Immunoadjuvant activity of pyran copolymer: I. Evidence for direct stimulation of T-lymphocytes and macrophages

A single injection of pyran copolymer has been shown to greatly increase the number of hemolytic plaque forming cells to sheep erythrocytes (sRBC). Pyran given from 1 day before to 2 days after sRBC inoculation increased both specific activity and plaques/spleen, suggesting that macrophage activation was probably not responsible for the enhancement seen. In addition, pyran given 1 day prior to the primary injection of sRBC was found to increase the secondary response to SRBC given alone. As similar experiments using thymectomized irradiated bone marrow reconstituted mice showed no increase in specific activity following pyran administration, it was unlikely that pyran was acting directly on B cells. Furthermore, experiments measuring the antibody response to Escherichia coli lipopolysaccharide, a thymic independent antigen, pyran did not increase the response to this antigen. In contrast to the above, pyran delayed and depressed cell mediated cytotoxicity to the allogeneic DBA/2 P815 mastocytoma. However, no difference in the titers of cytotoxic antibody against mastocytoma cells was seen between pyran-treated and normal animals. Pyran was mitogenic for spleen cells in vitro. However, following the administration of pyran in vivo, mitogen induced blastogenesis in vitro to PHA and LPS was inhibited and this inhibition was determined to be macrophage-dependent. These results are consistent with a model in which the immunoregulatory effects of pyran act through macrophages and T-lymphocytes.     

Stimulation of gastrin secretion from the perfused rat stomach by somatostatin antiserum

The effect of a high capacity somatostatin antiserum on antral gastrin secretion was examined in an isolated vascularly perfused rat stomach preparation. Infusion of somatostatin antiserum diluted 1:1 and 1:9 with Krebs buffer solution produced significant increases in gastrin secretion throughout the period of infusion. Neither infusion of somatostatin antiserum diluted 1:99 nor infusion of control rabbit serum had any effect on gastrin secretion. The data indicate that antral somatostatin excercises a continous restraint on gastrin secretion in the basal state.     

Comparison of antiviral and antitumor activity of activated macrophages.

The antiviral and antitumor activity in vitro of normal, stimulated, vaccinia virus “immune”, and activated peritoneal macrophages were compared. Activated (pyran or corynebacteria induced) PEC exhibited both antitumor and antiviral activity. Stimulated (thioglyocollate) and vaccinia virus “immune” PEC inhibited virus growth but did not possess antitumor activity. Normal (unstimulated) PEC were relatively ineffective in either activity. The antiviral activity was nonspecific, being expressed against herpes simplex and EMC viruses in addition to vaccinia. Although a possible role for interferon was suggested by the lack of activity of mouse PEC on vaccinia virus growth in heterologous FLK cells, definitive proof was not obtained. The activity was most pronounced against multiple cycles of viral infection initiated at a low multiplicity of infection. Single cycle virus growth was not affected, suggesting that the major inhibition was on subsequent cycles of virus growth.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP: I. Antigen-induced uptake of nucleotides and nucleosides by lymphocytes

