Determination of plasma leukotrienes in antigen-induced bronchoconstrictive guinea pigs.

Convenient extraction and radioimmunoassay methods for measurement of leukotrienes C4 and D4 (LTC4 and LTD4) in biological fluids are described. LTC4 or LTD4 in plasma was extracted with acetonitrile, and the extract was washed with dichloromethane then adjusted to pH 3.5 or 6.0, respectively. Each leukotriene was partially purified by using a C18-bonded silica cartridge and quantitated by radioimmunoassay. Amounts of LTC4 and LTD4 in the range of 0.025-1.6 ng could be assayed in plasma. This procedure was employed to examine the increase in plasma LTC4 (0.249 +/- 0.036 ng/ml) and LTD4 (1.399 +/- 0.235 ng/ml) of guinea pigs during intravenous challenge-induced anaphylactic bronchoconstriction, and the suppression of the increase of bronchoconstriction and leukotrienes by the administration of 5-lipoxygenase inhibitors such as E6080 (6-hydroxy-2-(4-sulfamoylbenzyl-amino)- 4,5,7-trimethylbenzothiazole hydrochloride), AA861 (2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone ) and phenidone. On the other hand, LTC4 and LTD4 were not detected in plasma after an inhaled challenge, though significant bronchoconstriction was provoked. It was concluded that the present study validates a new technique for quantitating plasma leukotrienes on the basis of pH and a suitable method for evaluating the pharmacological efficacy of 5-lipoxygenase inhibitors.     

Racemization in the Coupling Reaction with the Several Methods beside the Activated Ester Methods

The degree of racemization in the coupling reaction, Pro-Val + Pro, with the several other methods than the activated ester methods was measured and the results were compared with that in the coupling reaction, Leu-Phe + Val, as well as in the previous paper.¹⁾ In the azide and the mixed anhydride methods, no or almost no racemization was detected, whereas in the other tested methods of peptide synthesis (Pachornik-, DCCD- and phosphazo-methods) the significantly large racemization was observed. It can be attributed to the strong nucleo- philic N-atom in the penultimate amino acids (Pro) and the steric hindrance of C-terminal amino acid (Val), which are favourable to the formation of the oxazolone ring.

This assumption was further systematically confirmed in the synthesis of the other several tripeptides with the DCCD method. The separation of the diastereoisomers (LLL and LDL) of the resulting tripeptides by gas chromatography with a packed column was also here reported.     

RFP is a DNA binding protein associated with the nuclear matrix.

We reported that the RFP gene encodes a protein with putative zinc finger domains and was involved in the activation of the ret proto-oncogene. To further characterize the RFP protein, we developed a polyclonal antibody against the product synthesized from a fragment of the RFP cDNA expressed in Escherichia coli. Western blot analysis showed that RFP was identified as a 58 kDa protein in cell lysates from four human and rodent cell lines and from mouse testis. In addition, a unique 68 kDa protein was detected in the testis. Using AH7974 (rat ascites hepatoma) and Raji (human Burkitt lymphoma) cells, we demonstrated strong association of RFP with the nuclear matrix. Furthermore, RFP solubilized from the nuclear matrix had DNA-binding activity although it appears to bind more preferentially to double-stranded DNA than to single-stranded DNA. These results thus suggest that RFP may play a role in molecular processes which occur in the nuclear matrix.     

Incorporation of fatty acids by Streptococcus mutans

In a series of investigations into the cariogenicity of Streptococcus mutans, we studied the incorporation of exogenous fatty acids with reference to glucosyltransferase secretion and membrane fatty acid changes. When cells were grown with different fatty acids, both saturated and unsaturated fatty acids were readily incorporated into the membrane lipids and were biotransformed and elongated preferentially to the longer 16- and 18-carbon-chain fatty acids. This incorporation and chain-elongation led to significant changes in fatty acids composition. By adding fatty acids to the medium, it was possible to appropriately modify the degree of unsaturation and the relative ratio between specific fatty acids in the membrane lipids of S. mutans.     

Selectivity and contribution of lecithin: cholesterol acyltransferase to plasma cholesterol ester formation

Selectivity factors (Vm/Km) for human and rat lecithin: cholesterol acyltransferases (LCAT) for the transfer of various acyl groups from the 2-position of phosphatidylcholine were determined. By multiplying these values by the proportions of acyl groups at the 2-position of phosphatidylcholine, one can predict the proportions of molecular species of cholesterol ester which will be synthesized by LCAT. In human subjects fasted overnight, the molecular composition of plasma cholesterol ester was found to reflect the LCAT selectivity relatively accurately. This result supports the concepts that hepatic acyl-CoA:cholesterol acyltransferase (ACAT) does not contribute significantly to the synthesis of plasma cholesterol ester and that removal of cholesterol ester from plasma is not selective with respect to molecular species under these conditions. In contrast to the results with humans, the molecular composition of plasma cholesterol ester formed in spontaneously hypertensive rats fed a high-cholesterol diet and then fasted overnight differs from that which is predicted from LCAT selectivity and the proportion of various fatty acids at the 2-position of phosphatidylcholine: these results suggest that cholesterol ester is formed mainly via the ACAT reaction.     

