Body composition and estrous cyclicity responses of heifers of distinct body conditions to energy restriction and repletion.

Twenty Simmental x Angus, half-sibling, postpubertal heifers (initial BW of 443 +/- 9 kg) were allotted randomly to 2 treatments to evaluate if initial BCS affects how heifers respond to energy restriction and repletion. Dependent variables of interest were changes in BW, BCS, and reproductive status [determined by concentrations of serum progesterone (P(4))]. Empty body composition (EBC) was calculated using equations based on BCS. During a preliminary feeding period, diets were formulated so that each heifer in the designated treatment would reach a BCS of 5 (moderate condition; MOD) or a BCS of 7 (heavy condition; FAT). Once each heifer had reached the desired BCS, diets were formulated to supply 30% of NE(m) requirements until each heifer became anestrous (serum concentrations of P(4) < 1 ng/mL; restriction period). After anestrus, heifers were fed a high energy diet (1.43 Mcal/kg of DM) until estrous cycles resumed (serum concentrations of P(4) > 1 ng/mL; repletion period). Body weight, BCS, and EBC were determined on d 1 of each period, on d 43 of restriction, and d 44 of repletion, and when heifers were confirmed to have resumed estrous cycles (2 normal estrous cycles determined by P(4) > 1 ng/mL). Regression of BCS on BW determined individual BCS at anestrus and estrus. After 43 d of restriction, FAT condition heifers were heavier (P < 0.001), had greater BCS (P < 0.001), and had a greater proportion of empty body fat (P < 0.001). Heifers in FAT condition remained cyclic longer (P < 0.001) than those in MOD condition (148 vs. 61 d). In contrast, at the onset of anestrus, BW (P = 0.15), BCS (P = 0.54), and empty body fat were similar (P = 0.54) between treatments. At 44 d of repletion, BW (P = 0.46), BCS (P = 0.41), and empty body fat (P = 0.41) were similar between treatments. Heifers in both treatments recommenced estrous activity after similar (P = 0.43) number of days (54 d) of energy repletion, but near onset of estrous cycles, heifers in FAT condition were heavier (P = 0.002) and had greater BCS (P = 0.03) and empty body fat (P = 0.01) than those in MOD condition. Initial BCS influenced days to anestrus, but not BCS or EBC at onset of anestrus. Initial BCS had no effect on days to recommencement of estrous cycles, but did influence the degree of fatness required to resume estrous cycles.

Initial body condition score affects hormone and metabolite response to nutritional restriction and repletion in yearling postpubertal beef heifers.

Twenty Simmental x Angus, half-sibling, postpubertal heifers (initial BW of 443 +/- 9 kg) were allotted randomly into 2 treatment groups to evaluate if initial BCS affects response of the hypothalamic-pituitary-ovarian axis to metabolic signals elicited by energy restriction and repletion. During a preliminary feeding period, diets were formulated so that each heifer in the designated treatment would reach a BCS of 5 (moderate condition; MOD) or a BCS of 7 (heavy condition; FAT). Once each heifer had reached desired BCS, diets were formulated to supply 30% of NE(m) requirements until each heifer became anestrous (serum concentrations of progesterone < 1 ng/mL; restriction period). Blood collections took place on d 1 of each period, on d 43 of energy restriction and d 44 of energy repletion, and when heifers were confirmed to recommence estrous cycles. When heifers were cycling, their estrous cycles were synchronized to ensure hormone sampling occurred during late diestrus or early proestrus. Energy restriction resulted in decreased concentrations of LH (FAT, P = 0.02; MOD, P < 0.001), IGF-1 (FAT, P < 0.001; MOD, P = 0.003), and insulin (P < 0.001); in contrast, concentrations of GH (P < 0.001) and plasma urea nitrogen (P < 0.001) increased. During repletion, LH concentration increased (P = 0.03) in MOD condition heifers but was still less (P = 0.002) than d 1 of restriction, whereas LH concentration tended to increase in FAT heifers (P = 0.06) until it was similar (P = 0.40) to d 1 of restriction. Repletion also increased concentrations of IGF-1 (P < 0.001), insulin (P < 0.001), and glucose (P < 0.001), whereas concentrations of GH (P < 0.001), NEFA (P < 0.001), and plasma urea nitrogen (P < 0.001) decreased. For both treatments, concentrations of GH after repletion were similar (FAT, P = 0.88; MOD, P = 0.10) to those on d 1 of restriction. After repletion, FAT condition heifers had decreased concentrations of IGF-1 (P < 0.001), insulin (P < 0.05), and glucose (P < 0.001), but greater concentrations of acetate (P < 0.01) and butyrate (P < 0.05), than MOD heifers. Anestrus or resumption of estrous cycles seems to be activated gradually in response to dietary manipulation, unrelated to certain metabolite changes.

