Investigation of variation in phenotype and DNA content between single-conidium isolates of single Penicillium strains

Variation in phenotypic properties was examined in three strains of closely related fasciculate species of Penicillium using 114 morphological, physiological, and biochemical characters. Thirty-six of these characters showed variation within single-conidium isolates of the same strain. Conidial sizes and nuclear DNA contents were compared using flow microfluorimetry; these results suggested that significant differences in conidial DNA content are associated with phenotypic variation. The taxonomic significance of the results is discussed.     

Lymphokine-activated killer (LAK) cells. III. Characterization of LAK precursors and susceptible target cells within the murine thymus

In our search for a biologic role for lymphokine-activated killers (LAK), we examined their generation from murine thymocytes. Normal adult thymocytes were capable of generating LAK upon culture with relatively large doses (500 to 1000 U/ml) of interleukin 2. Normal thymocytes were fractionated into four subsets by virtue of their co-expression of the Lyt-2 and L3T4 markers: Lyt-2+ L3T4+ (2+4+); 2+4-; 2-4+; and 2-4-. None of these subsets had any natural killer activity. Upon examining the ability of these subsets to generate LAK, it was found that the 2-4- subset was the most potent and required the smallest relative amounts of interleukin 2. In addition to lysing tumor cells, thymus-derived LAK were capable of killing "fresh" 2+4+ thymocytes. Fresh 2+4-, 2-4+, 2-4-, and cortisone-resistant thymocytes were resistant to lysis by LAK. Upon mitogen stimulation, however, the cortisone-resistant thymocytes and 2+4- thymocytes became LAK-susceptible. These data demonstrate a possible mechanism for the elimination of the 2+4+ thymocyte subset which is generally believed to be a "dead-end" population. Moreover, these data suggest a possible biologic role for LAK in the process of thymocyte maturation and intrathymic selection.     

L-arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells

L-Arginine is required for expression of the activated macrophage cytotoxic effector mechanism that causes inhibition of mitochondrial respiration, aconitase activity, and DNA synthesis in tumor target cells. This effector mechanism is active in the presence of L-arginine even when the cocultivation medium lacks all other amino acids and serum. Cytotoxic activated macrophage-induced inhibition of mitochondrial respiration in target cells is proportional to the concentration of L-arginine in the medium. L-Arginine must be present during the cocultivation period. Pretreatment of cytotoxic activated macrophages with L-arginine or posttreatment of the target cells after cocultivation is not effective. D-Arginine does not substitute for L-arginine and at high concentrations is a competitive inhibitor of the L-arginine-dependent effector mechanism. Other analogues that could not replace L-arginine include agmatine, argininic acid, arginine hydroxamate, and tosyl-L-arginine methyl ester. L-homoarginine, however, can effectively substitute for L-arginine. NG-monomethyl-L-arginine is a potent competitive inhibitor of this effector mechanism. High concentrations of lipopolysaccharide do not reverse inhibition of the L-arginine-dependent effector mechanism by NG-monomethyl-L-arginine. However, inhibition of the effector mechanism by NG-monomethyl-L-arginine can be overridden by increasing the concentration of L-arginine in the culture medium. We compared NGNG-dimethyl-L-arginine and NGN1G-dimethyl-L-arginine with NG-monomethyl-L-arginine as inhibitors of the L-arginine-dependent effector mechanism. The results show that the inhibitory effect of these guanidino methylated derivatives of L-arginine is highly determined by structure. Guanidine is a weak competitive inhibitor of the L-arginine-dependent effector mechanism. The requirement for L-arginine does not appear to be for protein synthesis, creatine biosynthesis, polyamine biosynthesis, or ADP ribosylation reactions. Bacterial lipopolysaccharide is effective as a second signal only when the cocultivation medium contains L-arginine, and this strict L-arginine dependency is not overridden by increasing the concentration of lipopolysaccharide. Bovine liver arginase, by competing for L-arginine in the cocultivation medium, inhibits the L-arginine-dependent activated macrophage cytotoxic effector mechanism.     

Myasthenogenicity of human acetylcholine receptor synthetic alpha-subunit peptide 125-147 does not require intramolecular disulfide cyclization

