Interleukin 2 (IL-2) activity during tumor growth: IL-2 production kinetics,absorption of and responses to exogenous IL-2

A temporal study assessed the relationship between fibrosarcoma growth and immunologic encumberance due to the inability of BALB/c mouse splenocytes to elaborate the lymphokine Interleukin 2 (IL-2). Nylon-wool fractionation and antiserum treatments suggested the existence of a mildly nylon-wool-adherent, anti-Lyt 2-sensitive tumor-induced suppressor T (T_s,) cell which significantly decreased IL-2 activity. Absorption investigations indicated that ligand-activated tumor-bearing host (TBH) spleen cells were less receptive to IL-2 than their normal counterparts. When splenocytes were antiserum treated before absorption, removal of Lyt 2⁺ (suppressor T) cells resulted in greater IL-2 absorption by the remaining cells. Purified IL-2 only partially restored suppressed TBH spleen cell mitogen- or alloantigen-induced blastogenesis; whereas, normal host reactivity was significantly augmented. The collective data suggest that TBH spleen cells were capable of producing IL-2 and of responding to the IL-2 amplification signal when tumor-induced T_s cells were depleted.     

Pertussis toxin induces tachycardia and impairs the increase in blood pressure produced by alpha 2-adrenergic agonists

Administration of purified pertussis toxin to rats induced persistent tachycardia, (observed in conscious rats but not after pithing); as little as 0.05 microgram/100 g produced a significant effect. Pertussis toxin-treatment did not affected the pressor response produced in the pithed rats by the alpha 2-adrenergic agonist methoxamine but markedly diminished the pressor effect of the alpha 2-adrenergic agonists clonidine and azepexole. A role of adenylate cyclase inhibition in the action of postsynaptic vascular alpha 2-adrenergic receptors is suggested.     

Characterization of a monoclonal antibody reactive with rabbit T lymphocytes and neutrophils

An IgG1 monoclonal antibody, termed ACM-1, has been shown to react with rabbit T cells, but not Ig+ cells or macrophages. This antibody appears to recognize the same epitope as the previously described 9AE10 antibody and, together with 9AE10, has been used to obtain highly pure and fully functional T- and B-cell populations. However, the relevant epitope does not appear to be homologous to rodent Thy-1 since quantitative absorptions failed to show reactivity with rabbit brain. Furthermore, attempts to obtain in vivo T-cell depletion resulted in larger decreases in white blood cells than would be expected for simple T-cell removal. In vitro assays on enriched neutrophil preparations revealed that 80-95% of these cells were reactive with ACM-1 and 9AE10. Thus, it appears that in the rabbit, T cells and neutrophils share a major epitope.     

Comparison of an iterative microcalorimetric method and dialysis equilibrium for calculating thermodynamic parameters of a binding protein which presents a weak affinity for its substrate

An original iterative microcalorimetric method is used to study the interaction of 5-fluorouracil, a cancer chemotherapeutic agent, with human serum albumin. Two equivalent binding sites are demonstrated with an association constant and an enthalpy variation equal to 370 ± 30 m^?1 and ?10 ± 0,5 kcal/mol, respectively. By means of a competitive microcalorimetric method, the partially competitive binding of dipotassium chlorazepate and 5-fluorouracil is shown. This iterative microcalorimetric method can be applied to all techniques involving the measurement of a phenomenon which is proportional to the concentration of the complex.     

