Single-cell protein low in nucleic acids by alkaline treatment

Summary The effect of alkaline treatment on nucleic acid content and in vitro dry matter digestibility of C. utilis NRRL Y-660 has been evaluated using sodium and ammonium hydroxides for the treatment and finding the former preferable. Total dry matter and true protein recovery are also reported.     

Isolation and partial characterization of phages infectingCitrobacter intermedius C3

Several new bacteriophages infecting strain C3 ofCitrobacter intermedius have been isolated from fresh waters. The physicochemical properties, plaque morphology, growth characteristics, virion structure, and immunology of eight bacteriophage isolates are reported. The phages are classified into three groups according to their characteristics.     

A simple and rapid method for the isolation of peptides from sodium dodecyl sulfate-containing polyacrylamide gels

A rapid and simple method is described for the recovery of peptides from sodium dodecyl sulfate-containing polyacrylamide gels. It involves the electrophoretic concentration of a peptide in the stacking gel followed by elution into glycerol. The method requires no special equipment or chemicals, and the elution can be made using the same electrophoretic systems used in the separation step. The method is more rapid than normal extraction procedures, and simpler than most electrophoretic elution methods described. The method can be used for isolation of microgram as well as milligram quantities of an individual peptide with yields of approximately 100%.     

Modulation of Quinolinic and Kynurenic Acid Content in the Rat Brain: Effects of Endotoxins and Nicotinylalanine

Quinolinic acid, an endogenous excitotoxin, and kynurenic acid, an antagonist of excitatory amino acid receptors, are believed to be synthesized from tryptophan after the opening of the indole ring. They were measured in the rat brain and other organs using gas chromatography-mass spectrometry or HPLC. The enzyme indoleamine 2,3-dioxygenase, capable of cleaving the indole ring of tryptophan, was induced by administering bacterial endotoxins to rats, which significantly increased the brain content of both quinolinic and kynurenic acids. Nicotinylalanine, an analogue of kynurenine, inhibited this endotoxin-induced accumulation of quinolinic acid while potentiating the accumulation of kynurenic acid. The possibility of significantly increasing brain concentrations of kynurenic acid without a concomitant increase in quinolinic acid may provide a useful approach for studying the role of these electrophysiologically active tryptophan metabolites in brain function and preventing the possible toxic actions of abnormal synthesis of quinolinic acid.     

Reconstitution of sodium channels in large liposomes formed by the addition of acidic phospholipids and freeze-thaw sonication

Phosphatidylcholine (PC) alone or with phosphatidylethanolamine (PE) are sufficient for the reconstitution of Na+ channels in planar lipid bilayers. However, when Na+ channels were first reconstituted into liposomes using the freeze-thaw-sonication method, addition of acidic phospholipids, such as phosphatidylserine (PS), to the neutral phospholipids was necessary to obtain a significant toxin-modulated 22Na uptake. To further investigate the acidic phospholipid effect on reconstitution into liposomes, Na+ channels purified from Electrophorus electricus electrocytes were reconstituted into liposomes of different composition by freeze-thaw sonication and the effect of batrachotoxin and tetrodotoxin on the 22Na flux was measured. The results revealed that, under our experimental conditions, the presence of an acidic phospholipid was also necessary to obtain a significant neurotoxin-modulated 22Na influx. Though neurotoxin-modulated 22Na fluxes have been reported in proteoliposomes made with purified Na+ channels and PC alone, the 22Na fluxes were smaller than those found using lipid mixtures containing acidic phospholipids. Electron microscopy of negatively stained proteoliposomes prepared with PC, PC/PS (1:1 molar ratio), and PS revealed that the acidic phospholipid increases the size of the reconstituted proteoliposomes. The increment in size caused by the acidic phospholipid, due to the associated increase in internal volume for 22Na uptake and in area for Na+ channel incorporation, appears to be responsible for the large neurotoxin-modulated 22Na fluxes observed.     