Addition of N⁶, O²′-dibutyryl cAMP (DbcAMP) to keyhole limpet hemocyanin (KLH)-primed rabbit lymph node cells for 1 hr, followed by its removal and the addition of KLH, had no effect on the subsequent antibody response, whereas addition of KLH for 1 hr followed by DbcAMP resulted in a 100% enhancement of antibody synthesis. Addition of cholera enterotoxin (CT), which rapidly and irreversibly binds to lymphocytes and activates adenylate cyclase, either before or after the addition of antigen, elevated the antibody response by 100%. These results suggested that some antigen-induced event(s) may be required for DbcAMP to exert its enhancing effects on the antibody response. The effect of KLH on the uptake of DbcAMP by KLH-primed lymph node cells was investigated. One and one hundred micrograms of KLH, which induce optimal and supraoptimal antibody synthesis, respectively, promoted maximal uptake of DbcAMP. This induced uptake was first detectable about 12 hr after addition of KLH, and it peaked during 24–48 hr of culture. DbcAMP uptake induced by a brief exposure of KLH (0–1 hr) was equivalent to that observed with long-term KLH addition (0–24 hr). KLH-induced DbcAMP uptake required KLH-reactive lymphocytes and represented active transport. Antibody to rabbit T lymphocytes inhibited this antigen-induced uptake. The mitogens concanavalin A (Con A) (T cells) and goat anti-rabbit Fab' (anti-Fab') (B cells) also stimulated DbcAMP uptake, as did human serum albumin (HSA) and myoglobulin (Mb) when added to homologously primed cells, indicating the generality of the phenomenon. [³H]DbcAMP entered the cells as di- or monobutyryl cAMP with about 40% metabolized to 5′AMP. This uptake could be competitively inhibited by other adenine or guanine nucleotides and nucleosides.     

Mechanism for the differential induction of mutation by S9 activated benzo[a]pyrene employing either a glucose-6-phosphate-dependent NADPH-regenerating system or an isocitrate-dependent system

A significant difference in mutation frequency has been observed in CHO cells exposed to benzo[a]pyrene with alternative activation systems. Each system employed rat-liver S9 homogenate with one using isocitrate dehydrogenase to provide reduced NADP, while the other method uses glucose-6-phosphate dehydrogenase. Total aryl hydrocarbon hydroxylase (AHH) activity was greater for the isocitrate dependent system, however, this yielded a lower level of HGPRT mutants. It was ascertained that this reduced mutation frequency may result from sequestering of B[a]P substrate by crystals in the medium, possibly calcium phosphate, which decreased the effective substrate concentration. This sequestration enhances B[a]P internalization, which would explain the dichotomy between the AHH values and the mutation frequency data. The production of specific B[a]P metabolites was also examined by reverse phase HPLC quantitation of extracts of solutions in which the two activation systems were used. The levels of 7,8 dihydroxybenzo[a]pyrene produced by the glucose-6-phosphate protocol were consistently greater than with isocitrate. This may also be a contributing mechanism for elevating the mutation frequency with this procedure. These results demonstrate several interactions between test compound, cells, and metabolizing system which must be considered with in vitro activation systems.     

Rapid separation of low molecular weight solutes from liposomes without dilution

Liposomes can be separated from low molecular weight solutes on minicolumns of Sephadex G-50 made from the barrels of 1- or 5-ml plastic syringes. Excess fluid is first removed from the Sephadex beads by centrifugation and a mixture of liposomal entrapped and free solute is applied to the column bed. The centrifugation is repeated forcing the liposomal material through the column into a test tube while the free solute is quantitatively retained in the Sephadex. The procedure is applicable to a variety of solutes and 92 to 100% recovery is achieved for both charged and neutral liposomes. This technique has advantages over other methods for separating extraliposomal solutes from liposomes. Numerous samples can be processed simultaneously within minutes with no dilution of the liposomal preparation. Nonentrapped solute within the Sephadex can be easily recovered in a small volume of water or buffer.     

Macrophage heterogeneity in tumor resistance: Cytostatic and cytotoxic activity of corynebacterium parvum-activated and proteose peptone-elicited rat macrophages against Moloney sarcoma tumor cells

Peritoneal macrophages from proteose peptone and Corynebacterium parvum (CP)-treated Lewis and Brown Norway rats were separated into subpopulations by centrifugation on discontinuous gradients of Ficoll. Four macrophage subpopulations were prepared and tested for cytostatic and cytotoxic activity against syngeneic and allogeneic Moloney sarcoma tumor cells. Macrophages were cocultured with tumor cells for 48 hr, whereupon either the inhibition of [¹²⁵I]iododeoxyuridine uptake was measured (cytostasis) or the tumor monolayers were observed for cytotoxic effects. CP-Activated macrophages from heavy-density portions of the gradient (8–10% and 10%-pellet) were highly cytostatic and cytotoxic to both the syngeneic and allogeneic tumor cells while macrophages from the light-density portions (4–6 and 6–8% Ficoll bands) were not. Proteose peptone-stimulated macrophages from the heavy-density portions of the gradient were cytostatic but not cytotoxic to the tumor cells. The effector macrophages from the CP-activated pool were large, well-differentiated cells as determined by electron microscopic examinations and had enhanced phagocytic activity when contrasted with the noncytotoxic, less dense macrophages.     

