Stimulation of prolyl hydroxylase activity by chelating agents.

The activity of purified prolyl hydroxylase was enhanced several fold by addition of some chelating agents to the assay medium. Chelating agents could be classified into three groups. The chelating agents of Group I such as α, α′-dipyridyl were inactive until they reached equimolar concentration with ferrous ion in the assay mixture. The Group II agents, EDTA, diethylenetriaminepentaacetic acid, etc., stimulated the enzymatic activity 1.5- to 3-fold at equimolar concentration with ferrous ion. But the agents of both groups precipitously inhibited the enzymatic activity at concentrations greater than ferrous ion. On the other hand, Group III chelating agents, such as nitrilotriacetic acid, enhanced the enzymatic activity 5- to 10-fold at concentrations greater than ferrous ion. Nucleoside triphosphates, which also stimulate the enzymatic activity several fold and whose optimal concentrations are 1–3 × 10^?m, may be analogous to nitrilotriacetic acid of Group III.     

Role of tumour necrosis factor in the tumour-necrotizing activity of agents with diverging toxicity

Summary We investigated the ability of various tumournecrotizing agents with diverging toxicity to induce tumour necrosis factor (TNF) and cytostatic activity inPropionibacterium-acnes-primed Swiss and tumour-bearing BALB/c mice, and the capacity of anti-TNF antibodies to inhibit induction of tumour necrosis by the agents. Lipid A and especially its combination with muramyl dipeptide induced high TNF levels in Swiss mice, as measured in the serum. Lower levels were induced by detoxified lipid A and the nontoxic dsRNA, polyadenylic polyuridylic acid, either alone or combined with muramyl dipeptide. The toxic agents also appeared the strongest inducers of mediators with cytostatic activity against cultured endothelial cells and MethA tumour cells. Anti-TNF antibodies partially reduced the cytostatic activity of the sera against MethA cells. Tumour-bearing BALB/c mice produced only low levels of TNF and cytostatic factors in response to all agents. Recombinant mouse TNF hardly reduced the DNA synthesis of MethA cells, unless normal mouse serum was added. Serum fromP.-acnes-treated Swiss mice and tumour-bearing BALB/c mice, that were inhibitory on their own, failed to potentiate the action of TNF. Serum from Swiss mice treated with toxic, but not detoxified, lipid A caused extensive tumour necrosis upon injection into MethA-bearing BALB/c mice. This activity was completely abolished by pre-incubation of the serum with anti-TNF. The tumour-necrotizing activity of the agents could be partially reduced by prior injection of these antibodies. Results show that the capacity of the agents to induce TNF and cytostatic activity is not related to their antitumour potential. Although TNF is likely to be a crucial mediator of the tumour-necrotizing action of the toxic as well as the nontoxic agents, it is probably not the sole mediator. Data also indicate that induction of tumour necrosis does not require induction of high and, thus toxic, TNF levels in the serum.     

A review of anabolic therapies for osteoporosis

Osteoporosis results from a loss of bone mass and bone structure such that the bone becomes weak and fractures with very little trauma. Until recently, the approved osteoporosis therapies prevented more bone loss by altering osteoclast activity and lifespan. Recently, attention has turned away from osteoclast inhibition to agents that can stimulate the osteoblast to form new bone, or anabolic agents. This article reviews both approved and experimental anabolic agents that improve bone mass by improving osteoblast activity, or increasing osteoblast number. The use of the anabolic agents to improve bone mass and strength followed by agents that prevent the new bone mass from being lost may offer the ability to cure osteoporosis and reduce bone fracture healing time.     

Enhancement of glutathione levels in mouse peritoneal macrophages by sodium arsenite, cadmium chloride and glucose/glucose oxidase

Glutathione content of mouse peritoneal macrophages markedly increased when they were exposed to insulting agents like sodium arsenite, cadmium chloride, and glucose/glucose oxidase which generates hydrogen peroxide. This increase was attributed to the induction of the cystine transport activity by these agents. The transport activity for other amino acids was not induced, but rather diminished by these agents. Heat shock treatment did not induce the cystine transport activity, nor did it augment glutathione. Since glutathione protects cells against the cytotoxic effects of these agents, the induction of the cystine transport activity constitutes a protective mechanism related to the stress caused by the agents. The protein component(s) for cystine transport may fall into the category of the stress protein.     

