Metabolism of [H]Gibberellin A(5) by Immature Seeds of Apricot (Prunus armeniaca L.)

Immature seeds of apricot (Prunus armeniaca L.) were fed the native gibberellin A₅ (GA₅) as 1- and 1,2-[³H]GA₅ (5.3 Curies per millimole to 16 milliCuries per millimole) at doses (42 nanograms to 10.6 micrograms per seed) 2 to 530 times the expected endogenous level. After 4 days of incubation, seeds were extracted and free [³H]GA-like metabolites were separated from the highly H₂O-soluble [³H]metabolites. For high specific activity feeds the retention times (Rts) of radioactive peaks were compared with Rts of authentic GAs on sequential gradient-eluted → isocratic eluted reversed-phase C₁₈ high performance liquid chromatography (HPLC) -radiocounting (RC). From high substrate feeds (530 and 230 × expected endogenous levels) HPLC-RC peak groupings were subjected to capillary gas chromatography-selected ion monitoring (GC-SIM), usually six characteristic ions. The major free GA metabolites of [³H] GA₅ were identified as GA₁, GA₃, and GA₆ by GC-SIM. The major highly water soluble metabolite of [³H]GA₅ at all levels of substrate GA₅ had chromatographic characteristics similar to authentic GA₁-glucosyl ester. Expressed as a percentage of recovered radioactivity, low substrate [³H]GA₅ feeds (2 × expected endogenous level) yielded a broad spectrum of metabolites eluting at the Rts where GA₁, GA₃, GA₅ methyl ester, GA₆, GA₂₂, GA₂₉ (17, 14, 1.6, 7, 1.1, 0.5%, respectively) and GA glucosyl conjugates of GA₁, GA₃, GA₅, and GA₈ (33, 11, 1, 0.1%, respectively) elute. Metabolites were also present at Rts where GA glucosyl conjugates of GA₆ and GA₂₉ would be expected to elute (8 and 0.1%, respectively). Only 5% of the radioactivity remained as GA₅. Increasing substrate GA₅ levels increased the proportion of metabolites with HPLC Rts similar to GA₁, GA₆, and especially GA₁ glucosyl ester, primarily at the expense of metabolites with HPLC Rts similar to GA₃, GA₃-glucosyl ester, and a postulated conjugate of GA₆. There was evidence that high doses of substrate GA₅ induced new metabolites which often, but not always, differed from GA₁, GA₃, and GA₆ in HPLC Rt. These same metabolites, when analyzed by GC-SIM yielded m/e ions the same as the M⁺ and other characteristic m/e ions of the above GAs, albeit at differing GC Rt and relative intensities.     

Functional and phenotypic analyses of interleukin 2-activated tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TILs) were cultured with interleukin 2 (IL-2) to induce the activated killer cells possessing autologous tumor-killing activity, and analysed their cell surface phenotypes and assessed anti-tumor killing activity. Furthermore, the activated TILs were transferred into 7 patients adoptively resulting in complete remission in a patient with pancreatic cancer and partial remission in another patient with gastric cancer.The cytotoxic activities of activated TILs at 3 weeks-incubation was 72 ± 15, 42 ± 26, 27 ± 21 and 25 ± 15% against K562, Daudi, KATO-III and autologous tumor, respectively. The negative selection method, indicated that the killer cells recognizing autologous tumor cells consisted of CD4- or CD8-positive T lymphocytes and CD16- or CD56-positive natural killer cells. The activated TILs could not only lyse cultured tumor cell lines, but also autologous tumor cells.     

The promotive effect of interleukin 4 with interleukin 2 in the proliferation of tumor-infiltrating lymphocytes from patients with malignant tumor

In adoptive immunotherapy, the number of effector cells is one of the major factors relating to the therapeutic efficacy. We demonstrated that tumor-infiltrating lymphocytes (TILs) were stimulated to proliferate by incubation with interleukin 2 (IL-2) plus interleukin 4 (IL-4). TILs cultured with IL-2 plus IL-4 increased 3.1-fold more than TILs cultured with IL-2 alone. However, IL-4 did not alter the cytotoxic activity of TILs against autologous tumor cells and established tumor cell lines. It is suggested that IL-2 receptor is related to the mechanism of the proliferation of activated TILs cultured by combination with IL-2 and IL-4. Thus, the combination of IL-2 and IL-4 may increase the efficacy of adoptive immunotherapy using activated TILs.     

Identification of Gibberellins A(1), A(5), A(29), and A(32) from Immature Seeds of Apricot (Prunus armeniaca L.)

