Determination by HPLC fluorescence analysis of the natural enantiomers of sex pheromones in the New World screwworm fly, Cochliomyia hominivorax

Abstract.  Bioassays of six racemic synthesized candidate sex pheromone compounds against male New World screwworm Cochliomyia hominivorax (Coquerel) flies showed that the most potent bioactivity was found with 6-acetoxy-19-methylnonacosane and 7-acetoxy-15-methylnonacosane compared with four other isomeric acetoxy nonacosanes and a larger aliphatic ketone. As all these methyl-branched compounds have two asymmetric carbons and four possible enantiomers, characterization of the natural enantiomers was essential. All four enantiomers for the two most bioactive isomers of the natural sex pheromone were synthesized for bioassay. Hydrolysis and derivatization of these enantiomers with different fluorescent reagents was followed by column-switched high-performance liquid chromatography. The use of two linked, reversed-phase columns of different polarity held at sub-ambient temperatures allowed good separation of each enantiomer. This analysis applied to natural material was successful, as (6 R ,19 R )-6-acetoxy-19-methylnonanocosane, and (7 R ,15 R )- and (7 R ,15 S )-7-acetoxy-15-methylnonanocosane were detected in extracts of recently colonized female flies.     

Syntheses of four methyl-branched secondary acetates and a methyl-branched ketone as possible candidates for the female pheromone of the screwworm fly,Cochliomyia hominivorax

6-Acetoxy-19-methylnonacosane (1), 7-acetoxy-19-methylnonacosane (2), 8-acetoxy-19-methylnonacosane (3), 7-acetoxy-15-methylnonacosane (4), and 21-methyl-7-hentriacontanone (5) were synthesized as racemic and diastereomeric mixtures. These are new compounds isolated from an HPLC fraction of the female hexane extract which elicited mating responses in the male screwworm fly, Cochliomyia hominivorax.     

Syntheses of Four Methyl-branched Secondary Acetates and a Methyl-branched Ketone as Possible Candidates for the Female Pheromone of the Screwworm Fly,Cochliomyia hominivorax

6-Acetoxy-19-methylnonacosane (1), 7-acetoxy-19-methylnonacosane (2), 8-acetoxy-19-methylnonacosane (3), 7-acetoxy-15-methylnonacosane (4), and 21-methyl-7-hentriacontanone (5) were synthesized as racemic and diastereomeric mixtures. These are new compounds isolated from an HPLC fraction of the female hexane extract which elicited mating responses in the male screwworm fly, Cochliomyia hominivorax.     

Synthesis and Antimalarial Activity of Some New 1,2-Dioxolane Derivatives

The synthesis of 1,2-dioxolane derivatives in two different acetophenone series, as simplified models of natural coumarins is described. 2-Acetyl-3-acetoxy-4-(3-hydroperoxy-3-methylbut-1-enyl)phenyl acetate and 2-acetyl-5-acetoxy-4-(3-hydroperoxy-3-methylbut-1-enyl) phenyl acetate synthons are used as precursors to these structures. In vitro antimalarial activity of the 1,2-dioxolane derivatives has been investigated.     

Female mate choice in relation to heterozygosity in Tribolium castaneum

Abstract Female mate choice, both before and after copulation, is pervasive among insect species. It is often hypothesized that females would preferentially mate with males that are genetically dissimilar to promote the genetic variability of the offspring. We used various strains of red flour beetle, Tribolium castaneum, and tested the effect of male and female genetic backgrounds on precopulatory and post‐copulatory female mate choice. Simultaneous mate choice experiments using previously well established pheromone assays did not detect female preference for males of different strains. Post‐copulatory female mate choice was examined through paternity analysis. Two parameters were used to measure post‐copulatory female mate choice, including male defence capacity (P₁, proportion of offspring sired by the first male when a female mated with two males consecutively) and offence capacity (P₂, proportion of offspring sired by the second of two males to mate with a female). When female and male beetle strains were same, defence capacity was significantly higher than when female and male strains were different. However, such a pattern was not observed for offence capacity. The results suggest that female precopulatory mate choice is not affected by genetic background, but the outcome of post‐copulatory processes depends on the genetic background of male and female beetles.     

