Type IV pili, transient bacterial aggregates, and virulence of enteropathogenic Escherichia coli.

Type IV bundle-forming pili of enteropathogenic Escherichia coli are required for the localized adherence and autoaggregation phenotypes. Whether these pili are also required for virulence was tested in volunteers by inactivating bfpA or bfpT (perA) encoding, respectively, the pilus subunit and the bfp operon transcriptional activator. Both mutants caused significantly less diarrhea. Mutation of the bfpF nucleotide-binding domain caused increased piliation, enhanced localized adherence, and abolished the twitching motility-dispersal phase of the autoaggregation phenotype. The bfpF mutant colonized the human intestine but was about 200-fold less virulent. Thus, BfpF is required for dispersal from the bacterial aggregate and for full virulence.

Salient effects of L-carnitine on adenine-nucleotide loss and coenzyme A acylation in the diabetic heart perfused with excess palmitic acid. A phosphorus-31 NMR and chemical extract study.

The beneficial effects of L-carnitine perfusion on energy metabolism and coenzyme A acylation were studied in isolated hearts from control and diabetic rats. All hearts were perfused at a constant flow rate with a glucose/albumin buffer which contained 2.0 mM palmitate. 31P-NMR was utilized to assess sequential phosphocreatine and ATP metabolism during 1 h of recirculation perfusion. L-Carnitine (5.0 mM final concentration) was added after 12 min of baseline recirculation perfusion. Frozen samples were taken after 1 h of recirculation perfusion for spectrophotometric analysis of high-energy phosphates and the free and acylated fractions of coenzyme A. L-Carnitine perfusion of diabetic hearts attenuated or prevented the reduction of ATP observed in untreated diabetic hearts. It also attenuated the accumulation of long-chain fatty-acyl coenzyme A. Although L-carnitine improved myocardial function in diabetic hearts, this was independent of any direct effect on physiological indices. Thus, the salutory effect of acute perfusion with L-carnitine on energy metabolism in the isolated perfused diabetic rat heart appears to be a direct effect on lipid metabolism.

Lipid-mediated impairment of normal energy metabolism in the isolated perfused diabetic rat heart studied by phosphorus-31 NMR and chemical extraction.

The relationship between extracellular palmitate and the accumulation of long-chain fatty-acyl coenzyme A with that of high-energy phosphate metabolism was investigated in the isolated perfused diabetic rat heart. Hearts were perfused with a glucose/albumin buffer supplemented with 0, 0.5, 1.2 or 2.0 mM palmitate. 31P-NMR was used to analyze phosphocreatine and ATP metabolism during 1 h of constant-flow recirculation perfusion. At the end of perfusion, frozen samples were taken for chemical analysis of high-energy phosphates and the free and acylated fractions of coenzyme A and carnitine. Perfusion of diabetic hearts with palmitate, unlike control hearts, caused a time-dependent and concentration-dependent reduction in ATP, despite normal and constant phosphocreatine. Concentrations of acid-soluble coenzyme A, long-chain-acyl coenzyme A and total tissue coenzyme A were elevated in palmitate-perfused diabetic hearts, while the total tissue carnitine pool was decreased. Increases in long-chain-acyl coenzyme A correlated with the reduction in myocardial ATP. This reduction in ATP could not be adequately explained by alterations in heart rate, perfusion pressure or vascular resistance.

Potential anticoagulant drug interactions in ambulatory patients.

Computer-generated prescription drug purchase records for ambulatory patients receiving oral anticoagulants (OAC) were studied for concomitant use of other drugs which have been reported to induce clinically significant interactions. One third of 479 patients taking OAC were exposed to a potentially interacting drug at some time during this 6-month period. The percentage of patients with drug interaction exposure correlated directly with total drug use (p less than 0.0005). There were no significant differences when interaction exposure rates were compared in the cases of single : multiple pharmacy and single : multiple physician-patient groups. Warfarin was the most common anticoagulant (greater than 95%) and barbiturates the most common interacting drug.

Synthesis and biological evaluation of cyclic analogues of 1-carnitine as potential agents in the treatment of myocardial ischemia.

A series of cyclic rigid analogues of l-carnitine has been synthesized and examined for activity as substrates for the carnitine-acylcarnitine translocase, the enzyme that mediates transport of fatty acids into the mitochondria. Synthetic steps to seven of these analogues are described in this paper. Bioassay of these compounds is conducted in a preparation of isolated heart mitochondria that have been previously loaded with [14C]-l-carnitine. Efflux of radioactivity from the mitochondria is then monitored in the presence of the compound being evaluated in order to assess the amount of enzyme activity initiated. The palmityl ester of l-N,N-dimethyl-trans-2-carboxy-4-hydroxypyrrolidinium chloride elicited a 13.63 and 63.07% efflux of [14C]-l-carnitine at concentrations of 3 and 50 mM, respectively. This represents the first instance in which a nonnaturally occurring analogue of l-carnitine has been shown to undergo transport via this mitochondrial translocase, suggesting the possibility that cyclic carnitine analogues may find utility as agents in the treatment of myocardial ischemia.

