Heavy water labeling method for measuring the effect of genistein on mammary gland carcinogenesis

Heavy water labeling method was applied to measure the effect of genistein on mammary gland carcinogenesis by incoporating ²H from ²H₂O into the deoxyribose (dR) moiety of purine deoxyribonucleotides in dividing cells. In the present study, we followed the study design of Lamartiniere group to evaluate the efficacy of ²H₂O labeling on the measurement of mammary gland carconogenesis. Female Sprague–Dawley rats were fed estrogen-free AIN-93G diet starting 1 week before breeding and continuing through pregnancy and lactation. Female pups were assigned to the following groups on postnatal day 16 and fed AIN-93G diet: vehicle (dimethylsulfoxide) (DMSO), genistein, and estradiol benzoate (EB). On postnatal days 16, 18, and 20, female pups were injected subcutaneously with 500 μg genistein/g body wt, 500 ng EB/g body wt, or an equivalent volume of the vehicle. At day 50 postpartum, half of each group were gavaged with 60 mg dimethylbenz[a]anthracene (DMBA) in perila oil. After 1 week of DMBA treatment, all animals were labeled with ²H₂O by administration of 4% ²H₂O in drinking water after single intraperitonial bolus injection with 99.9% ²H₂O until sacrifice on postnatal day 81. The time-dependent weight gains were observed in all groups throughout the experimental period. The enrichment of body ²H₂O was attained at 1.84–2.47% through oral administration of ²H₂O. Mammary epithelial cell proliferation was measured by enrichment (EM1) of dA from rats. DMBA-treated groups showed higher fractional synthesis than DMBA non-treated groups. The group exposed only to genistein showed significantly lower EM1 (1.46 ± 0.87%) than those of control groups, i.e., the DMBA non-treated group (2.28 ± 0.29%) and the DMBA-treated group (2.32 ± 0.28%). Bromodeoxyuridine (BrdU) immunostaining of mammary tissue revealed that genistein reduced proliferation of the mammary epithelial, and the number of cells stained positive for BrdU both in DMBA-treated groups and DMBA non-treated groups. H&E staining of mammary epithelium also showed that the exposure to genistein decreased proliferation of the mammary epithelium. The epithelium in the rats treated with DMBA showed mostly multiple cell layers, in contrast to the mostly double layer shown in the DMBA non-treated rats. The exposure to genistein in the prepubertal period inhibited mammary epithelial cell proliferation. In conclusion, the ²H₂O labeling results were in good agreement with the results of BrdU incorporation and histomorphometry, which demonstrates that ²H₂O labeling can be used as a tool to measure carcinogenesis.     

A novel approach for assessing protein synthesis in channel catfish, Ictalurus punctatus

A comprehensive understanding of animal growth requires adequate knowledge of protein synthesis (PS), which in fish, has traditionally been determined by the flooding dose method. However, this procedure is limited to short-term assessments and may not accurately describe fish growth over extended periods of time. Since deuterium oxide (²H₂O) has been used to non-invasively quantify PS in mammals over short- and long-term periods, we aimed at determining if ²H₂O could also be used to measure PS in channel catfish. Fish were stocked in a 40-L aquarium with ~ 4% ²H₂O and sampled at 4, 8 and 24 h (n = 6 at each time period) to determine ²H-labeling of body water (plasma), as well as protein-free and protein-bound ²H-labeled alanine. The labeling of body water reflected that of aquarium water and the labeling of protein-free alanine remained constant over 24 h and was ~ 3.8 times greater than that of body water. By measuring ²H-labeled alanine incorporation after 24 h of ²H₂O exposure we were able to calculate a rate of PS: 0.04 ± 0.01% h^− 1. These results demonstrate that PS in fish can be effectively measured using ²H₂O and, because this method yields integrative measures of PS, is relatively inexpensive and accounts for perturbations such as feeding, it is a novel and practical assessment option.     

Determination of H-enrichment of rat brain interstitial fluid and rat plasma by headspace-gas-chromatography – quadrupole-mass-spectrometry

²H₂O as nonradioactive, stable marker substance is commonly used in preclinical and clinical studies and the precise determination of ²H₂O concentration in biological samples is crucial. However, aside from isotope ratio mass spectrometry (IRMS), only a very limited number of methods to accurately measure the ²H₂O concentration in biological samples are routinely established until now.     

A study of gramicidin using deuterium oxide

The single channel conductivity of the gramicidin channel has been measured for all the alkali ions using both H₂O and ²H₂O as a medium. Significant changes in conductivity with medium have been observed in all cases except lithium.     

