The role of mononuclear phagocytes in cardiac allograft rejection in the rat: II. Characterization of mononuclear phagocytes extracted from rat cardiac allografts

A method was developed for the extraction of leukocytes infiltrating rat cardiac allografts. Mononuclear phagocytes (MNP) comprised 52.4 ± 5.5% of the cells extracted from allografts at the time of rejection (Day 7). Day 4 allografts and Day 7 syngeneic grafts yielded considerably fewer MNP although numbers of lymphoid cells were similar in all three groups. Allograft MNP were phagocytic for latex particles but only very low numbers were adherent to a variety of surfaces. About 50% were positive for attachment and internalization of opsonized sheep red cells via the Fc receptor. However, fewer cells were able to internalize sheep red cells than were able to bind them when complement receptor-mediated phagocytosis was investigated. Large amounts of plasminogen activator were secreted by allograft MNP while cells from syngeneic grafts produced very little. The possible participation of MNP in the effector phase of a mechanism for allograft rejection similar to delayed-type hypersensitivity is discussed.     

The isolation and identification of a novel intermediate in ubiquinone-6 biosynthesis by Saccharomyces cerevisiae.

The reaction product obtained from HeLa cell nuclei incubated with [³H]NAD was specifically hydrolyzed with snake venom phosphodiesterase. Analysis of the hydrolyzed product revealed that it is a homopolymer consisting of 4–5 repetition of ADP-ribose units. The [³H]poly ADP-ribosylated histone fraction was anslyzed by urea-acetic acid polyacrylamide gel electrophoresis. The radioactive peak was clearly separated from the stained histone H1 band, while a slight overlap was observed. When chromatographed on a SP-Sephadex C-50 column, more than 90% of the radioactivity of [³H]poly(ADP-ribose) was eluted in accordance with histones but not with nonhistone contaminants. On a sodium dodecyl sulfate polyacrylamide gel electrophoresis, a major radioactive peak appeared at a position very close to the histone Hl band, which disappeared by the treatment with alkali prior to electrophoresis. A selective extraction of histone Hl with 5% perchloric acid showed that histone Hl contained about 85% of the radioactivity incorporated into whole histones.     

A new vasopeptide formed by the action of a Murphy-Sturm lymphosarcoma acid protease on rat plasma kininogen

A new vasoactive peptide, formed by the action of a Murphy-Sturm lymphosarcoma acid protease on rat plasma kininogen was purified by gel filtration on Sephadex G-50 (fine) and fractions assayed on the isolated rat uterus for smooth muscle stimulating activity. The most active fraction was purified further by CM-cellulose chromatography. High voltage electrophoresis showed the peptide to be one component (Mgly 2.49) with an electrophoretic mobility different from bradykinin, lysyl-bradykinin and methionyl-lysyl-bradykinin. The molecular weight of the peptide was estimated on Sephadex G-25 column to be 1460. The amino acid composition was determined and the carboxyl terminal sequence identified by carboxypeptidase Y treatment to be Pro-Phe-Arg-Leu. Dansyl-Edman procedure yielded an amino terminal sequence of Ile-Ser-Arg-Pro. The peptide produced a dose-dependent contraction of the isolated guinea pig anterior mesenteric vein and relaxed the rabbit superior mesenteric artery contracted by phenylephrine.     

Studies of nicotinic acetylcholine receptor protein from rat brain: II. Partial purification