Molecular connectivity. 6. Examination of the parabolic relationship between molecular connectivity and biological activity.

The topologically derived, nonempirical molecular connectivity index, chi, for several classes of compounds is shown to be parabolically related to the biological activities of these compounds. Similar nonlinear relationships were previously shown between the octanol-water partition coefficients, expressed as log P, of the compounds and their biological activities. These and previous studies indicate that many physiochemical properties presently used in structure-activity studies may be intermediaries between the nonempirical molecular structure encoded in chi and measured biological activities.

A model for thyroid hormone--receptor interactions.

Theoretical electronic structure calculations on the thyroid hormones and analogues, as well as model hormone--receptor interactions, have been carried out. These studies (a) support the concept that the 4'-OH group is a H-bond donor to the in vivo nuclear receptor and suggest that at the receptor this OH group is trans to the 3' (distal) substituent; (b) indicate that there is an important intramolecular interaction between 3' and 4' substituents, and those 3' substituents that most favor both 4' OH orientation trans to the 3' group and a more acidic OH group substantially increase binding and biological activity; and (c) support the concept that there is a direct correlation between the conformational free energy of the aromatic rings and biological activity.

Nitrous oxide availability.

Nitrous oxide (N2O) is marketed as an inhalation anesthetic and as a food ingredient (e.g., whipping cream propellant). In the human, inhalation has been associated with "highs," peripheral nerve damage, mitotic poisoning of bone marrow, psychosis, and mental impairment. Exposure to hypoxemic mixtures has resulted in death. The commercial N2O sources specifically studied were aerosol whipping cream containers (three brands) and 6.5-cm cylinders, or chargers (two brands). The gas content and N2O concentrations of these devices were measured. The aerosol cans, when not shaken, will dispense at least 3 liters of 87 to 90% N2O. Charger misuse may occur when they are substituted for identically designed carbon dioxide (CO2) chargers of a seltzer bottle; 4.3 to 5.0 liters of 93 to 98% N2O is expelled at a controllable rate. The toxicity of these inexpensive N2O products, their high potential for misuse, and the absence of labeling (chargers) argue that their distribution be discontinued.

Comparative effects of pair-feeding and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on various biochemical parameters in female rats.

Hypophagia is a common characteristic of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and may be responsible for some of the toxic manifestations. Pair-feeding has been used in control animals to compensate for the hypophagia, but relatively few studies have assessed biochemical changes associated with pair-feeding versus weight loss induced by TCDD. Rats were treated with TCDD and killed 7 days post-treatment while pair-fed animals received an amount of diet equivalent to TCDD-treated partner animals. Ad libitum-fed rats were also used. No correlations were seen in altered calcium and iron homeostasis between pair-feeding and TCDD administration relative to ad libitum-fed animals. Pair-feeding resulted in greater alterations than TCDD administration in the subcellular distribution of iron in mitochondria, microsomes and cytosol. Pair-feeding also resulted in greater accumulation of calcium in mitochondria and microsomes in pair-fed as compared to TCDD-treated animals. Greater lipid peroxidation was observed in whole liver and nuclei of rats receiving TCDD relative to pair-fed animals. A significantly greater incidence of DNA single strand breaks occurred in hepatic nuclei of TCDD-treated animals as compared to pair-fed and ad libitum-fed animals. Significantly greater inhibition of hepatic glutathione peroxidase activity and thymic involution were observed in TCDD treated animals as compared to the pair-fed group. Although some similarities existed between TCDD-treated animals and pair-fed rats, the overall biochemical changes which were observed following TCDD administration cannot be attributed to weight loss associated with hypophagia.

