Novel inhibitors of enkephalin-degrading enzymes. II: N5'-substituted-4-thioxohydantoic acids as aminopeptidase inhibitors

Some 2-substituted-(2'-aminophenyl)-4-thioxohydantoic acids (o-amino PTC-amino acids) have antinociceptive activity when administered (icv) alone (IC50 = 0.04-0.87 microM/animal) and show a striking prolongation of the antinociceptive action of (D-Ala-2 D-Leu5)-enkephalin (DADL) in combination. The effects are thought to be mediated via opioid receptors since they are naloxone-reversible. Although inhibitors of the enkephalin degrading puromycin-insensitive, bestatin-sensitive aminopeptidase (possibly aminopeptidase M) their action is weak (IC50 = 32 microM leucine, 536 microM, glycine) and they might be considered to have a direct antinociceptive effect on opioid receptors. The titled compounds constitute novel 'lead' compounds for the development of potent aminopeptidase M inhibitors.     

Why do males of the dance flyEmpis borealis refuse to mate? The importance of female age and size

Empis borealisfemales form swarms, and males carrying a nuptial gift come to swarms to mate. Males either mated with one of the females (accepted swarms) or left swarms without mating (refused swarms). Males mated with the younger (low wing-wear) and relatively larger females in accepted swarms. They seemed to be able to judge the relative size of the females but to ignore their absolute size. Visiting males stayed shorter in accepted swarms as female size variation increased. This probably reflects their greater ease in choosing a mate among females of relatively different sizes. Females in accepted swarms tended to be larger and to have less worn wings than females in rejected swarms.     

Food limitation and social regulation in a red fox population

This study evaluates a conceptual model on functional and numerical response to short-term fluctuating vole populations of a red fox ( Vulpes vulpes L.) population in south-central Sweden. The model assumes that this particular population is located in between socially regulated stable populations to the south and direct food-limited populations to the north. The model predicts: (1) food availability as the primary factor for limiting fox numbers, causing reduced rates of reproduction and survival during years of low vole densities, and (2) density-dependent regulation during years of increasing and high vole densities resulting in increased group sizes within territories of fixed dimensions. During 1973–1980 data were obtained from 1216 fox scats, 874 fox carcasses, 63 tagged foxes, nine radio-collared females and from yearly den counts in an area of 130 km², Eight predictions of the model were tested. These concerned the occurrence of small rodents in fox diet, fluctuations in the density of foxes, variations in the number of fox litters, the effect on reproduction of providing supplemental food during January–May, the proportion of vixens bearing a litter different years, dispersal of young males relative to that of young females throughout the vole cycle, and variations in mortality rates of young males and females. All tests were in favour of the conceptual model, and contradictory to alternative models.     

The influence of creosote compounds on the aerobic degradation of toluene

The inhibiting effect of 14 typical creosote compounds on the aerobic degradation of toluene was studied in batch experiments. Four NSO-compounds (pyrrole, 1-methylpyrrole, thiophene, and benzofuran) strongly inhibited the degradation of toluene. When the NSO-compounds were present together with toluene, little or no degradation of toluene was observed during 16 days of incubation, compared with a total removal of toluene within 4 days when the four compounds were absent. Indole (an N-compound) and three phenolic compounds (phenol, o-cresol, and 2,4-dimethylphenol) also inhibited the degradation of toluene, though the effect was much weaker that of the four NSO-compounds. O-xylene, p-xylene, naphthalene and 1-methylnaphthalene seemed to stimulate the degradation even though the influence was very weak. No effects of benzothiophene (an S-compound) and quinoline (an N-compound) were observed. Benzofuran (an O-compound) was identified as the compound that most inhibited the degradation of toluene. An effect could be detected even at low concentrations (40 g/l).Abbreviations bf benzofuran - bt benzothiophene - dmp 2,4-dimethylphenol - GC gas chromatograph - ind indole - mnap 1-methylnaphthalene - MAH monoaromatic hydrocarbons - mpyr 1-methylpyrrole - nap naphthalene - o-cre o-cresol - o-xyl o-xylene - phe phenol - pyr pyrrole - p-xyl p-xylene - tol toluene - thi thiophene - qui quinoline     

On possible different mechanisms subserving the release of arachidonic and di-homo-gamma-linolenic acids for the formation of monoenoic and bisenoic prostaglandins in uteri from ovariectomized rats