Discriminative stimulus properties of stereoisomers of cyclazocine in phencyclidine-trained squirrel monkeys

Squirrel monkeys were trained to discriminate 0.16 mg/kg phencyclidine (PCP) from saline in a two-layer drug discrimination task on a fixed-ratio 32 schedule of food presentation. After reliable discriminative control of lever choice was established, dose-response determinations for generalization to the training dose of PCP were made with several doses of PCP, a racemic mixture of cyclazocine and the pure (+)- and (-)-isomers of cyclazocine. Only PCP and the (+)-isomer produced dose-dependent PCP-appropriate responding. Neither the racemic mixture nor (-)-cyclazocine produced over 25% PCP-appropriate responding at any of the doses tested. (+)-Cyclazocine was eight times less potent than PCP in producing drug-lever appropriate responding. (-)-Cyclazocine was about 30 times more potent than PCP and over 200 times more potent than (+)-cyclazocine in overall response rate suppression. The potency of the racemic mixture for response-rate suppression was consistent with an additive effect of the isomers. Neither the PCP-lever appropriate responding produced by (+)-cyclazocine nor the response-rate suppression produced by (-)-cyclazocine were antagonized by naloxone. Thus, racemic cyclazocine is composed of two isomers with differing behavioral effects. The (-)-isomer is more potent, and the (+)-isomer has more specificity for PCP-like effects.     

Production of lysophospholipids and free fatty acids by a sarcolemmal fraction from canine myocardium.

The potential for injury of myocardial sarcolemma by endogenous lipases was studied. The sarcolemmal fraction was incubated for 30 min under conditions found optimal for hydrolysis of exogenous phosphatidylethanolamine (5 mM calcium, pH 7.0, 37°C). Incubation of the sarcolemmal fraction increased significantly the level of total free fatty acids (14.1 to 31.1 nmoles/mg protein, P < 0.001); in addition, production of arachidonic acid was increased significantly (P < 0.01). Lysophosphatidylcholine was increased significantly (P < 0.01) but the content of lysophosphatidylethanolamine was unchanged. A large proportion of the above free fatty acids (10.2 nmoles/mg protein) was derived from the hydrolysis of triacylglycerols. These data demonstrate that the sarcolemmal fraction preferentially hydrolyses endogenous membrane phosphatidylcholine at neutral pH in the presence of calcium with the formation of lysophosphatidylcholine and free fatty acids including arachidonic acid.     

Intracellular turnover of stable and labile soluble liver proteins

The cytosol proteins of mouse or rat liver were separated on the basis of charge and characterized with respect to molecular size, turnover in vivo, and several chemical properties. The basic proteins, which were synthesized and degraded slower than acidic proteins, were generally smaller, as multimers and subunits, than the acidic proteins. Charge and size, therefore, did not appear to be independent characteristics for soluble liver proteins. The amino acid composition of the smaller, basic, stable proteins was very similar to that of the larger, acidic, labile proteins. The charge differences between these two protein fractions could be attributed to the ratio of acidic to basic amino acid residues rather than to carbohydrate, nucleic acid, or phosphate content. The percentage of hydrophobic amino acid residues in the two protein fractions was the same. The acidic labile proteins were more vulnerable to proteolysis and associated with the lysosomal-mitochondrial fractions somewhat more extensively in vitro than the basic, stable proteins. These studies indicate that the information determining the half-lives of soluble enzymes is in the protein moiety of those enzymes and that the factors that correlate with turnover may not be independent variables for soluble mammalian proteins.     