Factors influencing the induction of DNA single strand breaks in rats by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

The ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce lipid peroxidation and DNA damage in rat hepatic nuclei was investigated. In addition, the role of iron in hepatic DNA damage was examined. Female Sprague-Dawley rats were treated with a single dose of 25--100 micrograms TCDD/kg orally, and sacrificed 3-14 days after treatment. Liver nuclei were isolated, and DNA single strand breaks (DNA-SSB) and lipid peroxidation were determined. Lipid peroxidation was assessed by measuring the content and production of thiobarbituric acid reactive substances (TBARS) while DNA-SSB were determined by the alkaline elution technique. The results demonstrate that TCDD dose and time-dependent increases in hepatic nuclear TBARS content and production occur in conjunction with an increase in DNA-SSB. The administration of the dithiolthione antioxidant oltipraz (30 mg/kg/day for 10 days) resulted in a significant decrease (47%) in the incidence of TCDD-induced DNA-SSB. Clofibrate administration (100 mg/kg/day for 12 days) and pair feeding had no effect on TCDD-induced DNA-SSB. The incubation of hepatic microsomes and mitochondria from TCDD-treated rats with nuclei from untreated animals for one hr at 37 degrees C resulted in enhanced DNA damage which was abolished by the addition of 0.10 mM desferrioxamine (DFX). Incubation with cytosol had no significant effect. Incubation of 0.10 mM Fe2+ or Fe3+ with isolated hepatic nuclei from untreated rats produced significant increases in DNA-SSB, which were abolished by the addition of 0.10 mM DFX to the incubation medium. TCDD may produce an increased bioavailability of iron which leads to enhanced DNA single strand breaks and lipid peroxidation in hepatic nuclei.

Possible mechanism of the synergistic effect of heparin and dihydroergotamine.

A combination of heparin and dihydroergotamine mesylate (Heparin-Dihydergot) has been shown to be more effective than either of these drugs alone in preventing postoperative venous thromboembolism. The possible mechanism for the synergistic effect of heparin and dihydroergotamine was assessed. Phlebograms obtained after administration of 0.5 mg of dihydroergotamine showed marked constriction of the tibial and soleal veins with a significant reduction in the mean diameter of the femoral vein. The effect on the coagulation mechanism was assessed in 28 patients. Blood samples were withdrawn from the femoral vein during varicose vein operations. A significant difference was observed in antithrombin III activity, kaolin-cephalin clotting time, beta-thromboglobulin, and fibrino-peptide levels between the control, dihydroergotamine, heparin, and the combination groups.

Diclofenac sodium (Voltaren) reduced exercise-induced injury in human skeletal muscle.

PURPOSE: To examine the effects of prolonged systemic administration of diclofenac sodium (Voltaren), a nonsteroidal anti-inflammatory drug, on objective indices of exercise-induced muscle damage in humans. METHODS: Fifty-four volunteers (mean age, 26.4 yr; range, 18-35) participated in this randomized double-blind, placebo-controlled trial. To achieve steady-state tissue levels, either placebo or diclofenac was orally administered two times a day for 27 consecutive days. A strenuous 20-min stepping exercise program, about which the subjects were unfamiliar, was conducted on day 15. Creatine kinase (CK) activities were measured immediately before the exercise session and on days 16, 18, and 27. Vastus lateralis muscle samples were obtained immediately before exercise and on day 27 for subsequent histological characterization of muscle inflammation. RESULTS: The preexercise muscle samples revealed no difference in muscle damage between the two groups. However, the postexercise muscle samples showed that the diclofenac-treated group demonstrated less muscle tissue damage than placebo-treated subjects (P = 0.002). The administration of diclofenac also resulted in a significant lowering of post-/pre-exercise CK ratios on days 18 (P = 0.03) and 27 (P = 0.02) compared with the placebo group, an indirect finding that supports the possibility of diclofenac reducing exercise-induced muscle damage. CONCLUSION: These findings demonstrate preadministration of diclofenac (in accordance with tissue half-life pharmacokinetics) significantly reduces quantitative indices of exercise-induced skeletal muscle damage in human muscle.

Long-term administration of heparin and heparin fractions and osteoporosis in experimental animals.

To test whether heparin-induced osteoporosis is influenced by the molecular weight of heparin, 24 male rabbits received single daily subcutaneous injections of either physiological saline (controls, n = 5), low molecular weight heparin (LMWH, n = 7), conventional heparin (UFH, n = 7) or high molecular weight heparin (HMWH, n = 6). Heparin was administered in supratherapeutic daily dosages for 120 days (750 anti-FXa units/kg for 90 days and 1500 anti-FXa units/kg for another 30 days). Studied variables were: serial analysis of serum calcium, albumin, phosphate and alkaline phosphatase, measurement of the cortical thickness of the femur (radiographically), tibial and trabecular bone density (both by cross-sectional analysis) and femoral fragility. Observed changes in blood biochemistry associated with bone metabolism were not correlated to any of the treatments. Compared with the controls, a reduction in cortical and trabecular bone density was seen with UFH (P < 0.05) and HMWH (P < 0.01) but not with LMWH. Femoral fragility was also significantly increased (P < 0.002) by HMWH. In conclusion, LMWH did not cause toxic skeletal effects as opposed to HMWH which clearly did, and UFH which induced some osteoporotic changes.

