Symmetry of the inhibitory unit of human alpha 2-macroglobulin

Human alpha 2-macroglobulin (alpha 2M) of Mr approximately 720,000 is a proteinase inhibitor whose four identical subunits are arranged to form two adjacent inhibitory units. At present, the spatial arrangement of the two subunits which form one inhibitory unit (the functional "half-molecule") is not known. Treatment of alpha 2M with either 0.5 mM dithiothreitol (DTT) or 4 M urea results in dissociation of the native tetramer into two half-molecules of Mr approximately 360,000. These half-molecules retain trypsin inhibitory activity, but in each case, the reaction results in reassociation of the half-molecules to produce tetramers of Mr approximately 720,000. However, when reacted with plasmin, the preparations of half-molecules have different properties. DTT-induced half-molecules protect the activity of plasmin from inhibition by soybean trypsin inhibitor (STI) without reassociation, while urea-induced half-molecules show no ability to protect plasmin from reaction with STI. High-performance size-exclusion chromatography and sedimentation velocity ultracentrifugation studies were then used to estimate the Stokes radius (Re) of alpha 2M and both DTT- and urea-induced half-molecules of alpha 2M. The Re of tetrameric alpha 2M was 88-94 A, while that of DTT-induced half-molecules was 57-60 A and urea-induced half-molecules 75-77 A. These results demonstrate that DTT- and urea-induced half-molecules have fundamentally different molecular dimensions as well as inhibitory properties. The hydrodynamic data suggest that the urea-induced half-molecule is a "rod"-like structure, although it is not possible to predict the three-dimensional structure of this molecule with the available data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of in vitro growth phase on the pathogenesis of Salmonella typhimurium in mice

The growth phase of a bacterial (Salmonella typhimurium) culture was shown to have pronounced effects on the pathogenic properties of the harvested bacteria. Salmonellae obtained from a culture in primary (exponential) growth phase (PP) were more readily cleared from the blood and more readily killed by phagocytes than were salmonellae obtained from a more slowly growing secondary growth phase (SP) culture. PP salmonellae were observed to cause death of mice sooner than SP salmonellae. This appeared to be because the more rapid growth of PP, as compared to SP, salmonellae continued in the liver and spleen for several hours following intravenous injection, and more than compensated for their high in vivo death rate. As a result, within 4 h there were approximately 10-fold more live salmonellae in the spleens and livers of mice that had received PP, as compared to SP, salmonellae. This 10-fold difference was maintained until the death of the mice, indicating that after the first 4 h post-inoculation, the net in vivo growth of the salmonellae was the same regardless of their growth phase in the inoculating culture. This transition between PP and SP salmonellae occurred long before a dense stationary phase culture was obtained. Salmonellae grown in minimal media exhibited the biological properties of SP salmonellae and never entered as rapid a growth phase as did salmonellae in complete media.     

Recombinant human interleukin 1 alpha: purification and biological characterization

Interleukin 1 (IL 1) is a polypeptide hormone produced by activated macrophages that affects many different cell types involved in immune and inflammatory responses. The cloning and expression of a murine IL 1 cDNA in Escherichia coli encoding a polypeptide precursor of 270 amino acids has been reported, and expression of the carboxy-terminal 156 amino acids of this precursor in E. coli yields biologically active IL 1. By using the murine IL 1 cDNA as a probe, we have isolated its human homolog from cDNA generated to lipopolysaccharide-stimulated human leukocyte mRNA. Nucleotide sequence analysis of this cDNA predicts a protein of analysis of this cDNA predicts a protein of 271 amino acids (termed IL 1 alpha) which shows congruent to 61% homology to its murine counterpart but only 27% homology to a recently characterized human IL 1 precursor (IL 1 beta). We have expressed the carboxy-terminal 154 amino acids of IL 1 alpha in E. coli, purified this protein to homogeneity, and have compared it with pure recombinant murine IL 1 in several different IL 1 assays based on murine and human cells. Recombinant IL 1 is capable of stimulating T cell and fibroblast proliferation and inducing fibroblast collagenase and prostaglandin production, thus proving that a single molecule has many of the activities previously ascribed to only partially purified IL 1 preparations. Our results indicate that there exists a family of at least two human IL 1 genes (alpha and beta) whose dissimilar protein products have similar biological activities.     

Properties of the pyruvate dehydrogenase kinase bound to and separated from the dihydrolipoyl transacetylase-protein X subcomplex and evidence for binding of the kinase to protein X

Studies were conducted on four pyruvate dehydrogenase kinase-containing fractions: purified pyruvate dehydrogenase complex, the dihydrolipoyl transacetylase-protein X-kinase subcomplex (E2.X.K), a kinase fraction (K fraction) prepared from the E2.X.K subcomplex, and a kinase fraction generated by limited trypsin-digestion of E2.X.K. We characterized the gel electrophoresis properties of dissociated subunits (one-dimensional and two-dimensional), the catalytic and ATP binding properties of kinase-containing fractions, and the subunit requirements for kinase binding to and being activated by the transacetylase-protein X subcomplex (E2.X). A significant portion of protein X was retained with the transacetylase core following release of virtually all the kinase. The K fraction had four major bands separated by sodium dodecyl sulfate-slab gel electrophoresis which corresponded to the dihydrolipoyl dehydrogenase, protein X, the trypsin-resistant catalytic subunit of the kinase and a chymotrypsin-resistant subunit which had a high pI and comigrated in one-dimensional systems with the chymotrypsin-sensitive alpha-subunit of the pyruvate dehydrogenase component. While purified kidney complex contained only about three molecules of kinase (determined by [14C]ATP binding), one molecule of E2.X subcomplex activated a large number (greater than 15) molecules of kinase associated with the protein X-containing K fraction. Sephadex G-200 chromatography of the K fraction in the presence of dithiothreitol led to coelution of protein X and kinase subunits. Limited trypsin digestion converted the transacetylase into subdomains and cleaved protein X and the high pI subunit of the kinase. Under those conditions, the intact catalytic subunit of the kinase did not bind to the large inner domain of the transacetylase but could be activated by untreated E2.X subcomplex. Thus, binding of the catalytic subunit of the kinase and its activation by E2.X required either protein X or the lipoyl-bearing outer domain of the transacetylase. In combination, our results suggest that protein X serves to anchor the kinase to the core of the complex.     

