Transport of Radioactivity in Relation to Bracts in the Cotton Plant

[¹⁴C]sucrose was applied to the leaf subtending 14-d-old boll(cotton fruit) with and without bracts, and to bracts alonein var. Suvin (Gossypium barbadense L.). The removal of bractssubstantially enhanced the transport of radioactivity from theleaf to the carpels and seed cotton. When bracts alone weretreated, still larger quantities of radioactivity were incorporatedin the carpels and seed cotton. The bracts appeared to nourishthe developing boll through their own photosynthate and regulatethe transport of assimilate from the leaf. Radioactivity, transport, bracts, boll, carpels, seed cotton     

Lipogenesis in Fatty Liver by Amino Acid Imbalance

Lipogenesis in the fatty liver of rat which was induced by feeding an amino acid-irnbalanced diet containing 8% casein supplemented with 0.3% dl-methionine has been investigated by measuring the incorporation of acetate-1-¹⁴C into various lipid fractions during in vitro incubation of liver slices.

In the incorporation of acetate-1-¹⁴C into the total lipid per one g of the slices, no significant difference for the imbalance group was observed. However, the total radioactivity of liver lipid per 100 g of the body and the incorporation into triglyceride in the lipids were significantly higher in the imbalance group than in the control group. Conversion of acetate-1-¹⁴C to CO₂ was not impaired in the imbalance group.

It is evident from these results that the induction of this type of fatty liver is due mainly to the synthesis of triglyceride.     

Participation of Mevalonate in the Biosynthetic Pathway of Ipomeamarone

1. The incorporation of mevalonate-2-¹⁴C into ipomeamarone in sweet potato root tissue infected by Ceratocystis fimbriata was demonstrated, but the rate was low when compared with acetate-2-¹⁴C. No dilution effect of mevalonate was noted during the incorporation of acetate-2-¹⁴C into ipomeamarone. This is very likely to result from the passive transfer of mevalonate into the cells.

2. No dilution effect of acetate during the incorporation of mevalonate-2-¹⁴C into ipomeamarone was noted. This indicates that mevalonate is not incorporated into ipomeamarone after its conversion to acetate.

3. Evidence for incorporation of acetate-2-¹⁴C into mevalonate was shown by the fact that the specific radioactivity of mevalonic acid benzhydrylamide was not lowered throughout repetitive crystallizations. These data also support the participation of mevalonate in ipomeamarone synthesis as an intermediate.

     

Biosynthesis of the 3-benzylchroman-4-one eucomin in Eucomis bicolor

Feeding experiments have demonstrated the specific incorporation of radioactivity from dl-phenylalanine-[1-¹⁴C], l-phenylalanine-[U-¹⁴C], sodium acetate-[2-¹⁴C] and l-methionine-[methyl-¹⁴C] into the 3-benzylchroman-4-one eucomin in Eucomis bicolor. The labelling patterns indicate that eucomin is biosynthesized by the addition of a carbon atom derived from methionine onto a C₁₅ chalcone-type skeleton. Radioactivity from 2′,4′,4-trihydroxy-6′-methoxychalcone-[methyl-¹⁴C] and 2′,4′-dihydroxy-4,6′-dimethoxychalcone-[6′-methyl-¹⁴C] was incorporated into eucomin, the latter compound being the better precursor, demonstrating the feasibility that 2′-methoxychalcones are biosynthetic precursors of the “homoisoflavonoids”. Possible biosynthetic relationships in this class of compounds are discussed.     

Biosynthesis of the sesquiterpenoid,capsidiol, in sweet pepper fruits inoculated with fungal spores

Capsicum frutescens fruits inoculated with spore suspensions of Monilinia fructicola incorporated 1–4% of sodium acetate-[2-¹⁴C] or RS-mevalonolactone-[2-¹⁴C] into the phytoalexin, capsidiol. Labelled capsidiol was characterized by GC-RC, TLC-RC, gel chromatography (in conjunction with liquid scintillation counting) and GC-MS. The mode of incorporation of sodium acetate-[1,2-¹³C₂] into capsidiol, as indicated by the pattern of ¹³C-¹³C coupling from ¹³C NMR data, supports the hypothesis that the angular methyl group of the capsidiol skeleton arises by migration from the C-10 position of a eudesmane-type intermediate.     