Mutagenic and carcinogenic actions of some polycyclic aromatic hydrocarbons]

The carcinogenic and mutagenic actions of benz(a)pyrene (BP) and its derivatives 6-methyl-, 6-formyl-, 6-chloro-, 6-hydroxy-, 6-acetoxy-, 6-methoxy- and 4(5)-methoxy-BP were studied in mice and bacteria Salmonella typhimurium TA-98 and TA-100, respectively. The potent carcinogenic agents BP, 6-methyl-, 6-formyl-BP proved also mutagenic in bacteria of both strains. Weak carcinogenic compounds (6-chloro-, 6-methoxy-, 4(5)-methoxy-BP) and noncarcinogenic ones (6-acetoxy-, 6-hydroxy-BP) either turned out nonmutagenic or mutagenic in one of two bacterial strains tested. The differences in carcinogenic and mutagenic actions of the substances under study are not relative to the rate of their oxidation in the enzymatic system.     

Synthesis of the enantiomers of 21-methyl-7-hentriacontanone and a stereoisomeric mixture of 5-acetoxy-19-methylnonacosane, candidates for the female sex pheromone of the screwworm fly Cochliomyia hominivorax

The enantiomers of 21-methyl-7-hentriacontanone (1), which might show weak bioactivity as the female sex pheromone of the screwworm fly (Cochliomyia hominivorax), were synthesized by starting from the enantiomers of citronellal. (+/-)-Citronellol was converted to a racemic and diastereomeric mixture of 5-acetoxy-19-methylnonacosane (2), which was considered to be a candidate for the female sex pheromone of C. hominivorax. Synthetic 2 exhibited no pheromone activity against male C. hominivorax.     

Synthesis of the four stereoisomers of 7-acetoxy-15-methylnonacosane, a component of the female sex pheromone of the screwworm fly, Cochliomyia hominivorax

The four stereoisomers of 7-acetoxy-15-methylnonacosane (1), a component of the female sex pheromone of the New World screwworm fly (Cochliomyia hominivorax) were synthesized. The stereogenic center at C-15 of 1 originated from that of the enantiomers of citronellal, and that at C-7 was generated by lipase-catalyzed asymmetric acetylation of (3RS,11R)- and (3RS,11S)-17-methyl-1-trimethylsilylpentacos-1-yn-3-ol (13). Three of the stereoisomers of 1 showed equivalent good pheromone activity, while the activity of (7R,15R)-1 was weak.     

Two farnesol derivatives from Cousinia adenostica

The aerial parts of Cousinia adenostica afforded, in addition to widespread compounds, cynaropicrin, chlorojanerin and janerin as well as two new farnesol derivatives, 5,8,12-trihydroxyfarnesol and 12-acetoxy-5,8-dihydroxyfarnesyl acetate.     

Synthesis and antimalarial activity of some new 1,2-dioxolane derivatives

The synthesis of 1,2-dioxolane derivatives in two different acetophenone series, as simplified models of natural coumarins is described. 2-Acetyl-3-acetoxy-4-(3-hydroperoxy-3-methylbut-1-enyl)phenyl acetate and 2-acetyl-5-acetoxy4-(3-hydroperoxy-3-methylbut-1-enyl) phenyl acetate synthons are used as precursors to these structures. In vitro antimalarial activity of the 1,2-dioxolane derivatives has been investigated.     