Pharmacological control of muscular activity in the sea urchin larva. III. Role of cyclic nucleotides.

1. The muscular activity of the sea urchin pluteus is strongly affected by dibutyryl-c-AMP, in a stimulatory or inhibitory manner depending on the concentration, the time of exposure, and the spontaneous level of activity. 2. Dibutyryl-c-GMP, like muscarinic agents and the guanylate-cyclase activators biotin and nitrite, keeps the activity low. 3. It is suggested that the effects of muscarinic agents is mediated by c-GMP, the effects of certain monoamines by c-AMP. 4. The two cyclic nucleotides appear to control the cellular influx of Ca2+ in opposite directions. They therefore interfere with the stimulatory and paralytic effects of nicotinic agents.     

Cartilage cyclic nucleotide phosphodiesterase: inhibition by anti-inflammatory agents

Soluble 3′,5′-nucleotide phosphodiesterase (PDE) activity is described in chicken epiphyseal and articular cartilage. Kinetic studies of these enzymes demonstrate a high and low K_m for the substrates, adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and guanosine 3′,5′-cyclic monophosphate (cyclic GMP). Epiphyseal and articular PDE activities are inhibited by those anti-inflammatory agents which are potent inhibitors of the enzyme, prostaglandin synthetase (PS). Specificity of this inhibition is indicated by the activity of these agents against the low K_m enzyme. Other anti-inflammatory agents with significantly less potency as PS inhibitors or with no activity against prostaglandin synthetase are found to be either inactive or relatively less potent as inhibitors of cartilage PDE activity. A variety of other anti-inflammatory or anti-rheumatic agents, which are not known to affect prostaglandin synthetase activity, are poor inhibitors of cartilage PDE activity. These data provide insight into the mechanism of action of certain anti-inflammatory agents and into the relationships between prostaglandins and inflammatory reactions.     

Evaluation of Some Trypanostatic Agents by Means of Herpetomonas culicidarum

SYNOPSIS. A rapid quantitative method for measuring trypanostatic activity by means of the nonpathogenic flagellate Herpetomonas culicidarum is described. Of the known trypanocidal agents tested, pentamidine was the most active; stilbamidine and propamidine somewhat less active. H. culicidarum is more resistant to these agents than are some pathogenic hemoflagellates. Two new antifungal antibiotics, nystatin and heptamycin (a candicidin-like antibiotic), had powerful trypanostatic activity in vitro. The potential trypanostatic activity of antifungal agents is noted.     

Pharmacology of airway afferent nerve activity

Afferent nerves in the airways serve to regulate breathing pattern, cough, and airway autonomic neural tone. Pharmacologic agents that influence afferent nerve activity can be subclassified into compounds that modulate activity by indirect means (e.g. bronchial smooth muscle spasmogens) and those that act directly on the nerves. Directly acting agents affect afferent nerve activity by interacting with various ion channels and receptors within the membrane of the afferent terminals. Whether by direct or indirect means, most compounds that enter the airspace will modify afferent nerve activity, and through this action alter airway physiology.     

Fibrin-collagen substance as a vehicle for local application of antibacterial agents]

Inhibitory activity of fibrin-collagen substance (FCS) impregnated by various antibacterial agents against various microorganisms was investigated. It was demonstrated that antibacterials bind well with FCS, providing its sufficient antibacterial activity. Determination of growth-inhibition zones showed that antibacterial agents diffuse into medium well and demonstrated the same activity as discs with antibiotics. For clinical practice it is recommended to use for impregnation the following drugs: gentamycin, meropenem, cefriaxone and ciprofloxacin.     

