H2O2对蛇足石杉离体培养物诱变效应的研究

为了获得高产石杉碱甲(Huperzine A,Hup A)的蛇足石杉[Huperzia serrata(Thunb.)Trev.]叶状体,对H_2O_2诱变后的叶状体进行了研究。结果表明,诱变后叶状体株系的Hup A含量显著提高,并获得高产株系SH42,其相对生长率和Hup A含量分别达到4499.28%和261.17μg g~(–1) DW,比起始叶状体分别提高了2.35倍和2.43倍;且株系间可溶性蛋白质谱带和SOD同工酶谱均存在差异,经过连续9代培养,变异叶状体可以稳定遗传。因此,H_2O_2对叶状体细胞具有良好的诱变效应,可以用于筛选高产Hup A株系。     

The discovery of novel 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamides as vasopressin V1A receptor antagonists

The discovery of a novel series of 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamide antagonists of the vasopressin V_1A receptor is disclosed. Compounds 47 and 48 were found to be high affinity, selective vasopressin V_1A antagonists.     

Interaction between subunit C (Vma5p) of the yeast vacuolar ATPase and the stalk of the C-depleted V1 ATPase from Manduca sexta midgut

Projection maps of a V₁-Vma5p hybrid complex, composed of subunit C (Vma5p) of Saccharomyces cerevisiae V-ATPase and the C-depleted V₁ from Manduca sexta, were determined from single particle electron microscopy. V₁-Vma5p consists of a headpiece and an elongated wedgelike stalk with a 2.1×3.0 nm protuberance and a 9.5×7.5 globular domain, interpreted to include Vma5p. The interaction face of Vma5p in V₁ was explored by chemical modification experiments.     

C60对体外培养癌细胞的光致作用研究

研究结果表明用C₆₀-磷脂酰胆碱与体外培养的HeLa细胞融合后,以4 000 lx光强度激发C₆₀(C₆₀浓度20 mg／L)对HeLa细胞具有显著的光致杀伤作用.生化测定证实光激发C₆₀导致膜蛋白巯基含量减少、膜脂过氧化、丙二醛含量增高,SDS-PAGE证明膜蛋白交联,荧光偏振显示膜流动性降低,电镜超微膜结构破坏,经MTT法检测,大部分细胞死亡.C₆₀的强烈光致作用证实了Arbogast等认为光激发C₆₀可产生单线态氧的观态.     

Food competition and social experience effects on V1a receptor binding in the forebrain of male Long-Evans hooded rats

The present study investigated the effect of social status in Long-Evans hooded rats established during food competition on V(1a) vasopressin receptor (V(1a)R) binding in the lateral septum (LS), medial preoptic area (MPOA), bed nucleus of the stria terminalis (BNST), anterior hypothalamus (AH), and central/basolateral amygdala (CeB). Serum concentration of testosterone (T) and corticosterone (CORT) was also measured. In Experiment 1, thirty-two lever-trained weight-matched rat pairs were placed in operant chambers where a single bar press provided access to milk reinforcement. A dominant-subordinate relationship, determined by the duration of drinking, was evident in 88% of the pairs. Sixteen rats were lever-trained but did not interact and served as no-treatment (NT) controls. In the LS, V(1a)R binding in the subordinate (SUB) group was significantly higher than in the dominant (DOM) group. V(1a)R binding was significantly higher in the LS, BNST, CeB, and AH in the NT group than in the other groups. The levels of CORT and T were not affected significantly by group membership. Experiment 2 investigated whether the binding effect in the LS was related to differences in fluid consumption. The results did not indicate a significant effect of fluid consumption. In the rat, V(1a)R binding in several forebrain areas seems to be affected by brief periods of social interactions, and, in the LS, it also appears to be related to dominance status.     

The α1-adrenergic receptor in human erythrocyte membranes mediates interaction in vitro of epinephrine and thyroid hormone at the membrane Ca-ATPase

Membrane Ca²⁺-ATPase activity was stimulated in vitro separately by T₄ (10⁻¹⁰ M) and by epinephrine (10⁻⁶ M). In the presence of a fixed concentration of T₄, additions of 10⁻⁸ and 10⁻⁶ M epinephrine reduced the T₄ effect on the enzyme. β-Adrenergic blockade with propranolol (10⁻⁶ M) prevented stimulation by epinephrine of Ca²⁺-ATPase activity, but did not prevent the suppressive action of epinephrine on T₄-stimulable Ca²⁺-ATPase. In contrast α₁-adrenergic blockade with unlabelled prazosin restored the effect of T₄ on Ca²⁺-ATPase activity in the presence of epinephrine. Like propranolol, prazosin prevented enhancement of enzyme activity by epinephrine in the absence of thyroid hormone. Neither prazosin nor propranolol had any effect on the stimulations by T₄ of red cell Ca²⁺-ATPase in the absence of epinephrine. Analysis of radiolabelled prazosin binding to human red cell membranes revealed the presence of a single class of high-affinity binding sites (K_d, 1.2 × 10⁻⁸ M; B_max, 847 fmol/mg membrane protein). Thus, the human erythrocyte membrane contains α₁-radrenergic receptor sites that are capable of regulating Ca²⁺-ATPase activity.     

