Effects of peptide YY and its analogues on chloride ion secretion in fed and fasted rat jejunum

The aim of our study was to determine whether a meal modifies the antisecretory response induced by PYY and the structural requirements to elicit antisecretory effects of analogue PYY(22–36) for potential antidiarrhea therapy. The variations in short-circuit current (Isc) due to the modification of ionic transport across the rat intestine were assessed in vitro, using Ussing chambers. In fasted rats, PYY induced a dose- and time-dependent reduction in Isc, with a sensitivity threshold at 5 × 10⁻¹¹ M (ΔIsc −2 ± 0.5 μA/cm²). The reduction was maximal at 10⁻⁷ M (Isc −23 ± 2 μA/cm²), and the concentration producing half-maximal inhibition was 10⁻⁹ M. At 10⁻⁷ M, reduction of Isc by PYY reached 90% of response to 5 × 10⁻⁵ M bumetanide. The PYY effect was partly reversed by 10⁻⁵ M forskolin (Isc +13.43 ± 2.91 μA/h·cm², p < 0.05) or 10⁻³ M dibutyryl adenosine 3′,5′ cyclic monophosphate (Isc +12 ± 1.69 μA/cm², p < 0.05). Naloxone and tetrodotoxin did not alter the effect of PYY. In addition, PYY and its analogue P915 reduced net chloride ion secretion to 2.85 and 2.29 μEq/cm² (p < 0.05), respectively. The antisecretory effect of PYY was accompanied by dose- and time-dependent desensitization when jejunum was prestimulated by a lower dose of peptide. The antisecretory potencies exhibited by PYY analogues required both a C-terminal fragment (22–36) and an aromatic amino acid residue (Trp or Phe) at position 27. At 10⁻⁷ M the biological activity of PYY was lower in fed than fasted rats (p < 0.001). Our results confirm the antisecretory effect of PYY, but show that the fed period is accompanied by desensitization, similar to the transient desensitization observed in the fasted period with cumulative doses. This suggests that PYY may act as a physiological mediator that reduces intestinal secretion.     

Effects of ethacrynic acid and furosemide on phosphory-lation reactions of kidney mitochondria. Inhibition of the adenine nucleotide translocase

Previous reports that ethacrynic acid and furosemide diminish mitochondrial P : O ratios and reduce (Na⁺ + K⁺)-ATPase activity suggested that these diuretics may inhibit mitochondrial phosphorylation reactions. This possibility was initially studied by determining the effects of ethacrynic acid and furosemide on [³²P]ATP exchange activity of rat kidney mitochondria. Concentrations of both drugs at 10⁻⁴ M or greater, significantly inhibited [³²P]ATP exchange. To investigate the mechanism of this inhibition, the effects of ethacrynic acid and furosemide on the ATPase activity of intact mitochondria and sonicated submitochondrial particles were determined. Both diuretics inhibited ATPase activity of intact mitochondria at 10⁻⁴ M. In contrast, ATPase of submitochondrial particles was significantly less susceptible to inhibition by the diuretics. These results suggested that ethacrynic acid and furosemide inhibit adenine nucleotide transport across the mitochondrial membrane. This was directly tested by determining the effects of the diuretics on the mitochondrial adenine nucleotide translocase. At 5 · 10⁻⁴ M, both ethacrynic acid and furosemide significantly inhibited adenine nucleotide transport. These findings suggest that ethacrynic acid and furosemide may diminish renal tubular solute reabsorption by direct inhibition of adenine nucleotide transport across the mitochondrial inner membrane.     

Actual and potential dark respiration rates and different electron transport pathways in freshwater aquatic plants

The rates of respiratory O₂ uptake have been studied in leaves, stems and whole shoots of several freshwater plants: 6 angiosperms, 2 bryophytes and one alga. For angiosperm leaves, rates varied widely with species (30–142 μmol O₂ (gDW)⁻¹ h⁻¹), were correlated with chlorophyll content and were higher than those of the stems (13–71 μmol O₂ (gDQ)⁻¹ h⁻¹). The rates for the shoots of bryophytes (53–66 μmol O₂ (gDW)⁻¹ h⁻¹) and for the alga Cladophora glomerata (L.) Kütz. (96 μmol O₂ (gDW)⁻¹ h⁻¹) were slightly higher than those of most angiosperm stems, but lower than those for most leaves.

