Activity of foot-and-mouth disease virus RNA-dependent RNA polymerase in vitro: inhibition by polyamines and poly(amino acid)s

Replication of foot-and-mouth disease virus RNA in vitro is inhibited by high concentrations of the following polyamines in decreasing order of effectiveness: putrescine, spermine, and cadaverine. The basic poly(amino acids), polylysine, polyornithine, and polyarginine, as well as the basic protein salmine, are several orders of magnitude more inhibitory than polyamines. The interaction between polyornithine and foot-and-mouth disease virus RNA and its inhibition of replicase activity are related to the ability of basic polypeptides to bind to the RNA template. The neutral polymer, polysarcosine, and the polyanions, polyglutamic acid and heparin sulfate, do not inhibit replication; however, both polyanions relieve the inhibition by polyamines and polyamino acids. The mode of inhibition by polyamines and poly(amino acids) and the antagonism by heparin is discussed.     

Polyamine binding sites in the rat brain hippocampus plasma membranes: MK 801 does not influence the binding process

The study was undertaken to characterize the polyamine binding sites in rat brain hippocampus plasma membranes. There were two types of binding sites for putrescine, Bmax 650 and 100 pmol/mg protein, with Kd1 = 39.2 and Kd2 = 6.7 microM, respectively, while those for spermidine (Spd) and spermine (Spm) represented only one type of population with Bmax 2.55 and 15 nmol/mg protein, respectively. The Kd values for Spd and Spm were 34 and 30.3 microM, respectively. The maximum binding of polyamines was found at pH 8.0. The binding capacity of these molecules was curtailed at 4 degrees C, indicating that the binding is an energy-dependent phenomenon. The specific binding was not appreciably influenced by the addition of MK 801, an antagonist of NMDA receptor, indicating that there are polyamine-specific binding sites that are different from those for MK 801. Glycine also did not significantly influence the binding of these biogenic amines. Interestingly, the addition of polyamino acids (polylysine, polyornithine, and polyglutamic acid) inhibited the polyamine binding to their receptor sites, supporting the notion that positive charge of polyamines could be important factor in the binding process.     

The preferential loss of the polylysine- or polyornithine-stimulated activity of Clostridium perfringens polynucleotide phosphorylase during proteolysis

1. An improved method for the purification of Clostridium perfringens polynucleotide phosphorylase (nucleoside diphosphate-polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is described. The product was stable and was highly stimulated by polylysine or polyornithine. 2. It migrated as a single enzyme during sucrose-density-gradient centrifugation, and no separation of polymerization and phosphorolytic activities was observed. 3. Trypsin digestion caused a rapid, preferential loss of the polylysine- or polyornithine-stimulated activity, which was prevented by low concentrations of polyornithine. 4. The protection by polyornithine was not specific. 5. It is concluded that charge effects on the clostridial polynucleotide phosphorylase itself are primarily responsible for the stimulation of this enzyme by polylysine or polyornithine.     

Effects of Poly-l-Lysine on Infectious Viral Nucleic Acid

Infectious ribonucleic acids (IRNA) of Venezuelan equine encephalitis and Eastern equine encephalitis viruses were observed to form noninfectious complexes with a basic polyamino acid, poly-l-lysine. Original infectivity was recovered from the complexes by digestion of the polylysine with Pronase, and partial recovery was effected by treatment with sodium dodecyl sulfate. Infectivity could not be recovered from the complexes containing polylysine of 100,000 molecular weight by changes in ionic strength, pH, or by treatment with phenol, deoxycholate, or digitonin. Masking of infectivity by polylysine was demonstrated in vivo as well as by plaque assay in tissue culture. Poly-l-lysine preparations of high molecular weight (44,000 to 100,000) were more effective than low molecular weight (3,000) materials in masking infectivity of IRNA. When complexes, in which infectivity had been masked by low molecular weight polylysine, were suspended in 1 m NaCl, some infectivity was recovered. Complexes of polylysine-IRNA differed from control IRNA alone in (i) resistance to inactivation by ribonuclease, (ii) sedimentation patterns in sucrose gradient centrifugation, and (iii) stability of recoverable infectivity during different physical treatments.     