Stimulation of prostaglandin E2(PGE2) secretion by benzylpenicilloyl (BPO) carrier conjugates in human peripheral blood lymphocytes

The conjugates of the main determinant of penicillin, benzylpenicilloyl (BPO), with various protein carriers can stimulate mononuclear cells from human peripheral blood lymphocytes (PBL) to increase PGE₂ secretion. Thus, BPO bound to either human γ-globulin (BPO-HGG), bovine γ-globulin (BPO-BGG), and keyhole limpet menocyanin (BPO-KLH) increased the PGE₂ level 8–20 times above the level produced by stimulation with the carriers alone. Since in previous studies it was shown that preincubation with BPO-HGG can suppress the thymidine uptake after subsequent stimulation with BPO-KLH, we investigated if the induction of this unresponsiveness could be caused by PGE₂. The results of this study show that: (a) although the amount of PGE₂ secreted by the stimulation of BPO-HGG was 20-fold greater than the basic level, this amount is not sufficient to suppress significantly the thymidine uptake; (b) preincubation of PBL with higher than physiological concentration of externally added PGE₂ caused significant suppression of thymidine uptake after stimulation with BPO-KLH, but the addition of physiological concentration of PGE₂ did not show the same effect; (c) preincubation with BPO-HGG had no effect on PGE₂ level after subsequent challenge with BPO-KLH; and (d) inhibitor of PGE fails to influence the suppression of BPO-KLH proliferation induced by BPO-KLH.     

The purification of peptides which contain methionine residues

A novel procedure for isolating peptides which contain methionine is described. It relies upon the reversible increase in charge which occurs upon the alkylation of methionine by iodoacetamide. A digest of the protein is reacted with lodo[¹⁴C]acetamide under conditions which direct the reaction exclusively to the methionine residues. In this way, methionine-containing peptides are rendered radioactive and gain one positive charge per methionine simultaneously. The digest is then separated on a cation exchange column, the peptides are located by their radioactivity, and they are separately collected. The carboxyamidomethylation is reversed by thiolysis, which eliminates the extra positive charge which each methionine-containing peptide bore, decreasing their charge selectively. A second chromatographic separation, performed on the same cation exchange column, is sufficient to produce the desired peptides in a high state of purity. Equine myoglobin and bovine ribonuclease were used as models to demonstrate the feasibility of this approach. Methionine-containing tryptic peptides were purified from digests of these proteins in yields which were equivalent to those of previously reported separations. The present procedure, however, is applicable to peptide mixtures of far greater complexity than those which were derived from the model compounds and can be applied with the same success to digests of very large proteins containing many methionine residues.     

Diphtheria toxin induces leakage of acidic liposomes

Diphtheria toxin (DT) induces the leakage of dipalmitoylphosphatidic acid (DPPA) membranes but not neutral dipalmitoylphosphatidylcholine (DPPC) membranes. Cholesterol incorporated into liposomes enhances the membrane leakage induced by DT in acidic DPPA membranes but not in neutral DPPC membranes. Membrane leakage was determined by assaying the release of TEMPOcholine, a cationic spin probe from the multilamellar vesicles by using electron spin resonance methods. The effect of DT on membrane leakage is noticeable at 3 micrograms/ml concentrations, and reaches a plateau of about 20% leakage at 20 micrograms/ml. This saturation phenomenon led to the postulation that DT binds to the first shell of DPPA membranes and induces the leakage of TEMPOcholine limited to this layer of DPPA multimellar vesicles.     

Isolation and characterization of two species of receptor which bind intrinsic factor

The intrinsic factor receptor from guinea pig ilea has been characterized following purification by affinity chromatography. The purified receptor complexed to intrinsic factor-cobalamin (holo-receptor) had an anhydrous molecular weight of 680,000, a Stokes radius of 10.9 nm, and a sedimentation coefficient of 15.1 S. In contrast, unsaturated receptor (apo-receptor) resolved into distinct "large" and "small" molecular species having, respectively, (i) molecular weights of 700,000 and 350,000; (ii) Stokes radii of 11.1 and 7.06 nm; (iii) sedimentation coefficients of 15.5 S and 11.9 S; (iv) association constants for binding the intrinsic factor-cobalamin complex of 7.3 and 2.5 X 10(10) liters mol-1; and (v) two isoproteins for the larger species (pI's 4.05 and 4.80), and one isoprotein for the smaller species (pI 4.90). A rabbit anti-receptor serum gave only one precipitation line in immunodiffusion against either the large or the small receptor, and each one of these two lines fused completely with the one of two lines which formed with the purified preparation containing both receptor species. Autoradiography of the precipitin lines obtained when the receptor was coupled to intrinsic factor-cyano[57Co]cobalamin demonstrated that both species of receptor were functional. The reaction of complete immunologic identity, the similar electrical properties and similar kinetics for binding to intrinsic factor, and the observed molecular weight differences indicate that the small and large apo-receptors are chemically interrelated, and suggest that the large receptor may consist of two small functional proteins.     