These plants had a significant cyanide-resistant respiration, suggesting the existence of an alternative pathway to the “classic” cytochrome system. This pathway was found to be active in all the species studied, as judged by responses to a specific inhibitor, SHAM (salicylhydroxamic acid). Measurement of electron-transport system (ETS) activity showed that there is a large electron-transport capacity which is not normally used by respiration in vivo.     

Adenine nucleotide translocation in yeast mitochondria. Effect of inhibitors of mitochondrial biogenesis on the ADP translocase

1. Optimal test conditions for adenine nucleotide translocation in Candida utilis mitochondria are a standard medium, consisting of 0.63 M mannitol, 2 mM EDTA (or ethylene glycol tetraacetic acid, EGTA), 10 mM morpholinopropane sulfonic acid (pH 6.8), and a temperature of 0 °C.

2. Adenine nucleotide translocation in C. utilis mitochondria is an exchange-diffusion process. The whole pool of internal adenine nucleotides is exchangeable, ADP being the most readily exchangeable nucleotide. The rate of mitochondrial ADP exchange, but not the K_m value, depends on growth conditions. At 0 °C, the rate is about 3 to 4 nmoles ADP/min per mg protein for mitochondria obtained from yeast grown in the presence of 1.5% glucose; it rises to 11.5 nmoles when glucose is replaced by 3% ethanol in the growth medium. The K_m value for ADP is 2 μM. The Q₁₀ is about 2 between 0 and 20 °C. Among other exchangeable adenine nucleotides are ATP, dADP and the methylene and the hypophosphate analogues of ADP. Unlike mammalian mitochondria, C. utilis mitochondria are able to transport external UDP by a carboxyatractyloside-sensitive process.

3. Under conditions of oxidative phosphorylation (phosphate and substrate present in an aerated medium), added ADP is exchanged with internal ATP. A higher ATP/ADP ratio was found in the extramitochondrial space than in the intramito-chondrial space. The difference between the calculated phosphate potentials in the two spaces was 0.9–1.7 kcal/mole.

4. Atractyloside, carboxyatractyloside, bongkrekic acid and palmityl-CoA inhibit mitochondrial adenine nucleotide translocation in C. utilis as they do in mammalian mitochondria, but 2 to 4 times less efficiently. The inhibition due to atractyloside or palmityl-CoA is competitive with respect to ADP whereas that due to bongkrekic acid and carboxyatractyloside is non-competitive. Carboxyatractyloside and atractyloside inhibitions are additive. The apparent K_d for the binding of [³⁵S]-carboxyatractyloside and [¹⁴C]bongkrekic acid is 10–15 nM and the concentration of sites 0.4–0.6 nmole/mg protein in both cases. [³⁵S]Carboxyatractyloside binding is competitively displaced by atractyloside and vice versa.

5. Binding of [¹⁴C]ADP has been carried out with mitochondria depleted of their endogenous adenine nucleotides by incubation with phosphate and Mg²⁺ at 20 °C. The amount of bound [¹⁴C]ADP which is atractyloside removable is 0.08–0.16 nmole/mg protein.

6. The rate of ADP transport is quite different in mitochondria isolated from C. utilis, according to whether it is grown on glucose, or on ethanol or in the presence of chloramphenicol; for instance, it decreases by 10 times when 3% ethanol in the growth medium is replaced by 10% glucose and by 5 times when chloramphenicol is added to the medium. These variations are accompanied by parallel variations in cytochrome aa₃. The number of atractyloside-sensitive ADP binding sites is not modified by the above conditions of culture, nor the number of [³⁵S]carboxyatractyloside binding sites. The affinity for ADP is apparently not significantly modified, nor the size of the endogenous adenine nucleotide pool. In contrast to glucose repression or chloramphenicol inhibition, semi-anaerobiosis in C. utilis lowers significantly the mitochondrial binding capacity for carboxyatractyloside. Strict anaerobiosis in S. cerevisiae results in a practical loss of the cytochrome oxidase activity, and also of the carboxyatractyloside and ADP binding capacity. Transition from anaerobiosis to aerobiosis restores the cytochrome oxidase activity and the ADP and carboxyatractyloside binding capacities.     