The pharmacological specificity of the binding of ¹²⁵I-labeled α-bungarotoxin to a 1% Emulphogene BC-720 extract of a rat brain particulate fraction has been investigated. The extract contains a component which possesses the binding characteristics of a nicotinic acetylcholine receptor protein. The crude soluble acetylcholine receptor protein was purified by affinity chromatography utilizing the α-neurotoxin of Naja naja siamensis as ligand and 1.0 M carbamylcholine chloride as eluant. A single, batch-wise, affinity chromatography procedure yields an average purification of 510-fold. When this purified material is treated a second time by affinity chromatography, purification as high as 12 600-fold has been obtained. Binding of ¹²⁵I-labeled α-bungarotoxin to this purified acetylcholine receptor protein is saturable with a K_d of 1·10^?8 M. Nicotine and acetylcholine iodide at concentrations of 10^?5 M inhibit ¹²⁵I-labeled toxin-acetylcholine receptor protein complex formation by 41 and 61% respectively. At 10^?4 M, carbamylcholine chloride and (+)-tubocurarine chloride give respectively 52 and 82% inhibition. Eserine sulfate and atropine sulfate have no effect on complex formation at a concentration of 10^?4 M. These data support the isolation of partially purified nicotinic acetylcholine receptor protein.     

Messenger-like RNA synthesis and DNA chromosomal puffs in the salivary glands of Rhynchosciara americana

RNA synthesis was studied mainly in the proximal sections of Rhynchosciara salivary glands in late fourth instar at two typical periods of development. These are characterized either by the absence or presence of the so-called “DNA puffs” in the salivary gland chromosomes. It was found that simultaneously with the appearance of the DNA puffs there is a great increase in the synthesis of all RNA species. The greatest increase was found to take place in the rate of synthesis of messenger-like RNA. Four main classes of messenger-like RNA were detected, having mobilities corresponding to 33, 23, 16, and 14 S RNA. There is a correlation between the abundance of the 16 S messenger-like RNA and the degree of opening of the B-2 DNA puff. This species might therefore be transcribed from this puff.     

Isolation and characterization of protease do from Escherichia coli, a large serine protease containing multiple subunits

A new cytoplasmic proteolytic enzyme in Escherichia coli, named protease Do, has been purified to near homogeneity. The enzyme is an endoprotease that degrades casein, denatured bovine serum albumin, and globin but shows little or no hydrolytic activity against insulin, growth hormone, native bovine serum albumin, or a variety of commonly used peptide substrates. The molecular size of the enzyme was large, and it could be isolated in different preparations in either of two forms. One showed a molecular weight of about 500,000 on gel filtration and a sedimentation coefficient of 15.9 S on sucrose gradient centrifugation. The other appeared to be about 300,000 and sedimented at 12.7 S. No interconversion between the two forms and no other difference in the properties was found. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) shows that both forms contain a major 54,000-dalton band and three additional minor polypeptides with molecular weights of 45,000, 44,000, and 42,000. These minor polypeptides appear to result from autolytic degradation of the major protein as demonstrated by peptide mapping with Staphylococcus aureus V8 protease. Thus, protease Do appears to contain a single subunit of 54,000, and can exist either as a decamer or as a hexamer or pentamer. The enzyme is a serine protease. It is sensitive to diisopropyl fluorophosphate (DFP) but not to metal chelating agents, sulfhydryl blocking groups, certain chloromethyl ketones, or various peptide aldehyde inhibitors. The enzyme covalently binds [3H]DFP, and the labeled subunit was visualized on SDS-polyacrylamide gels by fluorography. When cells growing in rich broth enter stationary phase, the relative concentration of protease Do increases more than twofold.     

Evidence for the presence of an acid protease and protease inhibitors in dormant embryos of Artemia salina.

Proteolytic activity has been implicated in several key processes in early development. In an attempt to correlate proteolytic activity with developmental events, a study of the protease(s) in undeveloped cysts of Artemia salina was initiated using 2,4,6-trinitrobenzenesulfonic acid to determine the release of amino groups upon protein hydrolysis. The versatility and sensitivity of this reagent made it possible to detect and characterize the proteolytic activity in small quantities of cysts of the brine shrimp. A protease with a molecular weight of 84,000, a pH optimum of 3.6, and a temperature optimum of 45°C was partially purified from Artemia cysts using ion-exchange chromatography and gel filtration. In addition, two acid protease inhibitors, one dialyzable and one nondialyzable, were found in crude extracts of the cysts. The latter was partially purified and found to have a molecular weight of between 10,000 and 20,000. The activity of the acid protease is not dependent on CaCl₂ or EDTA, but CaCl₂ in the reaction mixture increases the rate of inactivation of the nondialyzable protease inhibitor. The inhibitors may complex with the acid protease in the embryo and control its activity during development.     

