Inhibition of macrophage nitric oxide production by arachidonate-cascade inhibitors

We examined whether inhibitors of the arachidonic acid cascade inhibited nitric oxide (NO) production, as measured by nitrite concentration, either in macrophages or by their cytosolic fractions. Nitrite production by peritoneal macrophages from mice receiving OK-432 treatment was significantly inhibited by phospholipase A₂ inhibitors [dexamethasone and 4-bromophenacyl bromide (4-BPB)], lipoxygenase inhibitors [nordihydroguaiaretic acid (NDGA) and ketoconazole] and a glutathioneS-transferase (leukotrienes LTA₄-LTC₄) inhibitor (ethacrynic acid). However, caffeic acid and esculetin, inhibitors of 5- and 12-lipoxygenase respectively, were not inhibitory. On the other hand, indomethacin, a cyclooxygenase inhibitor, slightly inhibited whereas another inhibitor, ibuprofen, did not. Inhibition of the nitrite production by dexamethasone, 4-BPB, NDGA and ethacrynic acid was also demonstrated when the macrophages were restimulated ex vivo with OK-432 or with lipopolysaccharide. The inhibitory activity of dexamethasone, NDGA and ethacrynic acid was significantly reduced by ex vivo restimulation with OK-432, whereas that of 4-BPB was hardly affected. Furthermore, the inhibitory activity of dexamethasone, NDGA and ethacrynic acid was much higher when the macrophages were continuously exposed to the agents than when they were pulsed. Meanwhile, inhibition by 4-BPB was almost the same with either treatment. In addition, the inhibitory activity of these agents was not blocked withl-arginine, a substrate of NO synthases, or with arachidonate metabolites (LTB₄, LTC₄ and LTE₄). Ethacrynic acid and 4-BPB, but not dexamethasone and NDGA, also inhibited nitrite production by the cytosolic fractions from OK-432-restimulated peritoneal macrophages, and the inhibitory activity of 4-BPB was superior to that of ethacrynic acid. These agents, however, did not inhibit nitrite production from sodium nitroprusside, a spontaneous NO-releasing compound. These results indicate that dexamethasone, 4-BPB, NDGA and ethacrynic acid inhibited the production of NO by macrophages through at least two different mechanisms: one was inhibited by dexamethasone, NDGA and ethacrynic acid and the other by 4-BPB. Furthermore, 4-BPB and ethacrynic acid directly inhibited the activity of the NO synthase in macrophages, suggesting that the agents work by binding to the active site(s) of the enzyme.     

Enantioselective pharmacokinetics of homochlorcyclizine: III. Simultaneous determination of (+)- and (−)- homochlorcyclizine in human urine by high-performance liquid chromatography

A method is described for the simultaneous determination of (+)- and (−)-homochlorcyclizine (HCZ) in human urine by high-performance liquid chromatography on a chiral stationary phase of ovomucoid-bonded silica. The pH of the buffer and organic modifier in the mobile phase markedly affected the chromatographic separation. A mobile phase of methanol—0.02 M acetate buffer (pH 4.7) (25:75, v/v) at a flow-rate of 1.0 ml/min was used for the urine assays. The ultraviolet absorption was monitored at 240 nm, and diphenhydramine was employed as the internal standard for the quantitation. (+)-HCZ, (−)-HCZ and the internal standard were eluted at retention times of 15, 25 and 8 min, respectively. The limit of determination for HCZ enantiomers was ca. 50 ng/ml of urine. One of the metabolites in human urine, which was a quaternary ammonium-linked glucuronide, could also be determined in a manner similar to unchanged HCZ after β-glucuronidase hydrolysis. A pharmacokinetic study was conducted with three healthy volunteers, who each received a single oral dose of racemic HCZ (20 mg). Distinct differences were found between the two enantiomers, particularly in the metabolic process, that is, the urinary excretion as (−)-HCZ-glucuronide within 48 h was ca. four times higher than that of the (+)-isomer. This method should be very useful for enantioselective pharmacokinetic studies of HCZ.     

