Quercetin sensitizes fluconazole-resistant candida albicans to induce apoptotic cell death by modulating quorum sensing.

Quorum sensing (QS) regulates group behaviors of Candida albicans such as biofilm, hyphal growth, and virulence factors. The sesquiterpene alcohol farnesol, a QS molecule produced by C. albicans, is known to regulate the expression of virulence weapons of this fungus. Fluconazole (FCZ) is a broad-spectrum antifungal drug that is used for the treatment of C. albicans infections. While FCZ can be cytotoxic at high concentrations, our results show that at much lower concentrations, quercetin (QC), a dietary flavonoid isolated from an edible lichen (Usnea longissima), can be implemented as a sensitizing agent for FCZ-resistant C. albicans NBC099, enhancing the efficacy of FCZ. QC enhanced FCZ-mediated cell killing of NBC099 and also induced cell death. These experiments indicated that the combined application of both drugs was FCZ dose dependent rather than QC dose dependent. In addition, we found that QC strongly suppressed the production of virulence weapons-biofilm formation, hyphal development, phospholipase, proteinase, esterase, and hemolytic activity. Treatment with QC also increased FCZ-mediated cell death in NBC099 biofilms. Interestingly, we also found that QC enhances the anticandidal activity of FCZ by inducing apoptotic cell death. We have also established that this sensitization is reliant on the farnesol response generated by QC. Molecular docking studies also support this conclusion and suggest that QC can form hydrogen bonds with Gln969, Thr1105, Ser1108, Arg1109, Asn1110, and Gly1061 in the ATP binding pocket of adenylate cyclase. Thus, this QS-mediated combined sensitizer (QC)-anticandidal agent (FCZ) strategy may be a novel way to enhance the efficacy of FCZ-based therapy of C. albicans infections.

Colostrum hexasaccharide, a novel Staphylococcus aureus quorum-sensing inhibitor.

The discovery of quorum-sensing (QS) systems regulating antibiotic resistance and virulence factors (VFs) has afforded a novel opportunity to prevent bacterial pathogenicity. Dietary molecules have been demonstrated to attenuate QS circuits of bacteria. But, to our knowledge, no study exploring the potential of colostrum hexasaccharide (CHS) in regulating QS systems has been published. In this study, we analyzed CHS for inhibiting QS signaling in Staphylococcus aureus. We isolated and characterized CHS from mare colostrum by high-performance thin-layer chromatography (HPTLC), reverse-phase high-performance liquid chromatography evaporative light-scattering detection (RP-HPLC-ELSD), (1)H and (13)C nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS). Antibiofilm activity of CHS against S. aureus and its possible interference with bacterial QS systems were determined. The inhibition and eradication potentials of the biofilms were studied by microscopic analyses and quantified by 96-well-microtiter-plate assays. Also, the ability of CHS to interfere in bacterial QS by degrading acyl-homoserine lactones (AHLs), one of the most studied signal molecules for Gram-negative bacteria, was evaluated. The results revealed that CHS exhibited promising inhibitory activities against QS-regulated secretion of VFs, including spreading ability, hemolysis, protease, and lipase activities, when applied at a rate of 5 mg/ml. The results of biofilm experiments indicated that CHS is a strong inhibitor of biofilm formation and also has the ability to eradicate it. The potential of CHS to interfere with bacterial QS systems was also examined by degradation of AHLs. Furthermore, it was documented that CHS decreased antibiotic resistance in S. aureus. The results thus give a lead that mare colostrum can be a promising source for isolating a next-generation antibacterial.

Immunological and biochemical analysis of glycosylated surface antigens and lipophosphoglycan of Tritrichomonas foetus.