Action of endogenous steroid inhibitors of brain aromatase relative to fadrozole

To study mechanisms of aromatase inhibition in brain cells, a highly effective non-steroidal aromatase inhibitor (Fadrozole; 4-[5,6,7,8-tetra-hydroimidazo-(1,5-a)-pyridin-5-yl] benzonitrile HCl; CGS 16949A) was compared with endogenous C-19 steroids, known to be formed in the preoptic area, which inhibit oestrogen formation. Using a sensitive in vitro tritiated water assay for aromatase activity in avian (dove) preoptic tissue, the order of potency, with testosterone as substrate was: Fadrozole (K_i < 1 × 10⁻⁹ M) > 4-androstenedione 5-androstanedione > 5-dihydrotestosterone (K_i = 6 × 10⁻⁸ M) > 5β-androstanedione > 5β-dihydrotestosterone (K_i = 3.5 × 10⁻⁷ M) > 5-androstane-3, 17β-diol (K_i = 5 × 10⁻⁶ M) > 5β-androstane-3β,17β-diol. Five other steroids, 5β-androstane-3,17β-diol, 5-androstane-3β,17β-diol, progesterone, oestradiol and oestrone, showed no inhibition at 10⁻⁴ M. The kinetics indicate that endogenous C-19 steroids show similar competitive inhibition of the aromatase as Fadrozole. Mouse (BALB/c) preoptic aromatase was also inhibited by Fadrozole. We conclude that endogenous C-19 metabolites of testosterone are effective inhibitors of the brain aromatase, and suggest that they bind competitively at the same active site as Fadrozole.     

Cytochrome c interaction with the mitochondrial membrane: A spin label study

Horse-heart ferrocytochrome c has been labeled with N-(2,2,5,5-tetramethyl-3-pyrrolidinyl-1-oxyl) iodoacetamide at methionine-65. The paramagnetic resonance spectrum of labeled ferricytochrome c indicates a weak immobilization of the radical (τ_c = 9.3·10⁻¹⁰ sec) which becomes stronger upon binding of labeled cytochrome c to cytochrome c-depleted mitochondrial membranes (τ_c = 3.3·10⁻⁹ sec). The hyperfine coupling constant remains, however, unchanged (16.7 ± 0.1 gauss) indicating that the cytochrome c binding site is highly polar. The region where cytochrome c is bound to the membrane is insensitive to large variations of medium viscosity.     

Reduction to homozygosity is the predominant spontaneous mutational event in cultured human lymphoblastoid cells

Expression of recessive mutant phenotypes can occur by a number of different mechanisms. Inactivation of the wild-type allele by base-substitution mutations, frameshift mutations or small deletions occurs at both hemizygous and heterozygous cellular loci, while other events, such as chromosome level rearrangements, may not be detected at hemizygous loci because of inviabiltty of the resulting mutants. In order to assess the relative contribution of each type of mutational event, we isolated a human lymphoblastoid cell line that is heterozygous at the adenine phosphoribosyltransgerase (aprt) locus. The mutation rate for the expression of the mutant phenotype (aprt^+/−→aprt^−/−) was 1.3 × 10⁻⁵/cell/ generation. Molecular analysis of the DNA from 26 mutant clones revealed that 19% had undergone deletion of the entire wild-type allele. The aprt heterozygote carries a mutation in the coding sequence of the gene that results in the loss of a restriction site. Analysis of aprt^−/− mutants for this restriction fragment length difference reveales that 23% of the mutants contained point mutations or small ((< 100 bp) deletions. The remainder of the mutants (58%) resulted from reduction to homozygosity of the mutant allele. We suggest that, as in tumor cells in vivo, reduction to homozygosity is a major mechanism for the expression of recessive mutant phenotypes in cultured human cells.     