Peculiarities of the antioxidant and radioprotective effects of hydrated C60 fullerene nanostuctures in vitro and in vivo

Aqueous solutions of highly stable supramolecular donor–acceptor complexes of chemically nonmodified pristine C₆₀ fullerene molecules with H₂O molecules (hydrated C₆₀ fullerene–C₆₀HyFn) and their labile nano-sized clusters were examined for their antioxidant effects on removal of hydroxyl radicals (OH) and protecting DNA against oxidative damage induced by ionizing radiation in vitro. The suppressing influence of C₆₀HyFn on the formation of OH-radicals in water exposed to X-rays at doses of 1–7 Gy was assessed by determination of oxidation levels of coumarin-3-carboxylic acid. C₆₀HyFn demonstrates apparent antiradical activity in vitro in the range of concentrations of 10⁻¹¹–10⁻⁶ M. Paradoxically, the OH-removing efficacy of C₆₀HyFn was in reverse correlation with fullerene concentration. It was hypothesized that the antiradical action of C₆₀HyFn in water medium generally is due to a “nonstoichiometric” mechanism, supposedly to a hydrated free radical recombination (self-neutralization), which is catalyzed by specific water structures ordered by C₆₀HyFn. With the use of 8-oxoguanine as a marker of oxidative damage to DNA, it has been demonstrated that C₆₀HyFn in concentrations of 10⁻⁷–10⁻⁶ M protects nucleic acids against radical-induced damage. The second part of the present study was aimed to evaluate the overall radioprotective efficacy of C₆₀HyFn in doses of 0.1 or 1 mg/kg b.w. injected intraperitoneally to mice either 1 h before or 15 min after lethal dose exposure of the X-ray (7 Gy) irradiation. Survival rate of the mice was observed at 30 day intervals after irradiation, while the weight gains of experimental animals were monitored as well. The most significant protective effect was demonstrated when 1 mg/kg dosage of C₆₀HyFn was administered before irradiation. The outcome of the substance testing is 15% survival rate of irradiated animals at 30 days of observation, and prevention of noticeable weight loss characteristic for radiation impact, versus unprotected control animals. In conclusion, results of the study obviate that the apparent protective action of C₆₀HyFn in vivo is determined by its considerable ability to decrease X-ray-generated reactive oxygen species. Based on the results and that neat C₆₀ is nontoxic, actually in the hydrated form, without side effects and with sufficient radioprotective effects in low doses, C₆₀HyFn may be considered as a novel antioxidant agent, which substantially diminishes the harmful effects of ionizing radiation.     

Clusterin over-expression modulates proapoptotic and antiproliferative effects of 1,25(OH)2D3 in prostate cancer cells in vitro

Prostate cancer is the most commonly diagnosed cancer in the majority of western countries. Due to their antiproliferative and proapoptotic activity, vitamin D analogues have been introduced recently as an experimental therapy for prostate cancer. Clusterin (CLU) is a glycoprotein that has two known isoforms generated in human cells. A nuclear form of CLU protein (nCLU) is pro-apoptotic, and a secretory form (sCLU) is pro-survival. In this study, we analyzed whether proapoptotic and antiproliferative effects of 1,25(OH)₂D₃ on LNCaP prostate cancer cells are modulated by expression of sCLU. Using colony forming assay, we studied the effect of treatment with different doses of 1,25(OH)₂D₃ (10⁻⁶, 10⁻⁷, 10⁻¹⁰ M) on proliferation of LNCaP cells that were stable transfected and over-express sCLU (LNT-1) as compared to empty vector-transfected cells (LN/C). We also measured apoptosis using TUNEL assay. sCLU over-expression protected against both antiproliferative (30%) and proapoptotic (15%) effects of 1,25(OH)₂D₃, although this effect was statistically not significant. In conclusion, our findings demonstrate that expression of sCLU modulates growth regulatory effects of 1,25(OH)₂D₃ in prostate cancer indicating that CLU interferes with vitamin D signalling pathways.     