Molecular connectivity and steric parameters.

The possible relationship between molecular connectivity indexes, mchi, and Es, a thermodynamically derived parameter used to estimate steric effects in organic reactions and sometimes used in biological structure-activity relationships, was investigated. The extended chi terms, 2chi, 3chi, and 4chi, were correlated significantly to Es (r = 0.961). Aralkyl esters deviated from the correlations, possibly due to intramolecular interactions.

Acetylcarnitine and cholinergic receptors.

Acetylcarnitine, a naturally occurring compound found in high concentration in heart and skeletal muscle of vertebrates, bears structural resemblance to acetylcholine, and studies have shown that it has slight cholinergic properties. Acetylcarnitine was subjected to conformational analysis by extended Hückel theory (EHT) and complete neglect of differential overlap (CNDO/2) molecular orbital methods. The preferred conformations were examined with respect to their similarity to the Kier and Chothia-Pauling models of cholinergic receptor patterns. The preferred conformations of both isomers did not fit the receptor pattern described by Kier's model, although energy barriers to rotation are low enough to permit accommodation. The Chothia-Pauling model predicts activity for the S-isomer only. These studies partially explain the low cholinergic activity found for acetylcarnitine and the higher activity of (S)-acetylcarnitine compared to the R-isomer.

pKa determination of benzhydrylpiperazine antihistamines in aqueous and aqueous methanol solutions.

The pKa1 and pKa2 values of three benzhydrylpiperazine antihistamines, cyclizine (I), chlorcyclizine (II), and hydroxyzine (III), were determined at 24.5 +/- 0.5 degrees by potentiometric titration in aqueous solution to be 2.16 +/- 0.02 and 8.05 +/- 0.03, 2.12 +/- 0.04 and 7.65 +/- 0.04, and 1.96 +/- 0.05 and 7.40 +/- 0.03, respectively. The pKa2 values were also determined by titration in seven aqueous methanol solutions in the range of 11.5-52.9% (w/w) methanol. The apparent dissociation constants of I-III in the aqueous methanol solutions, psKa2, were plotted according to two linear regression equations from which the values in water, p omega Ka2, were extrapolated. The plotted variables were psKa2 versus methanol concentration (%w/w) and psKa2 + log (water concentration, M) versus 1000/epsilon, where epsilon is the dielectric constant of the aqueous methanol solution. The maximum difference between pKa2 and p omega Ka2 was observed in the case of II where p omega Ka2 was 5.23% higher. Statistical analysis of the linear regression data obtained from the plots showed that slightly better accuracy (p less than 0.13) and correlation (p less than 0.16) were obtained, but the precision was essentially equal with both methods. The observed ratio of Ka1/Ka2 in I-III, 2.75 X 10(5)-7.76 X 10(5), was attributed to solven- and space-mediated field effects and electrostatic induction between nitrogen atoms in the piperazine ring.

Molecular connectivity V: connectivity series concept applied to density.

The concept of the expanded series of the connectivity index, chi, is introduced and applied to a consideration of the density of three classes of molecules. Correlations are found using two terms in the expanded series. A preliminary reflection on the extended series terms is made. It is noted that the regression equation constant in the three studies is close to the phase volume, 0.7402, and the possible significance of this fact is discussed.

Molecular connectivity. I: Relationship to nonspecific local anesthesia.

A very significant linear correlation was found between a recently proposed connectivity index and molecular polarizability, cavity surface areas calculated for water solubility of alcohols and hydrocarbons, and biological potencies of nonspecific local anesthetics. The simplicity of calculation of the index from the connectivity in the molecular skeleton, together with the very significant correlation, indicates its practical value.

Molecular connectivity. II: Relationship to water solubility and boiling point.

The connectivity index, easily computed by arithmetic and based upon the degree of connectedness at each vertex in the molecular skeleton, is shown to give highly significant correlations with water solubility of branched, cyclic, and straight-chain alcohols and hydrocarbons as well as with boiling points of alcohols. These correlations are superior to those based on well-founded theory relating to solvent cavity surface area. An empirical modification to the connectivity index gave an improved correlation for both solubilities and boiling points.

Molecular connectivity. III: Relationship to partition coefficients.

The molecular connectivity index is shown to be linearly related to the octanol-water partition coefficients of a variety of monofunctional chemical classes including esters, alcohol, ketones, ethers, carboxylic acids, amines, and hydrocarbons. A modification of the connectivity index, taking into account the valency or degree of unsaturation of an atom, merges the data for all compounds except hydrocarbons. The connectivity index is also shown to be useful for correlating biological activity. These studies indicate that partition coefficients represent empirical quantities having intermediate significance between biological data and the more fundamental property of molecular connectivity.