Molecular versus conventional culture for detection of respiratory bacterial pathogens in poultry.

Acute respiratory tract infections are leading causes of morbidity in poultry farms allover the world. Six pathogens; Escherichia coli, Mycoplasma gallisepticum, Staphylococcus aureus, Pasteurella multocida, Mannheimia haemolytica and Pseudomonas aeruginosa were involved in respiratory infections in poultry. Herein, conventional identification procedures and polymerase chain reaction (PCR) were applied for detection of the most common respiratory bacterial pathogens in clinical specimens of poultry obtained from 53 Egyptian farms with various respiratory problems and the results were compared statistically. The analyzed data demonstrated a significantly higher rate of detection of the most recovered microorganisms (P<0.05) by PCR comparing to classical culture procedures. Further, multiplex PCR could detect E. coli, M. gallisepticum, S. aureus and Ps. aeruginosa in a single reaction, however, M. haemolytica was reported in a uinplex system. According to PCR results, the most commonly recorded bacterial pathogens in examined poultry farms were E. coli and Ps. aeruginosa (54.71% each), followed by M. haemolylica (35.85%) and M. gallisepticum (20.75%). In conclusion, PCR assay offered an effective alternative to traditional typing methods for the identification and simultaneous detection of the most clinically relevant respiratory pathogens in poultry.

Oxidative stress on land snail Helix aspersa as a sentinel organism for ecotoxicological effects of urban pollution with heavy metals.

The oxidative stress in the digestive gland of the land snail Helix aspersa was considered as a bioindicator for atmospheric pollution with heavy metals from several industries and vehicular traffic in Kafr El-Zayat city. Regional means of heavy metals concentration of all sites were 0.71, 7.09, 0.71, 2.68, 41.44 and 18.01 mg kg(-1) wet mass for Cd, Mn, Ni, Pb, Zn and Cu, respectively. In addition, the highest values of Cd concentrations were found 1.22 and 1.73 mg kg(-1) wet mass in S1 (Potato International Center) and S4 (The Nile bank), respectively. Lactate dehydrogenase (D-LDH(and recorded lipid peroxidation (LPO) levels were significantly high in S1 and S2 (Traffic station). On the other hand, the highest activity of catalase (CAT) was found in S2 (194.04% of control), while the activity of glutathione peroxidase (GPx) reached the highest significant value in S1. As a matter of fact, glutathione-S-transferase (GST) and glutathione reductase (GR) activities were significantly higher in polluted sites than in reference zone. In contrast, the glutathione (GSH) concentration of exposed animals showed significant decrease in all sites, with the lowest value in S1 (57.61% of control). However, metallothioneins concentration (MT) showed no significant difference in all sites except in S1 which accounted for 127.81% of control. Therefore, the overall results of this study showed the importance of H. aspersa as a sentinel organism for biomonitoring the biologic impact of atmospheric pollution in urban areas.

Risk factors associated with caries experience in children and adolescents with intellectual disabilities.

PURPOSE: The purpose of this study was to examine caries experience and associated risk factors in children and adolescents with intellectual disability (ID). METHODS: A total of 86 participants aged 3-13 years (33 with ID and 53 healthy) were included in the study. Participants received an oral examination and their caregivers completed a questionnaire. Caregivers were required to determine the "level of function" of their children with regards to performing self care daily activities (brushing teeth, feeding and self dressing, walking and performing toilet activities). Four levels of function were determined; (A) being completely independent, (D) completely dependent, (B) and (C) partially dependent on caregivers. RESULTS: In healthy participants the mean dft score was 8.83 +/- 4.99 whereas in those with ID the mean dft score was 6.81 +/- 6.11. The mean DFT score in healthy participants was 2.32 +/- 2.98 while the mean DFT in those with ID was 0.92 +/- 1.57. Both dft and DFT scores were significantly different between participants with ID and healthy ones (p = 0. 042, p = 0.044 respectively). Caries status was not associated with gender, age or caregivers' education in the study sample. Significant associations were found between caries experience in participants with ID and their type of school (p = 0.01), nature of diet (p = 0.001) and "level of function" (p = 0.007). CONCLUSIONS: The type of school, nature of diet and "level of function" may be considered as influential risk factors associated with caries experience in children and adolescents with ID.