Esteroproteolytic enzymes from the submaxillary gland. Kinetics and other physicochemical properties.

Two esteroproteolytic enzymes (A and D) have been isolated from the mouse submaxillary gland and shown to be pure by ultracentrifugation, immunoelectrophoresis, acrylamide-gel electrophoresis, and amino acid analyses. The enzymes have molecular weights of approximately 30,000 and are structurally and antigenically related. Narrow pH optima between 7.5 and 8.0 are exhibited by both enzymes. The “pK₁'s” are between 6.0 and 6.5 and the “pK₂'s” are near 9.0. A marked preference for arginine-containing esters is shown by both enzymes. The maximum specific activity of enzyme A on p-tosylarginine methyl ester (TAME) at pH 8 was 2500–3000 μm min^?1 mg^?1 and for enzyme D, 400–600 μm min^?1 mg^?1. With TAME as substrate, the K_m for enzyme A was 8 × 10^?4m at 25 °C and 6 × 10^?4m at 37 °C. For D, K_m was 3 × 10^?4 at 25 °C and 2 × 10^?4m at 37 °C.An apparent activation of enzyme D by tosylarginine (TA), a product of TAME hydrolysis, and all α-amino acids examined was due to removal of an inhibitor by chelation. This effect could be duplicated by 8-hydroxyquinoline and diethyldithiocarbamate but not by EDTA. Enzyme A was not affected by these substances to any remarkable extent. Several divalent ions proved to be potent inhibitors of enzyme D. Both enzymes are inactivated by the active site reagents diisopropyl phosphofluoridate and tosyllysine chloromethylketone but much less rapidly than is trypsin. Nitrophenyl-4-guanidionobenzoate reacts with a burst of nitrophenol liberation but with a rapid continuing hydrolysis. One active site per molecule is indicated. Enzyme D is inactivated by urea, reversibly at 10 m and with maximal permanent losses at 6 m. Autolysis of the unfolded form by the native enzyme when they coexist at intermediate urea concentrations appears to occur.Identity of enzyme D and the epithelial growth factor binding protein is demonstrated.     

The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons : II. Developmental aspects

Dissociated sympathetic neurons from the neonatal rat, grown in cell culture in the virtual absence of other cell types, can develop many of the properties expected of differentiated adrenergic neurons including the ability to synthesize and accumulate catecholamines (CA)². However, in the presence of high concentrations of appropriately conditioned medium (CM), the cultures develop the ability to synthesize and accumulate acetylcholine (ACh); correspondingly, their ability to synthesize CA decreases. In this paper several developmental aspects of the CM effect are described. The time course of development of cultures grown with or without CM was followed using synthesis and accumulation of [³H]CA from [³H]tyrosine and production of [³H]ACh from [³H]choline as assays for adrenergic and cholinergic differentiation. The ability to produce CA or ACh developed along parallel time courses in the two sets of cultures, rising primarily during the second week in vitro and reaching a plateau during the fourth week. When CM was used as a cholinergic developmental signal, the sympathetic neurons showed a decreasing response to addition of CM as they matured adrenergically; addition of CM during the third or fourth 10 days in vitro was not as effective in inducing ACh production as addition during the first or second 10 days. Similarly, removal of CM at various times from cultures previously grown in CM showed that the cholinergic induction caused by CM was not easily reversible in older cultures. Thus, as with the adrenergic decision, the cholinergic decision becomes less reversible as the phenotype becomes fully expressed.     