Inhibition of cellular transformation by berry extracts.

Recent studies have examined and demonstrated the potential cancer chemopreventive activity of freeze-dried berries including strawberries and black raspberries. Although ellagic acid, an abundant component in these berries, has been shown to inhibit carcinogenesis both in vivo and in vitro, several studies have reported that other compounds in the berries may also contribute to the observed inhibitory effect. In the present study, freeze-dried strawberries (Fragara ananassa, FA) or black raspberries (Rubus ursinus, RU) were extracted, partitioned and chromatographed into several fractions (FA-F001, FA-F003, FA-F004, FA-F005, FA-DM, FA-ME from strawberries and RU-F001, RU-F003, RU-F004, RU-F005, RU-DM, RU-ME from black raspberries). These extracts, along with ellagic acid, were analyzed for anti-transformation activity in the Syrian hamster embryo (SHE) cell transformation model. None of the extracts nor ellagic acid by themselves produced an increase in morphological transformation. For assessment of chemopreventive activity, SHE cells were treated with each agent and benzo[a]pyrene (B[a]P) for 7 days. Ellagic acid, FA-ME and RU-ME fractions produced a dose-dependent decrease in transformation compared with B[a]P treatment only, while other fractions failed to induce a significant decrease. Ellagic acid, FA-ME and RU-ME were further examined using a 24 h co-treatment with B[a]P or a 6 day treatment following 24 h with B[a]P. Ellagic acid showed inhibitory ability in both protocols. FA-ME and RU-ME significantly reduced B[a]P-induced transformation only when co-treated with B[a]P for 24 h. These results suggest that a methanol extract from strawberries and black raspberries may display chemopreventive activity. The possible mechanism by which these methanol fractions (FA-ME, RU-ME) inhibited cell transformation appear to involve interference of uptake, activation, detoxification of B[a]P and/or intervention of DNA binding and DNA repair.

[Studies on chemical constituents of Fragaria ananassa Duch].

OBJECTIVE: To study the chemical constituents from the fruit of Fragaria ananassa. METHOD: Using chromatographic methods to isolate compounds and chemical and spectral methods to elucidate their structures. RESULT: Three compounds, 9, 19-cyclolanost-24-en-3-ol(1), 14-methyl-stigmasta-7, 24(28)-dien-3-ol(2) and beta-sitosterol(3) were isolated from the freeze-dried powder. CONCLUSION: All of the compounds were obtained from this plant for the first time.

[Study on the constituents from freeze-dried power of blackberries (Rubus ursinus)].

Three Compounds were isolated from freeze-dried powder of Blackberries (Rubus ursinus L.) which showed an activity on inhibition of chemocarcinogen. The structures of them were identified as stigmasta-5,22-dien-3-ol],beta-sitosterol and beta-sitosterol-3 beta-D-glucose. All these compounds were isolated at the first time from the plant.

Studies on epimeric D-xylo-and D-lyxo-tetritol-1-yl-2-phenyl-2H-1,2,3-triazoles. Synthesis and anomeric configuration of 4-(alpha-and beta-D-threofuranosyl)-2-phenyl-2H-1,2,3-triazole C-nucleoside analogs.

