An evaluation of analgesia associated with the immobility response in laboratory rabbits

The immobility response (IR) was studied in rabbits to evaluate its analgesic properties and reliability as a method of restraint. The participation of the endogenous opioid system in IR was studied indirectly by evaluating the effects of the narcotic antagonist naloxone on this phenomenon. Twenty-four adult New Zealand White rabbits were subjected to six noxious stimuli while restrained by IR and while restrained under control conditions. Testing on each animal was repeated under both conditions following the administration of naloxone. The noxious stimuli consisted of three levels of electric shock (10 volts, 30 volts, and 50 volts) applied to the shaved forearm, and mechanical pressure applied to the pinna, front toe, and hind toe. Withdrawal and changes in blood pressure, heart rate, and respiration were used as indicators of pain perception. Distress associated with noxious electrical and pressure stimulation was significantly reduced by IR, which suggested that the phenomenon does have a significant analgesic component. However, the rabbits showed wide variability in their susceptibility to IR induction, and even animals which did not withdraw in response to noxious stimulation under IR sometimes exhibited physiological changes suggestive of distress. Therefore, IR should not be considered as a reliable or humane alternative to analgesic/anesthetic drugs for laboratory rabbits. Naloxone had little effect on IR or IR-associated analgesia.     

Contrasting responses of oligochaete communities to the abatement of eutrophication in Lake Geneva

Tubificid and lumbriculid worms were used to monitor, at depths of 150 m, the recovery of Lake Geneva (Switzerland) from eutrophication. As predicted from the decrease of phosphorus concentrations, relative abundance of oligotrophic species was higher from 1988 to 1993 than in 1983, i.e. before the abatement of eutrophication. However, this trend towards oligotrophication can be reversed, as indicated by a decrease of oligotrophic species, recorded in 1993. But this change corresponded to the effects of an increase of water temperature on the abundance of the mesotrophic species Potamothrix vejdovskyi rather than to a deterioration of the profundal. In addition to this short-term setback, oligochaete communities located at a depth of 150 m responded more slowly and less clearly to the decrease of phosphorus concentrations than those located at a depth of 40 m. However, the zoobenthos indicated more clearly the recovery of Lake Geneva than the phytoplankton.     

Molecular binding scaffolds increase local substrate concentration enhancing the enzymatic hydrolysis of VX nerve agent

Kinetic enhancement of organophosphate hydrolysis is a long-standing challenge in catalysis. For prophylactic treatment against organophosphate exposure, enzymatic hydrolysis needs to occur at high rates in the presence of low substrate concentrations and enzymatic activity should persist over days and weeks. Here, the conjugation of small DNA scaffolds was used to introduce substrate binding sites with micromolar affinity to VX, paraoxon, and methyl-parathion in close proximity to the enzyme phosphotriesterase (PTE). The result was a decrease in K_M and increase in the rate at low substrate concentrations. An optimized system for paraoxon hydrolysis decreased K_M by 11-fold, with a corresponding increase in second-order rate constant. The initial rates of VX and methyl-parathion hydrolysis were also increased by 3.1- and 6.7-fold, respectively. The designed scaffolds not only increased the local substrate concentration, but they also resulted in increased stability and PTE-DNA particle size tuning between 25 and ~150 nm. The scaffold engineering approach taken here is focused on altering the local chemical and physical microenvironment around the enzyme and is therefore compatible with active site engineering via combinatorial and computational approaches.     

Evaluation of Repellency Effect of Two Natural Aroma Mosquito Repellent Compounds,Citronella and Citronellal*

Repellent efficacies of two natural aroma compounds, citronella and citronellal, against mosquitoes, Culex pipiens pallens, were evaluated both in field and in vitro. In vitro, the experiment was conducted with three controlled bands impregnated with 30% citronella extract, 15% citronella extract and 30% citronellal extract, and with bands impregnated 30% citronella in field. Data was obtained by the means of counting numbers bitten by mosquitoes per unit time, namely human bait method. Percentage repellencies of above three controlled bands were calculated at 86%, 73%, and 78%, respectively in vitro, and 80% in field, showing high repellent effectiveness against mosquitoes. This estimation was also confirmed by t‐test compared between control group and each experimental group.     

Immunological identity of a 60 kd oncofetal protein induced in rats by chemical carcinogens and released by transformed cells

A 60,000 dalton (60 kd) oncofetal protein was previously shown to be produced by tumors in tumor-bearing rats and by target tissues within 3 weeks of carcinogen treatment. The factor is released to and accumulates in the blood in vivo and in the conditioned medium of cultured transformed cells in vitro. A polyclonal antibody produced against the 60 kd factor purified from the plasma of a rat carrying the N-2-fluorenylphthalamic acid-induced transplantable Hepatoma 7777, was tested against the 60 kd factor from various sources. Based on the results of immunoprecipitation of biochemical activity associated with the 60 kd factor, it was determined that these anti-60 kd antibodies cross-reacted with the factor released by a dimethylbenzanthracene-induced rat mammary carcinoma, with the factor in rat tumor cytosol and with rat spontaneous lymphoma cells, but not with a 60 kd factor isolated from pooled cancer patient plasma. Furthermore, these antibodies cross-reacted with the 60 kd factor induced within 21 days of treatment of the rats with a range of carcinogens from 8 chemical structural groups. The anti-60 kd factor antibodies did not cross-react with a 35 kd factor having similar biochemical activity found in normal adult cells.     

