The determination of equilibrium constants for heterogeneous macromolecular interactions. Systems forming 2:1 complexes.

In developing a method for analyzing the heterogeneous association nA + mB in equilibrium AnBm, we have specifically investigated the case of n = 2, m = 1 for both the specific case of no appreciable intermediates and the more general case allowing intermediates. Computer-simulated three-dimensional surfaces of the 2:1 model generated from total concentrations of species A and B and the resulting weight-average molecular weights were analyzed with a Gauss-Newton nonlinear least-squares minimization routine. The surfaces generated included normalized random error of varying standard deviations imposed upon both the concentrations and weight-average molecular weights. For comparison purposes, these surfaces were analyzed not only by using the correct 2:1 model, but also by an incorrect (1:1) model and by the other (incorrect) 2:1 model. Except for those situations where the 'experimental' noise was consistently higher than the concentration of one of the species, correct K values were obtained and the correct model was easily distinguished from the incorrect model. The computer routine similarly distinguished between data correctly described as 1:1 and the same data incorrectly analyzed as either 2:1 model. For those cases in which a microscopic Ki value predicts an association such that all species involved for that particular Ki are in appreciable amounts, the Ki value is returned correctly. Correct overall equilibrium constants are also converged upon as long as adequate amounts of A2B, B and A are present.

The determination of equilibrium constants for heterogeneous macromolecular interactions.

A method has been developed to determine the association constant for a heterogeneous association of the type A + B in equilibrium AB. This method requires knowledge of the two initial concentrations and of the resulting weight-average molecular weight for each data point. Computer simulations using Gaussian-distributed error on the measured parameters show that the researcher can readily determine whether the particular concentration range chosen is appropriate for the strength of binding and therefore how reliable the calculated constant might be. It is also shown that errors in measuring molecular weight have, in general, a more profound effect than do errors in concentration.

Shaping of the chick neuroepithelium during primary and secondary neurulation: role of cell elongation.

Cell elongation is a cardinal event in formation and shaping of the neuroepithelium during both primary and secondary neurulation. This study had three purposes. The first was to clarify the role of microtubules in maintaining the elongated configurations of chick neuroepithelial cells. Neuroepithelial cells of the neural plate (the rudiment of the primary neural tube) and medullary cord (the rudiment of the secondary neural tube) reduced their heights an average of approximately 25% when their microtubules were depolymerized, but most cells remained considerably elongated and columnar. Complete rounding up occurred only as cells entered metaphase where they arrested. These results suggest that microtubules as well as other factors are required to maintain the fully elongated configurations of cells composing epithelial sheets. The second purpose of this study was to evaluate the hypothesis that neuroepithelial cell elongation plays a major role in narrowing of the neural plate. To do this, the width of the neural plate was examined after microtubule depolymerization and repolymerization. As the heights of neuroepithelial cells decreased with loss of their microtubules, the width of the neuroepithelium increased roughly proportionately; subsequent repolymerization with concomitant cell elongation resulted again in neural plate narrowing. Thus, the hypothesis is supported. The third purpose of this study was to examine the roles of cell rearrangement and change in neuroepithelial cell or extracellular volume in neural plate narrowing and extension. Extensive cell rearrangement, resulting in net cell loss from the width of selected, representative levels of the neural plate, does not seem to play a major role in plate narrowing, but decreases in cell or extracellular volume are likely involved. Further studies are necessary to complete our understanding of the mechanisms driving neural plate shaping and bending.

Serial analysis of peripheral blood T-cell phenotypes and myelin basic protein reactivity in experimental allergic encephalomyelitis.

We have serially analyzed peripheral blood T-cell phenotypes and reactivity to myelin basic protein (MBP) during the course of acute experimental allergic encephalomyelitis (EAE) in the Lewis rat and have correlated changes with the onset of clinical disease. A reduction in total T cells (W3/13+), due primarily to a reduced helper/inducer (W3/25+) subpopulation, preceded the onset of EAE. Circulating MBP-reactive lymphocytes were only transiently present in the blood at the time EAE was clinically evident. Our findings demonstrate that in EAE, immunologic abnormalities in the peripheral blood are transient and can begin before clinical disease is evident.

Adoptive transfer of murine chronic-relapsing autoimmune encephalomyelitis. Analysis of basic protein-reactive cells in lymphoid organs and nervous system of donor and recipient animals.