Induction of female sex pheromone production in male houseflies by ovary implants or 20-hydroxyecdysone

Implanting ovaries or injecting 20-hydroxyecdysone into male houseflies induced sex pheromone production, including (Z)-9-tricosene (muscalure), 9,10-epoxytricosane and (Z)-14-tricosen-10-one, which normally occurs only in vitellogenic females. Control males did not produce detectable amounts of these compounds. Injection of 20-hydroxyecdysone (5 μg/insect per day) for 3 days resulted in the accumulation of 1.81 μg/insect of (Z)-9-tricosene, 0.97 μg/insect of 9,10-epoxytricosane and 0.12 μg/insect (Z)-14-tricosen-10-one. Multiple injections of 20-hydroxyecdysone at doses as low as 50 ng resulted in the accumulation of 23:1, C₂₃ epoxide and C₂₃ ketone; shifted the distribution of label within the alkenes from 27:1 to 23:1 and decreased the amount of label in the hydrocarbon fractions as alkenes. Structures of the C₂₃ alkene and epoxide produced by the males were verified by gas chromatography-mass spectrometry. Radioactivity from [1-¹⁴C] acetate was incorporated into the C₂₃ alkene, epoxide and ketone in male insects after ovaries were implanted or they were injected with 20-hydroxyecdysone. Synthesis of the C₂₃ pheromone components decreased rapidly within several days after the administration of 20-hydroxyecdysone ceased, indicating that the enzymes involved in sex pheromone production were not permanently induced by hormone treatment. Ecdysone was also effective in initianing pheromone production in males, whereas inokosterone and cholesterol were not effective. Data presented demonstrate that male houseflies possess the metabolic capability to produce the sex pheromone components, and this suggests that 20-hydroxyecdysone alters the production of cuticular hydrocarbons such that the C₂₃ sex pheromone components become major products.     

The Origin of Carbons of Dihydrofuran Ring and C-6 in the Biosynthesis of Rotenone

For the investigation of rotenone biosynthesis, acetate-2-¹⁴C, mevalonic acid-2-¹⁴C lactone and methionine-methyl-¹⁴C were administered to Derris elliptica plants, respectively, and the distribution of carbon-14 in the labeled rotenone was determined by degradation. When mevalonic acid-2-¹⁴C lactone was incorporated into rotenone, the radioactivity was found equally in the carbons at both C-7′ and C-8′, indicating that these carbons are derived from the carbon-2 of mevalonic lactone. In the case of methionine-methyl-¹⁴C about 80% of the total radioactivity was found to enter two methoxyl groups. This result demonstrates that methionine is an efficient precursor of the methoxyl group. Furthermore, it is also suggested that methionine may be a precursor of the carbon at C-6.     

Volatile constituents of male and female boll weevils and their frass

The volatile constituents of male and female boll weevils, Anthonomus grandis, and their frass were analysed by GLC-MS. The 4 previously identified components of the male pheromone were present only in the male volatile oil (3·9%) and in the male frass volatile oil (38·9%). The compounds found in one or both sexes and their frass include 26 carbonyls, 23 hydrocarbons, 12 alcohols, 6 phenols, 4 esters, 3 furans, 1 ether, and 1 lactone. Also found were 2 compounds containing nitrogen, 1 halogen, and 1 sulphur. There were 33 terpenes and 24 aromatic compounds. 3,7-Dimethyl-1-octanol comprised 15·6 per cent of the male frass oil. Carvone was found only in females (5·4%) and in female frass (6·8%). A series of monoterpene aldehydes (M⁺ 152) were found only in the female frass oil. A pheromone rôle for these components was suggested.     