Changes in levels of mRNAs of transforming growth factor (TGF)-β1, -β2, -β3, TGF-β type II receptor and sulfated glycoprotein-2 during apoptosis of mouse uterine epithelium

To examine the roles played by transforming growth factors (TGF)-β1, -β2, -β3, and TGF-β type II receptors in the induction of apoptosis in the mouse uterine epithelium after estrogen deprivation, we investigated the expression of their mRNAs and the mRNA of sulfated glycoprotein-2 (SGP-2). Pellets containing 100 μg estradiol-17β (E₂) were implanted into ovariectomized mice and removed four days later. Apoptotic indices (percentage of apoptotic cells) of both luminal and glandular epithelia increased after E₂ pellets were removed, but administration of progesterone (P), 5-dihydrotestosterone (DHT), or continued implantation of E₂ pellets suppressed this increase. Levels of mRNAs of TGF-β1, -β2, and -β3, and SGP-2 did not increase after estrogen deprivation. However, estrogen deprivation caused a gradual increase in the level of TGF-β type II receptor mRNA, and its level increased about six-fold six days later. Moreover, E₂, P, and DHT markedly decreased the level of TGF-β type II receptor mRNA. In situ hybridization demonstrated that mRNAs of TGF-β1, -β2, -β3 and TGF-β type II receptor were localized to the epithelium. Exogenous administration of TGF-β1 into the uterine stroma induced apoptosis in the epithelium, a finding that suggests that signals produced by TGF-βs can induce apoptosis. Therefore, the present results suggest that increased sensitivity of uterine epithelial cells to TGF-βs, as demonstrated by an increase in TGF-β type II receptor mRNA, is involved in the induction of apoptosis after estrogen deprivation, although signals produced by TGF-βs do not appear sufficient to induce apoptosis.     

Synechococcus sp. PCC7942 Transformed with Escherichia coli bet Genes Produces Glycine Betaine from Choline and Acquires Resistance to Salt Stress

Synechococcus sp. PCC7942, a fresh water cyanobacterium, was transformed by a shuttle plasmid that contains a 9-kb fragment encoding the Escherichia coli bet gene cluster, i.e. betA (choline dehydrogenase), betB (betaine aldehyde dehydrogenase), betI (a putative regulatory protein), and betT (the choline transport system). The expression of these genes was demonstrated in the cyanobacterial cells (bet-containing cells) by northern blot analysis, as well as by the detection of glycine betaine by 1H nuclear magnetic resonance in cells supplemented with choline. Endogenous choline was not detected in either control or bet-containing cells. Both control and bet-containing cyanobacterial cells were found to import choline in an energy-dependent process, although this import was restricted only to bet-containing cells in conditions of salt stress. Glycine betaine was found to accumulate to a concentration of 45 mM in bet-containing cyanobacterial cells, and this resulted in a stabilization of the photosynthetic activities of photosystems I and II, higher phycobilisome contents, and general protective effects against salt stress when compared to control cells. The growth of bet-containing cells was much faster in the presence of 0.375 M NaCl than that of control cells, indicating that the transformant acquired resistance to salt stress.     

Involvement of L-Type-Like Amino Acid Transporters in S-Nitrosocysteine-Stimulated Noradrenaline Release in the Rat Hippocampus

Abstract: Nitrogen oxides, such as nitric oxide, have been shown to regulate neuronal functions, including neurotransmitter release. We investigated the effect of S-nitroso-l -cysteine (SNC) on noradrenaline (NA) release in the rat hippocampus in vivo and in vitro. SNC stimulated [³H]NA release from prelabeled hippocampal slices in a dose-dependent manner. SNC stimulated endogenous NA release within 30 min to almost five times the basal level in vivo (microdialysis in freely moving rats). In a Na⁺-containing Tyrode's buffer, SNC-stimulated [³H]NA release was inhibited 30% by the coaddition of l -leucine. In the Na⁺-free, choline-containing buffer, SNC-stimulated [³H]NA release, which was similar to that in the Na⁺-containing buffer, was inhibited markedly by l -leucine, l -alanine, l -methionine, l -phenylalanine, and l -tyrosine. The effects of the other amino acids examined were smaller or very limited. The effect of l -leucine was stronger than that of d -leucine. A specific inhibitor of the L-type amino acid transporter, 2-aminobicyclo[2.2.1]-heptane-2-carboxylate (BCH), inhibited the effects of SNC on [³H]NA release in the Na⁺-free buffer. Uptake of l -[³H]leucine into the slices in the Na⁺-free buffer was inhibited by SNC, BCH, and l -phenylalanine, but not by l -lysine. The effect of SNC on cyclic GMP accumulation was not inhibited by l -leucine, although SNC stimulated cyclic GMP accumulation at concentrations up to 25 µM, much less than the concentration that stimulates NA release. These findings suggest that SNC is incorporated into rat hippocampus via the L-type-like amino acid transporter, at least in Na⁺-free conditions, and that SNC stimulates NA release in vivo and in vitro in a cyclic GMP-independent manner.     

