Old and new targets for innovative antimalarial compounds: the different strategies of the Italian Malaria Network.

Clinical treatment-failures to affordable drugs encouraged new investigation for discovery and development of new prophylactic and therapeutic interventions against malaria. The Drug Discovery Cluster (DDcl) of the Italian Malaria Network gathers several highly integrated and complementary laboratories from different Italian Institutions to identify, synthesise, screen in vitro and in vivo new antimalarial molecules directed against the intraerythrocytic stage of P. falciparum parasites and/or with transmission blocking activity to select lead compounds for further development. Complementary research activities, both in vitro and in the clinics, aim at investigating the pathogenetic mechanisms of severe malaria anaemia and the different manifestations of the disease in malaria-HIV co-infected patients to identify new therapies and improve survival.

Defective hydroxylation of phenformin as a determinant of drug toxicity.

The kinetics of phenformin and its metabolite, p-hydroxyphenethylbiguanide, was studied in eight diabetic patients with varying degrees of renal impairment. Plasma and urinary phenformin and p-hydroxyphenethylbiguanide levels were determined by the multiple selected ion monitoring technique. Phenformin half-lives were unrelated to the degree of renal impairment, whereas reduced renal clearances of insulin and creatinine were significantly correlated with a prolonged half-life of the metabolite. The excretion of p-hydroxyphenethylbiguanide was quite variable (between 4.9% and 27% of total urinary drug loss), probably due to a genetic polymorphism of hepatic mechanisms for hydroxylation. A reduced formation of the metabolite was concomitant with marked increases in the amount of circulating phenformin. A positive reciprocal correlation was detected between areas under the plasma curve of phenformin and both the renal clearance of the unchanged drug and the percentage of metabolite formation. A reduced hydroxylation of phenformin seems, therefore, to be responsible for the high plasma levels of the drug previously described in toxic patients.

Plasma lipoproteins and cholesterol metabolism in spontaneously hyperlipemic rats.

The aim of this study was to characterize the plasma lipoprotein pattern and some aspects of cholesterol metabolism in a line of hyperlipemic male rats. Plasma cholesterol and triglycerides were increased about 3-fold as compared to control animals (238 vs. 75 and 185 vs. 59 mg/dl respectively). The plasma lipoprotein distribution and the chemical composition of the isolated lipoproteins was unaffected. Plasma triglyceride production rate was increased (40%, P less than 0.01) and post-heparin lipoprotein lipase activity in plasma decreased (-28%, P less than 0.01) in the hyperlipemic rat. The activity of 3 enzymes involved in cholesterol metabolism (HMG-CoA reductase, cholesterol 7 alpha-hydroxylase, and acyl-CoA cholesterol-acyltransferase) did not differ from control values. 3H2O incorporation into digitonin-precipitable sterols, however, was significantly higher than in controls. This finding was due, in part, to an increased liver weight in the hyperlipemic animals. Furthermore kinetic data using 125I-LDL showed that the fractional catabolic rate of lipoprotein was within the normal range, while the synthetic rate of LDL protein was increased (0.67 vs. 0.3 mg/kg/h, P less than 0.01) in the hyperlipemic rat. These observations suggest that multiple metabolic defects underline the hyperlipemia observed in this animal model.

Relationship of the phenotypic expression of the A-IMilano apoprotein with plasma lipid and lipoprotein patterns.

The plasma lipoprotein and apolipoprotein profile of 29 adult A-IMilano (A-IM) carriers and 29 age- and sex-matched non-affected subjects of the same kindred was examined, in order to investigate linkages between the lipid and apoprotein abnormalities and the phenotypic expression of the biochemical disorder. Carriers (A-IM+) showed a higher prevalence of hypertriglyceridemia (12 out of 29); they also had lower plasma total cholesterol, esterified cholesterol and phospholipids, compared to non-carriers. Lipoproteins were characterized by a significant enrichment of triglycerides in low and high density fractions (LDL and HDL), and by the expected striking reduction of HDL mass and cholesterolemia. Conversely, no significant alterations of the major circulating apolipoprotein levels, except for apo A-I and apo A-II, were noted in the A-IM+. The increased free cholesterol/esterified cholesterol ratio in plasma (most marked in HDL), was accompanied by a significant reduction of the lecithin cholesterol acyl transferase molar activity. Several correlations pertaining to lipids, lipoproteins and apoproteins were examined: cholesterol and triglycerides in HDL and, more remarkably, apoprotein A-I and C-III levels in plasma were significantly correlated in the A-IM+. While there was no significant prevalence of specific apo E phenotypes, plasma triglycerides and apo C-II levels were highly correlated in the carriers. The A-IM subjects, while in the presence of severe lipoprotein risk factors, may have alternative mechanisms of cholesterol disposal, potentially responsible for the apparently low prevalence of atherosclerosis.