The Biosynthesis of Some Isothiocyanates and Oxazolidinethiones in Rape (Brassica campestris L.)

The incorporation of the radioactivity from acetate-1-¹⁴C, acetate-2-¹⁴C, dl-methionine-1-¹⁴C, dl-methionine-2-¹⁴C, dl-methionine-3,4-¹⁴C, dl-homomethionine-2-¹⁴C, dl-allyl-glycine-2-¹⁴C, and dl-2-amino-5-hydroxyvalerate-2-¹⁴C into the aglycones of progoitrin, gluconapin, and glucobrassicanapin of maturing rape plants (Brassica campestris L.) was investigated. Radioactivity from dl-methionine-2-¹⁴C, dl-methionine-3,4-¹⁴C, dl-homomethionine-2-¹⁴C, and acetate-2-¹⁴C were incorporated into the 3 major thioglucosides. The other organic compounds were poorly incorporated except for dl-allylglycine-2-¹⁴C into glucobrassicanapin. The results obtained suggest that the rape plant can synthesize amino acids by the condensation of acetate (as acetyl CoA) to α-keto acids to yield a homologue of the original amino acid. These newly formed amino acids are then employed to synthesize the 3 major thioglucosides.     

Alternate pathways of glycolate synthesis in tobacco and maize leaves in relation to rates of photorespiration

After a preliminary period in light, leaf disks floated on 10 mm α-hydroxy-2-pyridinemethanesulfonic acid to inhibit glycolate oxidase accumulate glycolate at average initial rates of 67 micromoles in tobacco and 8 micromoles per gram fresh weight per hour in maize under optimal conditions in air. In the presence of ¹⁴CO₂, the glycolate synthesized has a high specific radioactivity in illuminated tobacco and a low one in maize. Isonicotinic acid hydrazide also inhibits glycolate oxidation and causes a slow accumulation of glycolate in maize but not in tobacco, while it inhibits glycolate synthesis in tobacco but not in maize. Radioactive carbon in acetate-2-¹⁴C and especially pyruvate-3-¹⁴C is incorporated predominantly into the C-2 of glycolate in both species, but the specific radioactivity is much greater in maize. Glyoxylate-2-¹⁴C is readily converted to glycolate-2-¹⁴C in both species. The addition of phosphoenolpyruvate stimulated glycolate formation in maize and inhibited its synthesis in tobacco, and in the presence of ¹⁴CO₂ the specific radioactivity in glycolate-¹⁴C was decreased greatly by the added phosphoenolpyruvate only in maize.     

The Biosynthesis of Sepedonin

The origin of each carbon of anhydrosepedonin produced by Sepedonium chrysospermum has been elucidated by carbon-14 tracer method. At first, the degradation pathways adequate to such object was studied. The radioactivity incorporated in sepedonin and anhydrosepedonin was considerably high when acetate-1-¹⁴C, acetate-2-¹⁴C and formate-¹⁴C were used as the precursor. The feature of the labeling pattern of anhydrosepedonin was analogous to those of the other fungal tropolones. The carbon atom derived from formate was almost exclusively situated at C-7 position of the tropolone ring. As a whole, the labeling pattern demonstrated that anhydrosepedonin might be synthesized through the poly-ketomethylene pathway.     