Magnetic relaxation in V15 cluster: Direct spin-phonon transitions

In this article we propose a model of spin-vibronic relaxation in K₆[V^IV₁₅As₆O₄₂(H₂O)]·8H₂O, the so called V₁₅ cluster exhibiting the unique layered structure. The work is motivated by the recent observation of the Rabi oscillation [1] in this system and aimed to elucidate the nature of the relaxation processes. The model assumes that the spin-phonon coupling arises as a result of modulation of the isotropic and antisymmetric (Dzyaloshinsky-Moriya) exchange interactions in the central triangular layer of vanadium ions by the acoustic lattice vibrations. Within the pseudo-angular momentum representation the selection rules for the direct (one-phonon) transitions between Zeeman levels are derived and a special role of the antisymmetric exchange is underlined. The relaxation times related to one-phonon transitions in different ranges of the field are estimated within the Debye model for the lattice vibrations.     

Assembly of subunit d (Vma6p) and G (Vma10p) and the NMR solution structure of subunit G (G1-59) of the Saccharomyces cerevisiae V1VO ATPase

Understanding the structural traits of subunit G is essential, as it is needed for V₁V_O assembly and function. Here solution NMR of the recombinant N- (G_1-59) and C-terminal segment (G_61-114) of subunit G, has been performed in the absence and presence of subunit d of the yeast V-ATPase. The data show that G does bind to subunit d via its N-terminal part, G_1-59 only. The residues of G_1-59 involved in d binding are Gly7 to Lys34. The structure of G_1-59 has been solved, revealing an α-helix between residues 10 and 56, whereby the first nine- and the last three residues of G_1-59 are flexible. The surface charge distribution of G_1-59 reveals an amphiphilic character at the N-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The C-terminus exhibits a strip of negative residues. The data imply that G_1-59-d assembly is accomplished by hydrophobic interactions and salt-bridges of the polar residues. Based on the recently determined NMR structure of segment E_18-38 of subunit E of yeast V-ATPase and the presently solved structure of G_1-59, both proteins have been docked and binding epitopes have been analyzed.     

The effect of F0 inhibitors on the Vibrio alginolyticus membrane ATPase

The inhibition of membrane ATPase from the marine alkalotolerant bacterium Vibrio alginolyticus by DCCD, triphenyltin and venturicidin was studied. DCCD proved to be an irreversible inhibitor, while venturicidin and triphenyltin produced a reversible inhibitory effect. The DCCD-binding proteolipid was identified in the membrane preparations. The effect of the inhibitors on ATPase activity and ATP-dependent Na⁺-transport in V. alginolyticus subcellular vesicles is discussed.     

Influence of in vitro growth conditions on in vitro and ex vitro photosynthetic rates of easy- and difficult-to-acclimatize sea oats (Uniola paniculata L.) genotypes

Net photosynthetic rates (P _n) of easy (EK 16-3) and difficult-to-acclimatize (EK 11-1) sea oats genotypes were examined under the following culture conditions: (1) photoautotrophic [sugar-free medium, high photosynthetic photon flux (PPF), high vessel ventilation rates and CO₂ enrichment, (PA)]; (2) modified photomixotrophic [sugar-containing medium diluted with sugar-free medium over time, high PPF, and high vessel ventilation rates (PM)]; (3) modified photomixotrophic enriched [same as PM with CO₂ enrichment, (PME)]; or (4) conventional photomixotrophic [sugar-containing medium, low PPF, and low vessel ventilation rates (control)]. Regardless of genotype, plantlets cultured under PA conditions died within 2 wk, whereas under PM and PME conditions, plantlets increased their P _n. After 6 wk, P _n per gram dry weight was 1.7 times greater in EK 16-3 than EK 11-1 plantlets cultured under PME conditions. In vitro-produced leaves of EK 16-3 plantlets were elongated with expanded blades, whereas EK 11-1 produced short leaves without expanded blades, especially under control conditions. After in vitro culture, EK 16-3 PME plantlets exhibited the highest dry weights among treatments. EK 16-3 PME and EK 16-3 PM had similarly high survivability, shoot and root dry weights and leaf lengths ex vitro compared to EK 16-3 control and EK 11-1 PM and PME plantlets. Ex vitro growth, survivability and P _n per leaf area of either genotype were not affected by CO₂ enrichment under modified photomixotrophic conditions. These results suggest that growth and survivability of sea oats genotypes with different acclimatization capacities can be enhanced by optimizing culture conditions.     

