Effects of primary, secondary and tertiary amines on in vitro cellulose digestion and volatile fatty acid production by ruminal microorganisms.

Decanamine hydrochloride (a C10 primary amine), N-methylundecanamine (a C11 secondary amine) and N, N-dimethyldodecanamine (a C12 tertiary amine) were tested for their effects upon in vitro cellulose digestibility by unadapted ruminal microorganisms. The three amines exhibited similar patterns of inhibition of cellulose digestibility in 48-hr in vitro incubations. Cellulose digestion was depressed to 97, 89, 31, 11 and 12% of the control value by 5, 10, 25, 50 and 100 ppm of the amines, respectively. Total volatile fatty acid production was depressed to 89, 85, 61, 40 and 32% of the control value by 5, 10, 25, 50 and 100 ppm of the amines, respectively. Propionate production was inhibited to a greater extent than was acetate production, and acetate to propionate ratios increased in the presence of the amines. The results demonstrate that these primary, secondary and tertiary amines inhibit those microorganisms which digest cellulose in the rumen.

The ecdysteroids from young embryonated eggs of the tobacco hornworm.

26-Hydroxyecdysone, which is the major free recoverable ecdysteroid of older age groups of embryonated eggs of the tobacco hornworm was also the major component in 4- to 18-hour-old embryonated eggs. The other 3 beta-ecdysteroids, ecdysone, 20-hydroxyecdysone, and 20,26-dihydroxy-ecdysone, were also present and accounted for all the molting hormone activity; 26-hydroxyecdysone was devoid of molting hormone activity in the house fly assay. 20-Hydroxyecdysone was a minor component, which confirms the earlier observations that the main metabolic route for ecdysteroids during embryonic development is that leading to 26-hydroxyecdysone, whereas formation of 20-hydroxyecdysone is a minor pathway. A new 3 alpha-ecdysteroid, 3-epi-26-hydroxyecdysone, also devoid of molting hormone activity, was the second major ecdysteroid isolated from the eggs. 3-Epi-20,26-dihydroxyecdysone was detected in very minute amounts. In addition to the six 3 beta-and 3 alpha-ecdysteroids there were at least an equivalent number of unknown ecdysteroids all of which lacked molting hormone activity. Their physical properties including chromatographic behavior are discussed.

Mastitis: II. Evaluation of antimicrobial amines for use as teat dips.

Recent proposals by the Food and Drug Administration to regulate teat dips as drugs have led to a search for safer teat dip ingredients. Primary, secondary, and tertiary alkyl amines (carbon-10 to -18 chain length) inhibit growth of mastitic bacteria (Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus, Escherichi coli, Klebsiella pneumoniae) in a broth tube culture assay. Since carbon-13 compounds were active, a carbon-13 primary (tridecanamine hydrochloride), secondary methyl (N-methyltridecanamine), secondary ethyl (N-ethyltridecanamine), tertiary dimethyl amine (N, N-dimethyltridecanamine), and carbon-12 quaternary amine (N, N, -trimethyldodecaneammonium chloride) were tested for their ability to reduce experimentally applied populations of S. agalactiae or E. coli on the bovine teat surface. The five compounds were compared at concentrations of 100, 500, 1,000, 3,000, 7,000, and 10,000 ppm. Activity was greater against the gram-positive S. agalactiae than against the gram-negative E. coli. The tertiary amine was most active, producing a log reduction of 4 (reduction of bacterial number from 10(6) to 10(2)) at a concentration of 3,000 ppm in the teat dip. The relative order of effectiveness for the amines was: dimethyl tertiary greater than methyl secondary greater than ethyl secondary greater than primary = quaternary. The results suggest that these amines may be useful as potent, effective antibacterial agents for incorporation into teat dips.

The sterols of the khapra beetle, Trogoderma granarium Everts.

The khapra beetle, Trogoderma granarium Everts, does not dealkylate and convert dietary C28- or C29-Phytosterols to C27-sterols such as cholesterol. There is, however, an increase in the concentration of cholesterol and campesterol in its tissues relative to the dietary concentrations of these sterols, presumably as a result of selective uptake.

The ecdysteroids from the tobacco hornworm during pupal-adult development five days after peak titer of molting hormone activity.