Thermodynamics of the disproportionation of adenosine 5'-diphosphate to adenosine 5'-triphosphate and adenosine 5'-monophosphate, II. Experimental data

High-pressure liquid-chromatography and microcalorimetry have been used to determine equilibrium constants and enthalpies of reaction for the disproportionation reaction of adenosine 5′-diphosphate (ADP) to adenosine 5′-triphosphate (ATP) andadenosine 5′-monophosphate (AMP). Adenylate kinase was used to catalyze this reaction. The measurements were carried out over the temperature range 286 to 311 K, at ionic strengths varying from 0.06 to 0.33 mol kg⁻¹, over the pH range 6.04 to 8.87, and over the pMg range 2.22 to 7.16, where pMg = -log a(Mg²⁺). The equilibrium model developed by Goldberg and Tewari (see the previous paper in this issue) was used for the analysis of the measurements. Thus, for the reference reaction: 2 ADp³⁻ (ao) AMp²⁻ (ao)+ ATp⁻ (ao), K° = 0.225 ± 0.010, ΔG° = 3.70 +- 0.11 kJ mol ⁻¹, ΔH° = −1.5 ± 1. 5 kJ mol ⁻¹, °S ° = −17 ± 5 J mol⁻¹ K⁻¹, and ACP_p°≈ = −46 J mo1l⁻¹ K⁻¹ at 298.15 K and 0.1 MPa. These results and the thermodynamic parameters for the auxiliary equilibria in solution have been used to model the thermodynamics of the disproportionation reaction over a wide range of temperature, pH, ionic strength, and magnesium ion morality. Under approximately physiological conditions (311.15 K, pH 6.94, [Mg²⁺] = 1.35 × 10⁻³ mol kg⁻¹, and I = 0.23 mol kg⁻¹) the apparent equilibrium constant (K_A′ = m(ΣAMP)m(ΣATP)/[ m(ΣADP)]²) for the overall disproportionation reaction is equal to 0.93 ± 0.02. Thermodynamic data on the disproportionation reaction and literature values for this apparent equilibrium constant in human red blood cells are used to calculate a morality of 1.94 × 10⁻⁴ mol kg⁻¹ for free magnesium ion in human red blood cells. The results are also discussed in relation to thermochemical cycles and compared with data on the hydrolysis of the guanosine phosphates.     

Zooplankton feeding ecology: bacterivory by metazoan microzooplankton

Bacterivory by the rotifer Brachionus plicatilis Müller, nauplii and copepodites of the copepods Centropages Krøyer sp. and Acartia tonsa Dana, and the tintinnid Favella panamensis Kofoid & Campbell was examined using fluorescently labelled bacteria (FLB) and epifluorescence microscopy. FLB were < 1 μm in diameter, and were offered at environmental concentrations (1.47−9.08 × 10⁶ cells·ml⁻¹). FLB were visible within rotifers, nauplii, copepodites, and tintinnids, confirming ingestion. Rotifer clearance rates (32–418 μl·animal⁻¹·h⁻¹) exhibited no relation with FLB concentration. In some cases rates of clearance of FLB by rotifers were different with alternative phytoplankton food (Nanochloris Naumann sp.) than in replicates with FLB alone, whereas in other cases presence of alternative food exhibited no clear effects on rates of ingestion of FLB. Clearance rates for all six naupliar stages of A. tonsa nauplii (0–320 μl·animal⁻¹·h⁻¹) were stage-related, with higher rates by NIII-VI nauplii than NI-II nauplii. Nauplii had higher rates of clearance of FLB in the absence of alternative phytoplankton food (Isochrysis Parke sp.). Clearance rates of FLB by a single stage of Centropages sp. nauplii, A. tonsa CI copepodites and F. panamensis (each obtained at only a single food concentration of either 1.5 or 5.0 × 10⁶ cells·ml⁻¹) were within the range of 85–142 μl·animal⁻¹·h⁻¹. These ranges were similar to those of rotifers and A. tonsa nauplii. This is the first report of FLB ingestion by metazoan marine microzooplankton. Although rotifers and ciliates might be expected to ingest small particles such as FLB using ciliary induced feeding currents, the means by which nauplii and copepodites eat FLB is less clear. We propose that they may “eat” bacteria as they “drink” to osmoregulate.     

