Uptake of cloacin DF13 by susceptible cells: removal of immunity protein and fragmentation of cloacin molecules.

Monoclonal antibodies (MAb) directed against different epitopes on the equimolar complex of cloacin and immunity protein (cloacin DF13) were isolated, characterized, and used to study the uptake of cloacin DF13 by susceptible cells. Four MAbs recognized the amino-terminal part, one MAb recognized the central part, and three MAbs recognized the carboxyl-terminal part of the cloacin molecule. Three MAbs reacted with the immunity protein. Five MAbs inhibited the lethal action of cloacin DF13, but none of the MAbs inhibited the binding of cloacin DF13 to its purified outer membrane receptor protein or the in vitro inactivation of ribosomes. Binding of cloacin DF13 to susceptible cells cultured in broth resulted in a specific, time-dependent dissociation of the complex and a fragmentation of the cloacin molecules. Increasing amounts of immunity protein were detected in the culture medium from about 20 min after the addition of cloacin DF13. Cloacin was fragmented into two carboxyl-terminal fragments with relative molecular masses of 50,000 and 10,000. The larger fragment was detected 5 min after the binding of the bacteriocin complex to the cells. The smaller fragment was detected after 10 min. Both fragments were associated with the cells and could not be detected in the culture supernatant fraction. Cells grown in brain heart infusion were much less susceptible to cloacin DF13 than cells grown in broth, although they possessed a similar number of outer membrane receptor molecules. This decreased susceptibility correlated with a decreased translocation, dissociation, and fragmentation of cloacin DF13.     

Manganese reduction by a marine Bacillus species.

Mature dormant spores of marine Bacillus sp. strain SG1 catalyze the oxidation of Mn(II) to MnO2. We report that vegetative cells of the same strain reduced MnO2 under low-oxygen conditions. The rate of reduction was a function of cell concentration. The process had a pH optimum of 7.5 to 8.0 and was inhibited by HgCl2, by preheating of the cells at 80 degrees C for 5 min, by antimycin A, and by N-heptyl-hydroxy-quinoline-N-oxide. At a nonlimiting O2 concentration, little MnO2 reduction was observed. Under these conditions, the process could be induced by the addition of NaN3. Spectrophotometric analysis of the Bacillus cells indicated the presence of type b and c cytochromes. Both types can be oxidized in situ by addition of MnO2 to the cells.     

Siderophores and outer membrane proteins of antagonistic, plant-growth-stimulating, root-colonizing Pseudomonas spp

As an approach to understanding the molecular basis of the reduction in plant yield depression by root-colonizing Pseudomonas spp. and especially of the role of the bacterial cell surfaces in this process, we characterized 30 plant-root-colonizing Pseudomonas spp. with respect to siderophore production, antagonistic activity, plasmid content, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis patterns of their cell envelope proteins. The results showed that all strains produce hydroxamate-type siderophores which, because of the correlation with Fe3+ limitation, are thought to be the major factor responsible for antagonistic activity. Siderophore-negative mutants of two strains had a strongly decreased antagonistic activity. Five strains maintained their antagonistic activity under conditions of iron excess. Analysis of cell envelope protein patterns of cells grown in excess Fe3+ showed that most strains differed from each other, although two classes of similar or identical strains were found. In one case such a class was subdivided on the basis of the patterns of proteins derepressed by iron limitation. Small plasmids were not detected in any of the strains, and only one of the four tested strains contained a large plasmid. Therefore, it is unlikely that the Fe3+ uptake system of the antagonistic strains is usually plasmid encoded.     

Regulation of glycerol kinase by enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system.

Wild-type glycerol kinase of Escherichia coli is inhibited by both nonphosphorylated enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system and fructose 1,6-diphosphate. Mutant glycerol kinase, resistant to inhibition by fructose 1,6-diphosphate, was much less sensitive to inhibition by enzyme IIIGlc. The difference between the wild-type and mutant enzymes was even greater when inhibition was measured in the presence of both enzyme IIIGlc and fructose 1,6-diphosphate. The binding of enzyme IIIGlc to glycerol kinase required the presence of the substrate glycerol.     

Molecular recognition of siderophores in fungi: role of iron-surrounding N-acyl residues and the peptide backbone during membrane transport in Neurospora crassa.

Recognition of ferric siderophores in Neurospora crassa was found to depend on the number and kind of N-acyl residues that surrounded the iron coordination center. In the coprogen series, uptake decreased in the order of coprogen, neocoprogen I, and neocoprogen II, indicating that gradual replacement of the N-transanhydromevalonyl groups by N-acetyl groups had an adverse effect on uptake. The reverse effect was observed in the ferrichrome series, where uptake decreased in the order of ferrichrysin, asperchrome D1, asperchrome B1, and ferrirubin. Configuration of the anhydromevalonyl group (cis or trans) in ferrichromes was also an important determinant in the recognition process. On the basis of uptake and inhibition studies, it is proposed that in ferrichromes part of the molecule (iron configuration and the N-acyl groups) is responsible for binding, whereas another (cyclic peptide ring) is involved in the subsequent process of transport.     

