Carbohydrate nutrition of claviceps purpurea for alkaloid production related to the osmolality of media

Summary The effects of most frequently used carbohydrates and osmolalities of media on the growth and productivity ofClaviceps purpurea in submerged cultures were investigated. The maximum alkaloid yield was obtained with 20% mannitol as well as with 10% mannitol when 2% NaCl was added to the medium and the osmolality about 1 osmol/kg H₂O was established at the end of fermentation. Increased or decreased osmolality of the medium had a negative influence on fungal growth and alkaloid production. Almost the same effect was observed with sucrose as with mannitol, whereas with glucose only maximal growth was achieved.     

Gene mapping by fluorescent in situ hybridization

We describe a new method for the mapping of mammalian genes, utilizing in situ hybridization of mRNA to DNA of chromosomes. It involves the hydrogen bonding of the polyadenylic acid at the 3' end of hybridized mRNA to the polyuridylic acid tail of a highly fluorescent latex microsphere. The resultant double hybrid can be visualized by fluorescence microscopy. The chromosomal localization of human alpha + beta globin genes has been explored by this method. Our data point ot the long arms of chromosomes 4 and 5 as the loci for the human globin genes.     

Chronotropism and blood flow patterns following teratogenic doses of catecholamines in 5-day-old chick embryos.

In vivo heart rates of 5-day-old chick embryos were recorded from electrodes placed in close proximity to the heart. L-epinephrine (4X10(-10) mole), 1-norepinephrine (1X10(-9)mole) and 1-isoproterenol (1.6X10(-10)mole) in 5 microliter of isotonic saline transiently accerlerated the mean heart rate by almost 9 percent. L-phenylephrine (2X10(-9)mole/5microliter) and the experimental procedure produced no appreciable effect. The positive chronotropic effect of the catecholamines was found to be highly significant (P less than 0.0005) as computed by Student's t test. However, no direct relationship could be established between the chronotropic response and the aortic arch anomalies produced. A prolonged reduction of blood flow in the primitive heart tube and the sixth aortic arch after administration of epinephrine and isoproterenol is apparently related to the induction of hypoplastic right pulmonary artery with absent or hypoplastic right ductus arteriosus.     

Genetic control of the primary humoral response to Glu56Lys35Phe9

The primary humoral responses of mice to the linear random terpolymerl-Glu⁵⁶-l-Lys³⁵-l-Phe⁹ (GLø) were studied, utilizing the Farr antigen-binding technique and a new hemagglutination assay. This new hemagglutinin assay was easier and more convenient than the conventional Farr method, and was more sensitive in detecting early IgM responses. Following primary immunization, the majority of antibodies produced by responder strains were 2-ME-sensitive. These 2-ME-sensitive antibodies chromatographed at the same relative position as IgM on a Sepharose 6B column. On the other hand, no antibodies of either the IgM or IgG class could be detected in nonresponder strains. These data are consistent with the hypothesis that two complementingIr genes are required for the primary IgM response to GLø, in contrast to findings previously reported for (T,G)-A — L, anotherH-2-linked, complementing,Ir gene system. The implications of these differences are discussed.     

Polyadenylic acid synthesis activity of purified DNA-dependent RNA polymerase from Caulobacter

Characterization of purified DNA-dependent RNA polymerase (EC 2.7.7.6) of Caulobacter crescentus, strain CB15 has led to the conclusion that this enzyme catalyzes poly(A) synthesis in the absence of template. Poly(A) synthetase activity co-purifies with both holoenzyme and core polymerase on DNA-cellulose columns, and core polymerase purified to 98% homogeneity by glycerol gradient centrifugation is still capable of catalyzing poly(A) polymerization. Both RNA synthesis and poly(A) polymerization activities are sensitive to rifampicin. In addition, RNA polymerase purified from partially rifampicin-sensitive mutants exhibits the same partial sensitivity in vitro to the drug in the synthesis of RNA and poly(A). The enzyme used in these studies was prepared by a simple method which allows a high yield of pure RNA polymerase from large batches of exponential cells. The procedure includes high speed centrifugation of cell extracts, DEAE-cellulose column, DNA-affinity chromatography, and low salt glycerol gradient centrifugation. Holoenzyme can be resolved into core and sigma subunit by either DNA-cellulose chromatography or glycerol gradient centrifugation, and the latter step allows recovery of pure sigma factor.     

Rat brain adenylate cyclase. Further studies on its stimulation by a Ca2+-binding protein.