Freeze denaturation of enzymes and its prevention with additives

Freeze inactivation of LDH, MDH, ADH, G-6-PDH, and PK and its prevention with additives such as sodium glutamate and albumin were studied. LDH, MDH, ADH, G-6-PDH, and PK, each lost their activity during frozen storage at -20 degrees C. The speed of the inactivation differed in each. The stability of the enzymes increased with the increase of the enzyme concentration. Sodium glutamate and albumin prevented the freeze inactivation. While the activity of the LDH solution frozen without additives was almost lost during a day of frozen storage, those frozen with either glutamate (0.2 M) or albumin (0.1%) added decreased less quickly. The residual activity after 1 day was 50% the initial prefreeze value for the former and 10% for the latter, respectively. Combined use of glutamate and albumin prevented the inactivation the best and maintained the initial activity almost completely over 6 weeks. The enzymes tested lost some part of their activity when their solutions were diluted by the media. This inactivation was prevented to a significant extent by the addition of sodium glutamate and/or albumin to the diluting media.     

Morphine-Induced Changes in Histamine Dynamics in Mouse Brain

The effect of the acute morphine treatment on histamine (HA) pools in the brain and the spinal cord was examined in mice. Morphine (1-50 mg/kg, s.c.) administered alone caused no significant change in the steady-state levels of HA and its major metabolite, tele-methylhistamine (t-MH), in the brain. However, depending on the doses tested, morphine significantly enhanced the pargyline (65 mg/kg, i.p.)-induced accumulation of t-MH and this effect was antagonized by naloxone. A specific inhibitor of histidine decarboxylase, alpha-fluoromethylhistidine (alpha-FMH) (50 mg/kg, i.p.), decreased the brain HA level in consequence of the almost complete depletion of the HA pool with a rapid turnover. Morphine further decreased the brain HA level in alpha-FMH-pretreated mice. Morphine administered alone significantly reduced the HA level in the spinal cord, an area where the turnover of HA is very slow. These results suggest that the acute morphine treatment increases the turnover of neuronal HA via opioid receptors, and this opiate also releases HA from a slowly turning over pool(s).     

Regulation of Histamine Turnover via Muscarinic and Nicotinic Receptors in the Brain

To clarify the regulation of central histaminergic (HAergic) activity by cholinergic receptors, the effects of drugs that stimulate the cholinergic system on brain histamine (HA) turnover were examined, in vivo, in mice and rats. The HA turnover was estimated from the accumulation of tele-methylhistamine (t-MH) during the 90-min period after administration of pargyline (65 mg/kg, i.p.). In the whole brain of mice, oxotremorine, at doses higher than 0.05 mg/kg, s.c., significantly inhibited the HA turnover, this effect being completely antagonized by atropine but not by methylatropine. A large dose of nicotine (10 mg/kg, s.c.) also significantly inhibited the HA turnover. This inhibitory effect was antagonized by mecamylamine but not by atropine or hexamethonium. A cholinesterase inhibitor, physostigmine, at doses higher than 0.1 mg/kg, s.c., significantly inhibited the HA turnover. This effect was antagonized by atropine but not at all by mecamylamine. None of these cholinergic antagonists used affected the steady-state t-MH level or HA turnover by themselves. In the rat brain, physostigmine (0.1 and 0.3 mg/kg, s.c.) also decreased the HA turnover. This inhibitory effect of physostigmine was especially marked in the striatum and cerebral cortex where muscarinic receptors are present in high density. Oxotremorine (0.2 mg/kg, s.c.) and nicotine (1 mg/kg, s.c.) also decreased the HA turnover in the rat brain. However, these effects showed no marked regional differences. These results suggest that the stimulation of central muscarinic receptors potently inhibits the HAergic activity in the brain and that strong stimulation of central nicotinic receptors can also induce a similar effect.     

Effects of the Histamine H3-Agonist (R)-α-Methylhistamine and the Antagonist Thioperamide on Histamine Metabolism in the Mouse and Rat Brain

To study the feedback control by histamine (HA) H3-receptors on the synthesis and release of HA at nerve endings in the brain, the effects of a potent and selective H3-agonist, (R)-alpha-methylhistamine, and an H3-antagonist, thioperamide, on the pargyline-induced accumulation of tele-methylhistamine (t-MH) in the brain of mice and rats were examined in vivo. (R)-alpha-Methylhistamine dihydrochloride (6.3 mg free base/kg, i.p.) and thioperamide (2 mg/kg, i.p.), respectively, significantly decreased and increased the steady-state t-MH level in the mouse brain, whereas these compounds produced no significant changes in the HA level. When administered to mice immediately after pargyline (65 mg/kg, i.p.), (R)-alpha-methylhistamine (3.2 mg/kg, i.p.) inhibited the pargyline-induced increase in the t-MH level almost completely during the first 2 h after treatment. Thioperamide (2 mg/kg, i.p.) enhanced the pargyline-induced t-MH accumulation by approximately 70% 1 and 2 h after treatment. Lower doses of (R)-alpha-methylhistamine (1.3 mg/kg) and thioperamide (1 mg/kg) induced significant changes in the pargyline-induced t-MH accumulation in the mouse brain. In the rat, (R)-alpha-methylhistamine (3.2 mg/kg, i.p.) and thioperamide (2 mg/kg, i.p.) also affected the pargyline-induced t-MH accumulation in eight brain regions and the effects were especially marked in the cerebral cortex and amygdala. These results indicate that these compounds have potent effects on HA turnover in vivo in the brain.