Role of the Frank-Starling mechanism in maintaining cardiac output during increasing levels of treadmill exercise in beta-blocked normal men.

To determine the effects of beta blockade on hemodynamics during increasing levels of treadmill exercise, 10 healthy volunteers were studied after 1 week of placebo, and then after 1 week of treatment with oral propranolol, 80 mg twice daily, or dilevalol, 400 mg once daily. The study was randomized and double-blind, with a crossover sequence. Hemodynamics were measured by CO2 rebreathing at rest and at 25, 50, 75 and 100% of VO2 max. After placebo, cardiac output increased from 5.8 +/- 2.1 (rest), to 19.4 +/- 6.4 liters/min (100% VO2 max), mainly due to an increase in heart rate from 84 +/- 6 to 169 +/- 15 beats/min. Stroke volume increased from 70 +/- 27 (rest), to 137 +/- 65 ml (25% VO2 max), and then leveled off to 116 +/- 41 at 100% VO2 max. After both beta blockers, exercise cardiac output was maintained at 100% VO2 max: 20.1 +/- 9.3 liters/min with propranolol and 19.1 +/- 8.6 with dilevalol. However, a significant reduction versus placebo values was observed for cardiac output at 25% VO2 max, from 13.7 +/- 5.9 during placebo, to 9.4 +/- 2.5 during propranolol, and to 9.6 +/- 2.3 during dilevalol (both p less than 0.01 vs placebo). Maintenance of cardiac output with both beta blockers at higher levels of exercise came from an increased stroke volume (p less than 0.05 vs placebo), while heart rate (in beats/min) was greatly reduced (propranolol 61.6 +/- 9.4 rest, 90.1 +/- 10.7 at 100% VO2 max; dilevalol 70.8 +/- 6.4 rest, 99.2 +/- 11.8 at 100% VO2 max, p less than 0.01 vs placebo for each).(ABSTRACT TRUNCATED AT 250 WORDS)

HMGCoA reductase and cholesterol 7 alpha-hydroxylase in human liver.

The activities of HMGCoA reductase and cholesterol 7 alpha-hydroxylase were assayed in liver biopsies of patients with or without cholestyramine treatment. The active dephosphorylated form of HMGCoA reductase and the activity of cholesterol 7 alpha-hydroxylase were under the detection limits in untreated subjects. After cholestyramine treatment activities of both enzymes were stimulated and the active form of HMGCoA reductase became detectable in four out of the five tested patients. In two subjects who received cholestyramine, the effect of exogenously added [4-14C] cholesterol on cholesterol 7 alpha-hydroxylase was tested. In the presence of Tween 80, the detergent by which [14C]cholesterol was suspended, the enzyme activity was profoundly inhibited and synthesis of 7 alpha-hydroxycholesterol was extremely low.

The effect of cholestyramine on liver HMG-CoA reductase and cholesterol 7 alpha-hydroxylase in various laboratory animals.

The activity of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase was assayed in the liver of rats, rabbits, hamsters and guinea pigs at the minimum of the day cycle and after one night fasting. The amount of HMG-CoA reductase, as determined after its complete dephosphorylation in vitro was of the same order of magnitude in the tested species. The dephosphorylated active form of the enzyme was detectable only in the rat. Cholesterol 7 alpha-hydroxylase activity was also much higher in the rat. Cholestyramine treatment stimulated the activity of both enzymes. In particular, the ratio between active and inactive HMG-CoA reductase in rabbits, hamsters and guinea pigs became of the same order of magnitude of that found in rats.

Plasma lipoproteins and cholesterol metabolism in Yoshida rats: an animal model of spontaneous hyperlipemia.