Immunoaffinity-purified TF1.17 adhesin antigen was compared biochemically and antigenically to Tritrichomonas foetus (TF) lipophosphoglycan (LPG) and a soluble glycosylated antigen (SGA) released from T. foetus and implicated in pathogenesis and immunity. The monoclonal antibodies (Mabs TF1.15 and TF1.17) specific for a glycosylated TF1.17 antigen were previously shown to prevent adhesion of the T. foetus parasites to bovine vaginal epithelial cells and to mediate killing by bovine complement. SGA was isolated from T. foetus-conditioned buffer and purified by octyl-Sepharose hydrophobic column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of SGA showed a major SGA1 component (approximately 190 kDa) and a minor SGA2 component (50-70 kDa), which migrated close to TF-LPG and TF1.17. The carbohydrate and lipid compositional analyses of affinity-purified TF1.17 and SGA2 by high-performance liquid chromatography (HPLC) and gas-liquid chromatography revealed the presence of monosaccharides and fatty acids as found in TF-LPG. All antigens contained terminal fucose as determined by alpha-fucosidase digestion followed by HPLC. ELISA and western blots were used to further characterize these glycosylated antigens and to analyze their relationships. The Mabs TF1.15 and TF1.17 reacted very strongly to TF-LPG and SGA2. as well as TF1.17 antigen, indicating that these molecules share common epitopes. These Mabs did not react with the SGA1 component either in ELISA and western blot analyses. Also, the monosaccharide composition of SGA1 was very different from the other three antigen, suggesting SGA1 was different from LPG, SGA2 and TF1.17. Although LPG reacted with Mabs to native TF1.17 antigen, LPG did not induce an immune response in cattle with the same route and adjuvant used to produce strong antibody responses to the native antigen. The latter response suggests that the tightly bound peptide present in the immunoaffinity-purified antigen is necessary for induction of a response to (an) epitope(s) in TF-LPG and TF1.17. Furthermore, vaginal fluid from T. foetus-infected heifers and serum from a cow with a T. foetus-associated pyometra recognized both TF1.17 and TF-LPG in western blots. These results suggest that T. foetus LPG and SGA2 are related to TF1.17 antigen, which was previously shown to play an important role in the pathogenesis and host response in bovine trichomoniasis.

Use of calcium antagonists in ventricular dysfunction.

Calcium antagonists are now widely used in a variety of cardiocirculatory disorders, many of which are associated with varying levels of depressed myocardial function. Thus, the hemodynamic effects of calcium antagonists in patients with normal as well as depressed ventricular function are clinically relevant. None of the 3 agents verapamil, nifedipine or diltiazem exerts significant negative inotropic effects in patients with relatively normal myocardial function, although increases in left ventricular end-diastolic pressure may occur with verapamil and possibly diltiazem. In a setting in which ischemia, hypertension or arrhythmias contribute to cardiac failure, all 3 agents may ameliorate myocardial decompensation if they reverse the precipitating causes. In patients with depressed myocardial function, the effects of diltiazem are not known; verapamil may depress myocardial function, especially if the ventricular filling pressure is increased. Nifedipine generally has little depressant action in this setting and usually improves cardiac function, especially if the sympathetic reflexes are intact. However, hemodynamic deterioration after nifedipine administration has been reported. Thus, the available data do not support the use of calcium antagonists as afterload-reducing agents in heart failure and suggest caution in the use of these agents in patients with impaired ventricular performance.

Clinical applications of slow channel blocking compounds.

The slow-channel blockers constitute a structurally diverse group of drugs with varying mechanisms of action, propensities for site of greatest cardiovascular activity, and clinical efficacy. They share however the property of blocking the slow inward channel in heart muscle and of inhibiting calcium fluxes in smooth muscle. Their in vivo and in vitro actions must be distinguished. The overall actions represent a balance of direct and autonomically-mediated reflex actions interacting with the compounds' varying degrees of intrinsic non-competitive sympathetic antagonism. A knowledge of the pharmacodynamic differences between these drugs allows the physician to select the most appropriate agent for a given clinical situation. The central role of calcium in the cellular processes in the heart and the vascular system forms the basis for the utility of this class of drugs in a wide variety of cardiovascular disorders. Current intensive experimental and clinical investigations are likely to further define the roles of nifedipine, verapamil and diltiazem and their congeners in cardiovascular therapeutics. The prospect of development of newer compounds with greater selectivity of action is real. As pointed out by Braunwald (1982 a,b), with further clarification of the mechanisms of actions of these compounds and elucidation of the role of calcium fluxes throughout the body, more specific and potent agents may be developed. The apparent efficacy of the nifedipine congener nimodipine, in the treatment of cerebral vasospasm associated with subarachnoid hemorrhage (Allen et al., 1983) may simply be the first of a large number of 'specific' or targeted slow channel blockers. The development of such compounds may offer further therapeutic possibilities in the control of a variety of cardiocirculatory diseases.