Employing in vitro directed molecular evolution for the selection of α-amylase variant inhibitors with activity toward cotton boll weevil enzyme

Numerous species of insect pests attack cotton plants, out of which the cotton boll weevil (Anthonomus grandis) is the main insect in Brazil and must be controlled to avert large economic losses. Like other insect pests, A. grandis secretes a high level of α-amylases in the midgut lumen, which are required for digestion of carbohydrates. Thus, α-amylase inhibitors (α-AIs) represent a powerful tool to apply in the control of insect pests. Here, we applied DNA shuffling and phage display techniques and obtained a combinatorial library containing 10⁸α-AI variant forms. From this library, variants were selected exhibiting in vitro affinity for cotton boll weevil α-amylases. Twenty-six variant sequences were cloned into plant expression vectors and expressed in Arabidopsis thaliana. Transformed plant extracts were assayed in vitro to select specific and potent α-amylase inhibitors against boll weevil amylases. While the wild type inhibitors, used to create the shuffled library, did not inhibit the A. grandis α-amylases, three α-AI mutants, named α-AIC3, α-AIA11 and α-AIG4 revealed high inhibitory activities against A. grandis α-amylases in an in vitro assay. In summary, data reported here shown the potential biotechnology of new α-AI variant genes for cotton boll weevil control.     

An assessment of the life time biosynthesis potential of the male boll weevil

The four monoterpene compounds [1, (+)-cis-2-isopropenyl-1-methycyclobutaneethanol; II, (Z)-3,3-dimethyl-Δ^1,β-cyclohexaneethanol; III, (Z)-3,3-dimethyl-Δ^1,α-cyclohexaneacetaldehyde; and IV, (E)-3,3-dimethyl-Δ^1,α-cyclohexaneacetaldehyde] which comprise the pheromone of the male boll weevil, Anthonomus grandis, were determined daily throughout the lifespan of the insect by GLC analysis of the whole body and the frass. The total content of the components in the male is never greater than 210 ng, the average content in the frass produced during 1 day is 1268 ng, and the lifetime production is at least 40,000 ng. The ratio of the four components in frass is 6 : 6 : 2 : 1/I : II : III : IV. None of the four components was found in females, but female frass contained traces in three instances. These data can be used to compare the potential attractancy of insects from various geographical areas, at different seasons, in different nutritional states, and after chemosterilization. Such differences in potential insect attractiveness could be crucial to effective large-scale control programmes.     

Amo 1618 effects on incorporation of 14C-MVA and 14C-acetate into sterols in Nicotiana and Digitalis seedlings and cell-free preparations from Nicotiana

Incorporation of radioactivity from acetate-[¹⁴C] and MVA-[¹⁴C] into sterols and sterol precursors in tobacco was inhibited by Amo 1618; differing patterns of accumulation were obtained with the two precursors, suggesting more than one point of inhibition. This was borne out with cell-free preparations with which it was demonstrated that both HMG-CoA reductase and squalene-2,3-epoxide cyclase were inhibited, the latter more strongly than the former. GLC analysis of gross sterol and hydrocarbon fractions confirmed previous indications that incorporation of radioactivity into individual sterols was inhibited by Amo 1618. Finally, incorporation of MVA-[¹⁴C] into sterols and sterol precursors of Digitalis was significantly altered by the retardant, thus expanding the generality of the relationship between sterol (particularly 4-desmethylsterol) biosynthesis inhibition and retardant effect.     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

Biosynthesis of p-hydroxybenzoic acid in poplar lignin

Biosynthetic pathways to p-hydroxybenzoic acid in polar lignin were examined by tracer experiments. High incorporation of radioactivity to the acid was observed when shikimic acid-[1-¹⁴C], phenylalanine-[3-¹⁴C], trans-cinnamic acid-[3-¹⁴C], p-coumaric acid-[3-¹⁴C] and p-hydroxybenzoic acid-[COOH-¹⁴C] were administered, while incorporation was low from shikimic acid-[COOH-¹⁴C], phenylalanine-[1-¹⁴C], phenylalanine-[2-¹⁴C], tyrosine-[3-¹⁴C], benzoic acid-[COOH-¹⁴C], sodium acetate-[1-¹⁴C] and d-glucose-[U-¹⁴C]. Thus p-hydroxybenzoic acid in poplar lignin is formed mainly via the pathway: shikimic acid → phenylalanine → trans-cinnamic acid → p-coumaric acid → p-hydroxybenzoic acid.     

