Frequency of occurrence of cytomegalovirus and Chlamydia pneumoniae in lymphocytes of atherosclerotic patients.

Forty-three blood samples from atherosclerotic donors and 28 samples from normal individuals were analyzed to determine the frequency of occurrence of cytomegalovirus (CMV) and Chlamydia pneumoniae DNA sequences in lymphocytes of Saudi Arabian donors using Polymerase Chain Reaction (PCR). In non-atherosclerotic donors, no CMV DNA was detectable and only one sample was positive for C-pneumoniae DNA sequences. Of the 43 atherosclerotic patients, 22 were infected with CMV, 23 were infected with C-pneumoniae and 11 showed no infection. Thirteen of the 43 donors showed simultaneous infection with both CMV and C-pneumoniae. These results demonstrate that atherosclerotic patients are more frequently infected with CMV or C-pneumoniae or both.

Dual substrate model for novel approach towards a kinetic study of acetylcholinesterase inhibition by diazinon.

Limited reports as compared to other insecticides appear in the literature for acetylcholinesterase (AChE) inhibition by diazinon. In the current study, new kinetic parameters of AChE inhibition by diazinon have been investigated. The assay was done with bovine retinal AChE using two different substrate (ASCh) concentrations in the absence and presence of diazinon (0.08-1.28 mM). The optical density was monitored up to 25 min (reaction time) for the assay. New kinetic parameters k'(oms), K'(sms), k(oms), K(sms), K'(asms) and K(asms) ) were calculated from these experimental data.

Thermodynamic analysis of human retinal acetylcholinesterase inhibition using an anti-Alzheimer's drug, tacrine, through the development of a dual substrate and temperature model.

The present study determines the energy parameters, such as the Gibb's free energy change (deltaG), enthalpy change (deltaH), heat of activation (deltaH*), entropy change (deltaS), temperature coefficient (Q10) and activation energy (Ea), of human retinal acetylcholinesterase (AChE, EC 3.1.1.7) inhibition by tacrine. The stereo-frequency collisions factor (PZ, the number of sterically and energetically favorable collisions occurring between tacrine and AChE) was also studied in this investigation. Tacrine significantly increased the value of deltaG, deltaH, deltaH*, Q10, Ea and PZ factor, and decreased the value of deltaS for AChE. Since there is no known report on the inhibition of human retinal AChE by tacrine, these results were compared with the reported values for the energy parameters of camel retinal and chicken brain AChE inhibition by an anti-cancer drug, cyclophosphamide. The uniqueness of this approach lies in the development of the 'dual substrate and dual temperature' model, which may open up a new, more efficient avenue for the study of various enzyme catalyzed reactions.

Kinetic analysis of the toxicological effect of tacrine (Cognex) on human retinal acetylcholinesterase activity.

For the first time, kinetic parameters of the effect of tacrine, an anti-cholinesterase inhibitor of therapeutic potential in Alzheimer's disease has been studied on human retinal acetyl-cholinesterase (AChE). Tacrine inhibited the AChE activity in a concentration dependent manner, the IC(50) being about 45 nM. The Michaelis-Menten constant (K(m)) for the hydrolysis of acetylthiocholine iodide was found to be 0.120 mM and this value was increased by 4-52.8% in the presence of tacrine. V(max) was observed to be 2.23 micromol/h per mg protein for the control system, while it was decreased by 14.73-56.25% in the tacrine treated systems. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition was of the mixed type, i. e. a combination of competitive and noncompetitive inhibition. The values of K(i) and K(I) were estimated to be as 37.76 and 64.36 nM, respectively.

Kinetics of human acetylcholinesterase inhibition by the novel experimental Alzheimer therapeutic agent, tolserine.