Oxidation of Acetate by Various Strains of Bacillus popilliae

A number of strains of Bacillus popilliae were examined for their ability to oxidize acetate. Some of these would not sporulate in vitro, and some were oligosporogenous. The ability to oxidize acetate varied widely among the strains tested. A culture derived from spores of the parent strain produced in vivo and one of the asporogenous strains derived from it failed to produce any significant levels of (14)CO(2) from [(14)C]acetate. Oligosporogenous strains derived from the same parent culture all produced (14)CO(2) from both [1-(14)C] and [2-(14)C]acetate but at relatively low rates. The highest rates of acetate oxidation were observed with three strains which did not produce spores in vitro. When cultured under appropriate conditions, one of these strains displayed a secondary growth response concomitant with a decrease in the titratable acidity and an increase in the pH of the medium. The data indicate that B. popilliae has a complete citric acid cycle but that the activity of the cycle is strongly repressed in wild-type strains under the usual conditions used for in vitro cultivation.     

The hydrolytic activity of esterases in the yeast Saccharomyces cerevisiae is strain dependent

Ester precursors of fluorogenic or chromogenic probes are often employed in studies of yeast cell biology. This study was aimed at a comparison of the ability of several commonly used laboratory wild-type Saccharomyces cerevisiae strains to hydrolyse the following model esters: fluorescein diacetate, 2-naphthyl acetate, PNPA (p-nitrophenyl acetate) and AMQI (7-acetoxy-1-methylquinolinum iodide). In all the strains, the esterase activity was localized mainly to the cytosol. Considerable differences in esterase activity were observed between various wild-type laboratory yeast strains. The phase of growth also contributed to the variation in esterase activity of the yeast. This diversity implies the need for caution in using intracellularly hydrolysed probes for a comparison of yeast strains with various genetic backgrounds.     

Antifungal and antimicrobial activity of beta-ionone and vitamin A derivatives

The antifungal and antimicrobic activity of some derivatives of beta-ionon and vitamin A was studied. These compounds (citral, pseudo-ionon. beta-ionon aldehyde C14, ketone C18 and its derivatives--4,18-diketone, alcohol C18, semicarbazide ketone C18), as well as vitamin A and its derivatives--retinal, acetate, retinoic acid--differ in composition, structure and substituents of C-atoms in beta-ionon ring and in polyenoic chain. Fusarium solani, Botrytis cenerea and Verticillum dahliae II, race 447 were used as test organisms when studying the antifungal activity. When studying the antimicrobic activity, ketone C18 and alcohol C18 were tested using the museum strains Staphylococcus aureus 209 P, hemolytic Streptococcus pyogenes FF-38, Streptococcus sp. TOM-1606, Micrococcus luteus 2665, as well as the strains Staphylococcus aureus isolated from pyoderma patients. The antifungal activity was determined by inhibition of spore germination, and the antimicrobic activity--by the value of areas of growth inhibition of the test organisms on the agar medium. All the compounds mentioned above possessed the antifungal activity against all the phytopathogenes used. The degree of this activity depends on the composition and structure of both the cyclic part and polyenoic chain. on the number of conjugated unsaturations, substituents and end groups of C-atom. Ketone C18 is the most active one. It inhibits spore germination by 100-94% at concentrations ranging from 0.05 to 0.005% and 24 h exposure, and by 100% at the concentration of 0.1% and 72 h exposure. Alcohol C18 possesses almost the same antifungul activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coordinate induction of dissimilatory ammonification and fermentative pathways in rhizobia

Dissimilatory ammonification was indicated as the common feature of ten rhizobial strains representing six species and three genera. In the absence of external electron acceptors, all investigated strains were capable of ethanolic fermentation. However, induction of anaerobic nitrite reduction was shown to be coupled with a shift of fermentation towards acetate in all the strains tested. Three metabolic groups could be distinguished with regard to nitrite regulation of ethanolic fermentation. It was shown for Bradyrhizobium sp. strain USDA 3045 that nitrite is the signal for switching between fermentative pathways although both ammonia and acetate excretion could not accelerate until nitrate had been utilized first. In the absence of N oxyanions, ethanol was indicated as the main product of mannitol fermentation, five-fold more abundant than acetate. An inverse composition was found in nitrite-amended cultures, due to a four-fold increase in acetate excretion whereas ethanol was kept at low level. Nitrite-supported fermentation towards acetate has not been previously reported for rhizobia. This benefit of this pathway was a two-fold shorter doubling time on 1% mannitol and 2.5 mM nitrite compared to no-nitrite media variants but also enabled fermentation of the more reduced carbon compound glycerol.     

