Monoclonal antibodies to yeast catalase T

Hybridoma cell lines secreting antibodies directed against $\text{Saccharomyces cerevisiae}$ catalase T were constructed by fusing spleen cells of mice immunized with catalase T with P3×63Ag8 mouse myeloma cells. Culture supernatants were assayed for specific antibodies by incubation with ³⁵S-labelled yeast extracts, adsorption of the immune complexes to Protein A — carrying $\text{Staphylococus aureus}$ cells and analysis of the adsorbed yeast proteins by sodium dodecylsulfate gel electrophoresis. Two hybrid clones were isolated mediating adsorption of a protein with electrophoretic mobility of catalase T; one of them, showing considerably higher activity, was characterized further. Anti-bodies produced by this clone belong to the IgG class of immuniglobulins; they can be used for immunoadsorption, but not for direct immunoprecipitation and recognize authentic catalase T as well as catalase T apoprotein.     

Polypeptide composition and spectral properties of light-harvesting chlorophyll ab-protein complexes from intact and trypsin-treated chloroplast thylakoid membranes

The polypeptide composition and spectral properties of isolated light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complexes from intact and trypsin-treated thylakoid membranes of Hordeum vulgare and Vicia faba are compared. The LHCP complexes consist of four distinct polypeptides with molecular weights between 21 000 and 25 000 occurring in equal relative amounts in the whole polypeptide spectra of thylakoid membranes. It is shown indirectly that the two major polypeptides very probably belong to different chlorophyll-proteins. The loss of a small segment from both polypeptides during trypsin digestion of thylakoids does not substantially alter the spectral properties and cation-mediated aggregation of isolated LHCP complexes.     

In vitro maturation of ovine fetal adrenal cells adenylate cyclase: corticotropin-dependent and independent development of the response to corticotropin

Polyclonal antibodies against an estrogen induced 52 K protein released by human breast cancer cells have been developed by injecting rabbits with a crude cellular pellet of MCF₇ human breast cancer cells. The rabbit antisera have been tested against [³⁵S]Methionine labelled proteins released by the MCF₇ cells followed by separation of the immune complexes with Protein A Sepharose. In spite of their low specificity and titer, these antisera allowed us to investigate the release of the 52 K protein $\text{in vitro}$ by other mammary cancer or normal cells.     

Enhancement of immune response by the proteinaceous crystal of Bacillus thuringiensis var thuringiensis

The proteinaceous crystal of $\text{Bacillus thuringiensis}$ var $\text{thuringiensis}$ was found to enhance humoral immune response in rats and guinea pigs immunised with sheep red blood cells. The enhancement was due to the increased levels of both 19S and 7S antibodies in the sera of the treated animals. A novel synthesis of 7S haemolytic antibodies was observed in case of crystal treated animals.     

Anaphylactic properties of mouse monoclonal IgG2a antibodies

Mouse monoclonal antibodies (10 hybridoma antibodies specific for soluble antigens, 8 hybridoma antibodies specific for H-2 $\text{K}\text{D}$ antigens, and 9 myeloma immunoglobulins, among which 5 had a known specificity) of the IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM isotypes were studied for their ability to induce mouse mast cell degranulation in vitro, in the presence of specific antigen or after heat aggregation. Monoclonal IgG1 antibodies, as well as IgG2b, IgG3, IgA, and IgM behaved as polyclonal antibodies of corresponding classes: all IgG1 induced mast cell degranulation with typical characteristics of IgG-mediated anaphylactic reactions, whereas IgG2b, IgG3, IgA, and IgM did not. By contrast, 2 hybridoma IgG2a and 3 myeloma IgG2a induced intense mast cell degranulation that could not be explained by a contamination with IgG1 or IgG1-IgG2a hybrid molecules. IgG2a-mediated reactions were observed in four different situations: soluble antigen-hybridoma IgG2a complexes, specific H-2 antigen-bearing mast cells challenged with hybridoma IgG2a anti-H-2, heat-aggregated myeloma IgG2a, and soluble antigen-myeloma IgG2a complexes. The conclusion was reached that mouse mast cells could be activated by mouse monoclonal IgG2a antibodies through a noncytotoxic, complement-independent mechanism involving mast cell Fcγ receptors.     

OKY-1581: A selective inhibitor of thromboxane synthesis invivo and invitro

OKY-1581 is an effective inhibitor of thromboxane synthesis $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$ caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.     

Modification of hepatic microsomal oxidative drug metabolism in rats by the opiate maintenance drugs acetylmethadol, propoxyphene, and methadone.

The formation of cytochrome P-450 “metabolic intermediate” complexes $\text{in}$$\text{vivo}$ occurred with acetylmethadol and propoxyphene, but not methadone in both naive and phenobarbital-induced animals. The $\text{in}$$\text{vivo}$ formation correlated with the relative ability of these three compounds to form metabolic intermediate complexes and inhibit mixed-function oxidation reactions $\text{in vitro}$.     