Serum levels of TNF-alpha and antioxidant enzymes and placental TNF-alpha expression in unexplained recurrent spontaneous miscarriage.

It has been proposed that many factors have been implicated in the pathogenesis of unexplained recurrent spontaneous miscarriage (URSM). The objective of this study was to evaluate the levels of some antioxidants, tumor necrosis factor-alpha (TNF-alpha), luteinizing (LH) and follicle-stimulating hormone (FSH) in URSM. Serum levels of superoxide dismutase (SOD), catalase (CAT), LH, FSH as well as TNF-alpha in serum and the expression of TNF-alpha positive cells in placental tissues, were assayed in women suffering from unexplained first trimester miscarriage. Two groups were included, the first was represented by 16 women with URSM (number of abortions: 3-5) and the second one included 24 women with URSM (number of abortions > 5). The control groups included 20 women within their first trimester of pregnancy and 20 non pregnant healthy females within their follicular phase. The obtained results showed a highly significant decrease in serum levels of SOD and CAT, in the URSM groups compared to control groups (p < 0.05 for each comparison). Higher serum levels of TNF-alpha were detected in URSM groups compared to control groups (p < 0.05 for each comparison). A significant increase in serum levels of LH was encountered between URSM groups compared to control groups; on the other hand the mean levels of FSH expressed no significant changes among URSM groups as compared to first trimester pregnancies control group. A positive correlation was noticed between serum levels of TNF-alpha and the levels of LH (p < 0.05). We conclude that antioxidant enzymes (CAT, SOD), TNF-alpha, LH and FSH may play a major role in the pathogenesis of URSM. Much more work is required before the mechanisms, which lead to RSM, can be fully understood.

Serum levels of TNF-á and antioxidant enzymes and placental TNF-á expression in unexplained recurrent spontaneous miscarriage

It has been proposed that many factors have been implicated in the pathogenesisof unexplained recurrent spontaneous miscarriage (URSM). The objective ofthis study was to evaluate the levels of some antioxidants, tumor necrosis factor-á(TNF-á), luteinizing (LH) and follicle-stimulating hormone (FSH) in URSM. Serumlevels of superoxide dismutase (SOD), catalase (CAT), LH, FSH as well as TNF-áin serum and the expression of TNF-á positive cells in placental tissues, were assayedin women suffering from unexplained first trimester miscarriage. Two groups wereincluded, the first was represented by 16 women with URSM (number of abortions:3-5) and the second one included 24 women with URSM (number of abortions > 5).The control groups included 20 women within their first trimester of pregnancy and20 non pregnant healthy females within their follicular phase. The obtained resultsshowed a highly significant decrease in serum levels of SOD and CAT, in the URSMgroups compared to control groups (p< 0.05 for each comparison). Higher serumlevels of TNF- á were detected in URSM groups compared to control groups (p<0.05 for each comparison). A significant increase in serum levels of LH was encounteredbetween URSM groups compared to control groups; on the other hand themean levels of FSH expressed no significant changes among URSM groups as comparedto first trimester pregnancies control group. A positive correlation was noticedbetween serum levels of TNF-á and the levels of LH (p< 0.05). We conclude thatantioxidant enzymes (CAT, SOD), TNF- á, LH and FSH may play a major role inthe pathogenesis of URSM. Much more work is required before the mechanisms,which lead to RSM, can be fully understood(AU)

Organophosphorus pollutants (OPP) in aquatic environment at Damietta Governorate, Egypt: implications for monitoring and biomarker responses.