Gibberellins (GAs) A₁, A₅, and A₂₉ were identified, and also GA₃₂ was confirmed, as endogenous GAs of immature seeds (3-4 weeks after anthesis, 0.25-0.5 gram fresh weight) of apricot (Prunus armeniaca L.) based on capillary gas chromatography (GC), retention time (Rt), and selected ion monitoring (SIM), in comparison with authentic standards. Fractions subjected to GC-SIM were purified and separated using sequential solvent partitioning → paper chromatography → reverse phase C₁₈ high performance liquid chromatography (HPLC) → bioassay on dwarf rice cv Tan-ginbozu. Two other peaks of free GA-like bioactivity (microdrop and immersion dwarf rice assays) were eluted from C₁₈ HPLC at Rts where GA_4/7 and GA₈ (or other GAs with similar structures) would elute. Also, three unidentified GA glucoside-like compounds (based on bioactivity on the immersion assay, and no bioactivity on the microdrop assay) were noted. There were very high amounts of GA₃₂ (112 ng of GA₃ equivalents per gram fresh weight), and minor amounts (0.5 ng of GA₃ equivalents) for each of GA₁ and GA₅, respectively, based on the microdrop assay.     

Reversible conjugation of gibberellins in situ in maize

Gibberellins [³H]GA₄ (1.33 Curies per millimole) and [³H]GA₂₀ (2.36 Curies per millimole) were injected into the shanks of maize (Zea mays L.) cobs during rapid grain filling and mature seeds were subsequently harvested. Extracts of mature, dry seeds from 1980 feeds yielded only 20 to 30% of the ³H radioactivity in acidic, ethyl acetate-soluble form, and this was principally associated with the precursor, with lesser amounts of the major metabolite, [³H]GA₁ (putative identification based on sequential SiO₂ partition, and gradient-eluted reverse-phase C₁₈ high performance liquid chromatography [HPLC]). Most of the radioactivity in the dry seeds was associated with compounds having partition characteristics of, and co-chromatographing on, sequential SiO₂ partition and reverse-phase HPLC with glucosyl conjugates of the precursors (GA₄ or GA₂₀) and their probable major metabolite (GA₁). The majority of conjugate associated with the precursor GA₄ eluted coincidental with GA₄ glucoside. Subsequent acid or enzymic hydrolysis (β-glucosidase or cellulase) yielded the free GAs, putative identification being based on isocratic HPLC of each ³H-labeled conjugate → hydrolysis → isocratic HPLC of the ³H-labeled hydrolysate. Upon imbibition of the seeds, radioactivity associated with the conjugate fraction decreased; concomitantly, statistically significant increases in levels of free [³H]GA-like compounds were observed. Although the specific ratios of GA-like and GA-glucosyl conjugate-like substances varied substantially across years, hybrids, and even, in different plants from the same hybrid, this `reversible conjugation' (i.e. apparent conjugation during seed maturation followed by release of the GA moiety during germination), was reproducible for [³H]GA₂₀ in seed from two maize hybrids produced over 2 years.     

Metabolism of [3H]gibberellin A4 in somatic suspension cell cultures of carrot

The native gibberellin A₄ (GA₄), in radioactive form ([1,2-³H]GA₄, 1.06 Ci/mmol), was fed to carrot somatic cell cultures (suspension and immobilized cell systems) and its metabolism over a 48 hr period was investigated. It was found that the [³H]GA₄ was metabolized to at least two GAs, [³H]GA₁ and [³H]GA₈, six GA glucosyl conjugates, [³H]GA₁-0(3)-glucoside, [³H]GA₁-0(13)-glucoside, [³H]GA₁-glucosyl ester, [³H]GA₄-glucoside, [³H]GA₄-glucosyl ester, a [³H]GA₈ glucosyl conjugate(s) and a previously unknown [³H]GA₁ glucosyl conjugate ([³H]GA₁-0(3,13)-diglucoside-like compound). The GA₁-diglucoside-like compound was found only in extracts of cells and was present in significant amounts (33 % of total extractable radioactivity). All other metabolites were present in both cells and medium. For extracts of the medium, no differences between the suspension and immobilized cultures existed in types of [³H]GA₄ metabolites although quantitative differences were apparent.     