This study reports the synthesis of a disulfide-looped peptide corresponding to residues 125-147 (Cys 128-Cys 142) of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle, H alpha 125-147 (Lys-Ser-Tyr-Cys-Glu-Ile-Ile-Val-Thr-His-Phe-Pro-Phe-Asp-Glu-Gln- Asn-Cys-Ser-Nle-Lys Leu-Gly), and a nondisulfide-looped analogue, H alpha 125-147(S) (Lys-Ser-Tyr-Ser-Glu-Ile-Ile-Val-Thr-His-Phe-Pro-Phe-Asp-Glu- Gln-Asn-Cys-Ser-Nle-Lys-Leu-Gly), in which the amino acid Cys 128 was replaced with serine. Both peptides induced antigen-specific helper T cell responses, as evidenced in vitro by lymph node cell proliferation and in vivo by production of anti-AChR antibodies. Rats immunized with 100 micrograms of either synthetic peptide, without conjugation to a carrier, produced anti-peptide antibodies which bound to native AChR in immunoprecipitation assays and induced modulation of membrane-bound AChR from cultured human myotubes. Both peptides also induced electrophysiologic and biochemical signs of experimental autoimmune myasthenia gravis. Thus, region 125-147 of the AChR alpha-subunit is at least partly exposed extracellularly in human muscle and contains one or more autoantigenic sites capable of stimulating T cells and B cells. Disulfide-linkage between residues Cys 128 and Cys 142 is not essential for myasthenogenicity.     

Contribution of solvent drag through intercellular junctions to absorption of nutrients by the small intestine of the rat

Summary The lumen of the small intestine in anesthetized rats was recirculated with 50 ml perfusion fluid containing normal salts, 25mm glucose and low concentrations of hydrophilic solutes ranging in size from creatinine (mol wt 113) to Inulin (mol wt 5500). Ferrocyanide, a nontoxic, quadrupally charged anion was not absorbed; it could therefore be used as an osmotically active solute with reflection coefficient of 1.0 to adjust rates of fluid absorption,J _v , and to measure the coefficient of osmotic flow,L _p . The clearances from the perfusion fluid of all other test solutes were approximately proportional toJ _v . FromL _p and rates of clearances as a function ofJ _v and molecular size we estimate (a) the fraction of fluid absorption which passes paracellularly (approx. 50%), (b) coefficients of solvent drag of various solutes within intercellular junctions, (c) the equivalent pore radius of intercellular junctions (50 Å) and their cross sectional area per unit path length (4.3 cm per cm length of intestine). Glucose absorption also varied as a function ofJ _v . From this relationship and the clearances of inert markers we calculate the rate of active transport of glucose, the amount of glucose carried paracellularly by solvent drag or back-diffusion at any givenJ _v and luminal glucose concentration and the concentration of glucose in the absorbate. The results indicate that solvent drag through paracellular channels is the principal route for intestinal transport of glucose or amino acids at physiological rates of fluid absorption and concentration. In the absence of luminal glucose the rate of fluid absorption and the clearances of all inert hydrophilic solutes were greatly reduced. It is proposed that Na-coupled transport of organic solutes from lumen to intercellular spaces provides the principal osmotic force for fluid absorption and triggers widening of intercellular junctions, thus promoting bulk absorption of nutrients by solvent drag. Further evidence for regulation of channel width is provided in accompanying papers on changes in electrical impedance and ultrastructure of junctions during Na-coupled solute transport.     

Properties of a new crystal form of the complex of concanavalin A with methyl alpha-D-glucopyranoside

The complex of concanavalin A with methyl alpha-D-glucopyranoside crystallizes as regular rhombic dodecahedra containing 35% protein by weight. The crystal is of space group I23 with a = 167.8 A (1 A = 0.1 nm) and contains one concanavalin A dimer per asymmetric unit. It diffracts to a resolution of 1.9 A and is suitable for crystallographic investigation of the structure of the saccharide-binding site.     

Contribution of solvent drag through intercellular junctions to absorption of nutrients by the small intestine of the rat

The lumen of the small intestine in anesthetized rats was recirculated with 50 ml perfusion fluid containing normal salts, 25 mM glucose and low concentrations of hydrophilic solutes ranging in size from creatinine (mol wt 113) to Inulin (mol wt 5500). Ferrocyanide, a nontoxic, quadrupally charged anion was not absorbed; it could therefore be used as an osmotically active solute with reflection coefficient of 1.0 to adjust rates of fluid absorption, Jv, and to measure the coefficient of osmotic flow, Lp. The clearances from the perfusion fluid of all other test solutes were approximately proportional to Jv. From Lp and rates of clearances as a function of Jv and molecular size we estimate (a) the fraction of fluid absorption which passes paracellularly (approx. 50%), (b) coefficients of solvent drag of various solutes within intercellular junctions, (c) the equivalent pore radius of intercellular junctions (50 A) and their cross sectional area per unit path length (4.3 cm per cm length of intestine). Glucose absorption also varied as a function of Jv. From this relationship and the clearances of inert markers we calculate the rate of active transport of glucose, the amount of glucose carried paracellularly by solvent drag or back-diffusion at any given Jv and luminal glucose concentration and the concentration of glucose in the absorbate. The results indicate that solvent drag through paracellular channels is the principal route for intestinal transport of glucose or amino acids at physiological rates of fluid absorption and concentration. In the absence of luminal glucose the rate of fluid absorption and the clearances of all inert hydrophilic solutes were greatly reduced. It is proposed that Na-coupled transport of organic solutes from lumen to intercellular spaces provides the principal osmotic force for fluid absorption and triggers widening of intercellular junctions, thus promoting bulk absorption of nutrients by solvent drag. Further evidence for regulation of channel width is provided in accompanying papers on changes in electrical impedance and ultrastructure of junctions during Na-coupled solute transport.     

