Interaction of calmodulin with the particulate fraction of cardiac muscle

Tritiated calmodulin (T-CM) was bound to the EGTA-treated particulate fraction of cardiac muscle in a calcium-dependent manner with half-maximal binding occurring between 0.8 to 1.2 microM calcium. Binding exhibited high specificity at an optimum pH of 7.4-7.6. An excess of parvalbumin and other globular proteins did not displace T-CM. The Kd for the interaction was 2.5 +/- 0.83 microM. Binding was trypsin-sensitive, inhibited by high ionic strength and was heat inactivated at a midpoint of 48 - 50 degrees C. Competitive displacement of T-CM occurred with unlabeled troponin C and calmodulin over the same concentration range. The first-order rate constant of T-CM dissociation was 3.27 min-1. Calcium-dependent binding of T-CM was inhibited equally by both mepacrine and trifluoperazine with 50 percent inhibition occurring at 70 microM.     

The radiation response of a human colon adenocarcinoma grown in monolayer, as spheroids, and in nude mice

A human colon adenocarcinoma cell line, WiDr, has been grown in monolayer, as multicellular spheroids, and as xenografted tumors in immune-deprived mice. The growth and radiation responses of the cells under these different growth conditions were compared. The mean doubling time of monolayer cultures was 0.8 day and the initial volume doubling times of spheroids and xenografts averaged 1.2 and 6 days, respectively. The mean total viable cell plating efficiencies were 82, 63, and 7% for cells from monolayers, spheroids, and xenografted tumors, respectively. The radiation responses of single cell suspensions prepared from WiDr tumors (8-10 mm in diameter), exponentially growing monolayer cultures (5 days growth), and spheroids (1200 microns in diameter) irradiated in air at 4 degrees C were similar. Values for D0 were 1.5 Gy and for n between 3 and 5. Nitrogen curves were characterized by a D0 of 5 Gy and n between 3 and 6. Oxygen enhancement ratios were approximately 3.3. Both spheroids and tumors had radioresistant components to the 37 degrees C/air-breathing survival curves with estimated hypoxic fractions of 8 and 12%, respectively. The final portion of the survival curves for irradiations in nitrogen and under normal growth conditions were parallel for both tumors and spheroids. Thus WiDr spheroids appear to model accurately the radiation sensitivity of WiDr tumors.     

Protein phosphorylation in rat mammary acini and in cytosol preparations in vitro. Phosphorylation of acetyl-CoA carboxylase is unaffected by cyclic AMP.

Phosphorylation of soluble proteins in rat mammary acinar cells was investigated. When phosphorylation proceeded in intact cells, in the presence of [32P]Pi, the major non-casein phosphoproteins, including acetyl-CoA carboxylase, were unresponsive to incubation conditions that caused major increases in the intracellular concentration of cyclic AMP. The overall 32P specific radioactivity (c.p.m./microgram of protein) of acetyl-CoA carboxylase, assessed after affinity purification of the enzyme with avidin-Sepharose, was unchanged by incubation under such conditions. Furthermore, the distribution of 32P among tryptic phosphopeptides of the enzyme, resolved by reversed-phase h.p.l.c., was not altered by cyclic AMP-increasing treatments of the acinar cells. When cytosol fractions were incubated with [gamma-32P]ATP, some phosphoproteins responded to the addition of micromolar concentrations of dibutyryl cyclic AMP or cyclic AMP by undergoing an enhancement of phosphate incorporation. In these experiments in vitro, protein phosphatase activity did not make a major contribution to the net phosphorylation of individual phosphoproteins, and acetyl-CoA carboxylase was not prominent among the phosphoproteins identified after short (less than 1 min) incubations of cytosols with [gamma-32P]ATP. The resistance of protein phosphorylation to variations in the cyclic AMP concentration in intact mammary epithelial cells, demonstrated by this work, is one of several mechanisms that ensure the pleiotropic refractoriness of those cells to agents which normally cause a stimulation of adenylate cyclase activity in hormone-sensitive cells.     

Comparison of most probable number and pour plate procedures for isolation and enumeration of sulphite-reducing Clostridium spores and group D faecal streptococci from oysters

A comparative study of methods to enumerate sulphite-reducing Clostridium spores and Group D faecal streptococci in oysters demonstrated that pour plate solid agar techniques gave higher counts than liquid broth most probable number procedures. Reinforced clostridial broth with supplements to detect sulphite reduction was compared with pour plates of egg yolk-free tryptose sulphite cycloserine agar incubated at 37 degrees C for 24 h. Azide dextrose broth was compared with pour plates using Slanetz and Bartley (SB) agar or KF-streptococcus agar at 37 degrees C. Most probable number procedures used for both groups of organisms gave excessive numbers of improbable tube combinations. For enumeration of Group D faecal streptococci, a pour plate technique using SB agar incubated at 37 degrees C for 48 h is recommended.     

Aerobactin utilization by Neisseria gonorrhoeae and cloning of a genomic DNA fragment that complements Escherichia coli fhuB mutations.

Aerobactin, a dihydroxamate siderophore produced by many strains of enteric bacteria, stimulated the growth of Neisseria gonorrhoeae FA19 and F62 in iron-limiting medium. However, gonococci did not produce detectable amounts of aerobactin in the Escherichia coli LG1522 aerobactin bioassay. We probed gonococcal genomic DNA with the cloned E. coli aerobactin biosynthesis (iucABCD), aerobactin receptor (iutA), and hydroxamate utilization (fhuCDB) genes. Hybridization was detected with fhuB sequences but not with the other genes under conditions which will detect 70% or greater homology. Similar results were obtained with 21 additional strains of gonococci by colony filter hybridization. A library of DNA from N. gonorrhoeae FA19 was constructed in the phasmid vector lambda SE4, and a clone was isolated that complemented the fhuB mutation in derivatives of E. coli BU736 and BN3307. These results suggest that fhuB is a conserved gene and may play a fundamental role in iron acquisition by N. gonorrhoeae.     

The human glucocerebrosidase gene has two functional ATG initiator codons.

Gaucher disease is due to a deficiency in the activity of the enzyme glucocerebrosidase. Glucocerebrosidase is a lysosomal enzyme that presumably requires a signal peptide for transport across the membrane of the rough endoplasmic reticulum and glycosylation for transport into lysosomes. Human glucocerebrosidase cDNA contains two potential ATG start codons in its long open reading frame. The signal peptides that are initiated from each ATG are quite different in their hydrophobicity. We demonstrate that either ATG can function independently to produce active glucocerebrosidase enzyme in cultured fibroblasts. The glucocerebrosidase activity produced from translation products initiated at either ATG is found predominantly in the lysosomes.