Treatment of 4-(D-xylo-tetritol-1-yl)-2-phenyl-2H-1,2,3-triazole (1) with one mole equivalent of tosyl chloride in pyridine solution, afforded the C-nucleoside analog; 4-(beta-D-threofuranosyl)-2-phenyl-2H-1,2,3-triazole (2) in 55% yield, as well as the byproduct 4-(4-chloro-4-deoxy-D-xylo-tetritol-1-yl)-2-phenyl-2H-1,2,3-triazo le (4). Treatment of the epimeric 4-(D-lyxo-tetritol-1-yl)-2-phenyl-2H-1,2,3-triazole (6) with tosyl chloride in pyridine solution afforded the anomeric C-nucleoside analog; 4-(alpha-D-threofuranosyl)-2-phenyl-2H-1,2,3-triazole (7) in 29% yield, as well as the byproduct 4-(4-chloro-4-deoxy-D-lyxo-tetritol-1-yl)-2-phenyl-2H-1,2,3- triazole (9). Similar treatment of 1 and 6 with trifluoromethanesulfonyl chloride in pyridine solution afforded 2 and 7, respectively. The structure and anomeric configuration of these compounds were determined by acetylation, NMR, NOE, and circular dichroism spectroscopy, as well as mass spectrometry.

New cytotoxic 1-azaanthraquinones and 3-aminonaphthoquinone from the stem bark of Goniothalamus marcanii.

Guided by brine shrimp toxicity and human tumor cell toxicity, fractionation of the alcoholic extract from the stem bark of Goniothalamus marcanii led to the isolation of four new 1-azaanthraquinones: marcanines B (3), C (4), D (5), and E (6), along with two known derivatives: marcanine A and dielsiquinone. A new 5-hydroxy-3-amino-2-aceto-1,4-naphthoquinone (7), a possible 1-azaanthraquinone biosynthetic precursor, was also isolated. The structures of the compounds were elucidated by spectroscopic analyses, mainly 1D and 2D NMR techniques ((1)H, (13)C, NOEDS, COSY, HMQC, and HMBC), as well as comparison with literature data. All the compounds except 6 were evaluated for cytotoxic activity. They exhibited significant cytotoxicity against several human tumor cell lines, A-549, HT-29, MCF7, RPMI, and U251 with the ED(50) in the range of 0.04-3.03 microM.

Topoisomerase II inhibition by aporphine alkaloids.

The aporphine alkaloids (+)-dicentrine and (+)-bulbocapnine are non-planar molecules lacking features normally associated with DNA binding by intercalation or minor groove binding. Surprisingly, dicentrine showed significant activity as a topoisomerase II (EC 5.99.1.3) inhibitor and also was active in a DNA unwinding assay. The DNA unwinding suggests DNA intercalation, which could explain the inhibition of topoisomerase II. Bulbocapnine, which differs from dicentrine only by the presence of a hydroxyl group at position 11 and the absence of a methoxyl group at position 9, was inactive in all assays. Molecular modeling showed that dicentrine can attain a relatively planar conformation, whereas bulbocapnine cannot, due to steric interaction between the 11-hydroxyl group and an oxygen of the methylenedioxy ring. These observations suggest that dicentrine is an "adaptive" DNA intercalator, which can bind DNA only by adopting a somewhat strained planar conformation. The requirement of a suboptimal conformation to achieve DNA binding appears to make dicentrine a weaker topoisomerase II inhibitor than the very planar oxoaporphine alkaloid liriodenine. These results suggest that it may be possible to modulate DNA binding and biologic activity of drugs by modifications affecting their ability to adopt planar conformations.

DNA polymerase and topoisomerase II inhibitors from Psoralea corylifolia.

An ethanol extract of Psoralea corylifolia caused strong DNA polymerase inhibition in a whole cell bioassay specific for inhibitors of DNA replication enzymes. Bioassay-directed purification of the active compounds led to the isolation of the new compound corylifolin (1) and the known compound bakuchiol (2) as DNA polymerase inhibitors. On the basis of the structures of 1 and 2, resveratrol (3) was tested and found to be active as a DNA polymerase inhibitor in this bioassay. Neobavaisoflavone (4) was isolated as a DNA polymerase inhibitor, daidzein (5) as a DNA polymerase and topoisomerase II inhibitor, and bakuchicin (6) as a topoisomerase II inhibitor.