A series of murine interleukin molecules which stimulate both murine and human lymphocytes: I. Production by Phytolacca americana (pokeweed) lectin 2 (Pa-2)-stimulated thymus and thymus-derived cells

Cell-free supernatant fluid, from cultures of Phytolacca americana (pokeweed) lectin 2 (Pa-2)-pulsed murine spleen or thymus cells, contains factors which induce cultured lymphocytes to differentiate into IgM-secreting cells (assayed by a reverse plaque technique) and to proliferate (measured by the incorporation of tritiated thymidine) without the addition of mitogen. The factors in this supernatant fluid responsible for these activities have been designated as lymphocyte stimulating factors (LSF). LSF showed no genetic restrictions related to the major histocompatability complex; LSF made in one strain of mice worked in other strains. Indeed, LSF is not restricted by species barriers; human peripheral blood mononuclear cells were also stimulated by murine LSF to proliferate and differentiate into immunoglobulin-secreting cells without further addition of antigen or mitogen. Maximum production of LSF was achieved within 12 hr of culture and was independent of cell division. In contrast to TRF, no further production of LSF was detectable after 24 hr of culture. Unlike T-cell growth factor, this material stimulated increased mitosis of thymic, T, and B lymphocytes without the addition of mitogen or antigen. LSF also stimulated polyclonal B-cell differentiation into IgM-secreting cells. Maximal numbers of immunoglobulin-secreting cells were generated when LSF was added at the initiation of the culture. Indeed, unlike TRF, LSF needed to be present only during the first 6 hr of culture to achieve maximum stimulation, and did not require the presence of antigen. The production of LSF by a T-cell population in the spleen was shown by two independent methods. Spleen cells treated with anti-Thy 1 plus complement failed to produce detectable levels of LSF. On the other hand, purified populations of surface immunoglobulin-negative spleen cells produced LSF. Furthermore, the subset of thymocytes responsible for LSF production was the small population (approximately 10%) of cells in the thymus, which are not agglutinated by peanut agglutinin.     

Deoxyribonuclease I produces staggered cuts in the DNA of chromatin

The relationship of cuts made by deoxyribonuclease I (DNase I, EC. 3.1.4.5) on the two strands of DNA of chromatin has been investigated. DNA was extracted from a DNase I digest of rat liver nuclei and incubated with the large fragment of DNA polyrnerase I. Analysis of the products of this incubation indicates the cuts made by DNase I on opposite strands are staggered with respect to one another. A cut on one strand is about two bases in the 3′ direction or eight bases in the 5′ direction from the position on its own strand which is directly across from the cut on the other strand. A different result is obtained when a DNase I digest of native DNA is analyzed. Current models for the organization of DNA in the nucleosome are discussed with respect to these results.     

LH biosynthesis and secretion in rat anterior pituitary cell cultures: stimulation of LH glycosylation and secretion by GNRH and an agonistic analogue and blockade by an antagonistic analogue.

In rat anterior pituitary cell cultures GnRH (1nM) stimulated a progressive increase in LH release into the medium from 1 to 8 h of incubation, while cellular LH showed a corresponding decrease. GnRH (1nM) neither modified the uptake nor the incorporation of [³H]-glucosamine and [³H]-proline into total protein. The incorporation of [³H]-proline into cellular LH also was unaffected by GnRH. In contrast, GnRH stimulated a 3 to 4-fold increase in [³H]-glucosamine incorporation into cellular LH. The agonistic analogue, [des GlyNH₂¹⁰]-LHRH ethylamide, mimicked the GnRH effects and was 5 to 6 times more potent than GnRH. The antagonistic analogue, [D-Phe², D-Phe⁶]-LHRH blocked the GnRH-stimulated effects. These results suggest that GnRH and agonistic analogues may preferentially regulate turnover or synthesis of the carbohydrate moiety of LH.     