The uptake of arachidonic acid (AA) and of di-homo-gamma-linolenic acid (DGLA) and their incorporations into phospholipids (PLs) and into neutral lipids (NLs) of uteri isolated from spayed rats and the effect of inhibiting triglyceride (TG) metabolism with 4-pentenoic acid (4-PEA) on tissue TG levels and the output of prostaglandins (PGs), were explored. Attempts were also made to determine whether the acylation of labelled AA and of labelled DGLA into PLs and TGs is different and to confirm possible correlations between the synthesis of PGE1 and the degradation of TGs. Uterine PLs incorporated significantly less DGLA than AA (P less than 0.05). AA was acylated mainly into the phosphatidylinositol (PI) and into phosphatidylcholine (PC) subfractions of rat uteri, whereas the incorporation of DGLA into these two subfractions was significantly smaller than that of AA. The acylation of labelled DGLA into NL fractions, mainly into triacylglycerol, almost doubled that of labelled AA. The levels of TGs in isolated rat uteri suspended in glucose-free medium during a period of 60 minutes were significantly less than immediately after isolation (P less than 0.001). PGE1 released from uteri into the incubating solution, was significantly higher than that of PGE2. Moreover, the presence of 4-PEA (1.0 mM), added after tissue isolation, prevented the decrement of TGs observed following 60 minutes of incubation and simultaneously diminished significantly (P less than 0.001) the enhanced output of PGE1, without altering that of PGE2. Results presented herein suggest that PLs are not normal precursors for the synthesis of PGE1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opsonizing properties of natural antibodies of rainbow trout, Oncorhynchus mykiss (Walbaum)

In order to confirm previous observations in which a protective effect of rainbow trout natural antibodies against furunculosis was suspected, phagocytosis studies wereconducted in vitro , using combinations of rainbow trout sera with high or low levels of natural antibodies and active or inactivated complement as opsonizing factors. Opsonization was observed in all the cases where complement was present, and to a lesser degree with sera containing only natural antibodies. The results confirm the prime importance of the complement system and provide additional evidence for a possible role of natural antibodies in antimicrobial defences.     

Partial Purification and Characterization of the Vacuolar H+-ATPase of Mammalian Synaptic Vesicles

Several major proteins of synaptic vesicles from rat or cow brain sediment as a large complex on sucrose density gradients when solubilized in nonionic detergents. A vacuolar H(+)-ATPase identified by sensitivity to bafilomycin A1 appears to be associated with this oligomeric protein complex. Two subunits of this complex, synaptic vesicle proteins S and U, correspond to the 57-kDa (B) and 39-kDa accessory (Ac39) subunits, respectively, of bovine chromaffin granule vacuolar H(+)-ATPase as shown by Western immunoblot analysis. The five subunits of the oligomeric complex constitute approximately 20% of the total protein of rat brain synaptic vesicles. Taken together, these results strongly suggest that the abundant, multisubunit complex partially purified from brain synaptic vesicles by density gradient centrifugation is a vacuolar H(+)-ATPase. Bafilomycin A1 completely blocks proton pumping in rat brain synaptic vesicles as measured by [14C]methylamine uptake and also blocks catecholamine accumulation measured by [3H]dopamine uptake. Moreover, ATPase activity, [14C]methylamine uptake, and [3H]dopamine uptake are inhibited by bafilomycin A1 at similar I50 values of approximately 1.7 nmol/mg of protein. These findings indicate that the vacuolar H(+)-ATPase is essential for proton pumping as well as catecholamine uptake by mammalian synaptic vesicles.     

Pharmacological Characterization of Somatostatin Receptors in the Rat Cerebellum During Development