Six naturally occurring C27 ecdysteroids were isolated and identified from the tobacco hornworm during pupal-adult development five days after peak titer of molting hormone activity. In order of decreasing quantities the hormones were: 20,26-dihydroxyecdysone, 3-epi-20-hydroxyecdysone, 20hydroxyecdysone, 3-epi-20,26-dihydroxyecdysone, 3-epi-ecdysone, and ecdysone. 20-Hydroxyecdysone, in an earlier study, was the major molting hormone present at peak titer during pupal-adult development. The major ecdysteroid present during embryonic development in this insect, 26-hydroxyecdysone, was not detected. The copresence of all six of these ecdysteroids from a single developmental stage of an insect provides information on the metabolic interrelationships that exist among these steroids and on their possible function(s) in insects. The 3alpha-ecdysteroids were far less active than the 3 beta-epimers in the house fly assay. The significance of epimerization is discussed.

Mastitis: I. In vitro antimicrobial activity of alkyl amines against mastitic bacteria.

The activities of branched and straight chain amines (10 to 18 carbons chain length) were compared in inhibiting the growth of five microorganisms that cause about 95% of bovine mastitis. Three gram-positive (Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus) and two gram-negative (Escherichia coli, Klebsiella pneumoniae) bacteria were used in a trypticase soy broth tube culture growth assay. Sixty-two compounds were screened at concentrations of 200, 100, 50, 25, 10, 5, and 1 ppm in broth culture to determine the effective minimum inhibitory concentration. Alkyl secondary N-substituted monoethyl [CH3(CH2)nNHCH2CH3] and tertiary N,N-substituted dimethyl [CH3(CH2) nN(CH3)2] amines with chain lengths of 11 to 14 carbon atoms were active against both gram-positive and gram-negative organisms. Antimicrobial activity against gram-positive organisms increased with increasing chain length and carbon-14 to 18 amines were active at 1 to 5 ppm. The carbon-11 to 13 alkyl amines were most active against gram-positive organisms; longer chain amines (more than 14 carbons) were inactive. Branching of the alkyl chain caused a loss of activity against gram-negative but not against gram-positive bacteria. Antimicrobial testing of monoamines, polyamines, and the influence of order substituents were investigated to correlate structure-acitivity relationships.

Insect steroid metabolism.

Insects are unable to biosynthesize the steroid nucleus and generally require an exogenous source of sterols. Two salient areas of insect steroid metabolism are the dealkylation and conversion of dietary C28 and C29 plant sterols to cholesterol and other C27 sterols, and the biosynthesis and metabolism of the steroidal insect molting hormones. Certain azasteroids and nonsteroidal amines block this conversion of 24-alkyl sterols to cholesterol and/or disrupt molting and development in insects. These inhibitors have served in charting metabolic pathways for steroids in insects and are serving as models in developing selective pesticidal chemicals and chemotherapeutic agents for use against insects and other invertebrate pests and parasites. The mode of action of some of these inhibitors on molting and development has been investigated in vivo and in vitro. Certain of these inhibitors represent a new class of insect hormonal compounds with a novel mode of action-the disruption of molting hormone metabolism. Research on sterol metabolism in insects provides important information on the comparative biochemistry and physiological functions of steroids in living systems.

Isolation and identification of the metabolites of 22,25-dideoxyecdysone from cockroach fat body cultures.

Hydroxylation and conjugation were the principal pathways of metabolism of 22,25-dideoxyecdysone in cockroach fat body cultures. The major metabolite isolated and identified was the tetrahydroxy steroid 22-deoxyecdysone; other exdysteroids isolated, in order of decreasing quantities, were 22-deoxy-26-hydroxyecdysone, 22,25-dideoxy-26-hydroxyecdysone, and 22-deoxy-20-hydroxyecdysone. Cockroach fat body from late-instar nymphs appears to lack the mechanism for hydroxylating at C-22. Radioanalyses of the material obtained from enzymic hydrolysis of the conjugate fraction showed 65, 15 and 20% of tetraols, pentaols, and unhydrolyzed conjugates respectively, and no 22,25-dideoxyecdysone. An azasteroid and two nonsteroidal amines that effectively inhibit the activity of 22,25-dideoxyecdysone in the cockroach leg regenerate-fat body culture system enhanced the metabolism of 22,25-dideoxyecdysone, decreased the quantity of the pentaol fraction present, and caused an increase or accumulation of the tetraol and conjugate fractions in the fat body culture system.

Sterol composition and phytosterol utilization and metabolism in the milkweed bug.

Analysis of the sterols of the milkweed bug, Oncopeltus fasciatus (Dallas) and dietary sunflowerseeds revealed that there is little, if any, conversion of dietary C28 OR C29 phytosterols to cholesterol in this phytophagous insect. The dietary sterols are apparently utilized with little alteration both during development to the adult stage and egg production, and cholesterol comprises less than 1% of the sterols in either adult males and females or in the eggs. The significance of these findings are discussed in light of the recent discovery that the C28-ecdysone, makisterone A, is the predominant molting hormone inthe embryonated egg of the milkweed bug.