In vitro and in vivo pharmacology of CP-945,598, a potent and selective cannabinoid CB1 receptor antagonist for the management of obesity

Cannabinoid CB₁ receptor antagonists exhibit pharmacologic properties favorable for the treatment of metabolic disease. CP-945,598 (1-[9-(4-chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethylamino piperidine-4-carboxylic acid amide hydrochloride) is a recently discovered selective, high affinity, competitive CB₁ receptor antagonist that inhibits both basal and cannabinoid agonist-mediated CB₁ receptor signaling in vitro and in vivo. CP-945,598 exhibits sub-nanomolar potency at human CB₁ receptors in both binding (K_i = 0.7 nM) and functional assays (K_i = 0.2 nM). The compound has low affinity (K_i = 7600 nM) for human CB₂ receptors. In vivo, CP-945,598 reverses four cannabinoid agonist-mediated CNS-driven responses (hypo-locomotion, hypothermia, analgesia, and catalepsy) to a synthetic cannabinoid receptor agonist. CP-945,598 exhibits dose and concentration-dependent anorectic activity in two models of acute food intake in rodents, fast-induced re-feeding and spontaneous, nocturnal feeding. CP-945,598 also acutely stimulates energy expenditure in rats and decreases the respiratory quotient indicating a metabolic switch to increased fat oxidation. CP-945,598 at 10 mg/kg promoted a 9%, vehicle adjusted weight loss in a 10 day weight loss study in diet-induced obese mice. Concentration/effect relationships combined with ex vivo brain CB₁ receptor occupancy data were used to evaluate efficacy in behavioral, food intake, and energy expenditure studies. Together, these in vitro, ex vivo, and in vivo data indicate that CP-945,598 is a novel CB₁ receptor competitive antagonist that may further our understanding of the endocannabinoid system.     

An analog of 1α,25-dihydroxy-19-norvitamin D3 with the 1α-hydroxy group fixed in the axial position lacks biological activity in vitro

The relationship between the A-ring chair conformation of vitamin D compounds and their ability to bind the vitamin D receptor (VDR) has long attracted the attention of many researchers. It was established that in the crystalline complexes of hVDRmt with the natural hormone, 1α,25-dihydroxyvitamin D₃ (1), and its side-chain analogs the vitamins exist in β-chair form with an equatorial orientation of 1α-OH. However, with all these ligands the interconversion between both A-ring forms would be possible in solution. In an attempt to verify the conformation of vitamin D compounds required for binding the VDR we prepared analog 4, characterized by the presence of an axial 1α-hydroxy group. Since the additional ring connecting 3β-oxygen and C-2 prevents A-ring conformational flexibility, the synthesized vitamin 4 can exist exclusively in the α-chair form. The geometrical isomer 5 with a free 3β-OH group was also obtained. The analog 5 binds very poorly to VDR, whereas the vitamin 4 is practically devoid of binding ability. Both compounds also show very low HL-60-differentiating activity. When tested in vivo in mice the analogs 4 and 5 exhibit significant calcemic responses with analog 4 showing more activity than analog 5.     

Interactions between TGF-β1 and TGF-β3 and their role in medial edge epithelium cell death and palatal fusion in vitro

In recent decades, studies have shown that both TGF-β₁ and TGF-β₃ play an important role in the induction of medial edge epithelium (MEE) cell death and palatal fusion. Many of these experiments involved the addition or blockage of one of these growth factors in wild-type (WT) mouse palate cultures, where both TGF-β₁ and TGF-β₃ are present. Few studies have addressed the existence of interactions between TGF-β₁ and TGF-β₃, which could modify their individual roles in MEE cell death during palatal fusion. We carried out several experiments to test this possibility, and to investigate how this could influence TGF-β₁ and TGF-β₃ actions on MEE cell death and palatal shelf fusion. We double-immunolabelled developing mouse palates with anti-TGF-β₁ or anti-TGF-β₃ antibodies and TUNEL, added rhTGF-β₁ or rhTGF-β₃ or blocked the TGF-β₁ and TGF-β₃ action at different concentrations to WT or Tgf-β₃ null mutant palate cultures, performed in situ hybridizations with Tgf-β₁ or Tgf-β₃ riboprobes, and measured the presence of TUNEL-positive midline epithelial seam (MES) cells and MES disappearance (palatal shelf fusion) in the different in vitro conditions. By combining all these experiments, we demonstrate great interaction between TGF-β₁ and TGF-β₃ in the developing palate and confirm that TGF-β₃ has a more active role in MES cell death than TGF-β₁, although both are major inductors of MES disappearance. Finally, the co-localization of TGF-β₁, but not TGF-β₃, with TUNEL in the MES allows us to suggest a possible role for TGF-β₁ in MES apoptotic clearance.     