Frequency analysis of myelin basic protein (MBP)-reactive lymphocytes was performed in the chronic relapsing murine experimental allergic encephalomyelitis (EAE) model induced by the adoptive transfer of myelin basic protein (MBP)-primed lymphocytes to naive recipients. During the first attack, MBP-reactive cell frequencies were: 1/41,700 in spleen, 1/328,000 in lymph nodes, 1/64,500 in the peripheral blood. After recovery from a second attack, the frequencies were: 1/11,000 in spleen, 1/46,000 in lymph node, and 1/195,000 in the blood. In addition, lymph node cells obtained from animals following a second attack had increased encephalitogenic properties. CNS-derived lymphocytes analyzed during the first attack were 50% Lyt 1.2+ and 16% Lyt 2.2+. After recovery from the second attack, phenotypes were 20% Lyt 1.2+ and 49% Lyt 2.2+. There were only minimal responses to MBP in CNS-derived lymphocytes. Susceptibility to adoptively transferred EAE was in general predicted by whether a proliferative response to MBP occurred following immunization and was not solely H-2 linked. These studies demonstrate an accumulation of autoreactive cells in the spleen and lymph nodes and a shift of the phenotype of cells in the target organ as EAE becomes chronic and suggest there are dynamic immunologic processes, both in the peripheral immune system and target organ associated with relapsing EAE.

Interaction of reovirus with cell surface receptors. IV. The reovirus type 3 receptor is expressed predominantly on murine Lyt-2,3+ and human T8+ cells.

Reovirus type 3 binds to approximately 20% of murine and human T cells via the viral hemagglutinin, a small outer capsid polypeptide. By using purified viral particles as a ligand in a standard plate separation technique, we have been able to enrich human peripheral blood and murine splenic T cells for reovirus receptor-positive cells (reovirus 3+) to levels of 88 to 92%. Analysis of reovirus 3+ T cells with monoclonal antibodies that identify inducer and suppressor/cytotoxic cells demonstrated that in the mouse, 68% of reovirus 3+ cells were Lyt-2+, and in the human, 60% were T8+. In reciprocal experiments, when subpopulations of murine and human T cells were prepared with the use of monoclonal anti-T cell reagents, 16% of Lyt-1+ and 81% of Lyt-2+ cells bound reovirus, whereas 30% of T4+ and 65% of T8+ cells bound reovirus. To determine whether reovirus type 3 identified a functional as well as a phenotypic category of cells, an antigen-specific cytotoxic T cell assay was employed. There was complete loss of cytotoxic activity in the reovirus 3+ cell population and slight enhancement of cytotoxic activity in the cell population from which reovirus 3+ cells were removed. This suggested that reovirus was binding to functionally active suppressor cells. Furthermore, adoptive transfer of antigen-specific T cells that were enriched for reovirus 3+ cells demonstrated suppression of cytoxic T cell activity. These results suggest that reovirus type 3 may identify a structure common to a subclass of murine and human T cells and that by using the virus as a natural biologic probe for cell surface receptors, one may be able to functionally segregate murine cytotoxic from suppressor T cells.

Autoimmunity following viral infection: demonstration of monoclonal antibodies against normal tissue following infection of mice with reovirus and demonstration of shared antigenicity between virus and lymphocytes.

Splenic lymphocytes from adult C57BL/6 mice infected with purified reovirus type 1 or 3 particles were fused with NS1 myeloma cells. Approximately 300 clones were obtained from each fusion (type 1 or type 3) and the supernatants from these clones were screened by radioimmunoassay for their ability to bind virus, T lymphocytes, brain, liver, lung tissues and isolated oligodendrocytes and ependymal cells. Approximately 10% of clones (33 and 26 clones, respectively) were positive for each fusion. For reovirus type 1:21% of positive clones bound only virus, 64% bound one of the normal tissues but not virus, and 15% bound both virus and one or more of the normal tissues. For reovirus type 3: 19% of positive clones bound only virus, 73% bound normal tissue only, and 8% bound both virus and normal tissue. Only 3 positive clones were obtained from uninfected control animals. These experiments demonstrate that (a) during the course of an immune response to a virus, autoantibodies are generated which react with a large variety of normal tissues and that (b) there are shared antigenic structures between viral determinants and normal tissue that can be identified by monoclonal antibodies. Although these results suggest two mechanisms by which an autoimmune response may develop following viral infection, the biological significance of these autoreactive monoclonal antibodies remains to be elucidated.

Binding of 125I-labeled reovirus to cell surface receptors.

