Benzimidazoles (BZ) are widely used to treat parasitic nematode infections of humans and animals, but resistance is widespread in veterinary parasites. Several polymorphisms in beta-tubulin genes have been associated with BZ-resistance. In the present study, we investigated beta-tubulin isotype 1 sequences of 18 Haemonchus contortus isolates with varying levels of resistance to thiabendazole. The only polymorphism whose frequency was significantly increased in the resistant isolates was TTC to TAC at codon 200. Real-time PCR (using DNA from 100 third-stage larvae, L3s) and pyrosequencing (from DNA from 1000-10 000 L3s) were used to measure allele frequencies at codon 200 of these isolates, producing similar results; drug sensitivity decreased with increasing TAC frequency. Pyrosequencing was also used to measure allele frequencies at positions 167 and 198. We showed that such measurements are sufficient to assess the BZ-resistance status of most H. contortus isolates. The concordance between real-time PCR and pyrosequencing results carried out in different laboratories indicated that these tools are suitable for the routine diagnosis of BZ-resistance in H. contortus. The molecular methods were more sensitive than the 'egg hatch test', and less time-consuming than current in vivo- or in vitro-anthelmintic resistance detection methods. Thus, they provide a realistic option for routine molecular resistance testing on farms.
Renal medullary blood flow was well maintained for several hours after blood loss which produced hypotension. Renal cortical blood flow was altered by trauma and mild hemorrhage even though blood pressure remained normal; rate of blood flow through the subcapsular, peritubular capillaries decreased to the level of that in the outer medulla. With further hemorrhage and the development of hypotension, rate of blood flow in large cortical areas was reduced to that of the outer medulla. With prolonged hypotension, the rate of blood flow in most of the cortex approached outer medullary rate and a single exponential could describe the flow in this combined area. Small regions of even slower flow rate began to appear in the outer cortex; patches of tissue appeared to be completely ischemic. The progression of cortical ischemia noted in these experiments may provide additional evidence for the pathogenesis of the patchy tubular necrosis noted in hemorrhagic shock.
The antagonistic actions of the neurohumoral transmitters, norepinephrine and acetylcholine, on nodal, conducting and atrial tissue have been well established. 1 " 8 Evidence for their direct antagonistic effects on ventricular contractility, however, has been more difficult to obtain.*-0 Using direct intracoronary infusions of sympathomimetic and parasympathomimetic drugs, we have obtained unequivocal evidence for such antagonism in the left ventricular myocardium. Acetylcholine blocked the positive inotropic effects of stellate ganglion stimulation and infused catecholamines. In addition to this blocking action, acetylcholine evoked a positive inotropic response which became apparent only when infusion of the drug was stopped. MethodsFasted mongrel dogs (25 to 45 kg) were anesthetized with sodium pentobarbital (25 to 30 mgAg rv) and given additional doses as needed during the experiment. Artificial respiration was maintained by intermittent positive pressure through an endotracheal tube. The heart was exposed through a left thoracotomy, the pericardium opened, and the free margin sutured to the thoracic wall. Polyvinyl chloride catheters (O.D. 0.030 inch and I D . 0.015 inch) were implanted in the left anterior descending (LAD) and left circumflex (LC) coronary arteries ( fig. 1) by a modification of the method of Herd and Barger, 7 with a small, side orifice in the intravascular segment of the catheter. The former was placed distal to the origin of the septal artery branch and the latter beyond the atrial branches to avoid arrhythmias and changes in heart rate. To record contractile force, two cali-From the LAO / CATHETER STRAIN GAUGE STRAIN GAUGE FIGURE 1 Illustration of preparation used in these studies. A catheter is shown in the left anterior descending (LAD) coronary artery with strain gauge arch located within the area of distribution of the artery. A second catheter placed in the left circumflex (LC) coronary artery and a strain gauge arch in the region supplied by the LC provided control measurements of myocardtal blood flow and contractility.brated Walton-Brodie strain gauge arches were sutured to the left ventricular myocardium, one in the area supplied by the catheterized LAD artery (verified by intracoronary injection of indigo carmine) and the second in a region supplied by the LC artery. Femoral arterial blood pressure and heart rate were also recorded.The following drugs, freshly dissolved in 0.9% saline, were infused into the LAD coronary artery at 0.25 to 0.5 ml/min using a constant infusion pump: norepinephrine bitartrate, acetylcholine chloride, physostigmine (salicylate or sulfate), atropine sulfate, hexamethonium bromide, bretylium tosylate, pronethalol, and bradykinin. When applicable, the dose is expressed as weight of the free base per minute. In some experi-
The physical state of membrane lipids and relationships with the activity of Na+-K+-ATPase and alkaline phosphatase were studied in basolateral and brush border membranes of the dog kidney. Fluorescence polarization and electron spin resonance experiments demonstrate that basolateral membranes are much more fluid than brush border membranes. This can be accounted for by a difference in fluidity of the lipid part of the membranes. Broad (43-17 degrees C) thermotropic transitions are observed in liposomes made from total lipid extracts of brush border and basolateral membranes. Fluorescence data strongly suggest that thermotropic transitions also occur in intact membranes and that a change in membrane physical state may take place around the physiological temperature. A nonlinear Arrhenius plot for the Na+-K+-ATPase activity in basolateral membranes (breakpoint 21 degrees C) provides additional support for the existence of a lipid liquid leads to gel transition in antiluminal plasma membranes. A break in the Arrhenius plot of alkaline phosphatase activity is also observed but at a temperature significantly higher (26 degrees C) than that of the end of the thermotropic transition. "Room temperature" appears as a critical zone for lipid physical state and activities of both enzymes.
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