Bovine or rat brain adenylate cyclase (EC 4.6.1.1) solubilized by Lubrol-PX, a nonionic detergent, requires a Ca²⁺-binding protein activator for full activity (Cheung et al., 1975, Biochem. Biophys. Res. Commun.66, 1055–1062). We now show that particulate rat brain adenylate cyclase also required the activator for maximum activity. A brain particulate fraction was extracted with a hypertonic NaCl solution containing [ethyl-enebis(oxyethylenenitrilo)] tetraacetic acid. This procedure removed preferentially the activator, making adenylate Cyclase activator deficient and, consequently, dependent on an exogenous activator for maximum activity. The activator increased the V of adenylate cyclase without affecting its apparent K_m for ATP. In the presence of the activator, the enzyme was more stable against thermal inactivation, suggesting that the activator probably induced a conformational change to the enzyme. F^? and 5′-guanylylimidodi-phosphate [GMP-p(NH)p] greatly stimulated brain adenylate cyclase. Adenylate cyclase activity obtained in the presence of the activator and F^? was comparable to the summed activities of the two agents assayed separately, indicating that their effects were additive. Similarly, the effects of the activator and GMP-p(NH)p were additive. These results suggest that the action of the activator is independent of the other two ligands. Since the activator is present in excess over adenylate cyclase, the cellular flux of Ca²⁺ is believed to be important in modulating the enzyme activity. The role of the Ca²⁺/ activator is discussed with respect to cyclic AMP metabolism in brain.     

Failure of atractyloside to inhibit synaptosomal mitochondrial energy transduction

Studies on synaptosome mitochondrial respiration are complicated by “free” mitochondria. Veratridine stimulation of synaptosomal respiration was due to increased Na⁺ cycling at the synaptosome membrane associated with increased oxidative phosphorylation of intraterminal ADP and was inhibited by oligomycin, ouabain or Na⁺ free medium. Atractylate or carboxyatractyloside failed to block veratridine-stimulated respiration but inhibited exogenous-ADP-stimulated respiration. Protein synthesis in the synaptosome fraction was inhibited by oligomycin, valinomycin or 2,4-dinitrophenol but was unaffected by excess atractylate. No change in synaptosomal adenine nucleotide content was found in the presence of atractylate, although a significant decrease in the [ATP]/[ADP] was found with oligomycin, veratridine or valinomycin. These findings show that atractylate does not modify intraterminal mitochondrial energy transduction and indirectly suggest an impermeability of the synaptosome membrane to atractylate.     

Possible molecular detent in the DNA structure at regulatory sequences

A common feature that appears in a number of DNA sites where proteins interact is the sequence GTG/CAC. In the lac operator this sequence leads to a region with a higher imino proton exchange rate well below the optical melting temperature. It is suggested that this reflects a structural feature recognized by proteins that bind specific sites on the DNA molecule.     

Dynein ATPase is inhibited selectively in vitro by erythro-9-[3-2-(hydroxynonyl)]adenine

Dynein (ATP phosphohydrolase, EC 3.6.1.3) extracted from sea urchin sperm tails was inhibited by erythro-9-[3-2-(hydrosynonyl)]adenine in a dosedependent fashion; at the 50% inhibitory concentration, 0.23 mM, twelve other ATP-metabolizing enzymes were notsignificantly affected. Actomyosin and myosin ATPase activities were enhanced 1.5- to 2-fold by millimolar concentrations of erythro-9-[3-2-(hydroxynonyl)]adenine. Enzyme kinetic analysis supported a model of linear mixed-type inhibition, which suggests that the binding site for erythro-9-[3-2-(hydroxynonyl)]adenine on dynein is remote from the ATPase active site. As a selective inhibitor $\text{in}\text{vitro}$, erythro-9-[3-2-(hydroxynonyl)]adenine appears to offer a biochemical criterion for identifying dynein isozymes in tissue extracts.     

Eukaryotic DNA replication.

The past decade has witnessed an exciting evolution in our understanding of eukaryotic DNA replication at the molecular level. Progress has been particularly rapid within the last few years due to the convergence of research on a variety of cell types, from yeast to human, encompassing disciplines ranging from clinical immunology to the molecular biology of viruses. New eukaryotic DNA replicases and accessory proteins have been purified and characterized, and some have been cloned and sequenced. In vitro systems for the replication of viral DNA have been developed, allowing the identification and purification of several mammalian replication proteins. In this review we focus on DNA polymerases alpha and delta and the polymerase accessory proteins, their physical and functional properties, as well as their roles in eukaryotic DNA replication.