克隆植物结缕草的水分生理整合格局特征及其生态效应分析

以克隆植物结缕草为研究对象,采用18 O作为示踪元素,从克隆植株不同生长发育阶段的复合节根系引入H218 O,在"异质高水"、"均质低水"两种环境条件下,探测和分析结缕草克隆植株复合节根、匍匐茎、A和B分株叶各构件组分系列内的水分生理整合格局特征及其生态效应。结果表明:(1)在两种水分环境条件下,H218 O在克隆植株主匍匐茎内各构件组分系列中均表现出双向传输的趋势,但更倾向于向顶传输。(2)H218 O向顶传输时,在"异质高水"生境内,基部复合节根系吸收的H218 O呈先增加后降低的趋势,而中部复合节根系吸收的H218 O呈先降低后增加的趋势;在"均质低水"生境内,中部复合节根系吸收的H218 O呈持续增加趋势。(3)在两种生境的3种引入情况下,H218 O均向顶传输到尖端生长点。其中在"异质高水"和"均质低水"生境内H218 O在克隆植株中向基传输过程中,传输强度整体上呈下降趋势;H218 O在主匍匐茎中传输时18 O分配于分株叶片中的量较多;H218 O在二级匍匐茎中的传输都呈现出明显的向顶趋势,传输距离都到达了二级匍匐茎的顶端生长点。(4)在绝大多数情况下,A分株叶系列的18 O丰度均明显高于B分株叶系列,这与A、B分株系列的生长发育特征相一致;但在"异质高水"生境内,中部分株吸收的H218 O在二级匍匐茎中传输时,分配于B分株叶系列的18 O明显高于A分株叶系列,即A分株系列相对于B分株系列的比较优势并不是一成不变的,在某些情况下还可以发生逆转。     

Effects of naloxone on renal responses to noxious stimuli in the cynomolgus monkey

Behavioral influences on excretion of Na⁺ and H₂O were studied during saline diuresis in three unanesthetized adult female cynomolgus monkeys. During control infusions of isotonic saline, the average rates of urine and Na⁺ excretion were 210μl/kg/min and 36.1μl/kg/min, respectively, and the average rate of inulin clearance was 4.6 ml/kg/min. Intermittent exposure to an electrical stimulus applied to the monkey's tail for a 30-min period modestly reduced rates of excretion of Na⁺ and H₂O; these reductions were 58% and 56% of baseline values respectively during the first 10 min, but excretion rates returned to baseline values or exceeded them by the end of the 30-min period. The effects of naloxone hydrochloride (10 mg/kg), an opiate antagonist, were studied by administering the drug immediately before the period of electrical-stimulus delivery. After naloxone, the electrical-stimulus markedly reduced the rates of Na⁺ and H₂O excretion to 29% and 31% of baseline values during the first 10 min, and delayed the return to baseline values. Inulin clearance was not altered significantly by the electrical stimulus in the absence of naloxone, but was decreased to 32% of the baseline rate during the first 10 min of exposure to the electric stimulus in the presence of naloxone. Naloxone had similar effects on rates of Na⁺ and urine excretion in response to 30 min of 108 dBA noise. These results show that renal responses to noxious environmental stimuli (electrical stimulus or noise) can be altered by naloxone.     

Effects of D2O on permeation and gating in the Ca2+-activated potassium channel fromChara

We studied the effects of H₂O/D₂O substitution on the permeation and gating of the large conductance Ca²⁺-activated K⁺ channels inChara gymnophylla droplet membrane using the patchclamp technique. The selectivity sequence of the channel was: K⁺>Rb⁺≫Li⁺, Na⁺, Cs⁺ and Cl⁻. The conductance of this channel in symmetric 100mm KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D₂O as compared to H₂O. The blockade of channel conductance by cytosolic Ca²⁺ weakened in D₂O as a result of a decrease in zero voltage Ca²⁺ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D₂O primarily due to the change in Ca²⁺ binding to the channel during the activation step. The Hill coefficient for Ca²⁺ binding was 3 in D₂O and around 1 in H₂O. The values of the Ca²⁺ binding constant in the open channel conformation were 0.6 and 6 μm in H₂O and D₂O, respectively, while the binding in the closed conformation was much less affected by D₂O. The H₂O/D₂O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large as 60 mV with 1mm internal Ca²⁺.     

Scavenging of free radicals in gas-phase mainstream cigarette smoke by immobilized catalase at filter level

Catalase is well known as capable of inducing the decomposition of H₂O₂. In this study, a kind of immobilized catalase (entrapped in cross-linked chitosan beads) was dispersed in conventional acetate filter as an antioxidant additive. Quantitative estimation of the free radicals in mainstream cigarette smoke (MCS) was performed to address the effect of this modified filter. It was found that the levels of PBN adduct and NO^?/NO₂^? associated with the gas-phase mainstream cigarette smoke (GPCS) were efficiently decreased by ～40% through catalase filtering. Besides, the modified filter was found to lower the MCS-induced adverse biological effects including lipid peroxidation and mutagenicity. This was proved to be substantially attributed to the catalase-dependent breakdown of NO^?, which was stimulated by some of peroxides (most probably being H₂O₂), the dismutation products of tar particulate matters (TPM). These results highlighted a promising approach to reduce the smoking-associated health risks to passive smokers. Moreover, the mechanisms of catalase filtering may be helpful for the development of appropriate immobilized enzyme systems to be applied for reducing health risks associated with gaseous pollutants.     