The study was carried out from spring 1999 to spring 2001 to monitor the residue levels of organophosphorus pollutants (OPP) in aquatic environment of the drainage canal surrounding a pesticide factory at Damietta Governorate. Water, sediment, and fish samples were collected at six different seasonal periods. OPPs were analyzed by GLC and confirmed using GC-MS. Chlorpyrifos, chlorpyrifos-methyl, malathion, diazinon, pirimiphos-methyl and profenofos were detected in most samples. Chlorpyrifos was dominant in all water and sediment samples. It was ranged from 24.5 to 303.8 and 0.9 to 303.8 ppb in water and sediment samples, respectively. Diazinon level was slightly similar to chlorpyrifos in fish samples. Data based on the grand total concentration of OPP showed that the most polluted samples were collected either at spring 1999 or autumn 2000. They were 675.5 and 303.8 ppb in water samples and 43.0 and 52.2 ppb in fish collected at spring 1999 and autumn 2000, respectively. The obtained results are in parallel to that found in case of cholinesterase activity where the activity of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was declined at these seasonal period. The activity levels of AChE and BuChE were found to be 77.18% and 59.67% of control at spring 1999 and 78.62% and 85.80% of control, at autumn 2000, respectively. Thus, AChE and BuChE could be used as biomarkers for tracing and biomonitoring OPP pollution.

[Why patients do not comply with reference decision made by general practitioners?]. / Pourquoi les patients n'adhèrent-ils pas à la décision de référence faite par le médecin généraliste?

SUBJECT: A health system's efficacy depends on the efficacy of its different components (first-level health services and hospitals). It also depends on the system's ability to ensure the continuity of care among the various levels of the system. Health care officials in Settat Province, Morocco, found continuity in this province to be unsatisfactory. Depending on the health centre involved, only 31 to 52% of patients referred from the first to the second level of care reached the hospital. METHODS: The study was conducted in two rural and two urban health centres (HCs) covering a total population of around 94,000. The methodology consisted of two steps. First we analysed retrospectively various determinants (age, gender, distance, time until appointment) that might influence the compliance of patients referred by the four health centres in 1994. Then we observed curative medical consultations conducted in each of these health centres over a three-day period; the 38 patients referred to the hospital over this period were interviewed and the organisation of the hospital used on was analysed. RESULTS: The results revealed low compliance: only 43% (782/1807) of the patients referred actually consulted the hospital's departments. The compliance rates varied from one HC to the other and were lower in rural than urban areas taken as a whole (34% (207/607) versus 48% (575/1200), respectively). The interviews revealed that patients did not trust the last-year medical students who staffed the emergency rooms. Another organisational problem in the hospital was identified: patients referred to the hospital to consult a specialist were not seen immediately but given appointments at later dates, and these waiting times influenced the final success of the referral process. Thus, if the patients were seen immediately, compliance increased from 48 to 77% in the case of the urban HCs and from 34 to 67% in the case of the rural HCs. CONCLUSION: The most important determinants of compliance were above all associated with the way health services were organized and the quality of communication between health professionals and patients.

Characterization of a vertebrate neuromuscular junction that demonstrates selective resistance to botulinum toxin.

Botulinum toxin blocks transmitter release by proceeding through a series of four steps: binding to cell surface receptors, penetration of the cell membrane by receptor-mediated endocytosis, penetration of the endosome membrane by pH-induced translocation, and intracellular proteolysis of substrates that govern exocytosis. Each of these steps is essential for toxin action on intact cells. Therefore, alterations in cell structure or cell function that impede any of these steps should confer resistance to toxin. In the present study, screening for susceptibility to four serotypes of botulinum toxin revealed that the cutaneous-pectoris nerve-muscle preparation of Rana pipiens is resistant to type B botulinum toxin. Resistance was demonstrated both by electrophysiologic techniques and by dye-staining techniques. In addition, resistance to serotype B was demonstrated at toxin concentrations that were 2 orders of magnitude higher than those associated with blockade produced by other serotypes. In experiments on broken cell preparations, type B toxin cleaved synaptobrevin from frog brain synaptosomes. However, the toxin did not bind to frog nerve membranes. These findings suggest that resistance is due to an absence of cell surface receptors for botulinum toxin type B. The fact that cutaneous-pectoris preparations were sensitive to other botulinum toxin serotypes (A, C, and D), as well as other neuromuscular blocking agents (alpha-latrotoxin, beta-bungarotoxin), indicates that botulinum toxin type B receptors are distinct.