Polyamines and polyamino acids regulation of cytosolic tyrosine protein (Tyr-P) kinase from human erythrocytes.

In vitro regulation of cytosolic tyrosine protein (Tyr-P) kinase from human erythrocytes by polyamines, polyamino acids, negative charged compounds or by insulin using angiotensin II or poly (Glu-Tyr)4:1 as substrates was studied. All the three polyamines, putrescine (Put), spermidine (Spd) and spermine (Spm) stimulated the Tyr-P kinase activity in a dose dependent manner. Spm stimulated Tyr-P kinase activity higher than Put and Spd whether the substrate was angiotension II or poly (Glu-Tyr)4:1. Polyamino acids (polyornithine, polyarginine, polyglutamic acid and polyaspartic acid) did not affect significantly the Tyr-P kinase phosphorylation except polylysine which significantly stimulated the Tyr-P kinase activity. Negative charged compounds (chondroitin sulfate A, B and C) and heparin inhibited the Tyr-P kinase phosphorylation while insulin did not influence the enzyme activity in the presence of either substrates.     

Colorimetric determination of microgram quantities of polylysine by trypan blue precipitation

A simple and sensitive method for the determination of polylysine in solution is described. Polylysine is quantitatively precipitated with trypan blue. The absorbance of unbound dye in the supernatant is inversely proportional to the concentration of this polyamino acid. The precipitation is identical for all sizes of polylysine of molecular weight 13,000 or higher, and is prevented by the addition of either polyanions or serum. The measurable range of polylysine hydrobromide is between 1 and 10 micrograms/ml, which is about 10-fold lower than that by the published methyl orange precipitation method.     

Serum-induced inhibition of isotonic fluid absorption by the kidney proximal tubule. III. Further evidence that complement-mediated cell lysis is involved.

The results of our previous studies have suggested that serum-induced inhibition of proximal tubular fluid absorption is due to complement-induced lysis of the tubular cells. The present study provides further evidence in support of this idea as well as other information pertinent to the mechanism of complement activation in vivo. 1. The electrical resistance of the luminal brush border membrane is reduced drastically concomitantly with a drop in cell potential difference when serum is perfused intraluminally. 2. Human C1 inhibitor (30-50 units/ml) does not significantly affect the inhibitory activity of human serum on fluid absorption, suggesting the non-involvement of the classical pathway. 3. Reactive lysis reagents (C56, C7, C8 + C9) partially inhibit fluid absorption when infused into the lumen. 4. In contrast to our previous report (Sato, K. and Ullrich, K.J. (1974) Biochim. Biophys. Acta 354, 182-187), very fresh serum, 10-times diluted can inhibit fluid absorption if perfused for 10 min. 5. Both mouse and guinea pig serum, which are normally inactive, are activated to attack the tubular cells if 1/100 volume rat or rabbit serum is added to them No such activation occurs by mixing guinea pig serum and mouse serum. The available data suggest that the presence of the later complement components but not the heat-labile factor (Factor B) or C3PA or C1 in the added serum is a prerequisite for mouse and guinea pig sera to be activated to inhibit fluid absorption.     

Isolation and purification of poly(ADP-ribose) glycohydrolase from pig thymus

Poly(ADP-ribose) glycohydrolase has been purified about 12 300-fold from pig thymus with a recovery of 8.5%. The specific activity of the purified enzyme is 13.8 mumol min -1 mg protein -1. The molecular weight was estimated to be 59 000 by gel filtration through Sephadex G-100 in a non-denaturing solvent. Analysis of the final preparation by sodium dodecyl sulphate gel electrophoresis reveals two protein bands of molecular weight, 61 500 and 67 500. The Km value for poly(ADP-ribose) is estimated to be 1.8 microM monomer units. The enzyme preparation is free from phosphodiesterase, NADase and ADP-ribosyltransferase activities. The purified enzyme is inhibited by cyclic AMP, ADP-ribose, naphthylamine, histones H1, H2A, H2B, H3, polylysine, polyarginine, polyornithine and protamine. The inhibition by histone is relieved by an equal mass of DNA. Single-stranded DNA, poly(A), poly(I) and polyvinyl sulphate were inhibitory, but double-stranded DNA was not inhibitory.     