Peculiarities of sexual reproduction inFagus crenata forests in relation to annual production of reproductive organs

Annual production rates of reproductive organs inFagus crenata forests in the lower area of the species' range were studied using 10 litter traps in 1980–1986. The production rates of dispersed pollen were estimated by multiplying the number of fallen male inflorescences per ha per year by the mean amount of pollen per inflorescence before anthesis. Large annual fluctuations in the production rates of male and female inflorescences were recognized, whereas their annual trends were synchronized with each other. Pollen production rates were within the range 1.0–6900 (mean: 1630)×10⁹ha⁻¹ yr⁻¹, the maximum/minimum ratio attaining 7000.F. crenata was the lowest producer of pollen among seven tree species studied: the number of pollen grains equivalent to a single ovule was in the range 6.0–14×10⁴. Furthermore, the mean dry weight of a single pollen grain (3.77×10⁻⁵mg) was higher than for wind-pollinated species. Three factors seemed to cause the low seed fertility ofF. crenata. The dry-matter production rate in the best seed year reached 3252 kg ha⁻¹ yr⁻¹, of which pollen accounted for 259 kg ha⁻¹ yr⁻¹. Unproductive years with less than 10% of the maximum production occurred four times in a 7-yr period. In such years there were fewer male and female inflorescences, and more fruit dropped as a result of insect damage. Lower nut dissemination would play an important role in suppressing any increase in nut predators, and fewer flowers would be produced to avoid wastage of photosynthates in a cool-temperate climate.     

Studies on the Pungent Principle of Capsicum

The chemical constitutions of the pungent principle of Capsicum were investigated. These principles are assumed to consist of capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homodihydrocapsaicin and two or more analogues of these materials. Thin-layer chromatography and open tubular gas chromatography showed that the natural pungent mixture contains no cis-isomer of capsaicin. The chemical structure of nordihydrocapsaicin was determined as N-(4-hydroxy-3-methoxybenzyl)-7-methyloctanamide by gas chromatography, infrared spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy. In addition, homodihydrocapsaicin was identified as N-(4-hydroxy-3-methoxybenzyl)-9-methyl-decanamide. These identities were also proven by comparison with synthetic samples.     

Metabolism of Glycine and Threonine in Growing Rats at Various Dietary Protein Levels

The metabolic fates of the carbon skeletons of [U-¹⁴C]glycine and l-[U-¹⁴C]threonine were investigated in growing rats fed with diets containing different percentages of protein calories (0, 5, 10, 15, and 30PC%) at 4100 kcal of metabolizable energy per kg of diet.

The incorporation of ¹⁴C into the body protein at 12 hr after the injection of ¹⁴C-glycine was about 58% of the dose in rats fed with the 10 or 15 PC% diet, and the values were reduced in both the lower and higher PC% groups. A considerable amount of ¹⁴C was recovered in the soluble fraction, and it was attributed to labeled glycine and serine in the free amino acid pools of the tissues.

The incorporation of ¹⁴C into the body protein from ¹⁴C-threonine was extremely high in the dietary groups of 0 to 10 PC%, and it decreased in the 30PC% group. Conversely, the expired ¹⁴C0₂ production was much less until the dietary protein level reached at 10PC%, and it increased with higher PC% in the diets. The change in the activity of hepatic threonine dehydratase in rats fed diets with increasing protein levels was similar to that of the expired ¹⁴C0₂ production from ¹⁴C- threonine.

These results indicate that, though the metabolic patterns for glycine and threonine differ from each other, their responses to dietary protein levels change at 10 to 15 PC%, where the growth rate reached its approximate maximum.     

Metabolism of [3H] Gibberellin A5 in Cell Suspension Cultures of Pharbitis nil

Gibberellin A₅ (GA₅), a native GA of immature seeds of Pharbitis nil, was fed to Pharbitis nil cell suspension cultures as [C-l, ³H] GA₅ (3.1 Ci/mmol), and its metabolism over a 48 hr period was investigated. Radioactivity in free GA metabolites was 13.1%, with 79.9% in GA glucosyl conjugate-like metabolites. Only 7.0% of the radioactivity remained as [³H] GA₅. Tentative identifications were based on comparison with retention times of authentic free GAs and/or glucosyl conjugates after sequential chromatography on Si gel partition column → gradient-eluted C18 HPLC-radiocounting (RC) → isocratic-eluted C18 HPLC-RC, and showed that [³H] GA₅ was converted to [³H] GA₁ (2%), [³H] GA₃ (4%), [³H] GA₆ (2%), [³H] GA₂₂ (1%) and their glucosyl conjugates, and also to [³H] GA₈ glucoside, and [³H] GA₅ glucosyl conjugates. The major conjugate-like substances were [³H] GA₁ and [³H] GA₃ glucosyl esters, at 15% and 34%, respectively, of the total extractable radioactivity.