Inhibition of topoisomerase II by liriodenine.

The cytotoxic oxoaporphine alkaloid liriodenine, isolated from Cananga odorata, was found to be a potent inhibitor of topoisomerase II (EC 5.99.1.3) both in vivo and in vitro. Liriodenine treatment of SV40 (simian virus 40)-infected CV-1 cells caused highly catenated SV40 daughter chromosomes, a signature of topoisomerase II inhibition. Strong catalytic inhibition of topoisomerase II by liriodenine was confirmed by in vitro assays with purified human topoisomerase II and kinetoplast DNA. Liriodenine also caused low-level protein-DNA cross-links to pulse-labeled SV40 chromosomes in vivo, suggesting that it may be a weak topoisomerase II poison. This was supported by the finding that liriodenine caused topoisomerase II-DNA cross-links in an in vitro assay for topoisomerase II poisons. Verapamil did not increase either liriodenine-induced protein-DNA cross-links or catalytic inhibition of topoisomerase II in SV40-infected cells. This indicates that liriodenine is not a substrate for the verapamil-sensitive drug efflux pump in CV-1 cells.

Increased firing of neurons in the posterior hypothalamus which precede classically conditioned pupillary dilations.

Paralyzed cats were used as subjects in a classical conditioning experiment where each subject was exposed to 40 explicitly unpaired 1-s bursts of white noise and 0.5-s paw shocks. This training was followed by 60 trials of the two stimuli paired, where the white noise immediately preceded the paw shock. Following this training, the subjects were re-exposed to 40 trials of the explicitly unpaired procedure. The pupil was monitored as the behavior and electrodes implanted in the thalamus, the dorsal hypothalamus and the posterior hypothalamus recorded the activity of clusters of cells. Only the cells in the posterior hypothalamus showed robust changes in firing rates that preceded the pupillary behavior, both (a) on any particular trial and (b) as the learned association was being demonstrated behaviorally across trials.

Avoidance and classical conditioning of leg flexion in dogs.

This study of leg flexion conditioning in dogs, which were trained under both avoidance and classical contingencies consecutively, confirmed and extended the results from the Wahlsten and Cole study (In Classical Conditioning II: Current Research and Theory, Appleton Century-Crofts, New York, 1972). Briefly, dogs were trained to asymptotic behavioral levels under either avoidance or classical contingencies with a CS-US interval of either three (3) or five (5) seconds where the unconditioned stimulus (US) was shock to the foreleg and the conditioned stimulus (CS) was 1000 Hz tone. The dogs were then switched to the other contingency (without any modification in the stimulus situation other than the shock contingency) and trained to asymptotic behavioral levels. The CS remained on for the entire CS-US period and terminated with the end of the scheduled interval. Under the classical contingency, the US occurred as scheduled on every trial regardless of the dog's behavior. Under the avoidance contingency, the US was prevented from occurring if the subject responded with a criterion leg lift during the CS-US interval. The only feedback to the dog of a successful performance was the leg lift itself. The results indicated that there were two different conditioned responses produced, one just after CS onset under the avoidance contingency, and one just before US onset under the classical contingency for both CS-US intervals. The findings were interpreted as supporting a single-factor informational view of learning and a neural model was presented.

Grossamide and N-trans-caffeoyltyramine from Annona crassiflora seeds.

Grossamide, and N-trans-caffeoyltyramine, were isolated for the first time from the seeds of Annona crassiflora Mart, and in the Annonaceae family.

Posttranslational acylation of the transferrin receptor in LSTRA cells with myristate, palmitate and stearate: evidence for distinct acyltransferases.