Characterization of the kinetic parameters of tolserine, a novel acetylcholinesterase (AChE) inhibitor of potential in the therapy of Alzheimer's disease, to inhibit purified human erythrocyte AChE was undertaken for the first time. An IC(50) value was estimated by three methods. Its mean value was found to be 8.13 nM, whereas the IC(100) was observed to be 25.5 nM as calculated by single graphical method. The Michaelis-Menten constant (K(m)) for the hydrolysis of the substrate acetylthiocholine iodide was found to be 0.08 mM. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition was of the partial non-competitive type. The value of K(i) was estimated as 4.69 nM by the primary and secondary replots of the Dixon as well as secondary replots of the Lineweaver-Burk plot. Four new kinetic constants were also investigated by polynomial regression analysis of the relationship between the apparent K(i) (K(Iapp)) and substrate concentration, which may open new avenues for the kinetic study of the inhibition of several enzymes by a wide variety of inhibitors in vitro. Tolserine proved to be a highly potent inhibitor of human AChE compared to its structural analogues physostigmine and phenserine.

Investigation of total, free, peptide-bound, protein-bound, soluble and insoluble collagen hydroxyproline content in tissues from the Arabian camel (Camelus dromedarius).

This study was conducted to determine the concentration of total, free, peptide-bound, protein-bound, soluble and insoluble collagen hydroxyproline (Hyp) in tissues from the Arabian camel (Camelus dromedarius). Results indicated that there were significant differences in the concentration of total, free, peptide-bound, protein-bound, soluble and insoluble collagen Hyp in various tissues (P < 0.01). Camel kidney showed a significantly high concentration of total, free, peptide-bound and protein-bound Hyp and collagen content as compared to other tissues examined (P < 0.01). Kidney also showed a significantly high concentration of soluble collagen Hyp as compared to other tissues examined (P < 0.01). However, the concentration of insoluble collagen Hyp was significantly high in liver when compared to other tissues (P < 0.01). These variations may result from differences in the collagen structure and/or composition in this species.

Human erythrocyte acetylcholinesterase inhibition by cis-diamminediaquaplatinum (II): a novel kinetic approach.

The present work addresses the analyses of some novel kinetic parameters (k(t), K(v), t50, K(ir), t(c), m(c), IC50, IC99 and Ki) of human erythrocyte membrane-bound acetylcholinesterase (AChE, EC 3.1.1.7) inhibition by cis-diamminediaquaplatinum II (PDC). PDC is under a clinical trial for use as an antineoplastic drug. The authors recently reported that PDC and cisplatin have the ability to inhibit AChE activity in vitro. Therefore this study was designed to determine the estimation of time constant (k(t)), velocity constant (K(v)), 50% inhibition time (t50), inhibition rate constant (K(ir)), transition concentration (t(c)), meeting concentration (m(c)), 50% inhibition (IC50), 99% inhibition (IC99) and inhibition constant (Ki) by novel methods. The details are described in the text.

Kinetic constants for the inhibition of camel retinal acetylcholinesterase by the carbamate insecticide lannate.

We have designed this study to determine various kinetic parameters of camel retinal membrane-bound acetylcholinesterase (AChE; EC 3.1.1.7) inhibition by carbamate insecticide lannate [methyl N-[[(methylamino)carbonyl]oxy] ethanimidothioate]. All these kinetic constants were derived by simple graphical methods. The value of kinetic parameters was estimated as follows: 0.061 microM)(-1), 1.14 (microM)(-1), 0.216 microM, 0.016 min(-1), 0.0741 (gammaM min)(-1) 0.746 microM, and 4.42 microM for velocity constant (Kv), new inhibition constant (Knic), dissociation constant (Kd), carbamylation rate constant (k2c), overall carbamylation rate constant (k'2), 50% inhibition constant (K150), and 99% inhibition constant (K199), respectively. These unique methods may be used to estimate such kinetic parameters for time-dependent inhibition of enzymes by variety of chemicals, insecticides, herbicides, and drugs.

On the inhibition of camel retina acetylcholinesterase activity by cycloheximide in vitro.