Translational regulation of the synthesis of a major heat shock protein in HeLa cells

The synthesis of a major heat shock protein (HSP 70) was measured in HeLa cells incubated at 42.5 degrees C and then transferred to 37 degrees C or 30 degrees C. After 90 min, synthesis of HSP 70 decreased by 54 and 85%, respectively, whereas HSP 70 mRNA was reduced at most by 20%. Therefore, the reduced synthesis of HSP 70 could not be accounted for by mRNA turnover. HSP 70 was associated with large polyribosomes (6-10 ribosomes) in cells kept at 42.5 degrees C, but with medium or small polyribosomes in cells transferred to 37 degrees C or 30 degrees C (5-6 or 2-3 ribosomes, respectively). Addition of puromycin to these cells resulted in the release of all ribosomes from HSP 70 mRNA, indicating that they were translationally active. The regulation of HSP 70 synthesis was investigated in cell-free systems prepared from heat-shocked or control cells and incubated at 30 degrees C and 42 degrees C. After 5 min at 42 degrees C, the cell-free system from heat-shocked cells synthesized protein at 3 times the rate of the control cell-free system. This difference was in large part due to synthesis of HSP 70. Addition of HSP mRNA to the control cell-free system stimulated protein synthesis at 42 degrees C, but not at 30 degrees C. These findings suggest that translation of HSP 70 mRNA is specifically promoted at high temperature and repressed during recovery from heat shock by regulatory mechanisms active at the level of initiation.     

Molecular cloning of the cDNA for a growth factor-inducible gene with strong homology to S-100, a calcium-binding protein

We have identified a cDNA whose sequence is preferentially expressed when quiescent fibroblasts are stimulated to proliferate. The steady-state levels of the mRNA corresponding to this clone, called 2A9, are increased by serum, platelet-derived growth factor, and epidermal growth factor, but not by insulin or platelet-poor plasma. mRNA levels of 2A9 are also increased in human acute myeloid leukemia. The 2A9 cDNA has been molecularly cloned from an Okayama-Berg library, and its complete nucleotide sequence has been determined. It has an open reading frame of 270 nucleotides, which has a 55% homology with the coding sequence of the beta-subunit of the S-100 protein, a calcium-binding protein that belongs (like calmodulin and the vitamin D-dependent intestinal calcium-binding protein) to the family of calcium-modulated proteins and is found in abundance in several human tumors, including melanoma. The S-100 protein and the deduced aminoacid sequence of 2A9 are also partially homologous to the small subunit of a protein complex that serves as a cellular substrate to tyrosine kinase. The partial homology of 2A9 (whose RNA is inducible by growth factors and is overexpressed in human acute myeloid leukemias) to the S-100 protein, other calcium-modulated proteins, and the subunit of a substrate for tyrosine kinase, is particularly interesting in view of the role attributed to calcium and tyrosine kinases in the regulation of cell proliferation.     

Equilibrium binding of insulin to rat white fat cells at 15 degrees C

Equilibrium binding of insulin to isolated rat epididymal fat cells was investigated. A temperature of 15 degrees C was chosen for the study to minimize lysosomal degradation of insulin. Indeed, medium insulin lost only 1% of its precipitability in trichloroacetic acid during the 4-h incubation required to approach equilibrium. Binding was measured by a method that did not perturb the equilibrium of the system. A new formalism for analyzing binding data in general was introduced. A correction for trapping of insulin in the interstitial space of cell pellets was both necessary and sufficient to derive specific binding data from raw observations. Thus, so-called "nonspecific binding" was unmasked as a misnomer, and the expression "correction for trapping" was proposed as a substitute. Equations for one and two independent classes of binding sites were fit to the data by the method of maximum likelihood, and the best fit was selected based on Akaike's information criterion, as adapted for a constant fractional error. More than 99.7% of the binding sites were found to be describable by a simple binding isotherm with Kd,app = 8.8 multiplied by over divided by 1.3 nM. Less than 0.3% sites had a higher affinity (Kd approximately equal to 8 multiplied by over divided by 3 pM). There were 99,000 x/divided by 1.6 binding sites/cell. These equilibrium parameters are in agreement with values derived from a kinetic analysis, presented in the subsequent paper (Lipkin, E. W., Teller, D. C., and de Ha?n, C. (1986) J. Biol. Chem. 260, 1702-1711).     

Purification and characterization of two plasma membrane domains from ejaculated bull spermatozoa

Plasma membranes were detached from ejaculated bull spermatozoa by a brief sonication in a moderately hypotonic medium, and the released plasma membranes were partially purified by differential centrifugation. The resulting fraction was enriched 8- and 15-fold in alkaline phosphatase and 5' nucleotidase activities, respectively, compared with the starting sonicated spermatozoa. This total plasma membrane fraction was separated into two distinct fractions by equilibrium density centrifugation on a continuous linear sucrose gradient. Two peaks of light scattering material were formed at densities of 1.117 and 1.148 g/ml. The denser peak contained most of the protein of the plasma membrane fraction, whereas nearly all the concanavalin A binding activity was found in the lighter peak. The two bands had distinctly different polypeptide compositions when analyzed by SDS PAGE. Polyclonal antibodies were raised in rabbits against a major integral membrane glycoprotein of each fraction (Mr of 92,000 in the light peak and 98,000 in the dense peak). The two antigens were detected on the surface of intact spermatozoa by indirect immunofluorescence microscopy. The 92-kD protein (present in the lighter band) was detected only on the plasma membrane of the acrosomal and anterior postacrosomal regions of the head. The 98-kD antigen, present in the heavier band, was localized to the surface of the postacrosomal region of the head, to the principal piece of the tail, and to the connecting piece between the head and tail. The exclusive localization of the 92-kD polypeptide to the surface of the anterior portion of the head was confirmed by immunoelectron microscopy. These data show that the two fractions isolated on the sucrose gradient originate from different regions of the sperm cell plasma membrane.