Basic polycations activate the insulin receptor kinase and a tightly associated serine kinase.

The effects of cationic polyamino acids on phosphorylation of the insulin and insulin-like growth factor 1 receptor kinases were studied and the following observations were made. (a) Polylysine stimulated both tyrosine and serine phosphorylation of the insulin receptor and of additional proteins present in lectin-purified membrane preparations from rat liver. (b) Polylysine synergized with insulin to enhance phosphorylation of the insulin receptor and of additional proteins (pp40 and pp110). (c) Polylysine effects were more pronounced upon increasing the polylysine chain length. (d) The effect of polylysine was biphasic with an optimum at 100 micrograms/ml. (e) Polylysine was found ineffective in stimulating the phosphorylation of immobilized insulin receptors. Taken together, these findings support the notion that the action of polylysine involves conformational changes and presumably aggregation of soluble receptors. The same effects of polylysine were obtained with highly purified insulin receptor preparations. Under these conditions polylysine enhanced both serine and tyrosine phosphorylation of the insulin receptor, suggesting that polylysine stimulates the activity of the insulin receptor kinase, and of a serine kinase that is tightly associated with the insulin receptor.     

Stimulative Effect of a Casein Hydrolysate on Exocrine Pancreatic Secretion That is Independent of Luminal Trypsin Inhibitory Activity in Rats

We have previously demonstrated that proteins could stimulate pancreatic secretion independently of luminal bile-pancreatic juice (BPJ) in a BPJ-diverted rat. To determine whether luminal protease-independent pancreatic secretion occurs in normal rats with BPJ returned to the upper small intestine, we investigated the pancreatic secretory response to intraduodenal instillation of a casein hydrolysate or the synthetic trypsin inhibitor, FOY 305, at concentrations which could almost equally inhibit hydrolysis of the synthetic substrate for trypsin with the luminal content. FOY 305 at 10 μg/ml and casein hydrolysate solutions at both 100 and 200 mg/ml similarly inhibited approx. 80% of the tryptic activity in the luminal contents of the proximal small intestine. Intraduodenal administration of casein hydrolysate solutions (100 and 200 mg/ml) significantly increased pancreatic secretion in a dose-dependent manner. However, intraduodenal administration of FOY 305 (10 μg/ml) was ineffective for stimulating pancreatic secretion. These results demonstrate that dietary protein enhances pancreatic secretion independently of the masking of luminal trypsin activity in rats.     

Stimulative effect of a casein hydrolysate on exocrine pancreatic secretion that is independent of luminal trypsin inhibitory activity in rats.

We have previously demonstrated that proteins could stimulate pancreatic secretion independently of luminal bile-pancreatic juice (BPJ) in a BPJ-diverted rat. To determine whether luminal protease-independent pancreatic secretion occurs in normal rats with BPJ returned to the upper small intestine, we investigated the pancreatic secretory response to intraduodenal instillation of a casein hydrolysate or the synthetic trypsin inhibitor, FOY 305, at concentrations which could almost equally inhibit hydrolysis of the synthetic substrate for trypsin with the luminal content. FOY 305 at 10 micrograms/ml and casein hydrolysate solutions at both 100 and 200 mg/ml similarly inhibited approx. 80% of the tryptic activity in the luminal contents of the proximal small intestine. Intraduodenal administration of casein hydrolysate solutions (100 and 200 mg/ml) significantly increased pancreatic secretion in a dose-dependent manner. However, intraduodenal administration of FOY 305 (10 micrograms/ml) was ineffective for stimulating pancreatic secretion. These results demonstrate that dietary protein enhances pancreatic secretion independently of the masking of luminal trypsin activity in rats.     