The kinetic parameters of inhibition of camel retinal acetylcholinesterase (AChE) activity by cycloheximide (CH) were investigated. For the control system, the Michaelis-Menten constant (K(m)) for the hydrolysis of acetylthiocholine iodide was found to be 0.076 mmol/L and the Vmax was 0.547 mumol/min per mg protein. In contrast, these parameters were decreased in the CH-treated systems. Dixon and Lineweaver-Burk plots, and their secondary replots, indicated that the inhibition was of the linear mixed type, which seems to be a combination of partial competitive and pure noncompetitive inhibition. The values of K'i(slope) and KI(intercept) were estimated to be 3.50 and 5.68 mmol/L, respectively. Ki was greater than K'i, indicating that CH has a greater binding affinity for the peripheral site than the active site.

Kinetics of the inhibition of acetylcholinesterase in camel retina by cisplatin.

The inhibitory effect of cisplatin (CDDP) on camel retina acetylcholinesterase (AChE) was characterized. The CDDP effect was independent of the time of incubation with AChE before the addition of substrate, indicative of reversible inhibition. Moreover, dilution data prove that CDDP is a reversible inhibitor of camel retina AChE. Cisplatin inhibited AChE activity of camel retina in a concentration- and time-dependent manner, the IC50 values being 5.32 and 0.196 mM at 5 min and 24 h incubation times, respectively. The IC50 has dual components, i.e. directly proportional and inversely proportional to 0-1.5 h and 1.5-24 h incubation periods, respectively. The Michaelis-Menten constant (Km) for the hydrolysis of acetylthiocholine iodide (ASCh) was found to be 0.0796 mM and Vmax was 0.668 micromol/min/mg protein. Kmapp and Vmaxapp both decreased as the CDDP concentration increased. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition was of the pure uncompetitive type. The value of Ki was estimated as 0.811 mM by the primary and secondary replots of the Lineweaver-Burk and Dixon plots. Kiapp decreased while Vmaxiapp increased after increasing the ASCh concentration.

Kinetics of human erythrocyte acetylcholinesterase inhibition by a novel derivative of physostigmine: phenserine.

The effect of phenserine, a novel cholinesterase inhibitor, was assessed for the first time on kinetic parameters of human erythrocyte acetylcholinesterase (AChE). Phenserine (0.025-0.40 microM) inhibited the activity of human erythrocyte AChE in a concentration-dependent fashion, the IC50 was 0.0453 microM. The Michaelis-Menten constant (K(m)) for the hydrolysis of acetylthiocholine iodide was found to be 0.124 mM and the Vmax was 0.980 mumol/min/mg protein. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition was of the noncompetitive type. The value of Ki was estimated as 0.048 microM by the primary and secondary replots of the Dixon as well as secondary replots of the Lineweaver-Burk plot. A novel relationship between Ki and substrate concentration was also identified which permits more precise prediction of the specific type of noncompetitive inhibition of various enzymes by a wide variety of drugs, chemicals and, in some circumstances, by their own substrates.

Sensitivity of bovine retinal acetylcholinesterase (E.C. 3.1.1.7) toward tacrine: kinetic characterization.

This work addresses the kinetic analysis of the interaction of tacrine with bovine retina acetylcholinesterase (A ChE, E.C. 3.1.1.7). It was found that the tacrine effect was reversible in nature. Tacrine inhibited bovine retinal AChE activity in a concentration-dependent manner; IC50 was fo to be 8.07 nM. The Michaelis-Menten constant (Ka) for the hydrolysis of acetylthiocholine iodide (ASCh) by AChE was 0.061 mM in the control system, and this value was increased by 54-67% in the tacrine-treated systems. The Vmax was 0.701 mumole/min per milligram protein for the control system, but it was decreased by 26-69% in the tacrine-treated systems. The Lineweaver-Burk plot, Dixon plot, and their secondary replots indicated that the nature of the inhibition was of the partial mixed type, that is, a mixture of competitive and noncompetitive inhibition. The values of Ki and Kt were estimated to be as 4.475 and 8.517 nM, respectively.

The distribution of total, free, short-chain acyl and long-chain acyl carnitine in ocular tissues of the camel (Camelus dromedarius).

