The anti-Tac monoclonal antibody is known to bind to the p55 chain ofthe human interleukin 2 receptor and to inhibit proliferation of T cells by blocking interleukin 2 binding. However, use of anti-Tac as an immunosuppressant drug would be impaired by the human immune response against this murine antibody. We have therefore constructed a "humanized" antibody by combining the complementaritydetermining regions (CDRs) of the anti-Tac antibody with human framework and constant regions. The human framework regions were chosen to maximize homology with the anti-Tac antibody sequence. In addition, a computer model of murine anti-Tac was used to identify several amino acids which, while outside the CDRs, are likely to interact with the CDRs or antigen. These mouse amino acids were also retained in the humanized antibody. The humanized anti-Tac antibody has an affinity for p55 of 3 x 109 M-1, about
SummaryDifferential fluorescence induction (DFI) in Streptococcus pneumoniae was used as a method for the discovery of genes activated in specific growth environments. Competence stimulatory peptide (CSP) was used as the model inducing system to identify differentially expressed genes. To identify CSP-induced promoters, a plasmid library was constructed by inserting random pieces of S. pneumoniae chromosomal DNA upstream of the promoterless gfpmut2 gene in an Escherichia coli/S. pneumoniae shuttle vector. S. pneumoniae carrying the library were induced with CSP and enriched for green fluorescent protein (GFP)-expressing bacteria using fluorescenceactivated cell sorting. A total of 886 fluorescent clones was screened, and 12 differentially activated promoter elements were identified. Sequence analysis of these clones revealed that three were associated with novel competence loci, one of which we show is essential for DNA uptake, and six are known CSP-inducible promoters. We also explored whether competence proteins have a role in virulence and found that mutations in three CSP-inducible genes resulted in attenuated virulence phenotypes in either of two murine infection models. These results demonstrate the utility of DFI as a method for identifying differentially expressed genes in S. pneumoniae and the potential utility of applying DFI to other Gram-positive bacteria.
Differential fluorescence induction (DFI) technology was used to identify promoters of Streptococcus pneumoniae induced under various in vitro and in vivo conditions. A promoter-trap library using green fluorescent protein as the reporter was constructed in S. pneumoniae, and the entire library was screened for clones exhibiting increased gfp expression under the chosen conditions. The in vitro conditions used were chosen to mimic aspects of the in vivo environment encountered by the pathogen once it enters a host: changes in temperature, osmolarity, oxygen, and iron concentration, as well as blood. In addition, the library was used to infect animals in three different models, and clones induced in these environments were identified. Several promoters were identified in multiple screens, and genes whose promoters were induced twofold or greater under the inducing condition were mutated to assess their roles in virulence. A total of 25 genes were mutated, and the effects of the mutations were assessed in at least two different infection models. Over 50% of these mutants were attenuated in at least one infection model. We show that DFI is a useful tool for identifying bacterial virulence factors as well as a means of elucidating the microenvironment encountered by pathogens upon infection.The gram-positive pathogen Streptococcus pneumoniae is a major cause of community-acquired infections, including those of the upper and lower respiratory tract, otitis media, bacteremia, and meningitis (1, 4). The ability of this organism to disseminate from localized sites of infection to cause more serious invasive disease renders infections particularly difficult, yet imperative, to treat (24). Particularly vulnerable to pneumococcal infection are small children and the elderly, and the organism is usually a major cause of pneumonia and meningitis in these populations (7).Only a few pneumococcal virulence determinants have been associated with disease, including pneumolysin, autolysin, capsule, adhesins, and other surface molecules (1,5,6,15,21,24). The expression of these factors, for the most part, is unknown in vivo, and the temporal requirements for these factors during infection have not been determined. It has been hypothesized that surface factors play a role early in infection by preventing phagocytosis and allowing the bacteria to grow; later, continued growth in host tissues leads to the production of autolysin, and the subsequent release of pneumolysin results in inflammation. This progression of events during infection, culminating in high levels of inflammation, is probably the reason for the high morbidity and mortality for S. pneumoniae infections, even with antibiotic therapy (4). Indeed, in animals treated with pneumococcal cell wall components or with purified pneumolysin, similar levels of inflammation have been observed (17).Fortunately, relevant animal models are available which can mimic most diseases caused by pneumococci, including pneumonia, otitis media, meningitis, and bacteremia. These models allow ...