SCE evaluations in human lymphocytes after G0 exposure to mitomycin C Lack of expression of MMC-induced SCEs in cells that have undergone greater than two in vitro divisions

We conducted a series of experiments designed to determine whether DNA damage induced in G₀ lymphocytes by mitomycin C (MMC) would be expressed as sister-chromatid exchanges during the second and third post-treatment cell cycles. Lymphocytes from normal donors were exposed to MMC for 2 h prior to culture in the presence of phytohemagglutinin. MMC-treated and control cells were subsequently exposed to bromodeoxyuridine (BrdUrd) for the entire culture period (i.e. 48 h or 72 h) or for the terminal 24 h of 72-h cultures. We observed a 3–4-fold increase in SCEs in MII metaphases from lymphocytes treated with MMC and cultured in the presence of BrdUrd for the entire culture period. In contrast, in replicate cultures of MMC-treated lymphocytes that were exposed to BrdUrd for the terminal 24 h only, the SCE frequency in uniformly harlequinized metaphases was not significantly different from that observed in control cultures. We interpret these data as providing evidence that MMC-induced lesions (or alterations) in the DNA of G₀ lymphocytes are probably expressed as SCEs during the first period of mitogeninduced DNA synthesis, and that these lesions do not persist and give rise to SCEs in subsequent cell divisions.     

Human cardiac beta-adrenergic receptors: subtype heterogeneity delineated by direct radioligand binding

Human myocardial beta-adrenergic receptors were directly identified and characterized using the high affinity antagonist radioligand [125I]iodocyanopindolol. Beta 1 and beta 2 adrenergic receptors were found to coexist in both the left ventricle and right atrium. The relative proportions of the two receptor subtypes were determined by the use of competition radioligand binding and computer modelling techniques employing the subtype selective agents atenolol (beta 1 selective) and zinterol (beta 2 selective). The left ventricle contains 86 +/- 1% beta 1 and 14 +/- 1% beta 2 adrenergic receptors while the right atrium contains 74 +/- 6% beta 1 and 26 +/- 6% beta 2 adrenergic receptors. The direct demonstration of beta 2 adrenergic receptors in the human heart, with a higher proportion in the right atrium agrees with pharmacologic data and supports the notion that chronotropic effects of adrenergic agonists in man may be mediated by both beta 1 and beta 2 adrenergic receptors.     

pH-Sensitive mutagenic activity of ozone-treated 1,2-dimethylhydrazine in the Salmonella/microsome assay

Treatment with ozone inactivates the mutagenicity of many carcinogens in aqueous solution. The colon carcinogen, 1,2-dimethylhydrazine (DMH) has been reported an exception; ozone treatment convenrts dimethylhydrazine from a non-mutagen into a mutagen. In the Salmonella/microsone assay, the mutagenicity of ozone-treated dimethylhydrazine was dependent on pH. The ozonation product was a strong mutagen in acidic but was not mutagenic in basic solution. The mutagenicity of the acidic ozonation product was inactivated by raising the pH of the solution. Unlike untreated dimethylhydrazine, its ozonation product in basic solution was not converted to a mutagen in this ozone-low pH system.     