Carnitine (CA) plays an important role in the metabolism of fatty acids. Its presence is considerable in tissues that utilize fatty acids as an important source of energy, such as the heart and the skeletal muscle. The presence of free, short-chain acyl and long-chain acyl carnitine was shown for the first time in various tissues of the camel eye. The ratio of acyl carnitine (AC) to free carnitine (FC) was high in aqueous humor, indicating a predominance of AC, while it was low in vitreous humor, indicating a higher concentration of FC. The highest concentration of total carnitine (TC) was observed in the retina, followed by the cornea, while the lowest was found in lens nucleus. Among various forms, FC concentration was the highest in the cornea while it was the lowest in the lens cortex. In the cornea and retina, the proportion of short-chain acylcarnitines (SC) was two-fold when compared to long-chain acylcarnitines (LC), while in the lens it was the same. The ratio of AC to FC in the retina was high, indicating a predominance of AC, while in lens it was low, indicating a higher concentration of FC. In contrast, L-CA in the cornea was present equally between the free and the esterified forms. The quantitative differences in the distribution of L-CA in various tissues of the camel eye reflect that it may be involved in providing a source of readily available energy.

Thermodynamic investigation of camel retina acetylcholinesterase inhibition by cyclophosphamide.

The present work addresses the estimation of energy parameters such as Gibb's free energy change (delta G), enthalpy change (delta H); entropy change (delta S) and activation energy (E a) of acetylcholinesterase (AChE, EC 3.1.1.7) from camel retina in the absence and presence of the antineoplastic drug cyclophosphamide (CP). A spectrophotometric method was used for the determination of AChE activity, which was the basis for determination of these parameters. The PZ factor (number of sterically and energetically favorable collisions occurring between CP and AChE) have also been studied in this investigation. The energy parameters obtained in the present investigation were compared with the values reported elsewhere.

The inhibitory effect of the neuromuscular blocking agent, gallamine triethiodide, on camel retina acetylcholinesterase activity.

The present investigation addresses the mode of inhibition of the camel retinal acetylcholinesterase (AChE) activity by gallamine triethiodide, which is known to be a specific non-depolarizing neuromuscular blocking agent and polar cholinergic antagonist. This study gave the following results: it was found that gallamine (GA) reversibly inhibited the AChE activity in a concentration dependent manner, the IC50 being about 0.633 mM. The Km for the hydrolysis of acetylthiocholine iodide by AChE was found to be 0.0803 mM in the control system, and the value increased by 19-463% in the GA (0.125-1.0 mM) treated systems. The Vmax was 0.649 micromol/min per mg protein for the control as well as GA treated systems. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition is of the reversible competitive type. The Ki value was estimated as 0.160 mM. The Ki value increased with an increase in substrate concentration. The turnover number (Kcat) and specificity constant (Ksp) were 62.1 min(-1) and 7.73 x 10(5) (M x min)(-1) in the control system while the value for one parameter (Ksp) was decreased by 25-83% in the GA (0.125-1.0 mM) treated systems.

Estimation and correlation of IC50 for the inhibition of human erythrocyte acetylcholinesterase by cis-diamminediaquaplatinum (II).

The present work addresses the estimation and mode of aquiring fifty per cent inhibition of human erythrocyte membrane-bound acetylcholinesterase (AChE, EC 3.1.1.7) by cis-diamminediaqua-platinum II (DDP), which is presently in clinical trials for use as an antineoplastic drug. It has been recently reported that cisplatin itself has the ability to inhibit the AChE activity in vitro [Al-Jafari, et al, 1995; Kamal and Al-Jafari, 1996]. Therefore, this study was focused on the estimation of the IC50 of AChE inhibition by DDP, and its correlation with reaction times. It was found that 0.0-20.0% and 53.8-94.5% AChE inhibition takes place at 3.0 to 60 minutes after 0.025 and 0.40 mM DDP administration, respectively. The IC50 was proportional to the reaction period, and gave values of 0.057 to 0.918 mMat reaction times ranging from 3.0 to 60.0 minutes, respectively. The DDP has 1025 and 67 times higher inhibitory potency than cisplatin for human erythrocyte AChE at 3.0 and 60.0 minutes reaction time respectively. In the light of these findings, particular attention should be paid to DDP in tumor therapy and its inhibitory effect on AChE must be considered during the decision whether to use it as an antineoplastic drug.