Abstract: Somatostatin (SRIF) receptors (SRIF-Rs) are transiently expressed in a germinative lamina of the rat cerebellum, the external granule cell layer. The appearance of SRIF-Rs coincides with the expression of SRIF-like immunoreactivity in the cerebellum. However, the cellular location of SRIF-Rs does not overlap with the distribution of SRIF-like immunoreactivity, with the latter being restricted to ascending fibers arising from the brainstem, to perikarya within the white matter, and to some Purkinje cells. The characterization of SRIF-Rs in the immature (13–day-old) rat cerebellum was conducted by means of binding experiments in membraneenriched preparations and autoradiography, using two radioligands, [¹²⁵I-Tyr⁰,D-Trp⁸]SRIF-14 ([¹²⁵I-Tyr⁰,d -Trp⁸]S14) and ^I25I-SMS 204–090. The pharmacological profile of cerebellar SRIF-Rs was compared with that of adult cortical SRIF-Rs. Saturation studies performed in 13–day-old rat cerebellum showed that the A'_D values for [¹²⁵I-Tyr⁰,D-Trp⁸]S14 and ¹²⁵I-SMS 204–090 binding were 0.35 ± 0.04 and 0.39 ± 0.01 nM, respectively. The corresponding B_max values were 52.7 ± 4.8 and 49.9 ± 5.3 fmol/mg of protein, a result indicating that radioligands with high specific radioactivity (2,000 Ci/mmol) bind to a single class of high-affinity sites (SSI). Competition studies showed that different D-Trp-sub-stituted analogs displaced [¹²⁵I-Tyr⁰,d -Trp⁸]S14 binding with Hill coefficients >1, a finding indicating the existence of different subtypes of binding sites. When [Tyr⁰,d -Trp⁸]S14 was used as a competitor, two sites were resolved by Scatchard analysis in both 13–day-old cerebellum and adult cerebral cortex. The higher-affinity sites correspond to the SSI subtype identified in saturation experiments, whereas the lower-affinity sites most likely correspond to the SS2 subtype. Ionic supplementation studies showed that divalent cations were required to obtain maximal specific binding on the SSI sites. In particular, Mn²⁺ was the most efficient cation for promoting binding of [¹²⁵I-Tyr⁰,d -Trp⁸]S14. Addition of GTP to the incubation buffer induced a marked reduction of specific binding. The results obtained by membrane binding assays were similar to those obtained by quantitative autoradiography, a result indicating that the microenvironment of SRIF-Rs was preserved in both types of tissue preparations. Receptors expressed in the developing rat cerebellum exhibited the same K_D and similar pharmacological profile as those observed in the adult rat cortex. These results show that SRIF-binding sites transiently expressed in the external granule cell layer of the cerebellum of young rats are indistinguishable from adult rat brain SRIF-Rs. The extremely high density of SRIF-Rs found in the external granule cell layer in 13–day-old rats suggests that SRIF may play a pivotal role in the proliferation and/or differentiation of these germinative cells.     

Differences in low pH tolerance among closely related sunfish of the genus Enneacanthus

Synopsis Three closely related sunfish in the genus Enneacanthus were examined to determine if differences existed in their tolerance to low pH that could explain their contrasting distributions. Na fluxes of E. obesus, E. gloriosus, and E. chaetodon were measured during 12 h exposure to pH 4.0 and 3.5 (all species), and 3.25 (former 2 species only). All experienced ionic disturbances upon acid exposure resulting from inhibition of active Na influx and stimulation of passive Na efflux, but E. gloriosus and E. chaetodon experienced greater disturbances than E. obesus at all pH's tested. Body and plasma Na concentrations of E. gloriosus were measured after one week of exposure to a range of pH's for comparison with previously published data from E. obesus. Exposure to pH 4.0 and below caused a depression in body and plasma Na concentration of E. gloriosus, and only two of 10 fish survived the one week test period at pH 3.5; none survived at pH 3.25. In contrast, exposure to pH 4.0 for five weeks had no effect on body Na concentration of E. obesus, all 10 fish survived exposure to pH 3.5 for two weeks. Growth of E. gloriosus and E. obesus were measured separately during 12 weeks of exposure to a range of pH's. E. gloriosus exposed to pH 4.25 and 4.0 grew at a lower rate than those at higher pH's (4.5, 5.0, and 5.8), and body Na concentrations of fish at pH's 4.25 and 4.0 were significantly less than the others. With declining pH E. obesus did not exhibit reduced growth until pH 3.75 was reached; no depression in body Na concentration occurred at this pH. These results show that there are marked differences in low pH tolerance among closely related species of Enneacanthus which could affect their distributions and competitive interactions.     

Induction of rat hepatic N-nitrosodimethylamine demethylase by acetone is due to protein stabilization

The N-nitrosodimethylamine demethylase (P450I-IE1) is induced severalfold in liver by giving rats ethanol, acetone, pyrazole, and other related small molecular weight compounds. This induction is not the result of an increase in IIE1 mRNA, but could be due to either an increase in translation rate or a decrease in protein degradation. To determine the mechanism of induction, we measured IIE1 synthesis and degradation rates in untreated and acetone-treated rats. This was accomplished by immunopurification of radiolabeled IIE1 protein using a specific monoclonal antibody subsequent to in vivo labeling of total cellular protein with either NaH14CO3 or [3H]leucine. We found that in rats fed acetone, the rate of IIE1 synthesis was not changed; however, IIE1 degradation was markedly altered. In untreated rats, IIE1 protein was degraded via a biphasic pathway consisting of both a rapid and slow component with approximate half-lives of 7 and 37 h, respectively. However, in acetone-treated rats, only a monophasic curve with a half-life of 37 h was observed. The abolition of the rapid degradation component of the IIE1 turnover cycle indicates that induction of IIE1 by acetone is primarily due to specific stabilization of IIE1 protein. Since acetone is also metabolized by IIE1, we believe that this may be a substrate-induced enzyme stabilization.