Prostaglandin F and progesterone secretion by porcine endometrium and corpus luteum in vitro

Slices of porcine endometrium and corpus luteum tissue obtained from mature sows throughout the luteal phase of the oestrous cycle were incubated in culture medium which was analysed at regular intervals over a period of 8 hours for prostaglandin F and progesterone. Prostaglandin F secretion was greatest by endometrium obtained during the mid III to late I luteal stage of the cycle and the increased levels secreted by this tissue were paralleled by high levels of secretion from corpus luteum tissue. The addition of indomethacin (10 μg/ml) to the culture medium completely abolished prostaglandin F secretion by both endometrium and luteal tissue indicating that the high levels of the prostaglandin were due to synthesis. Progesterone secretion by the corpus luteum was maximal from early luteal tissue and had declined to considerably lower levels by late stage tissue when prostaglandin secretion was greatest. The possible physiological significance of luteal prostaglandin F secretion is discussed.     

Fc fragment of human immunoglobulin G: Reduction and carboxymethylation of inter-chain disulfide bonds enhances complement activating capacity

The complement activating capacity of the Fc fragment of human immunoglobulin G increases after selective reduction and carboxymethylation of the inter-chain disulfide bonds. Differences in the circular dichroism spectra of intact and modified fragment suggest that minor conformational changes have occurred. Our results are discussed in relation to the molecular requirements for complement activation.     

Characterization of bovine brain calmodulin-dependent protein phosphatase

Calmodulin-dependent protein phosphatase of bovine brain exhibited a pH optimum of 7 and appeared to require sulfhydryl groups for activity. Phosphatase activity was inhibited by both NaF and ZnCl2, but was stimulated approximately 2-fold by MnCl2. The enzyme exhibited broad substrate specificity, dephosphorylating casein, troponin I, protamine, histone, and phosvitin, and was not phosphorylated by cAMP-dependent protein kinase. With 32P-labeled casein as a substrate, phosphatase was activated 15-fold by calmodulin; the dissociation constant of phosphatase for calmodulin was 30 nM. Activation of the enzyme by calmodulin as a function of Ca2+ was highly cooperative; the Hill coefficient was 4.9. At a saturating concentration of calmodulin, half-maximal activation of phosphatase was obtained at 0.3 microM Ca2+. Calmodulin increased the Vmax from 1.7 to 41 nmol mg protein-1 min-1 with no significant change in its Km. Formation of a Ca2+-dependent complex between calmodulin and the phosphatase was demonstrated by a calmodulin-Sepharose affinity column, gel-filtration chromatography, and sedimentation on a sucrose density gradient. The rate of formation and dissociation of the calmodulin X phosphatase complex was rapid and readily reversible in response to changes in Ca2+ concentration. The calmodulin X phosphatase complex consists of 1 mol of calmodulin and 1 mol of phosphatase.     

Control of insulin secretion in the developing pancreatic rudiment.

The embryonic rat pancreas, removed on the 14th day of gestation and cultivated in vitro, accumulates differentiated levels of exocrine enzymes and insulin. In the period corresponding to days 16–22 in vivo, 99% of the final insulin content accumulates. During this period we have studied the development of competence for insulin secretion, the regulation of this secretion by glucose and other secretatogues, and the rate of synthesis following a secretory challenge. Our results demonstrate that the capacity for insulin secretion develops in parallel with the accumulation of insulin in secretory granules since β granules appear at day 16. On day 16, after 48 hr of culture, both glucose and caffeine are required for detectable insulin secretion. At later stages, insulin release can be effectuated by glucose alone. In the fetal pancreas at day 20 of development, glucose is ten times more efficient than caffeine and fourfold more efficient than caffeine combined with either glucagon, cholera toxin or dibutyryl cyclic AMP. Glucagon, cholera toxin or cyclic AMP in the presence of caffeine increases equally (about tenfold) both the “basal” and the glucose-induced level of secretion. This suggests that glucose and caffeine act independently but synergistically. The integrity of the cells is maintained under the stimulation conditions, and there is a selective increase in insulin synthesis measured during 18 hr following stimulation of insulin release.     