Expression of botulinum toxin binding sites in Xenopus oocytes.

The binding of iodinated botulinum toxin type B to nerve membranes was studied by using rat and mouse preparations. The toxin was examined both in the single-chain and in the proteolytically processed dichain form, and binding sites both in the spinal cord and in various brain regions were assayed. Rat and mouse brains possessed specific binding sites for botulinum toxin type B. The average Kd values for the various rat and mouse membrane preparations examined were 4.2 +/- 0.7 nM and 3.7 +/- 0.9 nM, respectively. The average Bmax values for the same tissue preparations were 7.3 +/- 0.7 pmol/mg of protein and 7.5 +/- 1.9 pmol/mg protein, respectively. The binding of botulinum toxin type B to rat brain membranes was not antagonized by a polyclonal antibody against the cytosolic domain of synaptotagmin 1 or by a monoclonal antibody directed against the luminal domain of synaptotagmin 1. In addition, these antibodies did not protect the mouse phrenic nerve-hemidiaphragm from toxin-induced neuromuscular blockade. Extraction of whole-brain mRNA and injection into Xenopus oocytes led to expression of binding sites for botulinum toxin. Extraction and injection of cerebellar mRNA led to expression of a higher density of binding sites. The number of binding sites was not diminished when oocytes were pretreated with antibodies against the cytosolic and luminal domains of synaptotagmin 1. These findings are likely to aid in the isolation, characterization, and reconstitution of toxin binding sites.

In vitro characterization of botulinum toxin types A, C and D action on human tissues: combined electrophysiologic, pharmacologic and molecular biologic approaches.

Human exposure to botulinum toxin typically occurs in two settings: 1) as an etiologic agent in the disease botulism and 2) as a therapeutic agent for the treatment of dystonia. Epidemiologic studies on botulism suggest that the human nervous system is susceptible to five toxin serotypes (A, B, E, F and G) and resistant to two (C and D). In the past, these epidemiologic findings have been used as the basis for selecting serotypes that should be tested as therapeutic agents for dystonia. Epidemiologic data have been utilized because there are no studies of botulinum neurotoxin action on isolated human nerves. In the present study, electrophysiologic techniques were used to monitor toxin effects on neuromuscular transmission in surgically excised human pyramidalis muscles, ligand binding studies were done to detect and characterize toxin receptors in human nerve membrane preparations, and molecular biologic techniques were used to isolate and sequence a human gene that encodes a substrate for botulinum neurotoxin. The results demonstrated that stable resting membrane potentials (-61.5 mV; S.E.M. +/- 0.7) were maintained in individual fibers of pyramidalis muscle for up to 6 hr at 33 degrees C. The rate of spontaneous miniature endplate potentials was low in physiologic solution (0.14 sec(-1)) but increased in response to elevations in extracellular potassium concentration. In keeping with epidemiologic findings, botulinum toxin type A (10(-8) M) paralyzed transmission in human preparations (ca. 90 min). In contrast to epidemiologic findings, serotype C (10(-8) M) also paralyzed human tissues (ca. 65 min). Iodinated botulinum toxin displayed high-affinity binding to receptors in human nerve membrane preparations (serotype A high-affinity site: K(d) = 0.3 nM, B(max) = 0.78 pmol/mg protein; serotype C high-affinity site: K(d) = 1.96 nM, B(max) = 8.9 pmol/mg protein). In addition, the human nervous system was found to encode polypeptides that are substrates for botulinum neurotoxin types A (synaptosomal-associated protein of M(r) 25,000) and C (syntaxin 1A). These data have important implications bearing on: 1) the development and administration of vaccines against botulism and 2) the testing of toxin serotypes for the treatment of dystonia.

Inhibition of vacuolar adenosine triphosphatase antagonizes the effects of clostridial neurotoxins but not phospholipase A2 neurotoxins.