Physiological and genetic modifications of the expression of the yeast mitochondrial adenosine triphosphatase inhibitor

1. The oligomycin-sensitive ATPase activity of submitochondrial particles of the glycerol-grown “petite-negative” yeast: Schizosaccharomyces pombe is markedly stimulated by incubation at 40°C and by trypsin activations are treatment. Both increased in Triton-X 100 extracts of the submitochondrial particles.

2. A trypsin-sensitive inhibitory factor of mitochondrial ATPase with properties similar to that of beef heart has been extracted and purified from glycerolgrown and glucose-grown S. pombe wild type, from the nuclear pleiotropic respiratory-deficient mutant S. pombe M126 and from Saccharomyces cerevisiae.

3. ATPase activation by heat is more pronounced in submitochondrial particles isolated from glycerol-grown than from glucose-grown S. pombe. An activation of lower extent is observed in rat liver mitochondrial particles but is barely detectable in the “petite-positive” yeast: S. cerevisiae. No activation but inhibition by heat is observed in the pleitotropic respiratory-deficient nuclear mutant S. pombe M126.

4. The inhibition of S. pombe ATPase activity by low concentrations of dicyclohexylcarbodiimide dissapears at inhibitor concentrations above 25 μM. In Triton-extract of submitochondrial particles net stimulation of ATPase activity is observed at 100 μM dicyclohexylcarbodiimide. The pattern of stimulation of ATPase activity by dicyclohexylcarbodiimide in different genetic and physiological conditions parallels that produced by heat and trypsin. A similar mode of action is therefore proposed for the three agents: dissociation or inactivation of an ATPase inhibitory factor.

5. We conclude that “petite-positive” and “petite-negative” yeasts contain an ATPase inhibitor factor with properties similar to those of the bovine mitochondrial ATPase inhibitor. The expression of the ATPase inhibitor, measured by ATPase activation by heat, trypsin or high concentrations of dicyclohexylcarbodiimide, is sensitive to alterations of the hydrophobic membrane environment and dependent on both physiological state and genetic conditions of the yeast cells.     

Production d'NH4 par la crevette Crangon crangon L. dans deux écosystémes côtiers. approche expérimentale et étude de l'influence du sédiment sur le taux d'excrétion

Estimation of the ammonia production of the shrimp C. crangon in two littoral ecosystems (oligotrophic sand and eutrophic mud) was determined in winter and summer conditions from laboratory observations in experimental microcosms. The ammonia excretion rate of C. crangon was not influenced by either the sediment type or the ammonia concentration of the overlying water; on the other hand, the mean excretion rate and the response to initial handling stress increased markedly as shrimp were deprived of soft substratum.

The daily ammonia production of C. crangon was 16 μmol NH₃ · g ⁻¹ wet wt · day ⁻¹ in winter and 40 μmol in summer. A gross production of 12 μmol NH₃ · m⁻² · day ⁻¹ and 300–700 μmol μ m⁻² · day⁻¹, respectively, could be expected in the two ecosystems studied. This would account for 5% (winter) and 2–4% (summer) of the total NH⁺₄ flux at the sediment-water interface. The contribution of the excretion of all macrofauna to the NH⁺₄ flux from the sediment is discussed.     

Bradykinin dilates rat middle cerebral artery and its large branches via endothelial B2 receptors and release of nitric oxide

Ring segments of rat middle cerebral artery (MCA) were prepared for measurement of isometric force and precontracted with 10⁻⁴ M uridine triphosphate (UTP). Concentration-effect curves (CEC) were constructed for bradykinin (BK, 10⁻⁸–10⁻⁵ M) in segments with functionally intect (E+) or denuded (E−) endothelium. E− segments did not dilate to BK. The BK receptor was characterized by application of specific B₁ or B₂ antagonists [des-Arg⁹-Leu⁸] BK (10⁻⁵ M) and [ -Arg⁰-Hyp³-Thi⁵- -Tic⁷-Oic⁸] BK (HOE140,3 × 10⁻⁷ M), respectively, or B₁ agonist [des-Arg⁹] BK (10⁻⁸–10⁻⁴ M). Involvement of nitric oxide (NO) was tested with N^G-nitro- -arginine (LNNA, 10⁻⁴ M). BK induced concentration-dependent relaxation with a maximal effect (E_max) of 40.86 ± 1.50% at 10⁻⁶ M and a pD₂ (−log₁₀ EC₅₀) of 6.818 ± 0.044. This relaxation could be prevented with HOE140 or LNNA, but was not influenced by [des-Arg⁹-Leu⁸] BK. [des-Arg⁹] BK did not induce any effect. These results demonstrate that BK induced relaxation via endothelial B₂ receptors and release of NO in isolated rat MCA.     