The purpose of this study was to characterize the lipoprotein profile and cholesterol metabolism in Yoshida rats, a strain of inbred genetically hyperlipemic animals. For comparison, Brown Norway rats were used as control animals. Plasma cholesterol and triglycerides were higher in Yoshida as compared to Brown Norway, the elevation of cholesterol being due to a rise in HDL fraction. Triglyceride distribution among lipoproteins showed an increase in VLDL fraction. Hyperlipemia was not related to diabetes, hypothyroidism or nephropathy. Plasma triglycerides production was increased in Yoshida rats, while lipoprotein and hepatic lipases were similar in the two groups. Hypercholesterolemia was associated with a defect of lipoprotein receptor activity and with elevated HMG-CoA reductase and cholesterol 7 alpha - hydroxylase; conversely ACAT activity was lower in Yoshida as compared to Brown Norway rats. Sterol fecal excretion was comparable in the two groups and hypercholesterolemia in Yoshida rats was not associated to an increase of cholesterol saturation of the bile. We suggest that lipoprotein overproduction is the main cause for hyperlipidemia in this strain of rats.

3-Hydroxy-3-methylglutaric acid (HMGA) reduces dietary cholesterol induction of saturated bile in hamster.

3-Hydroxy-3-methylglutaric acid (HMGA) is a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR) and strongly reduces cholesterol biosynthesis both in vitro and in vivo. Since the effects of this compound on biliary lipid composition are hitherto unknown, we have investigated whether it prevents dietary cholesterol induction of saturated bile in the hamster. Female Golden Syrian hamsters have been divided into four groups and treated for 10 weeks as follows: I) Standard diet, containing 0.8 mg cholesterol/g food; II) Standard diet plus HMGA (100 mg/kg b.w./day per os); III) Lithogenic diet containing 2.4 mg cholesterol/g food; IV) Lithogenic diet plus HMGA as above. The results indicate that HMGA is effective in reducing both bile cholesterol supersaturation and hypercholesterolemia. Inhibition of hepatic cholesterogenesis at the level of mevalonate synthesis by HMGCoAR and reduction of intestinal cholesterol absorption may be responsible for these effects.

Effect of natural and structurally modified bile acids on cholesterol metabolizing enzymes in rat liver microsomes. II.

The effect of ursodeoxycholic acid analogues bearing modifications at the side-chain moiety of the molecule was tested on cholesterol 7 alpha-hydroxylase and HMG-CoA reductase in rat liver microsomes. The compounds included 23 R,S mixture and the single isomers 23R and 23S of 23 methylursodeoxycholic acid (23-methyl UDCA), the isomeric mixture (cis + trans) of 3 alpha,7 beta-dihydroxy-20,22-methylen-5 beta-cholan-23-oic acid (norcypro-UDCA) and the corresponding single isomers. Each steroid was added to liver microsomes as the sodium salt, at concentrations ranging from 25 to 200 microM. Isomers 23R and 23S of 23-methyl-UDCA inhibited cholesterol 7 alpha-hydroxylase in a concentration-dependent manner. The inhibitory capacity was similar for the two isomers. The extent of inhibition of the analogues was greater than that of the parent compound UDCA. Shortening of the side-chain in norcypro-UDCA resulted in a partial loss of the inhibitory effect, as compared to cypro-UDCA (3 alpha,7 beta-dihydroxy-22,23-methylen-5 beta-cholan-24-oic acid). None of these bile acid derivatives affected the activity of the enzyme HMG-CoA reductase.

Effect of natural and structurally modified bile acids on cholesterol metabolizing enzymes in rat liver microsomes.