Stimulation of clonal growth of normal fibroblasts with substrata coated with basic polymers

Improved media have reduced the amount of serum protein required for clonal growth of normal human and chicken fibroblast-like cells. In the presence of limiting amounts of serum protein, attachment of colonies to tissue culture plastic surfaces is weak. Treatment of the culture surface with polylysine or other basic polymers causes the cells to adhere much more tightly. Growth is also improved on the surfaces treated with basic polymers, and further reductions in the concentration of serum as possible. At sufficiently low protein concentrations, growth of some types of cells is totally dependent on the use of a treated surface. Several different types of normal human and chicken fibroblast-like cells show improved growth on polylysine- coated surfaces, but no improvement was obtained in growth of a line of SV-40 transformed WI-38 cells. Acidic and neutral polymers are generally inactive. Collagen and gelatin improve growth slightly, but the effect is much less than that obtained with basic polymers. Both natural and synthetic polymers with an excess of basic groups are active, including histone, polyarginine, polyhistidine, polylysine, polyornithine, and protamine. The only critical requirement appears to be a polymer that carries a positive charge at a physiological pH.     

A rapid method for separating small vesicles from suspension.

Techniques for rapidly aggregating suspensions of small vesicles made either of natural biological membrane or of phospholipid or phospholipid-protein mixtures by addition of one of the basic polypeptides, polylysine or protamine, have been investigated. Either filtration or centrifugation may be used to rapidly (15 to 30 s) and completely (over 98%) separate the vesicular aggregate from the suspending medium. At low values of the polylysine-to-vesicle weight ratio, aggregation is observed to increase with increasing polylysine concentration. At high values of this same ratio, aggregation decreases with increasing polylysine concentration. With protamine, like polylysine, aggregation increases with protamine concentration at low values of the weight ratio. At high values of the ratio, aggregation induced by protamine does not rapidly decrease with increasing protamine concentration as it does with polylysine. Explanations are given for these observations. While leakage induced by polylysine may be troublesome under some conditions with phospholipid vesicles or reconstituted systems, protamine aggregation has been found to induce much less leakage than polylysine aggregation. The leakage rate induced by protamine was not found to be significantly different from the control leakage rate for the first few minutes after addition of protamine under any of the conditions tested. Since this provides ample time for filtration, the protamine aggregation-filtration technique seems to be the method of choice for separation of many types of small vesicles from the suspending medium. It combines the advantages of rapid separation, complete separation, low rate of vesicle leakage, and versatility in being able to separate the vesicles from uncharged molecular components such as sugars where ion-exchange techniques will not work (1), as well as from ionic components. Low cost and simplicity are further advantages of the technique.     

Flash photolysis of caged nitric oxide inhibits proximal tubular fluid reabsorption in free-flow nephron

A nonobstructing optical method was developed to measure proximal tubular fluid reabsorption in rat nephron at 0.25 Hz. The effects of uncaging luminal nitric oxide (NO) on proximal tubular reabsorption were investigated with this method. Proximal fluid reabsorption rate was calculated as the difference of tubular flow measured simultaneously at two locations (0.8-1.8 mm apart) along a convoluted proximal tubule. Tubular flow was estimated on the basis of the propagating velocity of fluorescent dextran pulses in the lumen. Changes in local tubular flow induced by intratubular perfusion were detected simultaneously along the proximal tubule, indicating that local tubular flow can be monitored in multiple sites along a tubule. The estimated tubular reabsorption rate was 5.52 +/- 0.38 nl.min(-1).mm(-1) (n = 20). Flash photolysis of luminal caged NO (potassium nitrosylpentachlororuthenate) was induced with a 30-Hz UV nitrogen-pulsed laser. Release of NO from caged NO into the proximal tubule was confirmed by monitoring intracellular NO concentration using a cell-permeant NO-sensitive fluorescent dye (DAF-FM). Emission of DAF-FM was proportional to the number of laser pulses used for uncaging. Photolysis of luminal caged NO induced a dose-dependent inhibition of proximal tubular reabsorption without activating tubuloglomerular feedback, whereas uncaging of intracellular cGMP in the proximal tubule decreased tubular flow. Coupling of this novel method to measure reabsorption with photolysis of caged signaling molecules provides a new paradigm to study tubular reabsorption with ambient tubular flow.     