Quantitative studies of 125I-labeled reovirus binding at equilibrium to several cell types was studied, including (1) murine L cell fibroblasts; (2) murine splenic T lymphocytes; (3) YAC cells, a murine lymphoma cell line; and (4) R1.1 cells, a murine thymoma cell line. Competition and saturation studies demonstrated (1) specific, saturable, high-affinity binding of reovirus types 1 and 3 to nonidentical receptors on L cell fibroblasts; (2) high-affinity binding of type 3 reovirus to murine splenic lymphocytes and R1.1 cells; (3) low-affinity binding of reovirus type 1 to lymphocytes and R1.1 cells; and (4) no significant binding of either serotype to YAC cells. Differences in the binding characteristics of the two reovirus serotypes to L cell fibroblasts were found to be a property of the viral hemagglutinin, as demonstrated using a recombinant viral clone. The equilibrium dissociation constant (Kd) for viral binding was of extremely high affinity (Kd in the range of 0.5 nM), and was slowly reversible. Experiments demonstrated temperature and pH dependence of reovirus binding and receptor modification studies using pronase, neuraminidase, and various sugars confirmed previous studies that reovirus receptors are predominantly protein in structure. The reovirus receptor site density was in the range of 2-8 X 10(4) sites/cell. These studies demonstrate that the pseudo-first-order kinetic model for ligand-receptor interactions provides a useful model for studying interactions of viral particles with membrane viral receptors. They also suggest that one cell may have distinct receptor sites for two serotypes of the same virus, and that one viral serotype may bind with different kinetics depending on the cell type.

Age dependent susceptibility to Reovirus type 3 encephalitis: role of viral and host factors.

Reovirus type 3 inoculated intracerebrally or subcutaneously into newborn mice induced an acute necrotizing and uniformly fatal encephalitis. Subsequently, between the eighth and tenth day of age, reovirus type 3 infection changed to a nonlethal infection. This pattern of virulence was directly correlated with the ability of the virus to replicate in the brain and histologically was accompanied by a gradual diminution in the intensity and extent of encephalitis such that histological abnormalities were absent in the brains of virus-injected adult animals. Some animals injected at ages 10 to 18 days developed brainstem and diencephalic lesions, indicating a nonuniform resistance of neurons to viral infection with aging. Neither a changing immune response pattern nor a modification or loss of viral receptors appears to explain either the susceptibility of newborn or the resistance of adult animals to reovirus type 3 encephalitis. Splenic mononuclear cells from mice younger (but not older) than 7 days also permitted reovirus type 3 replication in vitro. Thus, the age dependent resistance to reovirus type 3 encephalitis appears related to an intrinsic resistance to viral replication by neuronal cells, a resistance that may occur simultaneously in nonneuronal cell populations.

Stepwise sequence determination from the carboxyl terminus of peptides.

The thiocyanate method for stepwise degradation of peptides from their COOH termini [Stark, G. R. (1968) Biochemistry 7, 1796] has been investigated. The method involves first the reaction of the COOH-terminal residue with thiocyanate in an activation solvent of acetic acid and acetic anhydride and then cleavage of the COOH-terminal residue as its 2-thiohydantoin by acetohydroxamate in aqueous solution. The two steps of the degradation have been studied by using model peptides, and conditions have been developed for the rapid efficient removal and identification of the COOH-terminal residue of short peptides. The methods have been applied to peptides that have been covalently attached to insoluble supports. In this solid phase version of the degradation, a highly substituted porous glass activated with N,N'-carbonyldiimidazole has been prepared for use as the insoluble support. A number of peptides have been coupled to the porous glass, and several rounds of the degradation have been performed on immobilized peptides. High-pressure liquid chromatography provides a rapid, sensitive identification method for the 2-thiohydantoins. In addition, gas-liquid chromatography of the amino acid 2-thiohydantoins and reconversion to the parent amino acid have been used to identify the cleaved residues. The method of sequential degradation has been applied to a number of short model peptides such as Gly-Leu-Tyr, Met-enkephalin, and Val-Leu-Ser-Glu-Gly and has been used to determine the COOH-terminal sequence of 4 residues of a 22-residue cyanogen bromide fragment of pygmy sperm whale myoglobin.

Identification of a hemagglutinin-specific idiotype associated with reovirus recognition shared by lymphoid and neural cells.

A xenogeneic antiserum raised to antireovirus immunoglobulin was used to define an idiotypic determinant present on antibodies to reovirus type 3 hemagglutinin. The same idiotype was identified on nonimmune lymphoid cells and on neuronal cells that specifically bind the hemagglutinin of type 3 reovirus. This idiotypic determinant, called Id3, is shared by (a) a monoclonal antibody to the neutralization site of hemagglutinin from type 3 reovirus; (b) BALB/c serum antibodies to the hemagglutinin of reovirus type 3; (c) R1.1, a murine thymoma cell line that binds reovirus type 3; (d) primary cultures of murine neuronal cells. The presence of an idiotype shared by antihemagglutinin antibodies and by structures on nonlymphoid cells suggests a general relationship between disparate receptors that recognize a common determinant. Furthermore, this suggests a novel approach for the study of viral receptor interactions and for analysis of mechanisms of autoimmune responses.

Interaction of reovirus with cell surface receptors. III. Reovirus type 3 induces capping of viral receptors on murine lymphocytes.