Evidence for 5,12-dihydroxy-6,8,10,14-eicosatetraenoate as a mediator of human neutrophil aggregation

The human polymorphonuclear neutrophil (PMN) aggregation responses to 5(S),12(R)-dihydroxy-$\text{cis}$-6,14-$\text{trans}$-8,10-eicosatetraenoate (diHETE), C5a, N-formyl-methionyl-leucyl-phenylalanine (FMLP), and 1-$\text{0}$-alkyl-2-$\text{0}$-acetyl-$\text{sn}$-glycero-3-phosphocholine (AAGPC) were desensitized by preincubating the cells with small amounts of diHETE. Desensitization developed rapidly, persisted in washed cells, and was not due to stimulus inactivation. The desensitized cells exhibited normal aggregation responses to ionophore A23187 and phorbol myristate acetate (PMA). Thus, responsiveness to diHETE appears necessary for the aggregation response to C5a, FMLP, and AAGPC. Endogenous diHETE, which forms rapidly in cells challenged with these latter stimuli, may mediate their aggregating actions.     

Ribosomal proteins L7/L12 localized at a single region of the large subunit by immune electron microscopy.

Ribosomal proteins $\text{L}\text{7}\text{L}\text{12}$ have been mapped by immune electron microscopy. These multiple copy proteins are located at a single region extending from the large subunit, known as the $\text{L}\text{7}\text{L}\text{12}$ stalk. The $\text{L}\text{7}\text{L}\text{12}$ stalk is approximately 100 Å long, about 40 Å wide and extends at an angle of approximately 50 ° from one side of the central protuberance of the large subunit. In the monomeric 70 S ribosome, the portion of the $\text{L}\text{7}\text{L}\text{12}$ stalk proximal to the 50 S subunit is located in the vicinity of the 30 S-50 S interface.Anti-$\text{L}\text{7}\text{L}\text{12}$ antibody binding to the stalk was shown to be solely dependent upon the presence of $\text{L}\text{7}\text{L}\text{12}$ by the following experiments. Sucrose gradient analysis was used to demonstrate that large subunits depleted of $\text{L}\text{7}\text{L}\text{12}$ were unable to bind anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies and that re-incorporation of $\text{L}\text{7}\text{L}\text{12}$ restored the ability of $\text{L}\text{7}\text{L}\text{12}$-depleted cores to react with anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies. Anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies pre-absorbed with $\text{L}\text{7}\text{L}\text{12}$ did not react with 50 S subunits.Anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies used in these experiments reacted only with the $\text{L}\text{7}\text{L}\text{12}$ stalk and with no other region of the subunit. This was shown by electron microscopy and by immune electron microscopy in the following ways. Electron microscopy of 50 S subunits, $\text{L}\text{7}\text{L}\text{12}$-depleted 50 S cores, and reconstituted 50 S subunits was used to demonstrate that stripping removes the $\text{L}\text{7}\text{L}\text{12}$ stalk from more than 95% of the subunits, and that re-incorporation of $\text{L}\text{7}\text{L}\text{12}$ into depleted cores restores the $\text{L}\text{7}\text{L}\text{12}$ stalk. Double-labelling experiments, using monomeric subunits with two or more attached anti-$\text{L}\text{7}\text{L}\text{12}$ immunoglobulins, were used to demonstrate, independently of 50 S subunit morphology, that $\text{L}\text{7}\text{L}\text{12}$ are located only on the $\text{L}\text{7}\text{L}\text{12}$ stalk.     

Polyamines and platelet aggregation

High blood concentrations of the naturally occurring polyamines have been reported in leukemia, psoriasis, cystic fibrosis and polycythemia rubra vera. Spermidine and spermine inhibit $\text{in}$$\text{vitro}$ plate-let aggregation of platelet rich plasma preparations in which ADP and Ristocetin are the agglutinating agents. The proposal is made that these organic cations may modulate $\text{in}$$\text{vivo}$ platelet agglutinability.     

Anti-glycosyl antibodies: Preparation and characterization of rabbit anti-galactose and anti-lactose antibodies

Anti-galactose and anti-lactose antibodies have been isolated from the antisera of rabbits immunized with non-viable cells of $\text{Streptococcus}$$\text{faecalis}$, strain N containing an antigenic diheteroglycan of glucose and galactose in the cell wall. The anti-galactose antibodies are specific for the galactosyl moiety while the anti-lactose antibodies are specific for the lactosyl moiety of the diheteroglycan. Hapten inhibitions with galactose and lactose, the sedimentation constant, the immunoglobulin type, the carbohydrate content, the electrophoretic mobility and the amino acid composition have been determined for the two new types of anti-glycosyl antibodies.     