Bafilomycin A1, an inhibitor of vacuolar adenosine triphosphatase, was tested for its ability to antagonize botulinum neurotoxins (serotypes A-G), tetanus toxin and phospholipase A2 neurotoxins (notexin, beta-bungarotoxin, taipoxin and textilotoxin) on the mouse phrenic nerve-hemidiaphragm preparation. Bafilomycin itself produced concentration-dependent blockade of neuromuscular transmission without blocking nerve action potentials or muscle action potentials. This effect may have been due to inhibition of the proton pump that regulates acetylcholine transport into vesicles. At submaximal concentrations, bafilomycin was very effective in delaying the onset of paralysis due to all clostridial neurotoxins, but it had no protective effect against phospholipase A2 neurotoxins. Experiments were done to determine which of the three steps in clostridial neurotoxin action was antagonized by bafilomycin (e.g., binding, internalization and intracellular poisoning). Both pharmacological experiments and ligand-binding experiments showed that the drug did not block toxin binding to the plasma membrane. Similarly, pharmacological experiments on the time-dependent effects of bafilomycin showed that the drug did not antagonize the intracellular actions of toxins. The data indicated that bafilomycin acted at the intermediate step of internalization. This is in keeping with the facts that: 1) bafilomycin inhibits vacuolar adenosine triphosphatase, which in turn leads to inhibition of acidification in endosomes and 2) clostridial neurotoxins depend upon acidification of endosomes for translocation to the cytosol. The finding that bafilomycin antagonizes tetanus toxin may provide important clues for understanding how this toxin can act locally to produce flaccid paralysis. The finding that bafilomycin is a universal antagonist that protects against all clostridial neurotoxins may have important implications for developing therapeutic drugs.

Chelation of zinc antagonizes the neuromuscular blocking properties of the seven serotypes of botulinum neurotoxin as well as tetanus toxin.

Botulinum neurotoxin types A, B (unactivated and activated), C, D, E, F and G, as well as tetanus toxin, paralyzed transmission in mouse phrenic nerve-hemidiaphragm preparations. Toxin-induced blockade of transmission was antagonized by chelators [e.g., ethylenediamine tetraacetic acid, tetrakis(2-pyridylmethyl)ethylenediamine or diethylene-triaminepentaacetic anhydride], but this effect was dependent on incubation conditions. Pretreatment of toxin with chelators failed to produce antagonism, but pretreatment of tissues did produce antagonism. Of the various chelators tested, tetrakis(2-pyridylmethyl)ethylenediamine produced the greatest effect. Antagonism of toxin-induced neuromuscular blockade could be partially reversed by washing chelators from tissues and could be fully reversed by adding an excess of zinc. The ability of chelators to antagonize clostridial neurotoxins was specific and did not extend to phospholipase A2 neurotoxins. Ligand-binding studies with radioiodinated toxin and brain membrane preparations showed that chelators did not antagonize toxicity by inhibiting toxin association with receptors. Similarly, pharmacological experiments with unlabeled toxin- and type-specific antibodies demonstrated that chelators did not act by blocking receptor-mediated internalization of toxin. The chelators appeared to exert their effects by antagonizing the intracellular actions of clostridial neurotoxins. Electrophysiological studies showed that chelators, at concentrations relevant to antagonism of botulinum neurotoxin and tetanus toxin, did not enhance transmitter release.(ABSTRACT TRUNCATED AT 250 WORDS)

Low molecular weight poly(A)+ mRNA species encode factors that modulate gating of a non-Shaker A-type K+ channel.