Effect of Ethrel and Ceratocystis fimbriata on the Synthesis of Fatty Acids and 6-Methoxy Mellein in Carrot Root

The rate of incorporation of ¹⁴C from acetate-1-¹⁴C into fatty acids by carrot root discs, 18 hours after inoculation with Ceratocystis fimbriata, was 9-fold greater than that in freshly cut discs. The rate in discs treated with water or Ethrel was 3-fold greater. The rate of incorporation of ¹⁴C from glucose-U-¹³C into fatty acids was 3-fold greater 18 hours after any of the above treatments. The rate of ¹⁴C incorporation from malonate-2-¹⁴C into fatty acids 24 hours after inoculation with C. fimbriata or treatment with water was 25 and 60%, respectively, of that in freshly cut discs. Linoleic acid was the principal fatty acid in carrot root, but incorporation of ¹⁴C from acetate-1-¹⁴C into the acid was low until 18 hours after inoculation with C. fimbriata or treatment with Ethrel. Turnover rates of the fatty acids appeared low and were similar for all treatments.     

Biosynthesis of monoterpenes by Ceratocystis moniliformis

Ceratocystis moniliformis produced and excreted monoterpenes when grown on potato-dextrose broth. Geraniol, nerol, citronellol, linalol, α-terpineol, geranial and neral were identified by GC-MS. Their production commenced with the depletion of nitrogen in the growth medium and their combined concentration peaked at about 50 μg/ml on the 5th day of growth. The pathway for the biosynthesis of the identified monoterpenes was studied by supplying the radioactive precursors mevalonic acid-[2-¹⁴C], l-leucine-[4,5-³H(N)], and acetate- [2-¹⁴C] to C. moniliformis. For each precursor, the extent of incorporation into the above monoterpenes and the distribution of radioactivity in geraniol was determined. It was concluded that monoterpenes were formed via the mevalonate pathway, previously established for higher terpenes in other organisms. This represents the first information available on the biosynthetic pathway for free monoterpenes in a microbial system.     

The Incorporation of Leucine-U-14C into Ipomeamarone

1. It was demonstrated by silica gel thin layer chromatography that leucine-U-¹⁴C was incorporated into furanoterpenes, e. g. ipomeamarone, in sweet potato root tissue infected by Ceratocystis fimbriata.

2. Further proof for ipomeamarone synthesis from leucine-U-¹⁴C was obtained by the constancy of the specific radioactivity of ipomeamarone semicarbazone through repetitive crystallization.

3. The synthetic pathway of ipomeamarone from leucine was found to be connected with the synthetic pathway from acetate at least at some steps.

4. Leucine-U-¹⁴C was incorporated into both saponifiable and non-saponifiable materials in the same way as acetate-2-¹⁴C.     

Concurrent modulation of neuronal and behavioural olfactory responses to sex and host plant cues in a male moth

Mating has profound effects on animal physiology and behaviour, not only in females but also in males, which we show here for olfactory responses. In cotton leafworm moths, Spodoptera littoralis, odour-mediated attraction to sex pheromone and plant volatiles are modulated after mating, producing a behavioural response that matches the physiological condition of the male insect. Unmated males are attracted by upwind flight to sex pheromone released by calling females, as well as to volatiles of lilac flowers and green leaves of the host plant cotton, signalling adult food and mating sites, respectively. Mating temporarily abolishes male attraction to females and host plant odour, but does not diminish attraction to flowers. This behavioural modulation is correlated with a response modulation in the olfactory system, as shown by electro-physiological recordings from antennae and by functional imaging of the antennal lobe, using natural odours and synthetic compounds. An effect of mating on the olfactory responses to pheromone and cotton plant volatiles but not to lilac flowers indicates the presence of functionally independent neural circuits within the olfactory system. Our results indicate that these circuits interconnect and weigh perception of social and habitat odour signals to generate appropriate behavioural responses according to mating state.