Characteristics of macrophage cytotoxicity induced by IgE immune complexes

In earlier studies, the specific adherence of normal rat macrophages to Schistosoma mansoni schistosomula, followed by macrophage cytotoxicity against the larvae, was shown to be induced by incubation of the macrophages with serum from infected rats containing complexes of IgE antibody and circulating schistosome antigens. By the use of a chromium-51 release assay, it is pointed out that this cytotoxic process is a two-step phenomenon. The first step, i.e., activation of normal unstimulated macrophages induced by incubation of the cell with IgE complexes in immune rat serum, is a nonspecific mechanism which may also be elicited by various other macrophage activators. The second step, i.e., immune adherence and cytotoxicity of activated macrophages against S. mansoni schistosomula, is a specific process which imperatively needs the presence of S. mansoni IgE immune complexes. Aggregated myeloma IgE does not activate adherent peritoneal cells into cytotoxic effector cells unless the further participation of these specific IgE immune complexes is provided. The necessary preincubation of macrophages with immune rat serum before adding schistosomula accounts for the inefficiency of the incubation of the target itself with serum to elicit macrophage cytotoxicity. Serum dilution also appears as a critical factor since immune rat serum is inefficient when diluted more than $\text{1}\text{25}$. Aggregated rat IgG neither induces macrophage activation nor inhibits the activation by IgE immune complexes. Though the binding of IgE to the macrophage appears to be isotype specific, homologous immune complexes of IgE antibody and schistosome antigens are required to induce killing of S. mansoni larvae. The possible mechanism of this new model of macrophage activation and cytotoxicity is discussed.     

Synexin protein is non-selective in its ability to increase Ca2+-dependent aggregation of biological and artificial membranes

Synexin, a soluble protein which increases the specificity of Ca²⁺ to aggregate isolated bovine chromaffin granules was prepared from bovine adrenal medullary tissue by the method of Creutz, Pazoles and Pollard (J. Biol. Chem. $\text{253}$, 2858–2866, 1978). We also find that synexin increases both the initial rate and final amplitude of Ca²⁺-promoted aggregation of granule membranes. This effect is Ca²⁺-specific. However in contrast to Creutz $\text{et}\text{al}$, we find that synexin also potentiates aggregation of adrenal medulla and liver mitochondria and microsomes as well as phosphatidylserine vesicles. This lack of membrane specificity argues against the suggestion of Creutz $\text{et}\text{al}$ that synexin specifically binds the granule to the plasma membrane prior to exocytosis $\text{in}\text{vivo}$.     

Association of α2-macroglobulin-thrombin and α2-macroglobulin-plasmin complexes with isolated hepatocytes

¹²⁵I-labelled $\text{α}{}_2\text{-}\text{macroglobulin}$ complexed with thrombin or plasmin bound to hepatocytes in a concentration-and time-dependent manner. The apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values calculated from displacement experiments were 7.9 · 10^?8 M for $\text{α}{}_2\text{-}\text{macroglobulin-thrombin}$ and 8.5 · 10^?8 M for $\text{α}{}_2\text{-}\text{macroglobulin-plasmin}$. Association of these complexes was only partially reversible; after a 180 min incubation period, 50–60% of the bound radioactivity was internalized by the cells. $\text{α}{}_2\text{-}\text{Macroglobulin}$ itself bound also to hepatocytes, but the affinity of the $\text{α}{}_2\text{-}\text{macroglobulin}$ complexes was higher than that of the inhibitor alone, and $\text{α}{}_2\text{-}\text{macroglobulin}$ was not internalized, either. ¹²⁵I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their $\text{α}{}_2\text{-}\text{macroglobulin}$ complexes. The $\text{α}{}_2\text{-}\text{macroglobulin-thrombin}$ complex competed with the $\text{α}{}_2\text{-}\text{macroglobulin-plasmin}$ complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for $\text{α}{}_2\text{-}\text{macroglobulin-proteinase}$ and antithrombin III-proteinase complexes are different.     

Effect of synthetic estrogen on platelet aggregation and vascular release of PGI2-like material in the rabbit

Treatment of adult female New Zealand white rabbits with ethinyl estradiol, the synthetic estrogen used in many oral contraceptives, results in a significant increase in $\text{in vivo}$ aggregation. This alteration in platelet behavior is accompanied by diminished vascular release of antiaggregatory PGI₂ (prostacyclin)-like material. Addition of a progestin prevents the change in platelet aggregation seen with the estrogen alone. Diminished vascular PGI₂ release may be an important factor in the pathogenesis of thrombotic occurrences experienced by some oral contraceptive users. $\text{In vivo}$ platelet aggregation may be of value in identifying individuals at risk of developing thrombotic disturbances while taking oral contraceptives.     

Effects of lysolecithin on aggregation of human platelets induced by arachidonic acid and A23187 role of prostaglandin intermediary metabolism.