Pharmacological basis for the therapeutic applications of slow-channel blocking drugs.

Excitation-contraction coupling in cardiac muscle as well as in smooth muscle is mediated by the transmembrane fluxes of calcium. In the case of cardiac muscle, this transfer occurs through the slow-inward channel. Agents that selectively inhibit the myocardial slow-channel also block calcium entry in smooth muscle cells, particularly in arteries. Thus, such selective inhibitors of the slow channel, exemplified by verapamil, nifedipine, and diltiazem, produce a marked negative inotropic effect in cardiac muscle; in whole animals or in man, such a propensity is largely nullified or even reversed by the profound vasodilator effects of these compounds. The drugs known as calcium antagonists are chemically heterogeneous and they may exhibit associated pharmacological properties such as noncompetitive sympathetic inhibition while having varying potencies for inhibiting the calcium influx in smooth muscle and in the heart and nodal tissues. These similarities and differences influence the net electrophysiologic and hemodynamic effects of calcium antagonists in man. Electrophysiologically, the main effect is a depressant one on the AV node in which most agents of the class lengthen AV conduction and enhance refractoriness, a property that is relevant in the termination of PSVT and to the slowing of the ventricular response in atrial flutter and fibrillation. The effective refractory periods of atrial, ventricular, and His-Purkinje tissues or the bypass tracts are not altered by calcium antagonists, but conduction may be improved in ischemic tissues. On the surface ECG, the only effect is the short-term lengthening of the PR interval with no change in the QRS or Q-Tc intervals. The sinus frequency is variably affected relative to the competing influences of the direct effect, reflex response to hypotension and of sympathetic antagonism. The sinus node recovery time is affected little normally, but may be prolonged dramatically in the sick sinus syndrome. Hemodynamically, as a class of drugs, calcium antagonists produce a complex interplay of simultaneous changes in preload, afterload, contractility, coronary flow, and heart rate. The net hemodynamic effect that becomes apparent will be dependent on the agent used, on the cardiac condition and the level of ventricular function present, on the intactness of the autonomic nervous system, and on the route of drug administration. An appreciation of the electrophysiological and hemodynamic actions of calcium antagonists relative to their individual pharmacologic properties permits the rational choice of the appropriate agent in the control of a wide spectrum of cardiocirculatory disorders.

Local anaesthetic and anti-arrhythmic actions of alprenolol relative to its effect on intracellular potentials and other properties of isolated cardiac muscle.

1. Alprenolol, a beta-adrenoceptor blocking drug reported to have the same potency as propranolol in vitro and in vivo, was found to be four times more active than procaine as a local anaesthetic on frog sciatic nerve.2. At doses of 0.125 mg/kg and above alprenolol protected anaesthetized guinea-pigs against ouabain-induced ventricular fibrillation.3. In isolated rabbit atria, resting potentials were unchanged, and the duration of the action potential was not prolonged, by concentrations of alprenolol up to 10.5 x 10(-6)M. The maximum rate of depolarization (MRD), however, was reduced by 30% at a concentration of 0.525 x 10(-6)M. This was 214 times less than the concentration which reduced the frog action potential height by 25%.4. The concentration of alprenolol required to produce more than 15% decrease in contraction or maximum driven frequency was 3.5 x 10(-6)M.5. As a test of the direct action of alprenolol on isolated cardiac muscle, MRD was also a more sensitive test than the measurement of electrical threshold or conduction velocity.