Temperature Dependence of Drug Interaction with the Platelet 5-Hydroxytryptamine Transporter: A Clue to the Imipramine Selectivity Paradox

Although [3H]imipramine is a selective radioligand for the 5-hydroxytryptamine (5-HT) transporter in human platelets, its affinity for binding to the 5-HT transporter complex at 0 degrees C (0.6 nM) is significantly higher than its potency for inhibition of [3H]5-HT uptake at the physiological temperature of 37 degrees C (Ki = 29 nM). As this apparent discrepancy could be related to the assay temperature, we studied the thermodynamics of drug interaction with the 5-HT transporter at assay temperatures between 0 degrees C and 37 degrees C, using as radioligands [3H]imipramine (0 degrees C and 20 degrees C) and [3H]paroxetine (20 degrees C and 37 degrees C), a newly available probe for the 5-HT transporter. At 20 degrees C, Ki values of 14 tricyclic and nontricyclic drugs for inhibition of [3H]imipramine and [3H]paroxetine binding to human platelet membranes were highly significantly correlated (r = 0.98, p less than 0.001), validating the use of these two radioligands to study the 5-HT transporter over a temperature range larger than was previously possible with [3H]imipramine alone. The affinity of imipramine for the 5-HT transporter is progressively enhanced with decreasing incubation temperature, thus favoring the selectivity of [3H]imipramine for the 5-HT transporter at 0 degrees C. At 37 degrees C, the Ki of imipramine for inhibition of [3H]paroxetine binding is 32 nM, and equals its Ki value for inhibition of 5-HT uptake into human platelets. With the exception of chlorimipramine, other tricyclic 5-HT uptake inhibitors showed a temperature sensitivity in their interaction with the 5-HT transporter similar to that of imipramine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoradiographic Localization of [3H]Zolpidem Binding Sites in the Rat CNS: Comparison with the Distribution of [3H]Flunitrazepam Binding Sites

The regional distribution of [3H]zolpidem, a novel imidazopyridine hypnotic possessing preferential affinity for the BZD1 (benzodiazepine subtype 1) receptor, has been studied autoradiographically in the rat CNS and compared with that of [3H]flunitrazepam. The binding of [3H]zolpidem to rat brain sections was saturable, specific, reversible, and of high affinity (KD = 6.4 nM). It occurred at a single population of sites whose pharmacological characteristics were similar to those of the benzodiazepine receptors labeled with [3H]flunitrazepam. However, ethyl-beta-carboline-3-carboxylate and CL 218,872 were more potent displacers of [3H]zolpidem than of [3H]flunitrazepam. The autoradiographic brain distribution of [3H]zolpidem binding sites was qualitatively similar to that previously reported for benzodiazepine receptors. The highest levels of [3H]-zolpidem binding sites occurred in the olfactory bulb (glomerular layer), inferior colliculus, ventral pallidum, nucleus of the diagonal band of Broca, cerebral cortex (layer IV), medial septum, islands of Calleja, subthalamic nucleus, and substantia nigra pars reticulata, whereas the lowest densities were found in parts of the thalamus, pons, and medulla. Comparative quantitative autoradiographic analysis of the binding of [3H]zolpidem and [3H]flunitrazepam [a mixed BZD1/BZD2 (benzodiazepine subtype 2) receptor agonist] in the CNS revealed that the relative density of both 3H-labeled ligands differed in several brain areas. Similar levels of binding for both ligands were found in brain regions enriched in BZD1 receptors, e.g., substantia nigra pars reticulata, inferior colliculus, cerebellum, and cerebral cortex lamina IV. The levels of [3H]zolpidem binding were five times lower than those of [3H]flunitrazepam binding in those brain regions enriched in BZD2 receptors, e.g., nucleus accumbens, dentate gyrus, and striatum. Moreover, [3H]zolpidem binding was undetectable in the spinal cord (which contains predominantly BZD2 receptors). Finally, like CL 218,872 and ethyl-beta-carboline-3-carboxylate, zolpidem was a more potent displacer of [3H]flunitrazepam binding in brain regions enriched in BZD1 receptors than in brain areas enriched in BZD2 receptors. The present data add further support to the view that zolpidem, although structurally unrelated to the benzodiazepines, binds to the benzodiazepine receptor and possesses selectivity for the BZD1 receptor subtype.