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Four new cardenolide monoglycosides, cardenolides N-1 (1), N-2 (2), N-3 (3), and N-4 (4), were isolated from Nerium oleander, together with two known cardenolides, 5 and 12, and seven cardenolide monoglycosides, 6-11 and 13. The structures of compounds 1-4 were established on the basis of their spectroscopic data. The in vitro anti-inflammatory activity of compounds 1-13 was examined on the basis of inhibitory activity against the induction of the intercellular adhesion molecule-1 (ICAM-1). Compounds 1, 5, 6, and 11-13 were active at an IC50 value of less than 1 microM. The cytotoxicity of compounds 1-13 was evaluated against three human cell lines, normal human fibroblast cells (WI-38), malignant tumor cells induced from WI-38 (VA-13), and human liver tumor cells (HepG2). Compounds 1, 4, 6, and 11-13 were active toward V-13 cells, and compounds 1, 11, and 12 were active toward HepG2 cells at IC50 values of less than 1 microM. Compounds 4, 5, 10, and 12 showed selective cell growth inhibitory activity toward V-13 tumor cells compared with that of parental normal WI-38 cells. The MDR-reversal activity of compounds 1-13 was evaluated on the basis of the amount of calcein accumulated in MDR human ovarian cancer 2780AD cells in the presence of each compound. Compounds 4, 9, and 10 showed significant effects on calcein accumulation, compound 4 showing stronger activity than that of verapamil.

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Neutron diffraction analysis studies reported an isolated hydronium ion (H3O+) in the interior of d‐xylose isomerase (XI) and phycocyanobilin‐ferredoxin oxidoreductase (PcyA). H3O+ forms hydrogen bonds (H‐bonds) with two histidine side‐chains and a backbone carbonyl group in PcyA, whereas H3O+ forms H‐bonds with three acidic residues in XI. Using a quantum mechanical/molecular mechanical (QM/MM) approach, we analyzed stabilization of H3O+ by the protein environment. QM/MM calculations indicated that H3O+ was unstable in the PcyA crystal structure, releasing a proton to an H‐bond partner His88, producing H2O and protonated His88. On the other hand, H3O+ was stable in the XI crystal structure. H‐bond partners of isolated H3O+ would be practically limited to acidic residues such as aspartic and glutamic acids in the protein environment.

myo-Inositol biosynthesis has been examined in hypophysectomized and thyroidectomized male rats. After hypophysectomy, inositol-l-phosphate synthase [IL-myo-inositol-lphosphate lyase (isomerizing), EC 5.5. 1.4] Among the many tissues capable of synthesizing myo-inositol, the testis ranks as the richest mammalian source of inositol-1-phosphate synthase [lL-myo-inositol-l-phosphate synthase, EC 5.5.1.4], the NAD+-dependent enzyme that isomerizes glucose 6-phosphate to myo-inositol 1-phosphate (1) referred to here as simply inositol 1-phosphate. Because the testis is the target organ for various hormones and is itself the site of androgen biosynthesis, we were led to inquire into possible hormonal control of inositol biosynthesis. Although there have been sporadic reports on the effects of various physiological and pathological states on the levels of free inositol and inositol-l-phosphate synthase in certain tissues (2-7), there has been no systematic study of hormonal control of the enzyme. Here we report the effect in male rats of hypophysectomy, adrenalectomy, and thyroidectomy on tissue levels of inositol-l-phosphate synthase and preliminary experiments in the reversal of these effects by gonadotropic and thyroid hormones. MATERIALS AND METHODSMale Sprague-Dawley rats weighing approximately 200 g were obtained from Taconic Farms (Germantown, NY) and maintained as follows. Normal animals received regular tap water; hypophysectomized, 5% dextrose; thyroidectomized, 1% calcium lactate; and adrenalectomized, isotonic saline. Sham-operated rats were given the same fluids; all animals received regular laboratory chow ad lib. Tissues for enzyme assay were removed and weighed immediately after killing by decapitation. Seminal vesicles were washed with cold isotonic saline after removal of seminal fluid through an incision. Brain, liver, spleen, thymus, pancreas, kidney, testis, and ventral prostate were homogenized in a Potter-Elvehjem homogenizer with 2-3 vol of 0.154 M KCI/0.2 mM dithiothreitol. Epididymis and seminal vesicles were minced and then homogenized in a Polytron homogenizer with 3-5 vol of the same solution. The homogenates were centrifuged 20 min at 27,000 X g, and the supernatants were heated at 60'C for 2 min exactly, chilled in ice, and centrifuged at 105,000 x g for 1 hr. Solid ammonium sulfate was added to 40% of saturation (0.226 g/ml). The precipitate was collected by centrifugation, dissolved in 0.6-0.7 ml of 50 mM Tris acetate, pH 7.4/0.2 mM dithiothreitol and dialyzed against the same buffer overnight. The suspension was freed of insoluble material by centrifugation and assayed for inositol-1-phosphate synthase by incubation with glucose 6-phosphate (5 mM) and NAD' (1 mM); the resulting inositol 1-phosphate was determined as inorganic phosphate by the periodate method of Barnett et al. (8). Protein was determined by the method of Lowry et al. (9), with correction for the effect ofTris.

The presence of two types of /^-cyanoalanine synfhase in germinating seeds and fheir responses to ethylene. -Physiol. Plant. 93: 71.3-718.Germinafine seeds of many species contain fwo fypes of /i-cyanoalanine synthase (CAS. EC 4.4.1.9) thaf conveii HCN fo /?-cyanoa'lanine. One is cyfoplasmic CAS (cyt-C.AS), which is precipitated by 50 lo 609r (NHai^SO^ and has a pH optimum of 10.5. Cvtoplasmic C.^S is present at high levels in dry seed and its activity does nof increase during imbibition. The activity ot cyt-CAS is uot affected by exogenously applied efhylene (CjHji. except in riee (Or\~a sativa cv. Sasanishiki). The second fype of CAS ftiuud in seed is mitochondrial CAS (mit-CAS I. which is precipifated by 60 fo 1()''« (NHjl:SO4 and has a pH optimum of 9.5. Mitochondrial CAS is present at low levels iu dry seed, and its activity increases greatly during imbibition in the seeds of ali species fested. Exposure fo C:H^ stimulated mit-C.i^S activity in seeds of rice, barley (Hordeum yidgare cy, Hadakamugii, cucumber (Cucumis satiyus cv. Kagafushinari) and cocklebur {Xanihhmi pennsyhamcum). The increase in fhe mit-CAS activity in cocklebur in response to C^Hj ccmimeticed after a fag period of 2 to 3 h when fhe duration of soaking was short (16 hi. but commenced without a lag period when fhe seeds were soaked lor Ihree mouths. Application ot bofh chioramphenicol aud cycloheximide to the axial and eotyledonary tissues of cocklebur seeds strongly inhibifed growth as well as fhe increase in mit-CAS acfivity. It is postulated fhal the mit-CAS is syuthesized de noi-o dtiring imbibition and thai ifs acfivity is regulated by C;H4. CO;, vvhich also promotes seed genninatiou in some species. v»as ineffective in stimulating tnif-C.AS activity iu cocklebur seeds.

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