Voltage-dependent K+ channels control repolarization of action potentials and help establish firing patterns in nerve cells. To determine the nature and role of molecular components that modulate K+ channel function in vivo, we coinjected Xenopus oocytes with cRNA encoding a cloned subthreshold A-type K+ channel (mShal1, also referred to as mKv4.1) and a low molecular weight (LMW) fraction (2-4 kb) of poly(A)+ mRNA (both from rodent brain). Coinjected oocytes exhibited a significant (fourfold) increase in the surface expression of mShal1 K+ channels with no change in the open-channel conductance. Coexpression also modified the gating kinetics of mShal1 current in several respects. Macroscopic inactivation of whole oocyte currents was fitted with the sum of two exponential components. Both fast and slow time constants of inactivation were accelerated at all membrane potentials in coinjected oocytes (tau f = 47.2 ms vs 56.5 ms at 0 mV and tau s = 157 ms vs 225 ms at 0 mV), and the corresponding ratios of amplitude terms were shifted toward domination by the fast component (Af/As = 2.71 vs 1.17 at 0 mV). Macroscopic activation was characterized in terms of the time-to-peak current, and it was found to be more rapid at all membrane potentials in coinjected oocytes (9.9 ms vs 13.5 ms at 0 mV). Coexpression also leads to more rapid recovery from inactivation (approximately 2.4-fold faster at -100 mV). The coexpressed K+ currents in oocytes resemble currents expressed in mouse fibroblasts (NIH3T3) transfected only with mShal1 cDNA. These results indicate that mammalian regulatory subunits or enzymes encoded by LMW mRNA species, which are apparently missing or expressed at low levels in Xenopus oocytes, may modulate gating in some native subthreshold A-type K+ channels.

Lectins from Triticum vulgaris and Limax flavus are universal antagonists of botulinum neurotoxin and tetanus toxin.

Lectins from Anguilla anguilla, Artocarpus integrifolia, Canavalia ensiformis, Datora stramonium, Glycine max, Limax flavus, Ricinus communis and Triticum vulgaris were tested for their abilities to antagonize the binding of botulinum neurotoxin and tetanus toxin to rat brain membranes and to antagonize the ability of these toxins to block neuromuscular transmission in mouse phrenic nerve-hemidiaphragm preparations. Lectins from Limax flavus and Triticum vulgaris, both of which have affinity for sialic acid, were antagonists of the various serotypes of botulinum neurotoxin and tetanus toxin. When tested against the high affinity binding site for botulinum neurotoxin type B, the lectin from Limax flavus had a Ki of 3.1 x 10(-7) M and the lectin from Triticum vulgaris had a Ki of 3.75 x 10(-7) M. When tested against the high affinity binding site for tetanus toxin, the lectins from Limax flavus and Triticum vulgaris had Ki values of 1.5 x 10(-7) and 1 x 10(-6) M, respectively. In all cases the lectins behaved as competitive antagonists. In reverse experiments, neither botulinum toxin nor tetanus toxin was a very effective antagonist of lectin binding to brain membranes. Studies on isolated neuromuscular preparations showed that the lectin from Triticum vulgaris did not affect transmission at concentrations of 10(-6) to 10(-3) M, but at a concentration of 3 x 10(-5) M the lectin produced highly statistically significant antagonism of the neuromuscular blocking properties of botulinum neurotoxin types A, B, C, D, E and F as well as tetanus toxin. The lectin did not antagonize beta-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Tetanus toxin and neuronal membranes: the relationship between binding and toxicity.

Tetanus toxin labeled by the Bolton-Hunter technique possesses high specific activity and retains substantial biological activity. This material can be used to characterize tetanus toxin binding to receptors in brain membrane preparations. In experiments aimed at measuring the absorption of labeled toxin, the displacement of labeled toxin by unlabeled toxin and the on-rate and off-rate constants, the data revealed two binding sites. The high affinity site had a Kd of 0.033 to 0.070 nM and a Bmax of 0.26 to 0.4 pmol/mg of protein; the low affinity site had a Kd of 0.89 to 6.9 nM and a Bmax of 1.55 to 3.0 pmol/mg of protein. The binding of tetanus toxin to brain membranes was enhanced greatly by low pH and ionic strength. Similarly to tetanus toxin, botulinum neurotoxin could be labeled by the Bolton-Hunter technique, and its binding to brain membranes was also enhanced by low pH and ionic strength. In studies with a neutralizing monoclonal antibody against tetanus toxin, the antigen-antibody interaction was not significantly altered by media with low ionic strength and pH. On the other hand, the ability of the antibody to block toxin binding to brain membranes was reduced substantially in nonphysiologic media. In a bioassay aimed at determining the effect of pH and tonicity on tissue association by toxin, low pH and ionic strength did not enhance toxicity. The biological activity of tetanus toxin was unaffected and that of botulinum neurotoxin was greatly diminished. The present findings confirm the widely reported observation that low pH and ionic strength promote tissue association by tetanus toxin, but they challenge the premise that this binding is relevant to the normal process of cell poisoning.