Biotransformation of acetophenone and its halogen derivatives by Yarrowia lipolytica strains

The ability of 16 strains of Yarrowia lipolytica to biotransform acetophenone and its derivatives has been studied. Thirteen of these strains were derived from a wild-type strain Y. lipolytica A-101; six had the invertase gene (SUC2) from Saccharomyces cerevisiae integrated into their genome, as well as the damaged or undamaged gene encoding orotidine-5′-phosphate decarboxylase (URA3), three had integrated the damaged URA3 gene into their genome and three were UV acetate-negative mutants, not able to growth on acetate as the sole carbon source. The other tested strains included two wild strains, A-101 and PMR-1, and an adenine auxotroph ATCC 32-338A. All strains were capable of reducing acetophenone to the R-alcohol in high enantiomeric excess (80–89 %). In all of the cultures tested, reversibility of the reduction was observed, which led to an increase in the enantiomeric excess. nantioselective reduction of the acetophenone halogen derivatives revealed that the nature and location of the halogen atom had a significant influence on the enantioselectivity of the reduction. In the culture of ATCC 32-338A, after a 3-day biotransformation of 2,4′-dibromoacetophenone the enantiopure R-alcohol was obtained at a rate of 100 % of substrate conversion. In conclusion, using these invertase-containing strains or uracyl auxotrophs provided no additional benefit in terms of biotransformation capacity over the parental strain.     

1'S-1'-Acetoxychavicol acetate as a new type inhibitor of interferon-beta production in lipopolysaccharide-activated mouse peritoneal macrophages

1'S-1'-Acetoxychavicol acetate from the rhizomes of Alpinia galanga was known to show potent inhibitory effect on the production of nitric oxide (NO) in lipopolysaccharide-activated mouse peritoneal macrophages. To clarify its mechanism of action, the effects of 1'S-1'-acetoxychavicol acetate on the expression of interferon-beta (IFN-beta) mRNA and activation of nuclear factor-kappaB (NF-kappaB), both of which participate in the induction of inducible NO synthase, were examined in lipopolysaccharide-activated macrophages. The results were compared with those of two inhibitors of the NF-kappaB activation, costunolide and caffeic acid phenethyl ester. 1'S-1'-Acetoxychavicol acetate inhibited IFN-beta mRNA expression as well as NF-kappaB activation, and two related compounds, (+/-)-1-acetoxy-1-(2-acetoxyphenyl)-2-propene and (+/-)-1-acetoxy-1-(4-acetoxyphenol)-3-butene, also inhibited IFN-beta mRNA expression. In addition, 1'S-1'-acetoxychavicol acetate inhibited the production of NO stimulated by poly(I:C) via Toll-like receptor 3.     

Selective isolation of Acetobacterium woodii on methoxylated aromatic acids and determination of growth yields

Anaerobic enrichments with methoxylated aromatic compounds as substrates (vanillate, syringate, trimethoxycinnamate) were inoculated from freshwater mud and sewage sludge samples. In 12 out of 16 cultures the same type of rod-shaped, motile bacteria was selectively enriched. Two strains, NZva16 and NZva24, were isolated in pure culture and recognized as Acetobacterium woodii by comparison with the type strain (DSM 1030).All three Acetobacterium strains were able to grow with all 10 of the tested aromatic compounds containing methoxyl groups. In the presence of bicarbonate, these substrates were used as sole organic electron donors and carbon sources. UV-absorption spectra revealed that the aromatic rings were not degraded, and that the corresponding hydroxy derivatives of the methoxylated compounds were formed. The only further fermentation product formed was acetate. When equimolar concentrations of the methoxylated benzoic acid derivatives were applied, the growth yields were proportional to the number of methoxyl groups per molecule. Methoxyl groups or methanol were metabolized by homoacetate fermentation: in the presence of bicarbonate 4 mol of acetate. In case of the methoxylated cinnamic acid derivatives less acetate was formed and the corresponding hydroxy derivatives of phenylpropionic acid appeared as a result of the double bond reduction in the acrylate side chain. In comparison to the benzoate derivatives with the same number of methoxyl groups, higher growth yields were obtained with the cinnamate derivatives.     