We have previously shown that there are receptors for the hemagglutinin of reovirus type 3 on a subset of both murine T and B cells. Using purified reovirus type 3 particles as a ligand, followed by FITC-labeled antiviral antibody we have now been able to demonstrate viral induced capping of the reovirus receptor on both B and T cells. Kinetic studies and inhibition experiments using cytochalasin B and colchicine demonstrate that reovirus-induced capping of the viral receptor on both B and T cells has characteristics identical to the capping of immunoglobulin on B cells by anti-immunoglobulin reagents. Removal of the viral receptor and reexpression after overnight culture was demonstrated after membrane treatment by proteolytic enzymes (trypsin, pronase) or after viral induced modulation of receptor. Protein synthesis was required for reexpression since cyclohexamide prevented the reexpression of receptor in culture. Co-capping studies utilizing 2-color immunofluorescence failed to identify and association between the reovirus receptor and a variety of known surface structures on either B or T cells, but demonstrated independent modulation of the viral receptor from other surface structures. These studies suggest that the lymphocyte surface binding site for reovirus type 3 is a distinct structure that behaves identically in capping studies of both B and T cells, is protein in nature, and is intimately linked to the cell cytoskeleton.

Absolute linkage of virulence and central nervous system cell tropism of reoviruses to viral hemagglutinin.

That the hemagglutinin (HA) of reovirus, encoded in the S1 gene, determines the central nervous system (CNS) cell tropism of reovirus type 1 and 3 was shown using recombinant clones containing nine genes from one serotype and the S1 gene from the other. Clone 1.HA3 contains nine genes from type 1 and the S1 gene from type 3; 3.HA1 is the reciprocal clone. Type 3 and 1.HA3 cause a fatal encephalitis in newborn mice with neuronal destruction but no ependymal cell damage, whereas type 1 and 3.HA1 cause a nonfatal ependymal infection but no neuronal damage. Immunofluorescent studies showed no viral antigen in ependymal cells of mice infected with type 3 or 1.HA3 or in neuronal cells of mice infected with type 1 or 3.HA1. With type 3 or clones containing the type 3 HA, maximal brain titers were 10(10) plaque-forming units; maximal titers were 10(8) plaque-forming units for type 1 or clones containing the type 1 HA. This pattern of reovirus virulence for CNS probably relates to the specific interaction of viral HA with neuronal or ependymal surface receptors.

A virilizing adrenocortical tumor in a female infant: in vivo and in vitro biochemical characteristics.

Normal adrenal and adrenal tumor cells from a female infant with a virilizing adrenal tumor were grown in tissue culture as monolayers for a period of 7 weeks. Half of the cultures were exposed to ACTH (0.1 U/ml). The cells grew well and continued to produce steroid hormones over the entire period in culture. Production of steroid hormones was measured by RIA of individual steroids in the culture medium. Unstimulated normal and tumor cells produced equivalent amounts of cortisol, 11 beta, 18, 21-trihydroxy-4-pregnene-3,20-dione, and 18,21-dihydroxy-4-pregnene-3,20-dione, but tumor cells produced lesser amounts of dehydroepiandrosterone (DHA), androstenedione, testosterone, and progesterone. Normal cells exposed to ACTH showed an increase in all steroids measured, whereas ACTH-exposed tumor cells showed an increase principally in DHA consistent with a deficiency in 3 beta-hydroxysteroid dehydrogenase activity. Circulating levels of DHA, androstenedione, and testosterone were elevated in the patient before removal of the adrenal tumor. The production of androgens by tumor cells in vitro resembled the pattern of circulating steroids in vivo. These studies demonstrate that tissue culture of human adrenal cells provides a means both to determine their biochemical characteristics and to investigate their responses to exogenous hormones.

A direct effect of testosterone on muscle cells in tissue culture.

A direct effect of physiological levels of testosterone on skeletal muscle cells in tissue culture is reported. The effect is a 25% stimulation of labeling index by radioautographic analysis of cultures following incubation with [3H]thymidine at the end of a 48-h exposure to 10(-8)M testosterone in 2% gelding serum. This stimulation was observed in primary myoblasts, an established myogenic cell line (Yaffe's L6 cells), and muscle fibroblasts. The effect was specific for testosterone: the labeling index did not change significantly from control values when estradiol, 5beta-pregnanediol, androstenedione, or dihydrotestosterone were added, although small increases were noted in the latter two cases. The effect on the labeling index was localized as a decrease in time spent in the G1 phase of the cell cycle, which decreased about 30% in muscle cells exposed to testosterone. This first report of an effect of testosterone on isolated muscle cells, coupled with the recent description of a receptor for anabolic steroids in muscle cytoplasm, indicates that the effects of male sex hormones result from a direct interaction with muscle rather than from a primary interaction with some other tissue.