Actions of the insecticide 2(nitromethylene)tetrahydro-1,3-thiazine on insect and vertebrate nicotinic acetylcholine receptors.

The nitromethylene heterocyclic compound 2(nitromethylene)tetrahydro)1,3-thiazine (NMTHT) inhibits the binding of [125I]alpha-bungarotoxin to membranes prepared from cockroach (Periplaneta americana) nerve cord and fish (Torpedo californica) electric organ. Electrophysiological studies on the cockroach fast coxal depressor motorneuron (Df) reveal a dose-dependent depolarization in response to bath-applied NMTHT. Responses to ionophoretic application of NMTHT onto the cell-body membrane of motorneuron Df are suppressed by bath-applied mecamylamine (1.0 x 10(-4) M) and alpha-bungarotoxin (1.0 x 10(-7) M). These findings, together with the detection of a reversal potential close to that estimated for acetylcholine, provide evidence for an agonist action of this nitromethylene on an insect neuronal nicotinic acetylcholine receptor. The binding of [3H]H12-histrionicotoxin to Torpedo membranes was enhanced in the presence of NMTHT indicating an agonist action at this vertebrate peripheral nicotinic acetylcholine receptor. NMTHT is ineffective in radioligand binding assays for rat brain GABAA receptors, rat brain L-glutamate receptors and insect (Musca domestica) L-glutamate receptors. Partial block of rat brain muscarinic acetylcholine receptors is detected at millimolar concentrations of NMTHT. Thus nitromethylenes appear to exhibit selectivity for acetylcholine receptors and exhibit an agonist action at nicotinic acetylcholine receptors.

Desensitization of the nicotinic acetylcholine receptor by diisopropylfluorophosphate.

The interaction of diisopropylfluorophosphate (DFP) with the nicotinic acetylcholine (ACh) receptor of Torpedo electric organ was studied, using [3H]-phencyclidine ([3H]-PCP) as a reporter probe. Phencyclidine binds with different kinetics to resting, activated, and desensitized receptor conformations. Although DFP did not inhibit binding of [3H]-ACh or 125I-alpha-bungarotoxin (BGT) to the receptor recognition sites and potentiated in a time-dependent manner [3H]-PCP binding to the receptor's high-affinity allosteric site, it inhibited the ACh- or carbamylcholine-stimulated [3H]-PCP binding. This suggested that DFP bound to a third kind of site on the receptor and affected receptor conformation. Preincubation of the membranes with DFP increased the receptor's affinity for carbamylcholine by eightfold and raised the pseudo-first-order rate of [3H]-PCP binding to that of an agonist-desensitized receptor. Accordingly, it is suggested that DFP induces receptor desensitization by binding to a site that is distinct from the recognition or high-affinity noncompetitive sites.

Direct actions of organophosphate anticholinesterases on nicotinic and muscarinic acetylcholine receptors.