Characterization by photoaffinity labeling of a steroid binding protein in rat liver plasma membrane

Summary The mechanism of steroid uptake by the cell remains controversial. [³H]R5020 was utilized to characterize by photoaffinity labeling the steroid binding site in plasma membrane. This binding was saturable, reversible and had one type of binding site (K _d = 33 ± 4 nm, B _max = 32 ± 2 pmol/mg). [³H]R5020 could be prevented from binding by a variety of steroids (cortisol, progesterone, deoxycorticosterone, and levonorgestrel); estradiol did not have affinity for this binding site. The kinetics of R5020 photoactivation was time dependent and saturable. SDS-PAGE showed a specific band which corresponded to a 53-kDa peptide. The sucrose density gradient analysis has revealed the existence of a protein with a sedimentation coefficient of 3.6 ± 0.2 S. This polypeptide shows different characteristics than cytosolic steroid receptor or serum steroid binding proteins. This binding protein could correspond to the steroid binding site previously found in the plasma membrane.This work was supported by grants PB85-0461 from the Comisión Asesora de Investigatión Científica y Técnica and PGV-8612 from the Departamento de Educatión, Universidades e Investigation del Gobierno Vasco. We thank Roussel-Uclaf (France) for the nonradioactive RU-steroids kindly provided.     

Disruption of the low affinity receptor-binding site in NGF allows neuronal survival and differentiation by binding to the trk gene product.

Nerve growth factor (NGF), like many other growth factors and hormones, binds to two different receptor molecules on responsive cells. The product of the proto-oncogene trk, p140trk, is a tyrosine kinase receptor that has been identified as a signal-transducing receptor for NGF, while the role of the low affinity NGF receptor, p75NGFR, in signal transduction is less clear. The crystal structure of NGF has recently been determined, although structures involved in receptor binding and biological activity are unknown. Here we show that Lys-32, Lys-34, and Lys-95 form a positively charged interface involved in binding to p75NGFR. Simultaneous modification of Lys-32 with either of the two other lysines resulted in loss of binding to p75NGFR. Despite the lack of binding to p75NGFR, these mutants retained binding to p140trk and biological activity, demonstrating a functional dissociation between the two NGF receptors.     

Cell suspension cultures ofOnonis natrix L. subspeciesramosissima: Establishment and culture conditions

Summary Explants from petioles, folioles or hypocotyls ofOnonis natrix have been used for calli initiation. Hypocotyls inoculated on MS medium supplemented with 2% sucrose and 0.5 mg.1^–1 2,4-D / 1 mg.1^–1 Kin showed to be the best primary explant. Cell suspension cultures were established in MS basal medium supplemented with 2% sucrose, 0.5 mg.1^–1 NAA or 2,4-D and 1 mg.1^–1 Kin. Different subculturing periods, inoculum density, hormonal supplementation and sucrose concentration were assayed in order to obtain the best culture growth conditions. The optimal conditions were achieved with cultures initiated with 40 g.1^–1 of initial inoculum, growing in MS basal medium supplemented with 4% sucrose, 0.5 mg.1^–1 NAA and 1 mg.1^–1 Kin subcultured every twelve days. Under these experimental conditions, the cultures showed a doubling time of 36.3 hours.     

Anoxic survival of the isolated cerebellum of the turtle Pseudemis scripta elegans

Intact turtle brain provides a useful model for the study of anoxia and potential survival strategies, since this tissue maintains transmembrane ion gradients and ATP levels during prolonged anoxia and recovers functional activity afterwards. Since isolated tissues offer experimental advantages, the present study sought to determine effects of anoxia on the isolated turtle cerebellum and to define relationships between anoxia survival and glucose supply. In normoxia, the extracellular potassium ([K+]o) activity and evoked potentials were maintained with 5 mM glucose, but 20 mM glucose was required to maintain adenosine triphosphate (ATP) levels and prevent significant increases in [K+]o during anoxia. Inhibition of glycolysis by iodoacetic acid (IAA) during anoxia provoked large increases in [K+]o at all glucose levels. These results demonstrate the usefulness of the isolated turtle cerebellum for studies of anoxic survival since this tissue can maintain ATP levels and [K+]o during prolonged anoxia with 20 mM glucose in the artificial cerebrospinal fluid medium. They also suggest the presence of a Pasteur effect at least during the transition to a hypometabolic state.