The biologic effects of allogeneic effect factor on T lymphocytes. I. The mitogenic activity and the autonomous induction of cytotoxic T lymphocytes by AEF

Studies presented herein illustrate the capacity of the soluble mediator, allogeneic effect factor (AEF), which is derived from histoincompatible cell interactions, to induce the in vitro differentiation of normal murine splenic lymphocytes into mature cytotoxic cells capable of exerting activity on H-2-identical target cells. This process requires the presence of T lymphocytes during the sensitization phase, and the lytic activity on tumor cells is mediated by cytotoxic T lymphocytes (CTL). The capacity of AEF to induce differentiation of such CTL does not require the presence of stimulating target cells in the sensitization phase. The induction of CTL requires the presence of AEF at the initiation of culture, although exposure to AEF as brief as 1 hr is sufficient to induce fresh spleen cells to differentiate into CTL during the subsequent 5 days in culture. In addition to its ability to induce CTL, AEF is highly mitogenic for T lymphocytes. However, the mitogenic and the CTL-inducing activities of AEF can be experimentally dissociated, indicating that different subpopulations of T lymphocytes may be involved in the response to AEF. In contrast to similar soluble helper factors derived from allogeneic cell interactions, AEF appears to be unique in its ability to autonomously induce a primary CTL response in vitro.     

Isolation and sequence determination of a peptide located in or near the active site of bovine muscle pyruvate kinase

Bovine muscle pyruvate kinase was inactivated by treatment with trinitrobenzenesulfonic acid; approximately one trinitrophenyl group was incorporated per subunit. ADP or Mg-ADP decreased the rate of inactivation but Mg⁺⁺ alone or phosphoenolpyruvate had no effect. The inactivated protein was treated with trypsin and the trinitrophenylated peptide isolated by gel filtration. Homogeneity of the isolated peptide was shown by high voltage electrophoresis and high pressure liquid chromatography. Amino acid analysis and sequence determination revealed the presence of an acidic peptide 34 amino acids long and containing ?-trinitrophenylated lysine.     

Cytochemistry and freeze-fracture of membranes isolated from the electrocyte of Electrophorus electricus (L.)

Summary Membranes were isolated from the main electric organ of Electrophorus electricus and studied by means of cytochemistry and freezefracture. The membrane fractions consisted of vesicles inside-in as determined by localization of anionic sites using colloidal iron and cationized ferritin particles. The anionic sites were not homogeneously distributed on the surface of the vesicle. Freeze-fracture showed the presence of intramembranous particles associated with either protoplasmic (P) or extracellular (E) faces of the membrane. Regions of the membrane without particles were observed. The results are discussed in relation to the existence of association between intramembranous particles and membrane receptors.For all correspondence     

Bovine pyruvate kinase isozymes and hybrid isozymes. Electrophoretic studies and tissue distribution.

Electrophoresis of various bovine tissue extracts revealed, in addition to the three major homotetrameric isozymes of pyruvate kinase (K₄, L₄, and M₄), numerous intermediate bands that behave electrophoretically as hybrid isozymes. Kidney, for example, contains both K-L and K-M hybrid sets. Representative hybrids from each set, tentatively identified as K₂L₂ and K₃M, were isolated from kidney by ionexchange chromatography and their subunit compositions were confirmed by dissociation and subsequent reassociation into new hybrid sets. All of the tissues examined that contain type K₄ also have substantial quantities of K-M hybrids, establishing the presence of the type M isozyme in a great many tissues other than striated muscle and brain, where it is most abundant. In addition, small quantities of K subunits apparently are produced even in striated muscle, which previously had been thought to contain only M₄. The pattern of hybrids and enzyme specific activities differ markedly within tissues from the same organ, as shown by dissection of the heart and great vessels. Aortic smooth muscle has a fairly uniform distribution of K-M hybrids, while cardiac muscle has mostly M₄ with a little KM₃. Connective tissue from heart valves, on the other hand, has a five-membered set dominated by K₃M, while Purkinje fibers have a five-membered set dominated by KM₃. The occurrence of K-M hybrids in these and many other tissues indicates that the distribution of mammalian pyruvate kinase isozymes is much more complex than previously reported.     