The role of mononuclear phagocytes in cardiac allograft rejection in the rat: III. The effect of cells extracted from rat cardiac allografts upon beating heart cell cultures

Cells extracted from rat cardiac allografts were able to bring about cessation of beating of heart cell culture monolayers nonspecifically. Nonadherent populations, depleted of macrophages, were consistently less potent than unseparated cells in this assay. Cells extracted from isografts were totally ineffective. Allogeneically stimulated peritoneal cells were also nonspecifically active. Again, nonadherent cells were less efficient than unseparated cells at stopping heart cell monolayers from beating, while adherent cells, enriched for macrophages, were more efficient. Activated bone marrow culture macrophages syngeneic or allogeneic to the heart cultures were also highly potent in beating heart cell assays. Thus in all cases the predominant effector cell type was adherent and nonspecific in its action and therefore presumably a macrophage. Supernatants from wells in which no beating cells remained following incubation with each type of effector population tested were transferred undiluted to fresh wells. In all cases there was no effect at all upon the beating of heart cell monolayers. Antirat heart antiserum plus complement was able to bring about the cessation of beating of heart culture monolayers at a dilution of 1:64. Alloantibody plus complement did not bring about cessation of beating at any dilution, although nonmyocardial cells were killed. The possibility that macrophages are the chief effector cell type in a DTH-like mechanism for cardiac allograft rejection is discussed.     

Macrophage-like suppressor cells in rats. II. Evidence for a quantitative rather than a qualitative change in tumour-bearer animals

In order to investigate the mechanisms of steroid-induced inhibition of concanavalin A stimulation, we have compared, in mouse lymphoid cells, the ability of various steroids to block transformation with their affinity for glucocorticoid receptors. Our results suggest several possible explanations for the inhibitory effects of the various steroids tested. The action of glucocorticoids, which are inhibitors at concentrations within the physiological range (10^?8–10^?7M) is likely to be mediated through an interaction with specific cytosolic binding sites leading to an overall inhibition of cell metabolism. In contrast, sex steroids only inhibit at pharmacological concentrations, equal to or higher than 10^?5M. These compounds, which do not bind to glucocorticoid receptors, probably act in a “nonspecific” manner at the level of the cell membrane. The effect of 25-OH-cholesterol, a selective inhibitor of cholesterol synthesis, suggests that the level of sterol formation controls in part the proliferative activity of the cells.     

The role of coenzyme Q10 for preservation of the rat kidney: a model experiment for kidney transplantation.

In an experimental model system for kidney transplantation, the ability of the rat kidney to resynthesize ATP after warm ischemic time and subsequent restoration of renal blood flow was found to determine tissue viability and survival of the animal. The administration of CoQ₁₀ prior to warm ischemia enhanced the rate and extent of ATP resynthesis by reflow following two-hour warm ischemia and this was accompanied by an increase in survival rate of the rats.     

The role of mononuclear phagocytes in cardiac allograft rejection in the rat: I. Ultrastructural and cytochemical features

Mononuclear phagocytes (MNP) have been identified in rejecting rat cardiac allografts by morphological and cytochemical criteria. Their accumulation has been quantitated and their distribution within the graft recorded. Lymphocytes were the major infiltrating cell type present 3 days after transplantation, but by Day 5 and Day 7 there were 2.5 to 3 times as many MNP as lymphocytes. In the later stages (Days 6 and 7) many MNP were closely adjacent to myocardial cells and frequently possessed pseudopodia which were indenting the myocardial cell membrane. Allograft recipients given 750 rads γ-irradiation and reconstituted with thoracic duct lymphocytes rapidly rejected the graft. As many MNP were present in such grafts as in unmodified recipients. A potent antimacrophage serum did not prolong graft survival or alter the numbers of MNP within rejecting grafts. We conclude that MNP must be considered strong candidates for effector cells in allograft rejection and that satisfactory depletion techniques for MNP are not yet available.     