Deuterium oxide effects on excitation-contraction coupling of skeletal muscle

²H₂O (99.8%) Ringer's solution greatly reduces the twitch and tetanus of frog sartorius muscle and, as specially shown here, slows the onset features of the mechanical output of the twitch by: (a) increasing the time (L_R) from stimulus to start of latency relaxation; (b) slowing the developmet of the latency relaxation, and (c) greatly decreasing the rate of onset of tension development. These changes reflect effects of ²H₂O on excitation-contraction coupling and they represent the critical direct effects of ²H₂O on muscle since it does not depress either the action potential or the intrinsic myofibrillar contractility. The increase in L_R is attributed to slowed inward electrical propagation in the T-tubule. But the critical effect of ²H₂O on frog muscle is to greatly depress mobilization of activator Ca²⁺. The depression of the Ca²⁺ mobilization and of its effects on the activation of contraction evidently result from (a) a lowered rate of release of Ca²⁺ from the sarcoplasmic reticulum, as indicated by the slowed development of the latency relaxation, (b) a decreased amount of Ca²⁺ released in a twitch, and (c) a reduced speed of diffusion of the Ca²⁺ to the contractile filaments. The depressed mobilization of Ca²⁺ is apparently the essential cause of ²H₂O's general depression of twitch and tetanus output.     

Hyperpigmented patches in the skin of the newt Notophthalmus viridescens

In the integument of the red-spotted newt there occasionally appear patches of skin which are at the same time melanistic and iridescent. Such hyperpigmented patches have been found on the back, on the tail and on the dorsal surface of both fore and hind limbs. Cytological examination of several such areas revealed the presence of large numbers of chromatophores distributed throughout the dermis. The majority of the chromatophores consisted of atypically large and dendritic melanophores, which contained typical pigment granules. The iridescence resulted from a high incidence of iridophores. Xanthophores also were found in considerable abundance. This extensive and apparently random intermingling of melanophores, iridophores and xanthophores in limited areas constitutes a striking exception to the usual distributional patterns of pigment cells in this animal.     

Storage-excretion of metallic cations in the adult housefly, Musca domestica

The objective of this investigation was to elucidate further the phenomenon of storage-excretion of metallic cations in the housefly. The sites for deposition of zinc, calcium and copper have been identified in this study. The metal intake of the flies was altered by raising one group on sucrose and tap water while in the experimental groups sucrose was supplemented with either 0.05% zinc sulphate, 0.05% calcium phosphate or 0.03% copper sulphate. There were no significant differences in the average life spans of the flies in different groups indicating physiological tolerance to the higher mineral intake. The Malpighian tubules, the midgut and the remainder of the body were analyzed for mineral content in houseflies ranging from 1 to 25 days posteclosion by atomic absorption spectrophotometry. There was a progressive age-associated increase in the total metal content of the flies with age. Zinc and calcium were primarily stored in the Malpighian tubules whereas copper was sequestered in the midgut. Microscopic examination of the epithelial cells of the Malpighian tubules and the midgut revealed a corresponding age-associated increase in mineralized concretions.     

Myocardial infarction in the guinea pig: cellular electrophysiology

The cellular electrophysiology of left ventricular preparations from guinea pig hearts was studied 1 hour, 24 hours, and 4-6 weeks after myocardial infarction produced by 6-8 single ties of the distal left coronary artery system or after sham operation. Microelectrode recordings were used to monitor cells from the endocardial surface of each preparation in tissue bath. All coronary ligated preparations displayed accelerated spontaneous activity compared to normal and sham operated preparations. Single and multiple premature ventricular depolarizations occurred frequently in coronary ligated and rarely in normal and sham operated preparations. Premature stimuli delivered to areas overlying and bordering the area of infarction, induced short bursts of self-terminating rapid repetitive ventricular activity in 4 of 8 (50%) acute (1-hour), 5 of 9 (55%) subacute (24-hour), and 14 of 20 (70%) healed (4-6-week) infarcted preparations. Such activity could not be induced in normal and sham operated preparations. The preparations with healed infarction were unique in that they demonstrated runs of self-terminating repetitive ventricular activity which occurred spontaneously or was inducible with premature stimulation. Recordings from multiple sites in acute, subacute, and healed preparations revealed a variety of transmembrane action potential abnormalities (i.e., reduced action potential amplitude and resting potential, decreased and increased action potential duration, and depressed maximum rates of phase 0 depolarization) in cells overlying and bordering areas of infarction. Only Purkinje fiber action potentials were recorded over the healed infarcts. These data demonstrate that a spectrum of electrophysiological alterations occur in response to ischemic injury and persist after healing of the injury in this new model of myocardial infarction utilizing the guinea pig.     