Crystal and molecular conformation of the fluorescent probe. 8-anilino-1-naphthalenesulfonic acid (ANS).

The crystal and molecular structure of the fluorescent probe 8-anilino-1-naphthalenesulfonic acid (ANS) has been determined as the ammonium monohydrate with two conformationally distinct molecules in the triclinic $P\overline{1}$ lattice. The angles between the aromatic rings and the CNC plane are ?9/22° and 112/22° respectively. There is an NH...O intramolecular hydrogen bond in each molecule indicating that hydrogen bond formation is not dependent on the anilino geometry. There are also short intramolecular H...H contacts involving the hydrogens which have anomalous proton shifts shown in a recent NMR study.     

Chemoimmunotherapy of advanced breast cancer: prolongation of remission and survival with BCG.

Forty-five patients with disseminated breast cancer were given a trial of combination chemotherapy consisting of fluorouracil, adriamycin, and cyclophosphamide (FAC) and immunotherapy with BCG given by scarification. The results were compared with those in a comparable group of 44 patients treated with FAC alone immediately before the chemoimmunotherapy study. The remission rates (73% and 76% for FAC and FAC-BCG respectively) were similar in both studies. The durations of remission for patients on FAC-BCG (medium 12 months) were longer than remissions achieved for patients given FAC alone (median 8 months) (P = 0.068). The most notable effect of BCG was on survival. Thus 21 out of 34 patients achieving remission on FAC-BCG were alive at the time of the last follow-up examination (median over 22 months) compared with 11 out of 32 patients achieving remission on FAC (median 15 months) (P = 0.01). Twenty-six of the 45 patients given FAC-BCG were alive at the time of the last follow-up examination (median over 22 months) compared with 12 of the 44 patients given FAC (median 15 months) (P = 0.005). Although the apparent benefit of BCG could be explained by a maldistribution of some prognostic factors, the data suggest that further trial of chemoimmunotherapy of breast cancer should be carried out.     

On the fine structure of the electrocyte of Electrophorus electricus L.

Summary The general ultrastructure of the electrocyte, the basic unit of the electric organs of Electrophorus electricus, is analyzed. Presented herein are detailed observations of the syncytial surface, its fibrillar coat, invaginations of the plasma membrane and synaptic terminals. Using Thiéry's method glycogen granules were identified in the syncytial cytoplasm and inside the synaptic terminals, their size and structure being compatible with the muscular origin of the electric organs, to which the filamentous meshwork found in the cytoplasm may be related. Among the perinuclear-organelles, are dense bodies with crystalline patterns. The mitochondrial matrix contains dense granules, their size and structure varying according to the organ to which they belong and to the fixation method used.This work has been supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Conselho de Ensino para Graduados da UFRJ and Banco Nacional de Desenvolvimento Econômico, FUNTEC-241     

The reversibility of skeletal muscle pyruvate kinase and an assessment of its capacity to support glyconeogenesis.

The kinetics of pyruvate phosphorylation by rabbit skeletal muscle pyruvate kinase (EC 2.7.1.40) has been studied with a coupled assay using P-enolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (EC 1.1.1.37). The reaction sequence is (See journal for formula). Although the equilibrium of the pyruvate kinase reaction by itself strongly favors pyruvate production, the over-all equilibrium of this coupled system favors the depletion of pyruvate, thus greatly reducing the problem of back reaction during the assay. In addition, the oxidation of NADH by malate dehydrogenase makes it possible to monitor the system with a spectrophotometer. The Michaelis constant of pyruvate kinase was found to be 0.9 mM for ATP and 7 mM for pyruvate, values that agree reasonably well with earlier studies using direct assays. However, the maximum velocity is about 6 mumol of pyruvate phosphorylated/min/mg of enzyme, which is very much faster than that indicated by earlier studies. These results suggest that the metabolic significance of the reverse reaction of muscle pyruvate kinase may have been underestimated. In particular, the data given here suggest that its rate in vivo is probably comparable to the observed rate of glycogen synthesis from lactate, making possible glyconeogenesis in muscle by pyruvate kinase reversal without the need for an enzymatic bypass of the kind employed by liver and kidney.     