Novel polymyxin derivatives are less cytotoxic than polymyxin B to renal proximal tubular cells

The emergence of very multiresistant Gram-negative bacterial strains has reinstated polymyxins (polymyxin B, colistin), pentacationic lipopeptides, in the therapy, in spite of their nephrotoxicity. Extensive tubular reabsorption concentrates polymyxin in proximal tubular cells. The novel polymyxin derivatives NAB739, NAB7061 and NAB741 have their cyclic part identical to that of polymyxin B, but their side chain consists of uncharged octanoyl-threonyl-d-serinyl, octanoyl-threonyl-aminobutyryl, and acetyl-threonyl-D-serinyl respectively. In this study, we compared the toxicities of NAB739, NAB7061 and NAB741 with that of polymyxin B by using the porcine renal proximal tubular cell line LLC-PK1 electroporated or incubated with the selected compound. Both the ability to cause cell necrosis (quantified as the leakage of lactate dehydrogenase) and the ability to cause apoptosis (as quantified by counting apoptotic nuclei) were assessed. In electroporated cells, polymyxin B induced total (>85%) necrosis of the cells at 0.016 mM, whereas an approx. 8-fold concentration of NAB739 and NAB7961 and an approx. 32-fold concentration of NAB741 was required for the same effect. In cells treated without electroporation (incubated), polymyxin B elicited a marked degree (approx. 50%) of necrosis at 0.5mM, whereas the NAB compounds were inert even at 1mM. Neither polymyxin B nor the NAB compounds induced apoptosis.     

Cationic lipids and cationic ligands induce DNA helix denaturation: detection of single stranded regions by KMnO4 probing

Cationic lipids and cationic polymers are widely used in gene delivery. Using 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as a cationic lipid, we have investigated the stability of the DNA in DOTAP:DNA complexes by probing with potassium permanganate (KMnO4). Interestingly, thymidines followed by a purine showed higher susceptibility to cationic ligand-mediated melting. Similar studies performed with other water-soluble cationic ligands such as polylysine, protamine sulfate and polyethyleneimine also demonstrated melting of the DNA but with variations. Small cations such as spermine and spermidine and a cationic detergent, cetyl trimethylammonium bromide, also rendered the DNA susceptible to modification by KMnO4. The data presented here provide direct proof for melting of DNA upon interaction with cationic lipids. Structural changes subsequent to binding of cationic lipids/ligands to DNA may lead to instability and formation of DNA bubbles in double-stranded DNA.     

Intestinal water absorption from select carbohydrate solutions in humans.

Eight men positioned a triple-lumen tube in the duodenojejunum. By use of segmental perfusion, 2, 4, 6, or 8% solutions of glucose (111-444 mM), sucrose (55-233 mM), a maltodextrin [17-67 mM, avg. chain length = 7 glucose units (7G)], or a corn syrup solid [40-160 mM, avg. chain length = 3 glucose units (3G)] were perfused at 15 ml/min for 70 min after a 30-min equilibration period. All solutions were made isotonic with NaCl, except 6 and 8% glucose solutions, which were hypertonic. An isotonic NaCl solution was perfused as control. Water absorption (range: 9-15 ml.h-1.cm-1) did not differ for the 2, 4, and 6% CHO solutions but was greater (P < 0.05) than absorption from control (3.0 +/- 2.2 ml.h-1.cm-1). The 8% glucose and 3G solutions reduced (P < 0.05) net water flux compared with their 2, 4, and 6% solutions, but 8% sucrose and 8% 7G solutions promoted water absorption equivalent to lower CHO concentrations. Water absorption was independent of [Na+] in the original solution. In the test segment, 1) Na+ flux correlated with net water flux (r = 0.72, P < 0.01), K+ (r = 0.78, P < 0.01), and [Na+] (r = 0.68, P < 0.001); 2) Na+ absorption occurred at luminal [Na+] as low as 50 mM; 3) glucose transport increased linearly over the luminal concentration range of 40-180 mM; and 4) net water flux was similar over a range of glucose-to-Na+ concentration ratios of 0.4:1 to 3.5:1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of mitogenic activity and cellular binding of basic fibroblast growth factor by basic proteins