The effect of chenodeoxycholic (CDCA), ursodeoxycholic (UDCA), tauroursodeoxycholic (TUDCA), cholic (CA), ursocholic (UCA) acids, analogues of CDCA and UDCA with a cyclopropyl ring at C22, C23 (cypro-CDCA and cypro-UDCA) and 23-methylursodeoxycholic acid (MUDCA) on cholesterol 7 alpha-hydroxylase was studied in rat liver microsomes. Cypro-analogues consisted of a mixture of four diasteroisomers, while MUDCA was the racemic mixture of two enantiomers. Each steroid was added to liver microsomes at concentrations ranging from 10 to 200 microM. With the exception of UCA and CA, all the bile acids inhibited cholesterol 7 alpha-hydroxylase activity. The inhibition shown by cypro-CDCA and cypro-UDCA was stronger than that observed with the corresponding natural compounds. 22S,23S cypro-UDCA exhibited an inhibitory effect which was more pronounced than that of the diasteroisomer mixture. The isomer 22R,23S was less effective and decreased cholesterol 7 alpha-hydroxylase activity in a manner comparable to that of UDCA. The effect of CDCA, UDCA and the cyclopropyl analogues was also tested with respect to HMG-CoA reductase and acylCoA cholesterol acyltransferase (ACAT) activities. ACAT was stimulated by the isomer 22S,23S cypro-UDCA but not affected by the other bile acids. No effect was observed as regards HMG-CoA reductase.

Cardiac output with CO2 rebreathing method in CAPD patients.

The aim of this study is to evaluate cardiac output (CO) with CO2 rebreathing method (RCO2) in patients (pts) on CAPD. We have studied 15 pts on CAPD from at least 6 months, the mean (+/- SD) age was 55 +/- 4 years, mean (+/- SD) hemoglobin was 10 +/- 2 gr/dl. The respiratory tests excluded obstructive or restrictive broncopneumopathies. Electrocardiograms and B-mode echocardiograms were normal. RCO2 was evaluated using the FICK formula: CO = VCO2/CvCO2 - CaCO2 where VCO2 is CO2 production; CvCO2 is the CO2 content in venous mixed blood; CACO2 is arterial CO2. VCO2 was obtained by collecting expired air into a Douglas bag during respiration at rest for 4 minutes. CvCO2 was obtained after 10-15 seconds of respiration in a mixture of 7% CO2 in O2. CaCO2 was obtained at CO2 end-tidal capnogram. RCO2 was performed in CAPD with full and empty abdomen. The mean (+/- SD) CO was 2.3 +/- 1.04 l/min with both full and empty abdomen, values below those theoretically calculated, taking into account the age and body surface (4.7 +/- 0.6 l/min P less than 0.0005). The reduction of CO is not induced by left ventricular insufficiency, but such phenomenon could be attributed to a redistribution of body fluid between intra and extracellular, in favour of the intracellular compartment. Therefore the increase in hematocrit and total plasma proteins can be fictitious.

In-silico studies in Chinese herbal medicines' research: evaluation of in-silico methodologies and phytochemical data sources, and a review of research to date.

The available databases that catalogue information on traditional Chinese medicines are reviewed in terms of their content and utility for in-silico research on Chinese herbal medicines, as too are the various protein database resources, and the software available for use in such studies. The software available for bioinformatics and 'omics studies of Chinese herbal medicines are summarised, and a critical evaluation given of the various in-silico methods applied in screening Chinese herbal medicines, including classification trees, neural networks, support vector machines, docking and inverse docking algorithms. Recommendations are made regarding any future in-silico studies of Chinese herbal medicines.

In vitro study of aortic valves treated with neo-chordae grafts: hydrodynamics and tensile force measurements.

Reparative surgery of the aortic root functional unit (ARFU) allows for a better preservation of the functionality of the native structure compared to prosthesis implantation. Post-operative results are satisfactory, whereas mid- and long-term results are challenging, for example in terms of cusps prolapse recurrence. At the Cardiothoracic Surgery Unit of the Sacco Hospital, a new surgical technique aimed at the stabilization in time of the results of standard ARFU repair operations has been applied. This technique, inspired by the mitral neo-chordae (NC) implantation, consists of implanting an e-PTFE suture thread between the prolapsed cusp and the sinotubular junction. Aim of this study was to assess the influence of NC implantation on the ARFU functioning by evaluating with an owned pulsatile in vitro apparatus the force magnitude acting on the NC and the dynamic behavior of porcine ARFUs treated according to the operating-room procedures. The maximum recorded values of the mean and peak diastolic forces were 0.064 and 0.186 N, respectively, and were linearly dependent upon the mean diastolic pressure across the valve. In addition, the measurements of the opening-closing times and valve leakage volumes, performed at pre- and post-surgeries, yielded that the valve functionality was not significantly influenced by NC implantation.