Mechanism of enhancement of microbial cell hydrophobicity by cationic polymers.

Polycationic polymers have been noted for their effects in promoting cell adhesion to various surfaces, but previous studies have failed to describe a mechanism dealing with this type of adhesion. In the present study, three polycationic polymers (chitosan, poly-L-lysine, and lysozyme) were tested for their effects on microbial hydrophobicity, as determined by adhesion to hydrocarbon and polystyrene. Test strains (Escherichia coli, Candida albicans, and a nonhydrophobic mutant, MR-481, derived from Acinetobacter calcoaceticus RAG-1) were vortexed with hexadecane in the presence of the various polycations, and the extent of adhesion was measured turbidimetrically. Adhesion of all three test strains rose from near zero values to over 90% in the presence of low concentrations of chitosan (125 to 250 micrograms/ml). Adhesion occurred by adsorption of chitosan directly to the cell surface, since E. coli cells preincubated in the presence of the polymer were highly adherent, whereas hexadecane droplets pretreated with chitosan were subsequently unable to bind untreated cells. Inorganic cations (Na+, Mg2+) inhibited the chitosan-mediated adhesion of E. coli to hexadecane, presumably by interfering with the electrostatic interactions responsible for adsorption of the polymer to the bacterial surface. Chitosan similarly promoted E. coli adhesion to polystyrene at concentrations slightly higher than those which mediated adhesion to hexadecane. Poly-L-lysine also promoted microbial adhesion to hexadecane, although at concentrations somewhat higher than those observed for chitosan. In order to study the effect of the cationic protein lysozyme, adhesion was studied at 0 degree C (to prevent enzymatic activity), using n-octane as the test hydrocarbon. Adhesion of E. coli increased by 70% in the presence of 80 micrograms of lysozyme per ml. When the negatively charged carboxylate residues on the E. coli cell surface were substituted for positively charged ammonium groups, the resulting cells became highly hydrophobic, even in the absence of polycations. The observed "hydrophobicity" of the microbial cells in the presence of polycations is thus probably due to a loss of surface electronegativity. The data suggest that enhancement of hydrophobicity by polycationic polymers is a general phenomenon.     

Kinetic characterization of early immunoreactive intermediates during the refolding of guanidine-unfolded Escherichia coli tryptophan synthase beta 2 subunits.

This paper deals with stopped-flow studies on the kinetics of the regain of immunoreactivity toward five distinct monoclonal antibodies during the folding of the guanidine-unfolded beta 2 subunit of Escherichia coli tryptophan synthase and of two complementary proteolytic fragments of beta, F1 (N-terminal; Mw = 29,000) and F2 (C-terminal; Mw = 12,000). It is shown that, while selected as being "specific" for the native protein, these antibodies are all able to recognize early folding intermediates. The two antigenic determinants carried by the F2 domain and the antigenic site carried by the hinge peptide linking F1 and F2 are present so early during the folding process that their kinetics of appearance could not be followed. On the contrary, the rate constants of appearance of two "native-like" epitopes, carried by F1, could be determined during the folding of beta chains. The rate constant of appearance of the epitope to antibody 19 was found to be k = 0.065 s-1 at 12 degrees C. This value is very similar to that we reported previously for the appearance of an early epitope to the same antibody during the folding of acid-denatured beta chains. Thus, in spite of the important structural differences between guanidine-unfolded and acid-denatured beta chains, the same early folding events seem to be involved in the appearance of this epitope. The rate constant was found to be significantly smaller (k = 0.02 s-1 at 12 degrees C) for the appearance of the epitope to antibody 9. This shows that the regain of immunoreactivity is not concerted within the F1 domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of pyruvate carboxylase in organic acid-producing Aspergillus strains.

The localization of pyruvate carboxylase (cytosolic or mitochondrial) was studied in nine different Aspergillus species (14 strains). In some species (A. aculeatus, A. flavus, A. foetidus, A. nidulans, A. ochraceus, and A. sojae), the pyruvate carboxylase activity could be detected only in the cytosolic fraction of the cells. Pyruvate carboxylase has been found only in the mitochondrial fraction of two strains of Aspergillus wentii. In Aspergillus oryzae and in five strains of Aspergillus niger, pyruvate carboxylase activity was detected both in the mitochondrial fraction and in the cytosol. There was no quantitative or qualitative correlation between the activities of pyruvate carboxylase in the mitochondrial and cytosolic fractions of the cells and the ability of the various Aspergillus strains to accumulate different organic acids.     

The Role of Chloride in Acetylcholine Metabolism

Abstract: The chloride dependence of acetylcholine (ACh) synthesis and release and of choline uptake was studied in synaptosomal preparations from rat brain. The substitution of propionate for chloride, in the presence of 35 m m -potassium, lowered the ACh content of the synaptosomes. However, in the presence of 5 m m -potassium, the ACh level in synaptosomes was reduced, but significantly less so. Propionate had no effect on choline acetyltransferase (EC 2.3.1.6) activity when measured in a standard chloride-containing medium. In the presence of propionate, the spontaneous release of ACh was unchanged, but potassium-stimulated release of ACh was markedly reduced as compared with a chloride-containing medium. The synthesis of ACh, as measured by the net increase in the amount of ACh in the synaptosomes and that released to the medium, was reduced with propionate at 5 m m -potassium and was totally inhibited when the potassium concentration was increased to 35 m m . Choline uptake studies revealed that with propionate only a low-affinity component of the choline transport system existed. Further, the V _max was markedly reduced when the potassium concentration was increased to 35 m m . The results suggest that under certain conditions choline transported by a low-affinity system might provide a substantial source of choline for ACh synthesis.     