Induction of alkaline phosphatase activity by synergistic action of dibutyryl cyclic adenosine monophosphate, prednisolone, butyrate and sodium chloride in cultured cells

Human urinary bladder carcinoma cells (JTC-32) retain a low alkaline phosphatase activity. Prednisolone or a hypertonic concentration of NaCl caused a moderate increase in the activity (10- to 15-fold of control), but dibutyryl cAMP or butyrate did not. Examination of the combined effect of these four agents revealed that they acted synergistically in any combination. When the cells were incubated with the four agents together, the enzyme activity increased 60- to 250-fold. Serum also contributed to this synergistic increase. These agents slightly inhibited cell growth and protein synthesis. The enzyme induction was completely inhibited by cycloheximide or actinomycin D. The synergistic effect of the four agents on the enzyme activity was also observed in other strains of carcinoma cells, human urinary bladder carcinoma cells (JTC-30) and monkey hepatocarcinoma cells (NCLP-6E). Thus, it is concluded that the coexistence of the four agents provides general and superior conditions for the induction of alkaline phosphatase in cultured carcinoma cells.     

Role of first stimulating agents in the production of tumor necrosis factor

Summary The conditions and kinetics of tumor necrosis factor (TNF) production were examined. For TNF production, dual stimulation is necessary. Priming agents such as BCG, Corynebacterium parvum, and zymosan, which can stimulate the reticuloendothelial system (RES), are good substances for TNF production with the aid of lipopolysaccharide. Wide differences are observed in TNF producibility among different priming agents. The producibility of TNF depends on the degree of stimulation of the RES by the priming agents. Those priming agents, e.g., Propionibacterium acnes and Corynebacterium anaerobium, that are able to induce substantial RES hyperplasia are also able to induce high levels of TNF activity. Following administration of large doses of BCG or zymosan, mice were found to produce TNF activity. However, PPD, OK 432, PSK, and Choreito were unable to induce TNF activity.     

The contribution of alkylation to the activity of quinone antitumor agents

Studies have shown that the quinone group can produce tumor cell kill by a mechanism involving active oxygen species. This cytotoxic activity can be correlated with the induction of DNA double strand breaks and is enhanced by the ability of the quinone compound to bind to DNA by alkylation. The cytotoxic activity and the production of DNA damage by model quinone antitumor agents were compared in L5178Y cells, sensitive and resistant to alkylating agents, to assess the contribution of alkylation to the activity of these agents. The resistant L5178Y/HN2 cells were found to be two fold and six fold more resistant to the alkylating quinones, benzoquinone mustard and benzoquinone dimustard, respectively, than parent L5178Y cells. In contrast, the L5178Y/HN2 cells showed no resistance to the nonalkylating quinones, hydrolyzed benzoquinone mustard and bis(dimethylamino)benzoquinone. The alkylating quinones produced approximately two fold less cross-linking in L5178Y/HN2 cells compared with L5178Y sensitive cells. DNA double strand break formation by hydrolyzed benzoquinone mustard and bis(dimethylamino)benzoquinone was not significantly different in sensitive and resistant cells. However, the induction of double strand breaks by the alkylating quinones benzoquinone mustard and benzoquinone dimustard was reduced by 5-fold and 15-fold, respectively, in L5178Y/HN2 cells. These results show that the alkylating activity of the alkylating quinones cannot directly explain all of the enhanced cytotoxic activity of these agents. Furthermore, they provide strong evidence that the enhanced formation of DNA double strand breaks by alkylating quinone agents is directly related to the ability of these agents to bind to DNA. This increased formation of strand breaks may account for the enhanced cytotoxic activity of the alkylating quinones.     

The role of NAD(P)H oxidoreductase (DT-Diaphorase) in the bioactivation of quinone-containing antitumor agents: a review

Bioactivation of quinone-containing anticancer agents has been studied extensively within the context of the chemistry and structure of the individual quinones which may result in various mechanisms of bioactivation and activity. In this review we focus on the two electron enzymatic reduction/activation of quinone-containing anticancer agents by DT Diaphorase (DTD). This enzyme has become important in oncopharmacology because its activity varies with tissues and it has been found to be elevated in tumors. Thus, a selective tumor cell kill can exist for agents that are good substrates for this enzyme. In addition, the enzyme can be induced by a variety of agents, a fact that can be used in chemotherapy. That is induction by a nontoxic agent followed by treatment with a good DT-Diaphorase substrate. A wide variety of anticancer drugs are discussed some of which are not good substrates such as Adriamycin, and some of which are excellent substrates. The latter category includes a variety of quinone containing alkylating agents.     

