label immunofluorescence studies. Nylon-adherent, SIg-positive spleen lymphocytes from BALB/c mice are capable of being stimulated by purified MMTV in lymphocyte transformation assays. Two possibilities were explored: the MMTV-positive cells are the responders to MMTV in the blastogenesis assay, or there exists two B lymphocyte subsets, one expressing the MMTV antigen(s) and the other responding to the virus. Depletion of MMTV-positive, nylon-adherent cells with anti-MMTV and complement resulted in no significant change in the blastogenesis to MMTV, indicating that the MMTV-negative lymphocytes are the responders in this reaction. These results were confirmed by positive selection experiments by using a fluorescence-activated cell sorter. Two populations of nylon-adherent cells, on the basis of the presence or absence of MMTV antigen in their surfaces, were obtained by a two-way sorting procedure and were used in lymphocyte transformation assays. MMTV-expressing lymphocytes were found to be nonresponsive to MMTV antigens, although high levels of [3H]thymidine incorporation were observed in the MMTV-negative, nylon-adherent cell cultures exposed to MMTV. These data indicate that in normal BALB/c mice, expression of endogeneous retrovirus genetic information is restricted to a nylon-adherent spleen cell subset that is different from the one responding in blastogenesis to the viral antigens.     

The binding of [3H]oestradiol-receptor complex to hypothalamic chromatin of male and female mice

Histones and masking acidic proteins were removed from hypothalamic chromatin in order to evaluate/measure the number of available acceptor sites for the [3H]oestradiol-receptor complex. This number increases after dehistonizing and unmasking and is lower than published values for comparable preparations. No sex-related difference in [3H]oestradiol-receptor binding to hypothalamic chromatin in vitro was observed. Failure to observe such a difference suggests that sexual differentiation and steroid sensitivity cannot be attributed to marked differences in the degree of chromatin masking.     

Heterogeneity of macrophage populations in human lymphoid tissue and peripheral blood

Human lymphoid tissue and peripheral blood leukocytes were stained with six monoclonal antibodies directed against monocyte/macrophage populations. The staining pattern described by each of these monoclonal reagents was compared with the distribution of morphologically distinguishable tissue macrophages. The results show that there exists considerable heterogeneity of tissue macrophages based on the expression of surface and/or cytoplasmic antigens; furthermore, the distribution of cells bearing particular antigenic determinants is associated with distinct regions in normal lymphoid tissue. Double staining methods demonstrated that these antibodies bind to different, as well as to identical, macrophage populations. OKM-1 antibody binds predominantly to sinus histiocytes and tingible body macrophages. The Leu M-1 reagent stains interdigitating reticulum cells, while the KiM-4 antibody labels follicular dendritic cells. Leu M-3 antibody identifies cells predominantly in the germinal center, and histiocytes lining the sinuses. Both CM-1 and BRL-M.1 appear to stain tissue macrophages distributed throughout the lymphoid tissue.     

A human monoclonal antibody to cytomegalovirus (CMV)

We report the development of a human monoclonal antibody to cytomegalovirus (CMV) produced from a human X human hybridoma. This hybrid was developed by fusion of an EBV-transformed cell line making antibody to CMV and a human lymphoblastoid cell line WI-L2. The antibody is directed to a CMV-specific antigen primarily in the nucleus of CMV-infected human fibroblasts. It cross-reacts with at least 10 different strains of CMV and may provide a method for the rapid in vitro diagnosis of CMV infections. The production of CMV-specific human man monoclonal antibodies from human-human hybridomas for future therapeutic use is now technically feasible with this specific method of production.     

Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis

Summary The 10-kb chromosomal fragment of Streptococus pneumoniae cloned in pLS80 contains the sul-d allele of the pneumococcal gene for dihydropteroate synthase. As a single copy in the chromosome this allele confers resistance to sulfanilamide at 0.2 mg/ml; in the multicopy plasmid it confers resistance to 2.0 mg/ml. The sul-d mutation was mapped by restriction analysis to a 0.4-kb region. By the mechanism of chromosomal facilitation, in which the chromosome restores information to an entering plasmid fragment, a BamHI fragment missing the sul-d region of pLS80 established the full-sized plasmid, but with the sul-s allele of the recipient chromosome.A spontaneous deletion beginning 1.5 kb to the right of the sul-d mutation prevented gene function, possibly by removing a promoter. This region could be restored by chromosomal facilitation and be demonstrated in the plasmid by selection for sulfonamide resistance. Under selection for a vector marker, tetracycline resistance, only the deleted plasmid was detectable, apparently as a result of plasmid segregation and the advantageous growth rates of cells with smaller plasmids. When such cells were selected for sulfonamide resistance, the deleted region returned to the plasmid, presumably by equilibration between the chromosome and the plasmid pool, to give a low frequency (10^-3) of cells resistant to sulfanilamide at 2.0 mg/ml. Models for the mechanisms of chromosomal facilitation and equilibration are proposed.Several derivatives of pLS80 could be transferred to Bacillus subtilis, where they conferred resistance to sulfanil-amide at 2 mg/ml, thereby demonstrating cross-species expression of the pneumococcal gene. Transfer of the plasmids to B. subtilis gave rise to large deletions to the left of the sul-d marker, but these deletions did not interfere with the sul-d gene function. Restriction maps of pLS80 and its variously deleted derivatives are presented.     

Comparative study of Agrobacterium biotypes 1, 2 and 3 by electrophoresis and serological methods

A comparative study by electrophoresis and serology of strains representing the three Agrobacterium biotypes was carried out. Thirteen Spanish isolates and strains from international collections were included. Ten antisera were prepared by using strains from the three biotypes and different types of antigens. The strains were studied by immunodiffusion, indirect immunofluorescence and indirect ELISA. Serological relationship among all the strains was observed, although serological heterogeneity within each of the biotypes occurred. Biotype 3 appears as serologically related to biotypes 1 and 2, having an intermediate position. This observation is in agreement with their biochemical characteristics. Electrophoretic analysis of the three biotypes showed that there was high variability. Three main bands appeared in the six strains studied. One specific band occurred in the biotype 1 strains and another in the biotype 3 strains.     

Purification of plasma membranes from mouse parotid gland and membrane reorganization in response to isoproterenol

Two highly purified plasma membrane fractions have been obtained from mouse parotid glands by a combination of differential centrifugation and isopycnic centrifugation in discontinuous sucrose gradients. The membranes were characterized by enzymic, chemical and morphological criteria. The effect of isoproterenol, which induces parotid acinar cells to proliferate, upon sialic acid and five different enzyme activities located in the plasma membrane phosphodiesterase (EC 3.1.4.1), Mg2+-ATPase (EC 3.6.1.4), leucine aminopeptidase (EC 3.4.1.1), protein kinase (EC 2.7.1.37) and sialyltransferase (EC 2.4.99.1), were quantified along the cell cycle. Plasma membrane sialic acid content falls 30% within 30 min and remains depressed for at least 6 h with the major restoration towards normal levels occurring between 12 and 16 h later. In contrast multiple daily isoproterenol injections lead to a more than 2-fold elevation of sialic acid content. Sialyltransferase activity rises 2-fold by 12 h after isoproterenol treatment and then rapidly falls. This enzyme has a pH optimum of 6.5, requires a divalent cation for activity and is inhibited by Triton X-100. Other enzyme activities showed markedly different changes after isoproterenol stimulation, either increasing, decreasing or remaining unaltered. These continuous functional modifications suggest an active role of the plasma membrane in the control of the proliferative cycle.