Comparison of the control and pathways for degradation of the acetylcholine receptor and average protein in cultured muscle cells

In the studies reported here, we investigated whether the degradation of the acetylcholine receptor (AChR) in cultured muscle cells involves similar mechanisms as and is controlled in a manner similar to, the catabolism of the bulk of cell protein. We compared these processes after labeling cell protein with radioactive leucine or phenylalanine for 24 hours, or labeling the acetylcholine receptor with (¹²⁵I)-bungarotoxin. The apparent average half-life of cell protein was 38 ± 2 hours and that of the receptor-toxin complex was 25 ± 1 hours. Incubation in media lacking serum and embryo extract accelerated the degradation of both average protein and the receptor-toxin complex. Insulin reduced the rate of catabolism of both average protein and the receptor-toxin complex toward levels seen in the presence of serum. However, although these two degradative processes seem to be controlled similarly, they probably involve different mechanisms. The protease inhibitors leupeptin and chymostatin, which slowed overall proteolysis in nongrowing muscles and hepatocytes, reduced the degradation of the ACh receptor by 2–11-fold, but had no, or only slight, effects on the catabolism of average protein, even when overall proteolysis was accelerated by omitting serum and embryo extract. Chloroquine, an inhibitor of lysosomal function, also reduced the degradation of AChR, by about 10-fold, but decreased overall protein breakdown by only 20–30%. Incubation of myotubes at lower temperatures reduced both degradative processes, but affected the breakdown of the receptor to a greater extent. Thus the rate-limiting steps in these processes have different activation energies. Incubation with 2-deoxyglucose, an inhibitor of glycolysis, decreased the breakdown of average protein but not that of the receptor-toxin complex. However, the two degradative processes were sensitive to azide, an inhibitor of oxidative phosphorylation. Although the lysosome is the primary site for AChR degradation and perhaps for degradation of other surface proteins, the breakdown of most proteins in myotubes seems to involve a distinct proteolytic system requiring metabolic energy.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

The function of tail fibers in triggering baseplate expansion of bacteriophage T4

Normal particles of bacteriophage T4 have six long tail fibers attached to a hexagonal baseplate. T4 particles having various complements of tail fibers were prepared by in vitro addition of fibers to fiberless particles, and the infectivity of the particles was determined. Particles having fewer than six fibers (partially fibered) were found to have a decreased probability of infection. Partially fibered particles having T4 fibers were completed by addition of T6 fibers, and the infectivity was determined on a host that lacked the T6 tail fiber receptor. Attachment of the additional fibers increased the infectivity even though the T6 fibers could not bind to the host cell. The infectivity of particles having mixtures of T4 and T6 fibers was determined on cells having only one type of receptor. The results indicated that particles bound by only three fibers have a low probability of infection. The effect of thermolabile baseplate mutations was also examined. Studies of partially fibered particles and particles with mixtures of fibers indicated that particles with altered baseplates have a less stringent requirement for binding of the tail fibers for infection.     

Effects of starvation for potassium and other inorganic ions on protein degradation and ribonucleic acid synthesis in Escherichia coli.

Starvation of Escherichia coli for potassium, phosphate, or magnesium ions leads to a reversible increase in the rate of protein degradation and an inhibition of ribonucleic acid (RNA) synthesis. In cells deprived of potassium, the breakdown of the more stable cell proteins increased two- to threefold, whereas the hydrolysis of short-lived proteins, both normal ones and analog-containing polypeptides, did not change. The mechanisms initiating the enhancement of proteolysis during starvation for these ions were examined. Upon starvation for amino acids or amino acyl-transfer RNA (tRNA), protein breakdown increases in relA+ (but not relA) cells as a result of the rapid synthesis of guanosine-5'-diphosphate-3'-diphosphate (ppGpp). However, a lack of amino acyl-tRNA does not appear to be responsible for the increased protein breakdown in cells starved for inorganic ions, since protein breakdown increased in the absence of these ions in both relA+ and relA cultures, and since a large excess of amino acids did not affect this response. In bacteria in which energy production is restricted, ppGpp levels also rise, and protein breakdown increases. The ion-deprived cultures did show a 40 to 75% reduction in adenosine-5'-triphosphate levels,l similar to that seen upon glucose starvation. However, this decrease in ATP content does not appear to cause the increase in protein breakdown or lead to an accumulation of ppGpp. No consistent change in intracellular ppGpp levels was found in relA+ or relA cells starved for these ions. In addition, in relX mutants, removal of these ions led to accelerated protein degradation even though relX cells are unable to increase ppGpp levels or proteolysis when deprived of a carbon source. In the potassium-, phosphate-, and magnesium-deprived cultures, the addition of choramphenicol or tetracycline caused a reduction in protein breakdown toward basal levels. Such findings, however, do not indicate that protein synthesis is essential for the enhancement of protein degradation, since blockage of protein synthesis by inactivation of a temperature-sensitive valyl-tRNA synthetase did not restore protein catabolism to basal levels. These various results and related studies suggest that the mechanism for increased protein catabolism on starvation for inorganic ions differs from that occurring upon amino acid or arbon deprivation and probably involves an enhanced susceptibility of various cell proteins (especially ribosomal proteins) to proteolysis.