Lysolecithin at non-cytotoxic concentrations (30–500 uM) was found capable of completely inhibiting the $\text{aggregation of human platelets}$ induced by arachidonic acid in the absence of any effect upon total platelet production of malondialdehyde, an end-product of platelet $\text{prostaglandin intermediary metabolism}$, and to inhibit platelet aggregation stimulated by the calcium ionophore, A23187. As the induction of platelet aggregation by arachidonic acid is dependent upon an intact prostaglandin biosynthetic pathway while that of A23187 is not and since lysolecithin-induced inhibition of arachidonic acid-stimulated platelet aggregation was evident in the absence of an effect upon platelet malondialdehyde production, it is suggested that lysolecithin inhibits the platelet release reaction and irreversible aggregation by a mechanism separable from a major affect upon prostaglandin intermediary metabolism.     

Phylogeny of immune responsiveness in invertebrates

Existence of essential immunocompetence at different levels of complexity has recently been demostrated among diverse phylums of invertebrates. $\text{Immunorecognition}$ or capacity for non-self recognition of allogeneic tissue followed by incompatibility reactions is already found in Coelenterates as shown in colonial hydroids, gorgonians, and hard corals. Antagonistic reactions reveal convincing specificity, with compatible fusion preceding manifestations of incompatibility, but no memory component has been demonstrated. Allogeneic contact incompatibilities found in protochordates as well as mixed lymphocyte culture reactions between allogeneic cells of various vertebrates may tentatively be assigned to this “sipmle” immunorecognition category. $\text{Primordial Cell-Mediated Immunity}$ (PCMI) represents a higher level of immunoevolution revealed by specific allograft reactions with at least short-term memory as found in Annelids and Echinoderms. PCMI is inherent in populations of leukocytes and definitely has an immune memory component. $\text{Integrated Cell-Mediated and Humoral Antibody Immunity}$ may be regarded as the highest level of immunoevolution characteristic of all vertebrates from primitive fishes to mammals. Vertebrate-type immunoglobulin antibodies have yet to be demonstrated in any invertebrate species, although cell-surface receptors akin to antibodies are apparent on leukocytes of advanced invertebrates. The less elaborate immunoresponsiveness evident among invertebrates awaits definitive characterization.     

Ribosomal proteins L1, L17 and L27 from Escherichia coli localized at single sites on the large subunit by immune electron microscopy

Three-dimensional locations have been determined for Escherichia coli ribosomal proteins L1, L17 and L27 by immune electron microscopy using antibodies directed against these proteins. From the positions of immunoglobulin G attachment, observed in two characteristic projections, it was determined that these three proteins are located at single sites in different regions on the surface of the large subunit. In the quasisymmetric projection, L1 maps on the side opposite the “$\text{L}\text{7}\text{L}\text{12}$ stalk,” named the L1 ridge; protein L17 maps at the base of the subunit opposite the “central protuberance” (toward the $\text{L}\text{7}\text{L}\text{12}$ side of the subunit); and protein L27 is found on the central protuberance (on the side distal to the $\text{L}\text{7}\text{L}\text{12}$ stalk). In the asymmetric projection, proteins L1 and L27 are found on the surface of the subunit contracting the small subunit and protein L17 is on the surface of the subunit distal to the small subunit; i.e. on the cytoplasmic surface of the large subunit. Antibody binding at all three sites was eliminated when the immunoglobulin G molecules were preabsorbed with their specific proteins.     

Orientation and linear dichroism of chloroplasts and sub-chloroplast fragments oriented in an electric field

Whole or broken spinach chloroplasts, bacterial chromatosphores and CPI chlorophyll · protein complexes in aqueous suspensions at room temperature can be oriented in externally applied electric fields. The orientation is observed by monitoring the electric field induced linear dichroism (LD). With whole chloroplasts a detectable LD signal is observed using voltages as low as 2–3 V (50 Hz alternating voltage) across an 0.3 cm electrode gap, and nearly complete orientation is observed at fields of 30 V · cm^?1. The wavelength dependence of the LD signals using either orienting electric fields ($\text{E}$) alone, or magnetic fields ($\text{B}$) alone, are similar but opposite in sign with $\text{E}$ and $\text{B}$ pointing in the same direction. The chloroplasts tend to orient in such a way that the membrane planes are parallel to $\text{E}$. The CPI complexes and bacterial chromatophores require much higher electric fields for orientation than whole chloroplasts (for CPI complexes E > 2000 V · cm^?1); rectangular, millisecond duration, voltage pulses are utilized for the observation of electric field induced LD spectra in these cases. Oriented CPI complexes exhibit LD maxima of the same sign at 685 and at 440 nm. The oriented chromatophores exhibit an LD spectrum of either positive or negative sign, depending on the wavelength. The mechanisms of the orientation are discussed.