Is the expression of kinin B1 receptor related to intrahepatic vascular response?

Bradykinin elicits an intrahepatic vascular response (IHVR) mediated by the constitutive B₂ receptor (B₂R). The biological effects of kinins may also be mediated by the inducible B₁ receptor (B₁R). Aim: To verify if the hepatic B₁R expression modulates IHVR to kinins. Method: We evaluated the ability of bradykinin and B₁R agonists to elicit an IHVR in normal rats and in those submitted to acute or chronic inflammatory stimuli, fibrosis, cirrhosis, or hepatic regeneration. Results: Bradykinin-induced IHVR was similar in all groups. B₁R agonists did not elicit in any of them either a hypertensive or a hypotensive response. B₁ receptor induction was observed in all experimental groups (Western blot), except for the acute inflammatory group. Conclusion: B₁R hepatic expression did not modulate IHVR to kinins.     

Attenuation of ischemic efflux of endogenous amino acids by the novel 5-HT1A/5-HT2 receptor ligand adatanserin

Using sodium azide (NaN3)-induced anoxia plus aglycaemia as a model of chemically-induced ischemia in the hippocampal slice, we have evaluated the effects of the novel 5-HT(1A) partial agonist/5-HT(2) receptor antagonist adatanserin and the 5-HT(1A) receptor agonist BAYx3702 on the efflux of endogenous glutamate, aspartate and GABA. BAYx3702 (10-1000 nM) produced a significant (P<0.05) dose-related attenuation of ischemic efflux of both glutamate and GABA with maximum decrease being observed at 100 nM (73 and 69%, respectively). This attenuation was completely reversed by the addition of the 5-HT(1A) antagonist, WAY-100635 (100 nM). Similarly, adatanserin (10-1000 nM) produced a significant (P<0.05) dose-related attenuation in glutamate and GABA efflux with a maximum of 72 and 81% at 100 nM, respectively. This effect was completely reversed by the 5-HT(2A/C) receptor agonist, DOI but unaffected by WAY-100635. The 5-HT(2A) receptor antagonist MDL-100907 produced a comparable attenuation of glutamate when compared to adatanserin, while the 5-HT(2C) receptor antagonist, SB-206553, had no effect on ischemic efflux. None of these compounds significantly altered aspartate efflux from this preparation. In conclusion, the 5-HT(1A) receptor partial agonist 5-HT(2) receptor antagonist, adatanserin is able to attenuate ischemic amino acid efflux in a comparable manner to the full 5-HT(1A) agonist BAYx3702. However, in contrast to BAYx3702, adatanserin appears to produce it effects via blockade of the 5-HT(2A) receptor. This suggests that adatanserin may be an effective neuroprotectant, as has been previously demonstrated for full 5-HT(1A) receptor agonists such as BAYx3702.     

The role of fitness on VO2 and VCO2 kinetics in response to proportional step increases in work rate

The purpose of this study was to determine the effect of fitness and work level on the O2 uptake and CO2 output kinetics when the increase in work rate step is adjusted to the subject's maximum work capacity. Nine normal male subjects performed progressive incremental cycle ergometer exercise tests in 3-min steps to their maximum tolerance. The work rate step size was selected so that the symptom-limited maximum work rate would be reached in four steps at 12 min in all subjects. Oxygen consumption (VO2) and carbon dioxide production (VCO2) were calculated breath by breath. For the group, the time (mean, SEM) to reach 75% of the 3-min response (T0.75) for VO2 increased significantly (P less than 0.01) at progressively higher work rate steps, being 53.3 (5.5) s, 63.5 (4.6) s, 79.5 (5.0) s, and 94.5 (5.8) s, respectively. In contrast, T0.75 for VCO2 did not change significantly [74.9 (7.4) s, 75.6 (5.0) s, 85.1 (5.3) s, and 89.4 (6.3) s, respectively]. VCO2 kinetics were slower than VO2 kinetics at the low fractions of the subjects' work capacities but were the same or faster at the high fractions because of the slowing of VO2 kinetics. The first step showed the fastest rise in VO2. While VO2 kinetics slowed at each step, they were faster at each fraction of the work capacity in the fitter subjects. The step pattern in VO2 disappeared at high work rates for the less fit subjects.(ABSTRACT TRUNCATED AT 250 WORDS)