Evaluation of the nature of camel retinal acetylcholinesterase: inhibition by hexamethonium.

Acetylcholinesterase (AChE, EC 3.1.1.7) has been demonstrated in retinas of several species, however, the nature of the interaction of AChE with specific inhibitors are very limited in the literature and the mode of inhibition of camel retinal AChE by hexamethonium has been studied. Hexamethonium reversibly inhibited AChE in a concentration dependent manner, the IC50 value being c. 2.52 mM. The Km for the hydrolysis of acetylthiocholine iodide was found to be 0.087 mM and the Vmax was 0.63 mumol/min/mg protein. Dixon, as well as Lineweaver-Burk, plots and their secondary replots indicated that the nature of the inhibition is of the hyperbolic (partial) mixed type, which is considered to be a partial competitive and non-competitive mixture. The values of Ki(slope) and KI(intercept) from a Lineweaver-Burk plot were estimated as 0.30 mM and 0.17 mM, respectively, while Ki from a Dixon plot was estimated as 0.725 mM. The Ki was greater than KI indicating that hexamethonium has a greater affinity of binding for the active site than the peripheral site of the camel retina AChE.

Purification of carnitine acetyltransferase from skeletal muscle of the camel (Camelus dromedarius).

The enzyme carnitine acetyltransferase (CAT) catalyzes the reversible transfer of short-chain acyl groups between coenzyme A and L-carnitine, and hence, plays an important role in the beta-oxidation of fatty acids. Purification and characterization of CAT from desert animal species may help in explaining the involvement of secondary pathways for energy production in these species. In this paper, we report the purification and partial characterization of CAT from the Arabian camel. CAT was purified from the skeletal muscle of the Arabian camel by ammonium sulfate and acetone fractionation, followed by chromatography on DEAE-Sepharose, agarose-Co A and Superose 12 gel filtration columns. CAT was purified by 2937-fold to a specific activity of 94 Units mg-1. The purified CAT was a monomer of 59 kDa as judged by native and SDS-PAGE, and showed a pI of 5.2. The enzyme displayed maximum activity with propionyl-Co A. Apparent Km for acetyl-, propionyl- and butyryl-Co A were 27.7, 17.3 and 29 microM respectively, while palmitoyl-Co A was not a substrate.

Investigation of the influence of theophylline feeding on total, free, short-chain acyl and long-chain acyl carnitine levels in skeletal muscle and liver of rats.

L-carnitine, the obligate fatty acyl carrier across the inner mitochondrial membrane, is sensitive to biochemical changes that alter lipid metabolism. The present study describes the effects of oral theophylline on L-carnitine concentrations in the skeletal muscle and the liver of rats. A significant rise in total, free and long-chain acyl carnitine concentrations was observed in the skeletal muscle but not in the liver. The increases observed after the first week of theophylline feeding returned to the control concentrations after fifth week despite the continuation of treatment. The observed changes in the skeletal muscle may be due to a) increased acyl carnitines that are formed as a consequence of enhanced fatty acid mobilization and b) increased L-carnitine uptake by the tissue.

The inhibitory effect of cyclophosphamide on camel retina acetylcholinesterase activity.

Kinetic parameters for the effect of cyclophosphamide (CP) on the camel retina acetylcholinesterase (AChE) activity were investigated for the first time in the present study. It was found that 18 micrograms of retina protein and an incubation time of 4.0 min were suitable conditions for linear of AChE activity. The CP effect was independent of time of incubation with AChE before the addition of substrate, which shows it reversible action. Moreover, dilution data prove that CP is a reversible inhibitor of camel retina AChE. Cyclophosphamide (0.2-2.4 mM) inhibited activity of camel retina in a concentration-dependent fashion, the IC50 being about 1.17 mM. The Michaelis constant (K(m)) for the hydrolysis of acetylthiocholine iodide was found to be 0.106 mM and the Vmax was 0.765 mumol/min/mg protein. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition was of the pure noncompetitive type. The value of Ki was estimated as 0.763 mM by the primary Dixon and secondary replots of the Lineweaver-Burk plot.