Inactivation of rabbit muscle creatine kinase by reversible formation of an internal disulfide bond induced by the fungal toxin gliotoxin

The biological activity of gliotoxin is dependent on the presence of a strained disulfide bond that can react with accessible cysteine residues on proteins. Rabbit muscle creatine kinase contains 4 cysteines per 42-kDa subunit and is active in solution as a dimer. Only Cys-282 has been identified as essential for activity. Modification of this residue results in loss of activity of the enzyme. Treatment of creatine kinase with gliotoxin resulted in a time-dependent loss of activity abrogated in the presence of reducing agents. Activity was restored when the inactivated enzyme was treated with reducing agents. Inactivation of creatine kinase by gliotoxin was accompanied by the formation of a 37-kDa form of the enzyme. This oxidized form of creatine kinase was rapidly reconverted to the 42-kDa species by the addition of reducing agents concomitant with restoration of activity. A 1:1 mixture of the oxidized and reduced monomer forms of creatine kinase as shown on polyacrylamide gel electrophoresis was equivalent to the activity of the fully reduced form of the enzyme consistent with only one reduced monomer of the dimer necessary for complete activity. Conversion of the second monomeric species of the dimer to the oxidized form by gliotoxin correlated with loss of activity. Our data are consistent with gliotoxin inducing the formation of an internal disulfide bond in creatine kinase by initially binding and possibly activating a cysteine residue on the protein, followed by reaction with a second neighboring thiol. The recently published crystal structure of creatine kinase suggests the disulfide is formed between Cys-282 and Cys-73.     

Contribution of blue-green pigments to hemolytic activity of Pseudomonas aeruginosa cultural fluid

AIM: To assess the contribution of blue-green pigments of Pseudomonas aeruginosa to hemolytic activity of its cultural fluid. MATERIALS AND METHODS. Eight hospital strains and reference strain ATCC 15442 were used. Growth dynamics of strains as well as features of accumulation of hemolytic and phospholipase activity were studied. Purified samples of pyoverdin and pyocyanin were extracted by gel-chromatography and chloroform extraction methods. Hemolytic and lecitinase activities of the samples as well as effect of active oxygen scavengers and chelating agents on these activities were studied. RESULTS: Dynamics of accumulation of hemolytic activity significantly differed from that of phospholipase activity when strains were grown in liquid medium. Chromatographic separation of the pigments from cultural fluid supernatants sharply reduced its hemolytic activity. Purified samples of pyoverdin and pyocyanin were capable to lyse erythrocytes and chicken egg lecitin. These characteristics of the pigments were inhibited by nitroblue tetrazolium and sensitive to chelating agents. Conclusion. Pyoverdin and pyocyanin of pathogenic strains of P. aeruginosa are capable to lyse erythrocytes and suspension of purified chicken egg lecitin, they contribute to total hemolytic activity of pathogenic strains of Pseudomonas, which is not determined only by phospholipase C produced by microorganism. Lytic activity of the pigments is blocked by nitroblue tetrazolium and susceptible to some chelating agents. Apparently, this activity is mediated by superoxide radical and determined by presence of metals with transient valence in pigments' molecules.     

Autoimmunity as a special case of immunity: removing threats from within

The function of the adaptive immune response against exogenous (non-self) agents is to help the innate arm of the immune system (represented by phagocytic cells) to fight and eliminate these agents. We suggest that the body also protects itself against potentially harmful self components using mechanisms similar to those used for fighting and eliminating non-self agents, and that the protective immune activity against self-components competes with the activity of self-destructive compounds. Tolerance to self is thus not a lack of response to self, but the ability to tolerate an active defense response to self without developing an autoimmune disease.     

