Vacuoles from Sugarcane Suspension Cultures : I. ISOLATION AND PARTIAL CHARACTERIZATION

Vacuoles were isolated from suspension cultures of sugarcane (Saccharum sp.) cells by centrifugation of protoplasts at high g force against a 12% (w/v) Ficoll solution. Distribution of marker enzymes and Concanavalin A binding showed an 11% contamination of the vacuole preparation by cytoplasmic components, mitochondria, and endoplasmic reticulum, and 18% contamination by plasma membrane. Acid phosphatase, carboxypeptidase, protease, peroxidase, and ribonuclease activities were enriched in isolated vacuoles. Carboxypeptidase was tonoplast-bound, whereas the other enzymes were soluble. Sucrose, reducing sugars, and free amino acids were measured in protoplasts and vacuoles during growth of cells in suspension culture. Sucrose and reducing sugar content of vacuoles increased as the culture aged, while free amino acids decreased sharply.     

Intracellular phosphate pools show compartmentalization of intracellular pH in turtle urinary bladder

The urinary bladder of the fresh water turtle is capable of acidification and Na transport, in vitro, and it has been extensively used as a model of distal nephron of the kidney. In the course of measuring intracellular pH of stripped turtle bladder mucosa with phosphorus nuclear magnetic resonance, we observed the consistent presence of two inorganic phosphorus resonances under aerobic conditions, indicating the existence of a pH gradient possibly between cytosol and mitochondrion. This pH gradient was collapsed by addition of N₂ and could be restored by reintroduction of oxygen. These observations demonstrate the existence of a spontaneous pH gradient between cytosol and mitochondria of turtle bladder epithelial cells.     

Fast atom bombardment: A new mass spectrometric method for peptide sequence analysis

We have studied a selection of peptides using a new mass spectrometric ionisation technique - fast atom bombardment (FAB). We define the fragmentation pathways observed and comment on the utility in sequence analysis. A simple acetylation experiment is shown to aid rapid sequence assignment.     

Purification of glutamine synthetase from a variety of bacteria

We have developed two procedures which allow the very rapid purification of glutamine synthetase (GS) from a diverse variety of bacteria. The first procedure, based upon differential sedimentation, depends upon the association of GS with deoxyribonucleic acid in cell extracts. The second procedure, derived from the method of C. Gross et al (J. Bacteriol. 128:382-389, 1976) for purifying ribonucleic acid polymerase by polyethylene glycol (PEG) precipitation, enabled us to obtain high yields of GS from either small or large quantities of cells. We used the PEG procedure to purify GS from Klebsiella aerogenes, K. pneumoniae, Escherichia coli, Salmonella typhimurium, Rhizobium sp. strain 32H1, R. meliloti, Azotobacter vinelandii, Pseudomonas putida, Caulobacter crescentus, and Rhodopseudomonas capsulata. The purity of the GS obtained, judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was high, and in many instances only a single protein band was detected.     

Phosphorus-31 relaxation times of 2,3-diphosphoglycerate in intact human erythrocytes

The reciprocals of the spin-lattice relaxation times (T₁s) of the 2-P and 3-P nuclei of 2,3-diphosphoglycerate (DPG) increased linearly as percent DPG bound was raised in model hemoglobin solutions. The 2-P T₁ was slightly greater in intact erythrocytes than in model solutions under similar experimental conditions. The change in the 3-P T₁ with cellular deoxygenation was anomalous indicating that this nucleus should not be used to estimate DPG binding inside intact erythrocytes.     

The effect of ambient temperature on beta-adrenergic responsiveness in rats.

The responses of tail skin and colonic temperatures of female rats to ambient temperatures of 20, 22, 24, 26, 28, and 30 degrees C were measured. Within this range, colonic temperature was stable while tail skin temperature increased linearly with increasing ambient temperature. Administration of the beta-adrenergic agonist, d,l-isoproterenol, at 10.0, 25.0, and 62.5 micrograms/kg, sc, at each ambient temperature was accompanied by increases in tail skin and colonic temperatures that were dependent on both the dose of isoproterenol administered and the ambient temperature. The integrated responses of tail skin temperature following administration of the three doses of isoproterenol were maximal at an ambient temperature of 26 degrees C while the integrated responses of colonic temperature were maximal at 30 degrees C. The results suggest that tests of beta-adrenergic responsiveness using this technique should be performed at an ambient temperature of 26 degrees C for maximal sensitivity.     

Deletion mapping of the polA-metB region of the Escherichia coli chromosome.

A lambdacI857 prophage inserted into one of the genes of the rha locus was used to select deletions unambiguously ordering the markers polA-glnA-rha-pfkA-tpi-metBJF. Transduction with phage P1 indicates at least 70% linkage between glnA and polA. The order of the pfk and tpi markers is reversed from that previously published. Despite the relatively large distance separating the glnA and rha loci, deletions removing this entire region have no obvious phenotype. The isolation of Tn10 transposons integrated at different sites between rha and glnA greatly facilitated this work.     

Purification of pig synovial collagenase to high specific activity.

1. Pig synovium in tissue culture secretes a specific collagenase in a latent form. 2. The latent enzyme was concentrated by (NH4)2SO4 precipitation and activated with 4-aminophenylmercuric acetate, and the active enzyme was purified by chromatography on Ultrogel AcA44, DEAE-cellulose, heparin-Sepharose and a zinc-chelate medium to a specific activity of 53 400 units/mg. of protein. 3. The enzyme was shown to be essentially homogeneous by polyacrylamide-gel electrophoresis. 4. The purified collagenase digested collagen to give the characteristic three-quarter and one-quarter pieces.     

Relation between plasma hormone profiles,symptoms, and response to oestrogen treatment in women approaching the menopause.

Out of a consecutive series of 300 patients seen at a menopause clinic, 82 complained of symptoms generally associated with the climacteric, although they were still menstruating. Vasomotor disturbances were absent in 42 of these patients (group 1) and present in 40 (group 2). Headaches, insomnia, and dyspareunia were the most common complaints among the women with vasomotor symptoms, whereas loss of libido and depression predominated in those without. Conjugated equine oestrogens (Premarin) 1.25 mg daily given for three weeks out of four relieved nearly all symptoms in group 2, but in group 1 the response was disappointing. The mean plasma oestradiol concentration in women with vasomotor symptoms was significantly lower than that observed during days 1-10 of the menstrual cycle, but plasma testosterone values were not significantly different from those observed in younger women. Plasma follicle-stimulating hormone (FSH) and luteinising hormone (LH) concentrations were similar to those seen after the menopause. Concentrations of these hormones in the women without vasomotor symptoms were similar to those in the younger, regularly menstruating women. After six months of oestrogen treatment patients in group 2 had a 2.1-fold increase in mean plasma oestradiol concentration, and plasma FSH and LH concentrations were reduced to 39% and 66% of their pretreatment values respectively; in group 1, however, no such pronounced changes occurred. High concentrations of FSH were present in patients with oestrogen-responsive symptoms, 15 U/1 being the diagnostic cut-off point. This measurement in the presence of characteristic symptoms therefore constitutes the best method of selecting patients for oestrogen-replacement therapy.