The molting hormones from the embryonated EGG of the tobacco hornworm, Manduca sexta (l.).

26-Hydroxyecdysone is the predominant molting hormone in 24- to 44-hour-old embryonated tobacco hornworm eggs, accounting for approximately 80% of the ecdysones present at this stage of development. This molting hormone was previously shown to be the major ecdysone present in 48- to 64-hour-old embryonated eggs of this insect. During both of these periods of embryonic development in the hornworm 20-hydroxyecdysone is a minor component, in contrast to its presence as the major ecdysone in the hornworm during certain stages of post-embryonic development.

Ecdysome 20-hydroxylase from the midgut of the tobacco hornworm (Manduca sexta L.).

An ecdysone 20-hydroxylase enzyme system that converts alpha-ecdysone to 20-hydroxyecdysone was prepared from the midgut of the tobacco hornworm prepupa. This partially purified enzyme is NADPH dependent and is localized in the mitochondrial fraction of the midgut tissue.

Nematidical activity of secondary and tertiary alkyl amides and amines.

Several C11 to C15 amides and amines that disrupt growth in certain insects showed high nematicidal activity in direct contact tests. Two amides and 9 amines killed Panagrellus at 5-10 ppm. Of these, 1 amide and 3 amines killed Meloidogyne larvae at 20 ppm.

Makisterone A:a 28-carbon hexahydroxy molting hormone from the embryo of the milkweed bug.

Makisterone A is the predominant ecdysone in the 96 +/- 4-hour-old embryo of the large milkweed bug and it is the first molting hormone with a C-24 alkyl substituent of the side chain to be isolated and identified from an insect. In addition, unknown compounds that may represent other C28 ecdysones were detected in very low concentrations. The milkweed bug could well possess a biosynthetic-metabolic pathway for C28 molting hormones instead of or in addition to known pathways for the C27 ecdysones.

Inhibitive effects of structurally modified azasteroids and related nitrogen containing steroids on insect growth and development.

A number of azasteroids and other nitrogen containing steroids with a modified nucleus or side chain were prepared and tested for their inhibitory effects on the growth and development of several species of insects. Structure-activity studies showed that compounds with a structurally related steroid nucleus and side chain were approximately equal in inhibitory activity for a particular species. The replacement of the tertiary amino group in the side chain of the 5beta-steroid with other nitrogen substituents, such as nitro, cyano, acetylamino, or a quaternary ammonium salt, resulted in a considerable loss of inhibitive activity in the tobacco hornworm or the yellowfever mosquito. However, certain modifications of the azasteroid nucleus resulted in compounds that still retained high biological activity. As a result, a compound was synthesized that lacked the A and B rings of the steroid nucleus and that inhibited insect growth, molting, and metamorphosis and the delta24-sterol reductase enzyme system of the tobacco hornworm.

Unique pathways of sterol metabolism in the Mexican bean beetle, a plant-feeding insect.

Radiolabeled sterols, 14C-cholesterol, 14C-cholestanol, 3H-stigmasterol, 3H-stigmastanol, and 3H-sitosterol, were fed to larvae of the Mexican bean beetle, Epilachna varivestis Mulsant, bu coating soybean leaves. Free sterol and sterol ester fractions from treated insects were isolated and analyzed, and in each case nearly 30% or more of total radiolabeled sterols retained by the insect were found in the sterol ester fraction after 8 days. delta5-Dietary sterols were readily reduced to stanols, and C29-stanols thus produced were dealkylated to cholestanol. Significant amounts of labeled lathosterol were formed from delta5-C29 sterols; little, if any, radiolabeled cholesterol was detected in insects fed either of the labeled delta5-phytosterols, stigmasterol or sitosterol. Sterol metabolism of this insect thus differs considerably from that found for most phytophagous insects.

Nonsteroidal secondary and tertiary amines: inhibitors of insect development and metamorphosis and delta-24-sterol reductase system of tobacco hornworm.

Several new branched and straight chain secondary and tertiary amines were shown to have inhibitive effects upon development and metamorphosis and the delta-24-sterol reductase system in larvae of the tobacco hornworm similar to those previously observed with a number of azasteroids. Certain of the amines which are related structurally to compounds with juvenile hormone activity in insects also blocked development and metamorphosis in three other species of insects. These compounds are lethal or inhibit development in all larval stages and thus differ in action from compounds with juvenile hormone activity where the principal effect is to block the penultimate or ultimate molt.