Three-dimensional hair follicular differentiation of a trichilemmoma cell line in vitro

A cell line was established in vitro from a benign hair follicular tumor of human trichilemmoma. Individual and organized cellular differentiation of this cell line was studied. When these cells were cultured for a long time (more than 3 weeks) without subculture, they started to pile up spontaneously. A part of the pile became indented and simultaneously the opposite side of the indentation budded out. The bud slowly elongated 2 to 3 mm in length in 8 to 12 weeks in culture. Light and electron microscopy revealed the internal structure of piles and elongated buds to be a three-dimensional hair follicular structure. The cells in the outermost layer were least mature. These were cuboid in shape and contained glycogen. The cells in the middle layer were more differentiated with a decreased amount of glycogen and an increased number of tonofilaments and desmosomes. The cells in the innermost layer were most differentiated. Cells were flat in shape and highly convoluted. The cell membrane was thickened as observed in cornified cells in vivo. These organized differentiations were also confirmed by histochemical and immunocytochemical studies; using a fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide method, free sulfhydryl groups were detected but disulfide bonds were absent in the early cell culture. Disulfide bonds increased slowly and accumulated in the innermost layer of piles. Accumulation of keratin substances, detected by indirect immunofluorescence method using anti-human keratin antibody, was also observed specifically in the piles. These results suggest that an established cell line of human trichilemmoma spontaneously produced, without stromal influence, hair follicular structures as well as individual cell differentiations in vitro as do trichilemmal (hair follicular) cells in vivo.     

Characterization of ligand-induced states of maize homoserine dehydrogenase

The threonine-sensitive homoserine dehydrogenase (L-homoserine: NAD(P)+ oxido-reductase), isolated from seedlings of Zea mays L., is characterized by variable kinetic and regulatory properties. Previous analysis of this enzyme suggested that it is capable of ligand-mediated interconversions among four kinetically distinct states (S. Krishnaswamy and J. K. Bryan (1983) Arch. Biochem. Biophys. 222, 449-463). These forms of the enzyme have been identified and found to differ in oligomeric configuration and conformation. In the presence of KCl and threonine a rapid equilibrium among three species of the enzyme (B, T, and K) is established. Each of these species can undergo a unique slow transition to a steady-state form under assay conditions. Results obtained from gel-filtration chromatography and sucrose density centrifugation indicate that the B and steady-state forms are tetramers and the T and K states are dimers. Evidence is presented to indicate that the rapid conversion from one dimeric species to the other can only occur via formation of the tetrameric B state. Chromatography under reacting-enzyme conditions provides direct support for the slow formation of a common steady-state species from any one of the other forms of the enzyme. The rate of transition is influenced by threonine, homoserine, NAD+, and, for transitions involving association reactions, by enzyme concentration. Small, reproducible differences in the apparent size of the T and K forms, and the B and steady-state species, are attributed to changes in conformation. This conclusion is supported by differential susceptibility of the enzymic states to proteolytic inactivation, by different rates of inactivation by dithio-bis-nitrobenzoate, and by alterations in their thermal stability. In addition, the B, T, and K states of the enzyme exhibit unique intrinsic fluorescence spectra. Spectral changes are shown to closely parallel changes in kinetic and hysteretic properties of the enzyme. The results of diverse methods of analysis are internally consistent, and provide considerable support for the conclusion that this pleiotropic regulatory enzyme can exist in any of several physically distinct states.