Role of water receptor in the frog

To elucidate the role of the water receptor in the frog (Rana catesbeiana), reflex activities elicited by its excitation were studied. Application of tap water to the oral mucosa depressed the rhythmical movement of gorge (buccal) respiration, accompanied by an elevation of the inner pressure of the oral cavity (buccal pressure). Tonic reflex discharges were elicited in the nerves innervating the submental and submaxillary muscles, which close the nostrils, the pterygoid and the profound portion of the major masseter muscles, which produce a strong bite, and the geniohyoid and hyoglossus muscles, which elevate buccal pressure. These muscles, except for the pterygoid, also participate in the rhythmical movement of gorge respiration as expiratory muscles. Rhythmical movements in the minor masseter and sternohyoid muscles, which act as inspiratory muscles in gorge respiration, were depressed by the water stimulation of the oral mucosa. These findings indicate that the water receptor plays a role in the interruption of gorge respiratory movements, accompanied by an elevation of buccal pressure.     

Benthic foraminiferal fauna during the time of the Indian-Asian contact, in southern Balochistan, Pakistan

Cretaceous and early Paleocene benthic foraminifera were studied from one section along the western Gaj River, southern Balochistan, Pakistan, to reconstruct the paleoenvironment of the Tethys Sea during the Indian-Asian contact. We recognize three lithostratigraphic units in ascending order: the Mughal Kot Formation, the Pab Sandstone, and the Jamburo Group. Both the Maastrichtian Mughal Kot Formation, which consists of shale with grey marly limestone, and the Maastrichtian Pab Sandstone, which consists of quartzose sandstone, indicate an open ocean environment as they have diversified planktic and benthic foraminiferal assemblages. The Maastrichtian-Paleocene Jamburo Group, consisting of dark grey, calcareous shale and marlstone with some sulfide grains, is characterized by low diversities of benthic assemblages. The change to the lower diversities may be associated with the development of poor circulation of deeper water that was caused by narrowing of the Tethys Sea.The Trochammina spp. Assemblage from the Jamburo Group, which can be correlated with flysch-type agglutinated foraminiferal assemblages, has a low benthic species diversity, indicating an unfavorable condition for calcareous foraminifera because of the development of oxygen-depleted water. The absolute abundance of agglutinated specimens shows a remarkable change from low numbers in the Maastrichtian to high ones in the Paleocene. The benthic foraminiferal evidence supports the hypothesis that the collision of the Asian and Indian plates occurred near the end of the Cretaceous.     

Possible participation of Ca2+, Mg2+-dependent endonuclease in liver DNA fragmentation after N-methyl-N-nitrosourea treatment

We examined the fragmentation of DNA treated with N-methyl-N-nitrosourea under conditions in which Ca2+, Mg2+-dependent endonuclease is active. The molecular mass of DNA found in mouse liver slices treated with methylnitrosurea in the presence of Ca2+ plus Mg2+ was 4 X 10(5) Da. Similar results were obtained with a reconstituted system containing partially purified Ca2+, Mg2+-dependent endonuclease and methylnitrosurea-treated DNA. The enzyme extensively cleaved methylnitrosurea-treated DNA, compared with non-treated DNA. The methylnitrosurea-treated nuclear proteins obtained from mouse liver nuclei had no effect on the DNA fragmentation by the enzyme. Using closed-circular DNA treated with methylnitrosurea, the enzyme produced single-strand cuts in the DNA, as was seen in non-treated, closed-circular DNA, however, the rate of hydrolysis was increased. Ca2+, Mg2+-dependent endonuclease thus warrants further investigation, with regard to the precise mechanism of extensive degradation of DNA in cells treated with carcinogenic alkylating agents.     

UV Irradiation induces an activity which stimulates Simian virus 40 rescue upon cell fusion.

UV irradiation of African green monkey cells greatly stimulated efficiency of simian virus 40 induction from simian virus 40-transformed Syrian hamster cells after cell fusion. The maximum inducing activity was observed at 15 to 20 h after irradiation but remained only transiently. The addition of cycloheximide after UV irradiation eliminated the stimulation of the activity.     

Separation of keratinocytes by density gradient centrifugation for DNA cytofluorometry

Summary A method for the separation of guinea pig epidermal keratinocytes, in which the Feulgen-stainable material suffers minimal damage, has been investigated. The principal stage involves trypsin treatment of the epidermal sheet, stripped from the dermis with ethylenediamine tetraacetic acid. The epidermal cells thus isolated are separated into three groups by centrifugation on a continuous colloidal silica (Percoll) density gradient. The resulting arrangement of the keratinocytes in the centrifuge tube corresponds to their arrangement in situ, with basal cells at the bottom and the more differentiated cells above. By morphological examination, it can be shown that relatively pure fractions of basal cells, spinous cells, and granular cells are obtained by this method. With respect to DNA distribution pattern, there was good agreement between that of keratinocytes separated by the microdissection-ultrasonic irradiation method, or by the chymotrypsin method as reported previously by us, and that obtained by the present method.