Evaluation of chromosome painting to assess the induction and persistence of chromosome aberrations in bone marrow cells of mice treated with benzene

Intact pZ189 DNA was allowed to replicate in FL-FEN-1⁻ cell line that was established in this laboratory in which the expression of FEN-1 gene was blocked by dexamethasone-inducible expression of antisense RNA to FEN-1. E. coli MBM7070 was transfected with the replicated plasmid, and those with mutations in the supF gene were identified. The frequency of mutants that did not contain recognizable changes in the electrophoretic mobility of the plasmid DNA was scored. The frequency of such mutants was 19.1 × 10⁻⁴ (34/17781), significantly higher than those of 2.9 × 10⁻⁴ (4/13668) and 3.0 × 10⁻⁴ (3/9857) in the corresponding controls, respectively. Sequence analysis of the supF genes of these mutants showed that all (37/37) the base substitutions occurred at C:G base pairs; 68% (23/37) of the base substitutions were base transversions, while 32% (12/37) were transitions. Approximately 76% (23/37) of these base substitutions occurred frequently at nine positions; two of these sites contain triple pyrimidine (T or C) repeat upstream to the mutated base; four of these sites consist of 5′-TTN₁N₂ and mutations occurred at N₁ site sequence; another two sites have the characteristics of triple A flanked at both 5′ and 3′ side by TCT, with the base substitution occurring at C in the context sequence. These data suggested that these sites are the hot spot of mutagenesis in plasmid replicated in FEN-1-deficient cells. Besides the mutator phenotype of the FEN-1-deficient cell, it was also demonstrated that FEN-1-deficient cell exhibited an increased N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) sensitive phenotype.     

Quantification of illegitimate V(D)J recombinase-mediated mutations in lymphocytes of newborns and adults

We used a direct polymerase chain reaction (PCR) method for quantification of HPRT exons 2+3 deletions and t(14;18) translocations as a measure of illegitimate V(D)J recombination. We determined the baseline frequencies of these two mutations in mononuclear leukocyte DNA from the umbilical cord blood of newborns and from the peripheral blood of adults. In an initial group of 21 newborns, no t(14;18) translocations were detected (<0.049×10⁻⁷). The frequency of HPRT exons 2+3 deletions was 0.10×10⁻⁷ per mononuclear leukocyte, lower than expected based on the T-cell proportion of this cell fraction (55%–70%) and previous results using the T-cell cloning assay (2–3×10⁻⁷ per clonable T-cell). Phytohemagglutinin (PHA), as used in the T-cell cloning assay, was examined for its effect on the frequencies of these mutation events in mononuclear leukocytes from an additional 11 newborns and from 12 adults. There was no significant effect of PHA on t(14;18) translocations which were rare among the newborns (1 detected among 2.7×10⁸ leukocytes analyzed), and which occurred at frequencies from <1×10⁻⁷ (undetected) to 1.6×10⁻⁴ among the adults. The extremely high frequencies of t(14;18)-bearing cells in three adults were due mainly to in vivo expansion of two to six clones. However, PHA appeared to stimulate a modest (although not significant) increase in the frequency of HPRT exons 2+3 deletions in the leukocytes of the newborns, from 0.07×10⁻⁷ to 0.23×10⁻⁷. We show that both the direct PCR assay and the T-cell cloning assay detect similar frequencies of HPRT exons 2+3 deletions when calculations are normalized to blood volume, indicating that the apparent discrepancy is probably due to the different population of cells used in the assays. This direct PCR assay may have utility in characterizing the effects of environmental genotoxic agents on this clinically important recombination mechanism.     