The involvement of histone H1[0] in chromatin structure.

Micrococcal nuclease digestion and light scattering are used to compare native chromatins with various histone H1[0] contents. The experimental data show that the higher the H1[0] content, the greater the ability to form compact structures with increasing ionic strength, and the lower the DNA accessibility to micrococcal nuclease. On the contrary, reconstituted samples from H1-depleted chromatin and pure individual H1 fractions behave in such a way that samples reconstituted with pure H1 degree give rise to a looser structure, more accessible to nuclease than samples reconstituted with H1-1. This contradiction suggests that the effect of H1o on chromatin structure must originate from the interaction of this histone with other components in native chromatin among which other histone H1 subfractions are good candidates.     

Time course localization of immunoglobulin M monoclonal antibody and its fragments in leukemic tumor-bearing mice

Summary In vivo localization of a mouse monoclonal antibody (F₂-10.23 IgM) binding leukemic L 1210 cells was studied in DBA/2 mice bearing an L 1210 tumor. F(ab)₂ fragments were prepared and their specific binding to L 1210 cells was analyzed by flow cytofluorometry. Radiolocalization studies were performed by using ¹²⁵I- or ¹³¹I-labeled IgM monoclonal antibody or its F(ab')₂ fragments to ascertain their capacity to visualize the L 1210 tumor. F(ab)₂ fragments were cleared more rapidly than the whole IgM; the clearance was as fast in healthy as in tumor-bearing mice. The tumor-to-muscle ratio observed 24 h after injection of ¹²⁵I-radiolabeled F(ab)₂ fragments and ¹²⁵I-radiolabeled IgM was 10; the radioactivity level in the blood with F(ab)₂ fragments was lower than with IgM, and so -camera imaging was workable with F(ab)₂ fragments without background substraction. The tumor localization was studied over a period of 5 days by recording the distribution of the iodinated fragments in the tumor-bearing leg compared with that in the normal leg, and by computer analysis of the region of interest. F(ab)₂ fragments gave better results than intact IgM in tumor visualization. Nevertheless, the rapid clearance of this antibody or its F(ab)₂ fragments make them hardly suitable as carriers of toxic drugs. Abbreviations used are: MEM Minimum essential medium; SDS sodium dodecylsulfate; PAGE polyacrylamide gel electrophoresis     

Lectin-induced modulation of the antibody response to type III pneumococcal polysaccharide

Several lectins were tested for their capacity to alter the antibody response to type III pneumococcal polysaccharide (SSS-III). The antibody response was enhanced by concanavalin A (Con A), phytohemagglutinin (PHA), as well as lectins from Phytolacca americana (Pa-2), Pisum sativum (PSA), and Lens culinaris (LCH), when these lectins were given 2 days after immunization with SSS-III; however, suppression was obtained when Con A and Pa-2 were given at the time of immunization. By contrast the lectins from Vicia villosa (VVL) and Bauhinia purpurea (BPA) did not alter the antibody response. Since the lectins PSA and LCH bind to the same monosaccharide as Con A, whereas the other lectins bind to different monosaccharides, these findings indicate that there is no relationship between nominal monosaccharide specificity and the capacity to modulate the antibody response. Substantial increases in the magnitude of the IgG₁ antibody response was noted after the administration of Con A whereas profound enhancement of IgG_2a antibody response was noted after PHA was given.     

Differential effects of positive and negative proliferative stimuli on murine cytolytic and helper T-cell clones

Studies were performed to determine whether substances could be identified which exhibited differential regulatory effects--either positive or negative--on the growth of murine alloreactive cytolytic (Tc) and helper (Th) cloned T-cell lines. The following lines of evidence suggested that Tc and Th proliferate in response to the same growth factor (GF). (1) When GF-containing fluids from cultures of phorbol myristic acetate (PMA)-activated EL4 thymoma were fractionated by a variety of biochemical techniques. Tc and Th eluted together. (2) Absorption of GF-containing supernatants with either cloned Tc or cloned Th depleted GF activity for each to a similar extent, and GF eluted from either Tc or Th to which it had adsorbed supported the proliferation of Tc and Th equally well. (3) Lectin-depleted supernatants from cultures of concanavalin A (Con A)-activated Th stimulated the proliferation of Th as well as Tc. (4) Recombinant human interleukin (IL-2) supported the growth of Tc and Th with equal efficiency. On the other hand, the following observations indicated that Tc and Th differed in their responses to inhibitors of GF-driven proliferation. (1) Con A at greater than or equal to 0.3 micrograms/ml inhibited the GF-driven proliferation of each of three Th lines but not either of two Tc lines. To the contrary, Con A enhanced GF-dependent proliferation of Tc. (2) Like Con A, allogeneic splenocytes selectively depressed GF-driven proliferation of Th but not Tc. (3) A substance generated during the acid elution of GF from cells, possibly a modified fetal calf serum component, greatly reduced the GF-driven proliferation of Tc but not Th. These results suggest that differential control of the proliferation of Tc and Th in cellular immune responses may be achieved via negative regulatory signals and raise the possibility that substances which can selectively depress the proliferation of specific T-cell subsets might be found which would be of therapeutic value.