The mode of action of adenosine 3′:5′-cyclic monophosphate in mammalian islets of Langerhans. Effects of insulin secretagogues on islet-cell protein kinase activity

1. Protein kinase activity was measured in islets of Langerhans that had been incubated in the presence of agents known to affect insulin release. 2. Glucagon, theophylline, caffeine and 3-isobutyl-1-methylxanthine, agents that raise cyclic AMP concentrations in islet cells and stimulate insulin release, increased protein kinase activity. Adrenaline and diazoxide, agents that decrease cyclic AMP concentrations and inhibit insulin secretion, decreased the activity. 3. The increase in protein kinase activity produced by different concentrations of 3-isobutyl-1-methylxanthine was apparently related to the increase in intracellular concentrations of cyclic AMP. 4. The sulphonylureas, tolbutamide and glibenclamide, agents that increase insulin release, also increased the protein kinase activity; however, leucine, arginine and xylitol, which also stimulate insulin release, were without effect on the kinase activity. 5. Increasing the glucose concentration of the incubation medium from 2 to 20mm had no effect on protein kinase activity. Further, the ability of 3-isobutyl-1-methylxanthine to increase the protein kinase activity was not affected by the glucose concentration of the incubation medium. 6. These results suggest that agents which affect insulin secretion by altering cyclic AMP concentrations may exert their effects on hormone release by altering the activity of a cyclic AMP-dependent protein kinase in islet cells.     

cAMP mediated proteolysis of the catalytic subunit of cAMP-dependent protein kinase

The cAMP-dependent protein kinase from LLC-PK1 cells can be activated in vivo by calcitonin and vasopressin, or forskolin. Continuous treatment of cells with these agents results in a decrease of total cAMP-PK activity. The loss of kinase activity was enhanced when either of these three agents was incubated in the presence of isobutylmethylxanthine. Results obtained using affinity purified antibodies to the catalytic subunit show that the loss of kinase was due to specific proteolysis of this subunit.     

Mitochondrially targeted anti-cancer agents

Cancer is an ever-increasing problem that is yet to be harnessed. Frequent mutations make this pathology very variable and, consequently, a considerable challenge. Intriguingly, mitochondria have recently emerged as novel targets for cancer therapy. A group of agents with anti-cancer activity that induce apoptosis by way of mitochondrial destabilisation, termed mitocans, have been a recent focus of research. Of these compounds, many are hydrophobic agents that associate with various sub-cellular organelles. Clearly, modification of such structures with mitochondria-targeting moieties, for example tagging them with lipophilic cations, would be expected to enhance their activity. This may be accomplished by the addition of triphenylphosphonium groups that direct such compounds to mitochondria, enhancing their activity. In this paper, we will review agents that possess anti-cancer activity by way of destabilising mitochondria and their possible targets. We propose that mitochondrial targeting, in particular where the agent associates directly with the target, results in more specific and efficient anti-cancer drugs of potential high clinical relevance.     

Free fatty acids as feedback regulators of adenylate cyclase and cyclic 3':5'-AMP accumulation in rat fat cells.

Rat fat cells incubated with lipolytic agents released substances to the medium which acted as feedback regulators of cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. The feedback regulators were not removed by adenosine deaminase. Dialyzed medium that had previously been incubated with fat cells in the presence of norepinephrine markedly inhibited cyclic AMP accumulation by fresh cells, whereas dialyzed medium from control cells did not inhibit cyclic AMP accumulation. The effects of lipolytic agents could be mimicked by adding dialyzed medium previously incubated with fat cells in the presence of oleic acid. This suggested that free fatty acids were the nondialyzable and adenosine deaminase-insensitive inhibitors of cyclic AMP accumulation released to the medium by fat cells incubated with lipolytic agents. The regulatory function of free fatty acids was related to the molar ratio of fatty acid to albumin. Profound inhibition of both lipolysis and cyclic AMP accumulation was seen as the free fatty acid/albumin ratio exceeded 3. The inhibition of cyclic AMP accumulation by oleate was seen as soon as there was a detectable increase in cyclic AMP due to lipolytic agents. Protein kinase activity (in the presence of cyclic AMP) of the infranatant obtained after centrifugation of fat cell homogenates at 48,000 x g was inhibited by medium from cells incubated with lipolytic agents or added oleate. Adenylate cyclase activity of rat fat cell ghosts was also inhibited by dialyzed or nondialyzed medium that previously had been incubated with lipolytic agents or added fatty acids. The direct addition of oleate markedly inhibited adenylate cyclase activity as the free fatty acid/albumin ratio exceeded 2. These data suggest that the prolonged drop in cyclic AMP accumulation seen during the incubation of rat fat cells with lipolytic agents is due to the inhibition of adenylate cyclase. This occurs when the free fatty acid/albumin ratio exceeds 3.