Flufenamic acid, mefenamic acid and niflumic acid inhibit single nonselective cation channels in the rat exocrine pancreas.

The non-steroidal anti-inflammatory drugs, flufenamic acid, mefenamic acid and niflumic acid, block Ca2(+)-activated non-selective cation channels in inside-out patches from the basolateral membrane of rat exocrine pancreatic cells. Half-maximal inhibition was about 10 microM for flufenamic acid and mefenamic acid, whereas niflumic acid was less potent (IC50 about 50 microM). Indomethacin, aspirin, diltiazem and ibuprofen (100 microM) had not effect. It is concluded that the inhibitory effect of flufenamate, mefenamate and niflumate is dependent on the specific structure, consisting of two phenyl rings linked by an amino bridge.     

Selective inhibitors of KCl cotransport in human red cells

Two analogues of the loop diuretics furosemide and bumetanide have been identified as differential inhibitors of KCl and NaKCl cotransport systems, assayed by measuring K+ influx in 'young' human red cells. H25 inhibited both NaKCl and KCl cotransport, with I50% values of 0.03 and 30 microM respectively; H74 had no effect on NaKCl cotransport, even at 0.3 mM, but inhibited KCl cotransport with an I50% of 75 microM. These compounds are therefore useful for resolving the two transport systems.     

Ile-Ser-bradykinin is an aberrant permeability factor in various human malignant effusions

In this study we provide evidence for the presence of the aberrant peptide, Ile-Ser-bradykinin, in various human malignant exudates. The peptide was detected by deproteinisation of the effusion, application to reversed-phase HPLC, collection of the fractions containing Ile-Ser-bradykinin (retention time 6.90 min), degradation with carboxypeptidase B, and rechromatography of the resulting des-Arg-Ile-Ser-bradykinin (des-Arg-ISB) (retention time 13.5 min). In addition, all positive samples were confirmed by amino acid analysis and most of them (7/8) by amino-acid sequencing. In malignant effusions from 8 patients out of a group of 113 patients, Ile-Ser-bradykinin was found in concentrations between 12 and 520 mumol. In 44 malignant effusions, Ile-Ser-bradykinin was suspected, but could not be confirmed by the required additional methods (amino-acid analysis, sequencing) because of its low concentration. Sixty eight benign effusions were negative for Ile-Ser-bradykinin.     

Metabolism of the lipid peroxidation product 4-hydroxynonenal by isolated hepatocytes and by liver cytosolic fractions.

The metabolism of the lipid peroxidation product 4-hydroxynonenal and of several other related aldehydes by isolated hepatocytes and rat liver subcellular fractions has been investigated. Hepatocytes rapidly metabolize 4-hydroxynonenal in an oxygen-independent process with a maximum rate (depending on cell preparation) ranging from 130 to 230 nmol/min per 10(6) cells (average 193 +/- 50). The aldehyde is also rapidly utilized by whole rat liver homogenate and the cytosolic fraction (140 000 g supernatant) supplemented with NADH, whereas purified nuclei, mitochondria and microsomes supplemented with NADH show no noteworthy consumption of the aldehyde. In cytosol, the NADH-mediated metabolism of the aldehyde exhibits a 1:1 stoichiometry, i.e. 1 mol of NADH oxidized/mol of hydroxynonenal consumed, and the apparent Km value for the aldehyde is 0.1 mM. Addition of pyrazole (10 mM) or heat inactivation of the cytosol completely abolishes aldehyde metabolism. The various findings strongly suggest that hepatocytes and rat liver cytosol respectively convert 4-hydroxynonenal enzymically is the corresponding alcohol, non-2-ene-1,4-diol, according to the equation: CH3-[CH2]4-CH(OH)-CH = CH-CHO + NADH + H+----CH3-[CH2]4-CH(OH)-CH = CH-CH2OH + NAD+. The alcohol non-2-ene-1,4-diol has not yet been isolated from incubations with hepatocytes and liver cytosolic fractions, but was isolated in pure form from an incubation mixture containing 4-hydroxynonenal, isolated liver alcohol dehydrogenase and NADH and its chemical structure was confirmed by mass spectroscopy. Compared with liver, all other tissues possess only little ability to metabolize 4-hydroxynonenal, ranging from 0% (fat pads) to a maximal 10% (kidney) of the activity present in liver. The structure of the aldehyde has a strong influence on the rate and extent of its enzymic NADH-dependent reduction to the alcohol. The saturated analogue nonanal is a poor substrate and only a small proportion of it is converted to the alcohol. Similarly, nonenal is much less readily utilized as compared with 4-hydroxynonenal. The effective conversion of the cytotoxic 4-hydroxynonenal and other reactive aldehydes to alcohols, which are probably less toxic, could play a role in the general defence system of the liver against toxic products arising from radical-induced lipid peroxidation.