Inhibition of antennal esterases of the egyptian armyworm Spodoptera littoralis by trifluoromethyl ketones

A series of aliphatic and aromatic trifluoromethyl ketones has been tested as inhibitors of the antennal esterases of the Egyptian armyworm Spodoptera littoralis, by evaluation of the extent of hydrolysis of [1-³H]-(Z,E)-9, 11-tetradecadienyl acetate (1), a tritiated analog of the major component of the sex pheromone. The most active compounds with a long chain aliphatic structure were 3-octylthio-1,1,1-trifluoropropan-2-one (2) (IC₅₀ 0.55 μM) and 1,1,1-trifluorotetradecan-2-one (4) (IC₅₀ 1.16 μM). The aromatic compounds were generally less potent inhbitors than the coressponding aromatic ones, although β-naphthyltrifuloromethyl ketone (10) exhibited a remarkable inhibitory activity (IC₅₀ 7.9 μM). Compounds 2, 4 and 10 exhibit a competitive inhibition with K_i values of 2.51×10⁻⁵ M, 2.98×10⁻⁵ M and 2.49×10⁻⁴ M, respectively. Some of the trifluoromethyl ketones tested were slow-binding inhibitors and compounds 2 and 10 are described as inhibitors of the antennal esterases of a moth for the first time.     

Cortisol and testosterone: Inhibitory agents of the mineralocorticoid pathway. Is there a physiopathological meaning in adrenal tumors?

The authors incubated adrenal mitochondria to study the in vitro action of cortisol and testosterone on the transformation of corticosterone and 18-hydroxycorticosterone into aldosterone. The results show that cortisol at concentrations of 5 × 10⁻⁶ and 10⁻⁴ M inhibit the conversion of corticosterone into aldosterone by 23.6 to 90%; testosterone 5 × 10⁻⁵ and 10⁻⁴ M inhibit the reaction by 78.4 and 87.2%, respectively. The inhibition of the conversion of 18-hydroxycorticosterone into aldosterone is 12.5 to 91% by cortisol with concentrations ranging from 5 × 10⁻⁷ to 5 × 10⁻⁵ M and testosterone 5 × 10⁻⁵ and 10⁻⁴ M inhibits the reaction by 87.3 and 91%, respectively. Aldosterone (10⁻⁸ and 10⁻⁶ M) does not inhibit aldosterone biosynthesis from corticosterone or 18-hydroxycorticosterone. It thus appears that cortisol and testosterone have an effect on the aldosterone biosynthesis pathways in mitochondria. This action may be located at the binding site of the cytochrome P450 11β, which catalyzes all hydroxylation steps in the mineralocorticoid biosynthesis pathway. Because cortisol and testosterone may interfere with aldosterone biosynthesis, and since functional zonation is expected in adrenal carcinomas, the presence of these steroids in substantial amounts could explain the very low plasma aldosterone level usually observed, in adrenal carcinomas studies in our laboratory.     

Fluorescence yield kinetics in the microsecond-range in Chlorella pyrenoidosa and spinach chloroplasts in the presence of hydroxylamine

The kinetics of fluorescence yield inChlorella pyrenoidosa and spinach chloroplasts were studied in the time range of 0.5 μs to several hundreds of microseconds in the presence of hydroxylamine. Fluorescence was excited with a just-saturating xenon flash with a halfwidth of 13 μs (λ = 420 nm). The fast rise of the fluorescence yield which was limited by the rate of light influx, was, in the presence of 10⁻³–10⁻² M hydroxylamine, replaced by a slow component which had a half risetime of 25 μs in essence independent of light intensity. This slow fluorescence yield increase reflects a dark reaction on the watersplitting side of Photosystem II. Simultaneous oxygen evolution measurements suggested that a fast fluorescence component is only present in organisms with intact O₂-evolving system, whereas a slow rise predominantly occurs in organisms with the watersplitting system irreversibly inhibited by hydroxylamine.

The results can be explained by the following hypotheses: (a) The primary donor of Photosystem II in its oxidized state, P⁺, is a fluorescence quencher. (b) Hydroxylamine prevents the secondary electron donor Z from reducing the oxidized reaction center pigment P⁺ rapidly. This inhibition is dependent on hydroxylamine concentration and is complete at a concentration of 10⁻² M. (c) A second donor (not transporting electrons from water) transfers electrons to P⁺ with a half time of roughly 25 μs.     