When incubated with [3H]myristate or [3H]palmitate, LSTRA cells, a murine T cell line, incorporated radiolabel into a protein of 95 kDa as analyzed by SDS-polyacrylamide gel electrophoresis. This dually acylated protein was identified as the transferrin receptor by immunoprecipitation with a monoclonal anti-transferrin receptor antibody. Acylation of the transferrin receptor was posttranslational and occurred via ester or thioester linkages. Analysis of radiolabeled transferrin receptor protein from [3H]myristate-labeled cells by acid hydrolysis followed by thin layer chromatography revealed the exclusive presence of [3H]myristate. Labeled transferrin receptor protein from [3H]palmitate-labeled cells contained predominantly [3H]stearate and smaller amounts of [3H]palmitate. This is in contrast to the protein-tyrosine kinase p56lck, which in [3H]palmitate-treated LSTRA cells, incorporated primarily [3H]palmitate. An analog of myristic acid, 5-nonanyloxyfuran-2-carboxylic acid, inhibited the incorporation of [3H]myristate, but not [3H]palmitate or [3H]stearate into transferrin receptor protein, suggesting that these acylation events are distinct. These studies indicate that the murine transferrin receptor is acylated posttranslationally with myristate, palmitate and stearate and suggest that more than one acyltransferase activity is responsible for its acylation.

Mechanism of action of the antileukemic xanthone psorospermin: DNA strand breaks, abasic sites, and protein-DNA cross-links.

Psorospermin, a cytotoxic dihydrofuranoxanthone isolated from Psorospermum febrifugum, produced aberrant simian virus 40 DNA replication intermediates when added to lytically infected CV-1 monkey kidney cells. The aberrant viral intermediates showed dose-dependent DNA strand breaks and protein-DNA cross-links, as well as decreased electrophoretic mobility. Simian virus 40 DNA from psorospermin-treated cells was shown to contain numerous abasic (apyrimidinic/apurinic) sites. The density of abasic sites was a function of the psorospermin dose. We conclude that psorospermin causes extensive loss of DNA bases in vivo. Primary amine groups of cellular proteins are known to react with abasic sites to form covalent protein-DNA cross-links and DNA strand breaks. Cytochrome c cross-linked spontaneously to viral DNA prepared from psorospermin-treated cells but not to DNA from untreated cells. This suggests that the protein-DNA cross-links and many of the DNA strand breaks observed in vivo result from reactions between abasic sites and chromosomal proteins. It is likely that the protein-DNA cross-links and DNA strand breaks contribute to the cytotoxicity and antineoplastic activity of psorospermin.

Synthesis of myristoyl CoA analogues and myristoyl peptides as inhibitors of myristoyl CoA:protein N-myristoyltransferase.

To develop inhibitors of myristoyl CoA:protein N-myristoyltransferase (NMT), a series of myristoyl coenzyme A analogues and myristoyl peptides were synthesized, including S-(2-oxopentadecyl)-CoA (1), S-(2-hydroxypentadecyl)-CoA (2), S-(2-oxopentadecyl)-pantetheine (3), Myr-N-Gly-(L)-Phe (4), Myr-N-Gly-(L)-Tyr (5), and Myr-N-Gly-(L)-Asn-Ala- Ala-Ser-Ala-Arg-(NH2) (6). Biological evaluation of these compounds in an in vitro NMT enzyme assay revealed that the nonhydrolyzable acyl CoA analogue 1 was the most potent inhibitor [inhibitor dissociation constant (Ki) = 24 nM]. A preliminary structure-activity relationship study showed that the adenosine moiety and the 2-keto group in this nonhydrolyzable analogue were necessary for inhibitory activity. A possible mechanism for the inhibition of NMT by 1 was proposed, in which 1 might block the reaction at the stage of an acyl-CoA-NMT-peptide complex. Product analogues such as the myristoylated peptides 4-6 were poor inhibitors of NMT.

Ohioensins and pallidisetins: novel cytotoxic agents from the moss Polytrichum pallidisetum.