Four nerve agents and one therapeutic organophosphate (OP) anticholinesterase (anti-ChE) bind to acetylcholine (ACh) receptors, inhibit or modulate binding of radioactive ligands to these receptors, and modify events regulated by them. The affinity of nicotinic (n) ACh receptors of Torpedo electric organs and most muscarinic (m) ACh receptors of rat brain and N1E-115 neuroblastoma cultures for the OP compounds was usually two to three orders of magnitude lower than concentrations required to inhibit 50% (IC-50) of ACh-esterase activity. However, a small population of m-ACh receptors had an affinity as high as that of ACh-esterase for the OP compound. This population is identified by its high-affinity [3H]-cis-methyldioxolane ([3H]-CD) binding. Although sarin, soman, and tabun had no effect, (O-ethyl S[2-(diisopropylamino)ethyl)] methyl phosphonothionate (VX) and echothiophate inhibited competitively the binding of [3H]-quinuclidinyl benzilate ([3H]-QNB) and [3H]-pirenzepine ([3H]-PZ) to m-ACh receptors. However, VX was more potent than echothiophate in inhibiting this binding and 50-fold more potent in inhibiting carbamylcholine (carb)-stimulated [3H]-cGMP synthesis in N1E-115 neuroblastoma cells--both acting as m receptor antagonist. All five OPs inhibited [3H]-CD binding, with IC-50s of 3, 10, 40, 100, and 800 nM for VX, soman, sarin, echothiophate, and tabun, respectively. The OP anticholinesterases also bound to allosteric sites on the n-ACh receptor (identified by inhibition of [3H]-phencyclidine binding), but some bound as well to the receptor's recognition site (identified by inhibition of [125I]-alpha-bungarotoxin binding). Soman and echothiophate in micromolar concentrations acted as partial agonists of the n-ACh receptor and induced receptor desensitization. On the other hand, VX acted as an open channel blocker of the activated receptor and also enhanced receptor desensitization. It is suggested that the toxicity of OP anticholinesterases may include their action on n-ACh as well as m-ACh receptors if their concentrations in circulation rise above micromolar levels. At nanomolar concentrations their toxicity is due mainly to their inhibition of ACh-esterase. However, at these low concentrations, many OP anticholinesterases (eg, VX and soman) may affect a small population of m-ACh receptors, which have a high affinity for CD. Such effects on m-ACh receptors may play an important role in the toxicity of certain OP compounds.

Assessment of lead toxicity in traffic controllers of Alexandria, Egypt, road intersections.

Blood lead level (BPbL) was determined in forty-five traffic controllers working on Alexandria road intersections. Central nervous system dysfunction in the subjects studied was investigated by means of performance tests. Biochemical indicators related to lead exposure such as delta-aminolevulinic acid dehydratase and hemoglobin in their blood were also determined. Results indicated that most of the subjects studied have a comparably high BPbL. They also showed significantly poorer performance scores than that obtained in a previous study with a group of textile workers of the same age and educational levels. The mean of the BPbL in the traffic controllers was found to be 68.28 +/- 13.22 micrograms/dl. This is a very high level compared to an acceptable level of 30.00 micrograms/dl. All neurobehavioral symptoms demonstrated in the traffic controllers could be attributed to a high level of lead exposure.

Oxadiazolidinones: irreversible inhibition of cholinesterases and effects on acetylcholine receptors.

Inhibition of four acetylcholinesterases (AChE) and a butyrylcholinesterase (BuChE) by 3-(2,3-dihydro-2,2-dimethyl-benzofuran-'7-yl)-5-methoxy-1,3,4-oxadiaz ol-2(3H)-one (DBOX) and 3-(2-methoxyphenyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one (MPOX) was measured by the Ellman spectrophotometric method. Both oxadiazolidinones inhibited AChE and BuChE irreversibly and with quasi first order kinetics. DBOX was 2-3 orders of magnitude more potent than MPOX. Housefly brain AChE and horse serum BuChE were more sensitive than AChEs of red blood cells or eel and Torpedo electric organs. Aldicarb, a carbamate anticholinesterase, which protected Torpedo AChE against irreversible phosphorylation by DFP, also protected it against irreversible inhibition by DBOX and MPOX. It is suggested that the nonesteratic oxadiazolidinones are converted to carbanillates on the surface of the enzyme, then acylate the active site of ChEs, producing carbanillated enzymes. At higher concentrations, the two oxadiazolidinones also affected the specific binding of (125I) alpha-bungarotoxin (alpha-BGT) and [3H]perhydrohistrionicotoxin (H12-HTX) to Torpedo nicotinic ACh-receptors, but did not affect the specific binding of [3H]quinuclidinyl benzilate (QNB) to rat brain muscarinic ACh-receptors.