1-Hydroxy monocyclic carotenoid 3,4-dehydrogenase from a marine bacterium that produces myxol

A crtD (1-HO carotenoid 3,4-dehydrogenase gene) homolog from marine bacterium strain P99-3 included in the gene cluster for the biosynthesis of myxol (3^′,4^′-didehydro-1^′,2^′-dihydro-β,ψ-carotene-3,1^′,2^′-triol) was functionally identified. The P99-3 CrtD was phylogenetically distant from the other CrtDs. A catalytic feature was its high activity for the monocyclic carotenoid conversion: 1^′-HO-torulene (3^′,4^′-didehydro-1^′,2^′-dihydro-β,ψ-caroten-1^′-ol) was prominently formed from 1^′-HO-γ-carotene (1^′,2^′-dihydro-β,ψ-caroten-1^′-ol) in Escherichia coli with P99-3 CrtD, indicating that this enzyme has been highly adapted to myxol biosynthesis. This unique type of crtD is a valuable tool for obtaining 1^′-HO-3^′,4^′-didehydro monocyclic carotenoids in a heterologous carotenoid production system.     

Crystal and microparticle effects on MDCK cell superoxide production: oxalate-specific mitochondrial membrane potential changes

We have previously shown that crystals of calcium oxalate (COM) elicit a superoxide (O₂⁻) response from mitochondria. We have now investigated: (i) if other microparticles can elicit the same response, (ii) if processing of crystals is involved, and (iii) at what level of mitochondrial function oxalate acts. O₂⁻ was measured in digitonin-permeabilized MDCK cells by lucigenin (10 μM) chemiluminescence. [¹⁴C]-COM dissociation was examined with or without EDTA and employing alternative chelators. Whereas mitochondrial O₂⁻ in COM-treated cells was three- to fourfold enhanced compared to controls, other particulates (uric acid, zymosan, and latex beads) either did not increase O₂⁻ or were much less effective (hydroxyapatite +50%, p < 0.01), with all at 28 μg/cm². Free oxalate (750 μM), at the level released from COM with EDTA (1 mM), increased O₂⁻ (+50%, p < 0.01). Omitting EDTA abrogated this signal, which was restored completely by EGTA and partially by ascorbate, but not by desferrioxamine or citrate. Omission of phosphate abrogated O₂⁻, implicating phosphate-dependent mitochondrial dicarboxylate transport. COM caused a time-related increase in the mitochondrial membrane potential (Δψ_m) measured using TMRM fluorescence and confocal microscopy. Application of COM to Fura 2-loaded cells induced rapid, large-amplitude cytosolic Ca²⁺ transients, which were inhibited by thapsigargin, indicating that COM induces release of Ca²⁺ from internal stores. Thus, COM-induced mitochondrial O₂⁻ requires the release of free oxalate and contributes to a synergistic response. Intracellular dissociation of COM and the mitochondrial dicarboxylate transporter are important in O₂⁻ production, which is probably regulated by Δψ_m.     

Glutathione-proline activation of feeding behavior in the zoanthid Palythoa psammophilia Walsh & Bowers

Palythoa psammophilia Walsh & Bowers has a well coordinated, stereotyped feeding response, the culminating step of which is ingestion; this may be elicited by the synergistic effect of the tripeptide glutathione and the -imino acid, proline. Either activator acting separately causes responses only at high concentrations (above 10⁻⁵ M for glutathione; above 10⁻⁴ M for proline) in a reduced number of animals and at a low rate (5.00 ± 1.73 min in 5 × 10⁻³ M solutions of glutathione; 11.10±3.74 min in 5 × 10⁻³ M solutions of proline). Highest percentages of response were obtained in combinations where glutathione was at a concentration of 5 × 10⁻³ M and proline at 5 × 10⁻⁴ M or in combinations of glutathione at concentrations 5 × 10⁻⁶ M and proline at 5 × 10⁻⁵ M. The speed of ingestion is considerably enhanced when these activators are combined (1.17±1.18 min).