Hybrid tryptophan synthetase at and .3 polypeptides were produced by genetic .recombination between the trpB-trpA regions ofEscherichia coli and Salmonella typhimurium contained on compatible, multicopy plasmids. Intragenic recombination was decreased but still evident in recA cells. Genetic exchange occurred at many sites within trpA, but every recombinant gene produced a functional a polypeptide despite many amino acid differences from one or the other of-the parental polypeptides. The five hybrid tryptophan synthetase a subunits examined resembled the parental polypeptides in catalytic function but differed in thermostability. The stability differences suggest that, as amino acid changes occurred in these proteins during the course of evolution, subsequent changes were limited to those. that would allow retention of a desired protein conformation. The gene trpA ofEscherichia coli and Salmonella typhimurium encodes the a polypeptide subunit of the a2f82 tryptophan synthetase complex. Comparison ofthe nucleotide sequences ofthe trpAs of the two species reveals that there are 199 sequence differences in the 804 nucleotide pairs in this gene (1). The corresponding a polypeptides have 40 amino acid differences in their 268 residues. Thus, most of the nucleotide differences between the two trpAs are in synonymous codons. Despite the 40 amino acid differences between the two a subunits, the polypeptides are functionally interchangeable, both in vitro (2-4) and in vivo (3,5). In this and comparable examples, the question arises as to whether the nucleotide and amino acid sequence differences that are seen in homologous genes and proteins are selectively favored in their respective organisms.The functional significance of the amino acid differences in the two tryptophan synthetase a subunits can be assessed by constructing hybrid trpAs containing sequences from both species and examining the properties of the corresponding hybrid polypeptides. By using this approach, one class of hybrid tryptophan synthetase a polypeptides was isolated and studied (6). Unfortunately, such recombinants were extremely rare and were obtained only when trpA' recombinants were selected in crosses between trpA mutants of the two species. The single class of hybrid a subunits examined resembled the parental polypeptides in all the functional tests that were performed.In this investigation, the difficulty ofobtaining hybrid a sub--units was overcome by incorporating inactive trpB-trpA segments of the two species into compatible multicopy plasmids and introducing both plasmids into bacteria with the trpB-trpA region deleted. The two plasmids were constructed so that the region of nucleotide sequence homology was confined to the trpB-trpA segment and so that genetic recombination in this segment could reconstitute intact trpB and trpA. Using this approach, we readily recovered hybrid trpBs and trpAs containing varying segments from the parental genes. It was also possible to modify the selection procedure slightly so that we could dete...
We have carried out nanosecond fluorescence polarization studies of genetically engineered immunoglobulins to determine the structural features controlling their segmental flexibility. The proteins studied were hybrids of a relatively rigid isotype (mouse IgGl) and a relatively flexible one (mouse IgG2a). They have identical light chains and heavy chain variable regions and have the same combining sites for e-dansyl-L-lysine, a fluorescent hapten. The fluorescence of the bound dansyl chromophore was excited at 348 nm with subnanosecond laser pulses, and the emission in the nanosecond time range was measured with a single-photon-counting apparatus. The emission anisotropy kinetics of the hybrid antibodies revealed that segmental flexibility is controlled by the heavy chain constant region 1 (CH1) as well as by the hinge. In contrast, the CH2 and CH3 domains did not influence segmental flexibility. The hinge and CH1 domains must be properly matched to allow facile movement of the Fab units. Studies of hybrids of IgG1 and IgG2a within CH1 showed that the loop formed by residues 131-139 is important in controlling segmental flexibility. X-ray crystallographic studies by others of human IgG1 have shown that this loop makes several van der Waals contacts with the hinge.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.