Endocrine events during pre-diapause and non-diapause larval-pupal development of the tobacco hornworm, Manduca sexta

The haemolymph ecdysteroid titre and in vitro capacities of prothoracic glands and corpora allata to synthesize ecdysone and juvenile hormone, respectively, during the last-larval instar of diapause-destined (short-day) and non-diapause-destined (long-day) Manduca sexta were investigated. In general, the ecdysteroid titres for both populations of larvae were the same and exhibited the two peaks characteristic of the haemolymph titre during this developmental stage in Manduca. The only difference in the titre occurred between day 7 plus 12 h and day 7 plus 20 h, when the short-day larval titre did not decrease as quickly as the long-day titre. The in vitro synthesis of ecdysone by prothoracic glands of short- and long-day larvae during the pharate pupal phase of the instar were also essentially the same. Activity fluctuated at times which would support the idea that ecdysone synthesis by the glands is a major contributing factor to the changes in the haemolymph ecdysteroid titre. There was one subtle difference in prothoracic gland activity between the two populations, occurring on day 7 plus 2 h. By day 7 plus 10 h, however, rates of ecdysone synthesis by the short- and long-day glands were comparable. This elevated activity of the short-day glands occurred just prior to the period the haemolymph ecdysteroid titre remained elevated in these larvae. The capacities of corpora allata to synthesize juvenile hormone I and III in vitro were not markedly different in long- and short-day last-instar larvae. At the time of prothoracicotropic hormone release in the early pupa, activity of corpora allata from short- and long-day reared animals was low and also essentially the same. There were a few differences in the levels of synthesis at isolated times, but they were not consistent for both homologues. Overall, there are no compelling differences in the fluctuations of ecdysteroids and juvenile hormones between diapause-destined and non-diapause-destined Manduca larvae. Since these hormones do not appear to play any obviously significant role in the induction of pupal diapause in this insect, the photoperiodic induction of diapause in Manduca appears to be a predominantly brain-centred phenomenon not involving endocrine effectors.     

Application of the Hodgkin-Huxley equations to an electric field model for interaction between excitable cells

We previously described a model for the electrical transfer of excitation from one cell to the next which utilized the electric potential generated in the junctional cleft between the cells. Low-resistance connections between the cells were not used in the model, and it was assumed that the junctional membranes were excitable. This model was analyzed for the static case without capacitances and for the dynamic case in which capacitances were part of the circuit elements. For simplicity, the Na⁺ resistance (R_Na), after a threshold potential was exceeded, was allowed to decrease exponentially (to 1% of its initial value) within 0·25–1·0 ms, and possible changes in the K⁺ resistance were ignored. In this paper, we have incorporated the Hodgkin-Huxley equations into the operation of the lumped membrane units for the electrical equivalent circuit of the cell membrane. The parameters varied are the membrane capacitances, resistances, maximum Na⁺ conductance ($\text{g}{}_{\mathrm{<mtext>Na</mtext>}}$), and the radial cleft resistance (R_jc). We demonstrated that our model worked very well, i.e. the successful transfer of action potentials was achieved, with the membrane units following Hodgkin-Huxley dynamics for changes in g_Na and g_K. The calculations indicate that transmission is facilitated when the junctional units have a higher $\text{g}{}_{\mathrm{<mtext>Na</mtext>}}$ and a lower capacitance and when R_jc is elevated. Lowering the resistance of the junctional membrane units several fold, relative to the surface membrane units, also facilitated transmission; however, the absolute resistance of the junctional membrane was still well above the maximum value that would allow sufficient local-circuit current to flow to effect transmission. Thus, the electric field model provides an alternative means of cell-to-cell propagation between myocardial cells which is electrical in nature but does not require the presence of low-resistance connections between cells.