Kinetic properties of pyruvate kinase hybrids formed with native type L and inactivated type M subunits.

Bovine type M pyruvate kinase, which normally has hyperbolic kinetics with its substrates, was inactivated by treatment with trinitrobenzenesulfonic acid. The inactivation probably occurs through trinitrophenylation of the epsilon-amino group of a lysine residue in or near the ADP binding site. Although 90 to 95% of the enzymatic activity is lost by this treatment, the molecular weight and sedimentation coefficient of the trinitrophenylated enzyme are quite similar to values obtained with the native enzyme. The inactivated, trinitrophenylated type M pyruvate kinase was hybridized in vitro with the native bovine type L enzyme, which has sigmoidal kinetics with phosphoenolpyruvate but can be activated by fructose 1,6-diphosphate to give hyperbolic kinetics. Four enzymatically active species were produced, designated L4, L3M, L2M2, and LM3, according to their subunit composition. L4 and L3M have sigmoidal kinetics with phosphoenolpyruvate and are activated by fructose diphosphate. Little or no sigmoidicity was seen for L2M2, although this species is activated to a moderate degree by fructose diphosphate. LM3 appears to have hyperbolic kinetics and is activated only slightly by fructose diphosphate. The kinetic results obtained with hybrids containing trinitrophenylated type M subunits are quite similar to the results previously reported by Dyson and Cardenas ((1973) J. Biol. Chem. 248, 8482-8488) using native type M and type L subunits, indicating that the properties of a type L subunit are profoundly affected by the nature of the other subunits present in the tetramer. In fact, type L and type M subunits in a given hybrid seem to have similar kinetic responses toward phosphoenolpyruvate and fructose diphosphate.     

PYRUVATE KINASE ISOZYMES IN NEURONS, GLIA, NEUROBLASTOMA, AND GLIOBLASTOMA

Abstract– The distribution of pyruvate kinase isozymes (EC 2.7.1.40) was examined in cells and tissues from the central and peripheral nervous system of the rat. Most tissues contain significant quantities of both the K₄ (fetal type) and M₄ (skeletal muscle type) isozymes plus tetrameric hybrids comprised of various combination of the type M and type K subunits. Retina, for example, contains a five-mem-bered hybrid set weighted toward K₄, while sciatic nerve and spinal cord have patterns very similar to that of adult brain, consisting predominantly of M₄ with small amounts of K₄ and K-M hybrids. This adult pattern is achieved by a gradual shift from a hybrid set dominated by K₄ in fetal life, to the pattern at birth at which time the two most prominent bands were M₄ and K₂M₂, and finally to the adult pattern by about 28 days after birth. Neurons and glial cells were isolated from rat and mouse brains at the various developmental levels. The pyruvate kinase isozyme patterns in the two cell types were similar to each other and to the patterns seen in whole brain homogenates at all ages, indicating similar rates of isozymic maturation in the two cell types. The correlation of maturation with pyruvate kinase isozyme patterns was further tested in cultures of malignant cell lines. A K-M hybrid set, weighted toward K₄, was seen in two clonal lines of mouse neuroblastoma under normal culture conditions. However, lowering the serum concentration in the culture medium or adding bromodeoxyuridine caused a shift in the patterns toward type M as the cells differentiated, mimicking in part the in vivo maturation of normal cells. On the other hand, a rapidly growing and poorly differentiated line of rat glioblastoma had only K₄ under all conditions examined.