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.     

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.     

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.     

A lea-class gene of tomato confers salt and freezing tolerance when expressed in Saccharomyces cerevisiae

During periods of water deficit, plants accumulate late embryogenesis-abundant (LEA) proteins which are thought to protect cells from stresses associated with dehydration. One of these genes, le25, is expressed in tomato leaves and roots in response to water deficit and abscisic acid accumulation. To study the function of this protein and to test the effect of overproduction of the LE25 protein in Saccharomyces cerevisiae (Sc), a recombinant plasmid in which le25 is expressed under the control of the GAL1 promoter was constructed. The content of LE25 was high in Sc cells transformed with the recombinant plasmid. The transformant exhibited several stress-tolerant phenotypes. Growth of the transformant in a medium with 1.2 M NaCl was improved, as compared to a control strain. While the control strain showed a long lag phase of 40 h, le25-expressing cells showed a shortened lag phase of 10 h. However, no growth improvement was observed in a medium with 2 M sorbitol. In addition, the transformant had an increased survival rate after freezing stress, but not after high-temperature stress. These results, together with its predicted secondary structure, may indicate that LE25 functions as an ion scavenger.     

A new method of inhalation anesthesia with nasopharyngeal insufflation in rat experiment.

We have established a new method of anesthesia with nasopharyngeal insufflation for intraoral procedure in rodents. Twelve male Wistar rats weighing 330-390 g were used in this study. Insertion of a feeding tube 1.0 mm in diameter coated with 2% xylocaine jelly was inserted into the nasal cavity approximately 25 mm from the naris, and anesthetization with mixed gas of 100% oxygen with 3-4% enflurane at 0.25-0.5 l/min flow rate was achieved. Using this anesthetic method, a chronic experiment comprising 1-h/day experimental procedure was carried out for 14 days. This method enabled, 1) simple and safe operation of the induction, emergence and anesthetic depth, 2) experimental procedures on the dental/oral region, 3) avoidance of the dyspnea and tachypnea, and 4) avoidance of cumulative effects in daily anesthesia.     

Expression of an ABA-induced gene of tomato in transgenic tobacco during periods of water deficit

The gene le25 is an abscisic acid (ABA)-induced gene of tomatowhich is expressed both in wilted vegetative organs and developingseeds. Spatial and temporal expression was analysed in tobaccoplants transformed with a chimeric gene in which 5'-upstreamDNA sequences of le25 were fused to the E. coli uidA gene, whichencodes ß-glucuronidase (GUS). Histochemical stainingrevealed that GUS was expressed in all tissues of vegetativeorgans in response to water deficit. Exogenous ABA induced expressionto a lesser extent, even though ABA content was the same asdroughtstressed leaves, indicating a difference in responseto endogenous ABA compared to exogenous ABA. Water-deficit-inducedGUS expression in floral tissues was examined in pre-anthesisfloral buds from four different stages (I–IV; 11, 16,33, 49 mm bud length, respectively). While non-stressed floralorgans showed no GUS activity except in pollen at stages IIIand IV, GUS activity was water-deficit-induced in sepals ofall stages, petals of stage II, and stigmas of stage II andIII. In seeds, GUS activity was detected in both the embryoand endosperm at 15 d post-anthesis, which coincided with alarge increase in the concentration of ABA in the seed. In transgenicplants, the le25 5'-flanking DNA drove expression of GUS duringwater deficit in two modes: non-tissue-specific expression invegetative organs, and tissue-specific expression in reproductiveorgans. The location of GUS activity indicated that ABA concentrationis elevated throughout the tissues of the leaf during periodsof water deficit. Key words: Tomato, ABA, drought stress, lea gene, water deficit     

Racemization in the Coupling Reaction,Pro-Val+Pro,with the Activated Ester Methods

The degree of racemization in the several activated ester methods of the peptide synthesis was measured in using the critical racemization test, Pro-Val+Pro, with help of gas chromatography. The results were compared with that in the coupling reaction, Leu-Phe+Val, in which no racemization had been reported in the corresponding reaction conditions by F. Weygand et al., when the activated dipeptide esters had been prepared from Z-Leu+Phe-activated esters. The significantly higher racemization was observed in the methods of N-hydroxypiperidine ester and thiophenyl ester, even when the activated dipeptide esters were prepared from Z-Pro+Val-activated esters. On the other hand, almost no racemization was observed in the N-hydroxysuccinimide ester and p-nitrophenyl ester methods. A great extent of the racemization was detected when the activated dipeptide esters were prepared directly from Z-Pro-Val-OH.