Bioassay-directed fractionation of an EtOH extract of the moss Polytrichum pallidisetum (Polytrichaceae) led to the isolation of three novel benzonaphthoxanthenones, 1-O-methylohioensin B [6], 1-O-methyldihydroohioensin B [7] and 1,14-di-O-methyldihydroohioensin B [8], and two novel cinnamoyl bibenzyls, pallidisetin A [9] and pallidisetin B [10]. Their structures and relative stereochemistry were established by spectral analyses and chemical correlation. Compounds 6-10 exhibited cytotoxic activity against the human tumor cell lines RPMI-7951 melanoma and U-251 glioblastoma multiforme. These two types of compounds could hypothetically be derived from cinnamic acid and bibenzyls through different biogenetic pathways.

Treatment of T cells with 2-hydroxymyristic acid inhibits the myristoylation and alters the stability of p56lck.

N-Myristoylation of p56lck, a member of the Src family of protein-tyrosine kinases, is essential for its proper targeting to the plasma membrane. 2-Hydroxymyristic acid (HMA) is an analog of myristic acid that becomes metabolically activated in cells to form 2-hydroxymyristoyl-CoA, a potent inhibitor of myristoyl-CoA:protein N-myristoyltransferase (NMT), the enzyme that catalyzes protein N-myristoylation [Paige, L. A., Zheng, G.-q., DeFrees, S. A., Cassady, J. M., & Geahlen, R. L. (1990) Biochemistry 29, 10566]. In the presence of HMA, LSTRA cells, which overexpress p56lck, synthesized nonmyristoylated p56lck, which displayed a reduced electrophoretic mobility on SDS-polyacrylamide gels identical to that of a nonmyristoylated Gly2-->Ala2 mutant of p56lck. Treatment with myristic acid, 2-hydroxypalmitic acid, or 2-fluoromyristic acid did not result in the synthesis of nonmyristoylated p56lck. In contrast to the membrane-associated, myristoylated p56lck, nonmyristoylated p56lck was cytosolic. Although nonmyristoylated p56lck retained tyrosine kinase activity, it was not labeled in vivo with [32P]orthophosphate, indicating that a change in subcellular location altered its state of phosphorylation. A pulse-chase analysis revealed that cytosolic, nonmyristoylated p56lck was less stable than the myristoylated enzyme. In cell lines that do not overexpress p56lck, HMA treatment resulted in a reduction in the levels of both newly synthesized and total p56lck. Treatment of CD4+ cells with HMA caused a corresponding decrease in the amount of CD4-associated p56lck. Thus, chemical inhibition of protein N-myristoylation with HMA is an effective method for reducing the amount of p56lck available at the plasma membrane for signal transduction.

Reversible palmitoylation of the protein-tyrosine kinase p56lck.

The myristoylated protein-tyrosine kinase, p56lck, is expressed predominantly in T cells where it is believed to play a role in T cell activation. We observed a 56-kDa protein that became metabolically labeled in intact T lymphoid cells that were incubated with either [3H]myristate or [3H]palmitate. This protein was identified as p56lck based on its specific immunoprecipitation with polyclonal antisera to p56lck, by induction of a shift in its electrophoretic mobility following treatment of cells with 12-O-tetradecanoylphorbol-13-acetate and by co-chromatography with p56lck on protamine-agarose. Characterization of the two acylation events revealed that, in contrast to the p56lck-associated radioactivity from [3H]myristate-labeled cells, the p56lck-associated radioactivity from [3H]palmitate-labeled cells was susceptible to cleavage by neutral hydroxylamine and was not blocked by inhibitors of protein synthesis. Pulse-chase analyses revealed that the labeling of p56lck with [3H]palmitate, but not [3H]myristate, was reversible. The presence of covalently attached palmitate on p56lck from [3H]palmitate-labeled cells was verified by thin-layer chromatography following acid hydrolysis of the acylated protein. 2-Hydroxymyristate, which is metabolically activated to form a potent inhibitor of protein myristoylation, specifically inhibited the acylation of p56lck with [3H]myristate without affecting its labeling with [3H]palmitate. These studies indicate that p56lck is both a cotranslationally myristoylated and post-translationally palmitoylated protein.