The combinatorial distance geometry method for the calculation of molecular conformation. II. Sample problems and computational statistics

The performance of a branch and bound algorithm for molecular energy minimization is evaluated on a variety of test problems. Although not at present efficient enough for use in most practical situations, we show that it has distinct advantages over more conventional methods of global minimization. In addition, this study illustrates the technique on which the present algorithm is based, and the problems which must be overcome in developing an efficient algorithm based on similar principles.     

Presence of proenkephalin in chromaffin cells of the frog adrenal gland

Using a specific antiserum to bovine proenkephalin 1–77 (synenkephalin), the distribution of this peptide in the frog adrenal gland has been studied by means of the indirect immunofluorescence technique. Proenkephalin immunoreactivity was found in all chromaffin cells, which also demonstrated enkephalin- and vasoactive intestinal peptide-like immunoreactivity. No nerve endings containing proenkephalin-, enkephalin-, or vasoactive intestinal peptide-like material could be detected. These data suggest a precursor-product mode of biosynthesis for enkephalins in amphibian chromaffin cells. On a phylogenic point of view, they further indicate a high stability of the structure of proenkephalin during the evolution process.     

Different levels of glycosylation contribute to the heterogeneity of alpha 1(II) collagen chains derived from a transplantable rat chondrosarcoma

Three collagen fractions, each of which contain molecules composed of alpha 1(II) chains, have been isolated from pepsin-solubilized rat chondrosarcoma collagen. One fraction could be selectively precipitated from the pepsin digest at 0.7 M NaCl. Two additional fractions were obtained on chromatography of the collagen precipitating at 1.2 M NaCl on carboxymethyl cellulose under nondenaturing conditions. When chromatographed on carboxymethyl cellulose under denaturing conditions, each fraction contained components eluting in the position expected for alpha 1(II) chains. One of the fractions precipitating at 1.2 M NaCl contained the recently described 1 alpha and 2 alpha chains in addition to material eluting as alpha 1(II) chains. Comparison of the chains eluting as alpha 1(II) chains in the various fractions with respect to amino acid composition, carbohydrate content, and cyanogen bromide-cleavage products showed that they differed only in the number of glycosylated hydroxylysyl residues. In this regard, alpha 1(II) chains obtained from collagens precipitated at 1.2 M NaCl exhibited significantly higher levels of glucosylgalactosylhydroxylysyl residues than alpha 1(II) chains precipitated at 0.7 M NaCl. These results indicate that molecules composed of alpha 1(II) chains are heterogeneous with respect to levels of hydroxylysine-linked carbohydrate moieties and that the more highly glycosylated molecules require higher salt concentrations for precipitation from acidic solutions. The data also indicate that a proportion of the more highly glycosylated alpha 1(II) chains are involved in the formation of one or more molecular species with 1 alpha and 2 alpha chains.     

Neutral metal chelator-sensitive protease in insect moulting fluid

Proteolytic activity in moulting fluid from the sphingid Manduca sexta has at least two pH optima; these occur at pH 7 and at pH 7·7. The latter activity is shown to be trypsin-like in that it is susceptible to inhibition by diisopropylfluorophosphate. By contrast, the peak at neutral pH consists of proteolytic activity not hitherto described in invertebrates. This activity shows little or no inhibition with diisopropylfluorophosphate or p-hydroxymercuribenzoate but is strongly inhibited by chelators such as 1,10-phenanthroline, 8-hydroxyquinoline, and EDTA. The neutral metal chelator-sensitive activity requires calcium but the inhibitor data permit the conclusion that the metal ion inhibited by the chelators belongs to the first transition series and thus cannot be calcium. The neutral protease appears to be similar to proteases previously characterized from bacteria and snake venom. In moulting fluid from Manduca, proteolytic activity in vitro is very low in the presence of 1,10-phenanthroline at every pH studied except pH 7·7; in vivo, ecdysis is inhibited in Manduca larvae fed on diet containing a sufficient level (0·02 per cent or higher) of 1,10-phenanthroline. The metal chelator-sensitive proteolytic activity appears to be an essential moulting protease in Manduca.