Sniffing behaviour, copulatory readiness, copulatory performance and reproductive morphology of adult male rats weaned prematurely

Sexual behaviour of adult male rats weaned prematurely--on day 15 after birth--and of males weaned at the optimal age--on day 28--was compared at two life periods: at the age of 90 days and when the males were one year old. Sexual behaviour was evaluated by means of three characteristics: a) sniffing behaviour, b) copulatory readiness, c) copulatory performance. The differences in sniffing behaviour between prematurely and normally weaned males were assessed in terms of their responsiveness to sccents of unrelated adult males or oestrous females and from the point of view of both housing conditions after weaning and the animals' sexual experience. No differences were found in copulatory readiness as well as in copulatory performance between the two groups of males tested in their interaction with a female exhibiting complete precopulatory pattern. The morphological examination revealed no differences in the weight of reproductive organs and in spermatozoa development. We conclude that the sexual behaviour as well as reproductive organs of male rats are very resistant to early separation of pups from the mother which contrasts with some earlier findings.     

Studies on Lignin

Two new phenylpropane derivatives, 1-(4-hydroxy-3-methoxyphenyl)-propanediol-(1, 2) and 1-(4-hydroxy-3-methoxyphenyl)-propanediol-(1, 3) were synthesized as lignin model compounds. The former was prepared from 2-acetoxy-1-(4-acetoxy-3-methoxyphenyl)-propanone-(1), and the latter from 3-acetoxy-1-(4-acetoxy-3-methoxyphenyl)-propanone-(1) by lithium aluminum hydride reduction, respectively. The two diols were obtained as colorless syrup, their ultraviolet spectra were determined and crystalline derivatives also prepared.     

Heterosexual interactions in laboratory-housed stumptail macaques (Macaca arctoides): observations during the menstrual cycle and after ovariectomy.

Heterosexual interactions of pairs of stumptail macaques (Macaca arctoides) were studied in relation to the female menstrual cycle and after ovariectomy. Five intact male and 10 tubal-ligated female macaques were observed in laboratory pair tests of 20-mins duration, and data were obtained on various male and female behaviors. Each male was tested with the same two females during four 40-day observation periods. Males were tested daily and females were tested every other day. After two 40-day testing periods, one female partner of each male was ovariectomized and the other was sham-operated. Blood was collected regularly from the females during the course of observation and serum levels of estradiol and progesterone were determined by radioimmunoassay. The midcycle peak of estradiol was observed to occur approximately 18 days prior to menses. A distinct secondary peak in estradiol was observed to occur during the luteal phase of all cycles examined. Of 28 different male and female behaviors studied only female presentation to male sexual contact showed a significant midcycle peak related to the endogenous estradiol surge. After ovariectomy a significant decrease in the frequency of several male copulatory behaviors was observed, but most males continued to copulate regularly with their spayed partners throughout the period of this study. Thus, the pattern of copulatory behavior observed in stumptail macaques over the cycle of the female partner and subsequent to ovariectomy differs from that observed in other macaque species studied in the laboratory. It is concluded that cyclical fluctuations in the level of ovarian hormones are not significantly related to measures of sexual interactions in laboratory tests of this species, although the maintenance of copulation and associated behaviors at high levels depends to some degree upon the ovary.