Polycationic molecules were studied either for their ability to displace the binding of basic fibroblast growth factor (bFGF) to high- and low-affinity membrane interaction sites and/or to modulate bFGF-induced proliferation of fibroblasts. Heparin-binding polypeptides, such as polylysine, protamine, histones, and thrombin-displaced [125I]bFGF bound to bovine brain membrane receptors. The most displacing polypeptides were those with the strongest affinity to heparin. Two of these polypeptides, protamine and polylysine, inhibited (at 5 microM) by more than 90% the mitogenic effect induced by bFGF on Chinese hamster lung fibroblast cells (CCL39). At the same dose, no effect was observed with basic proteins that do not bind to heparin, such as cytochrome C and lysozyme. An interesting observation was that protamine at 1 microM potentiated by 1.5-fold the mitogenic activity of bFGF, while it acted as an inhibitor at higher concentration.     

Prenatal programming of rat proximal tubule Na+/H+ exchanger by dexamethasone

Prenatal administration of dexamethasone causes hypertension in rats when they are studied as adults. Although an increase in tubular sodium reabsorption has been postulated to be a factor programming hypertension, this has never been directly demonstrated. The purpose of this study was to examine whether prenatal programming by dexamethasone affected postnatal proximal tubular transport. Pregnant Sprague-Dawley rats were injected with intraperitoneal dexamethasone (0.2 mg/kg) daily for 4 days between the 15th and 18th days of gestation. Prenatal dexamethasone resulted in an elevation in systolic blood pressure when the rats were studied at 7-8 wk of age compared with vehicle-treated controls: 131 +/- 3 vs. 115 +/- 3 mmHg (P < 0.001). The rate of proximal convoluted tubule volume absorption, measured using in vitro microperfusion, was 0.61 + 0.07 nl.mm(-1).min(-1) in control rats and 0.93+ 0.07 nl.mm(-1).min(-1) in rats that received prenatal dexamethasone (P < 0.05). Na(+)/H(+) exchanger activity measured in perfused tubules in vitro using the pH-sensitive dye BCECF showed a similar 50% increase in activity in proximal convoluted tubules from rats treated with prenatal dexamethasone. Although there was no change in abundance of NHE3 mRNA, the predominant luminal proximal tubule Na(+)/H(+) exchanger, there was an increase in NHE3 protein abundance on brush-border membrane vesicles in 7- to 8-wk-old rats receiving prenatal dexamethasone. In conclusion, prenatal administration of dexamethasone in rats increases proximal tubule transport when rats are studied at 7-8 wk old, in part by stimulating Na(+)/H(+) exchanger activity. The increase in proximal tubule transport may be a factor mediating the hypertension by prenatal programming with dexamethasone.     

Mechanism of anticholinesterase activities of cardiotoxin, protamine and polylysine.

Cardiotoxin, protamine and polylysine are potent inhibitors of various cholinesterases. CaCl2 and MgCl2 overcome the inhibition. The order of addition of the inhibitor and the protecting agent (MgCl2) influences the final degree of the inhibition observed. These findings suggest that cardiotoxin, protamine and polylysine inhibit cholinesterases by the ionic binding of their basic groups with the anionic sites of cholinesterase molecules.