Involvement of aquaporin in thromboxane A2 receptor-mediated, G12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells

The physiological role of the thromboxane A₂ (TXA₂) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA₂ analogue. In the present study, we examined the detailed mechanisms of TXA₂ receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα_12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [³H]H₂O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA₂ receptor mediates water influx through aquaporins in astrocytoma cells via TXA₂ receptor-mediated activation of Gα_12/13, Rho A, Rho kinase and Na⁺/H⁺-exchanger.     

Growth yields and growth rates of Desulfovibrio vulgaris (Marburg) growing on hydrogen plus sulfate and hydrogen plus thiosulfate as the sole energy sources

Desulfovibrio vulgaris (Marburg) was grown on H₂ plus sulfate and H₂ plus thiosulfate as the sole energy sources and acetate plus CO₂ as the sole carbon sources. Conditions are described under which the bacteria grew exponentially. Specific growth rates () and molar growth yields (Y) at different pH were determined. and Y were found to be strongly dependent on the pH. Highest growth rates and molar growth yields were observed for growth on H₂ plus sulfate at pH 6.5 (=0.15h^-1; Y _{SO 4} ^2- =8.3g·mol^-1) and for growth on H₂ plus thiosulfate at pH 6.8 (=0.21h^-1; Y _{S 2}O ₃ ² =16.9g·mol^-1).The growth yields were found to increase with increasing growth rates: plots of 1/Y versus 1/ were linear. Via extrapolation to infinite growth rates a Y _SO4 ^2- /max of 12.2g·mol^-1 and a YS₂O ₃ ^2- /max of 33.5g·mol^-1 was obtained.The growth yield data are interpred to indicate that dissimilatory sulfate reduction to sulfide is associated with a net synthesis of 1 mol of ATP and that near to 3 mol of ATP are formed during dissimilatory sulfite reduction to sulfide.     

MeJA诱导的拟南芥保卫细胞H2O2积累与质膜H+-ATPase的关系

茉莉酸类物质(JAs)作为与昆虫啃噬及损伤相关的植物激素和信号分子在植物防御反应中起重要作用,但是茉莉酸引起的早期防御反应的机理仍不清楚。该研究以拟南芥叶片保卫细胞为材料,结合非损伤微测(NMT)及激光共聚焦技术探讨了茉莉酸诱导的保卫细胞中质膜H+-ATPase与H2O2积累的调控关系。结果表明:茉莉酸甲酯(MeJA)处理导致H+迅速跨膜外排和H2O2积累,H+外排和H2O2积累能够被钒酸钠抑制,而二苯基碘(DPI)处理则对MeJA诱导的H+跨膜外排无显著影响。研究结果证明,在MeJA诱导的早期信号事件中,质膜H+-ATPase的激活先于H2O2的产生。     

Effect of selenium in recovery of immunity damaged by H2O2 and60Co radiation

Con A stimulated lymphocytes proliferation was measured as [³H]thymidine incorporation and IgG was quantified by single radial immunodiffusion to study recovering or protecting effect of selenium (Se) on immunity attacked by exogenous active oxygen species, H₂O₂ and⁶⁰Co-radiation, respectively. Lipid peroxidation was also determined to observe the relation between antioxidation ability and protecting ability of Se. It was found that H₂O₂ injured lymphocytes immunocompetence deeply and⁶⁰Co-radiation decreased immune response capacity greatly, but that administration of Se counteracts this damage. The antioxidative ability of Se was correlated with its protecting ability.     

玉米叶表皮短细胞发育过程的形态特征及栓质细胞作用研究

采用大田试验,直接撕表皮或对叶片进行固定处理,结合单染、复染、荧光染色等多种细胞学显色方法,利用光学显微镜、荧光显微镜和扫描电子显微镜系统观察玉米叶表皮短细胞的发生时期、发育过程、分布规律以及形态结构特征,研究K⁺和H₂O₂在栓质细胞中的分布变化与表皮其它细胞中K⁺和H₂O₂的分布及气孔器开关的关系,为进一步挖掘短细胞的新功能提供细胞学依据。结果表明：（1）短细胞是同步发生在玉米多叶位新表皮组织形成过程中,所有植株从第7新生叶,大部分第6叶,极少数第5叶的基部同时开始发生短细胞,之后新生的高位叶也均发生短细胞,并随着叶位的升高叶片各部位短细胞密度均增大,所有植株的1~4叶（因不再生长）均无短细胞出现。（2）初期发育的叶表皮细胞进行不对称分裂,生成相互交替的长、短细胞,有的短表皮细胞横（垂直叶脉）分裂,形成栓质细胞和硅质细胞对;栓质细胞基部与叶肉细胞相邻,硅质细胞嵌在栓质细胞和表皮细胞间偏上。（3）有短细胞发生的叶片,宏观背面发亮且覆有蜡质层,微观表皮细胞的着色特性发生了变化;栓质细胞为面包形柱状细胞,硅质细胞为哑铃形扁细胞。（4）气孔器张开时,栓质细胞中没有K⁺和H₂O₂的积累;气孔器关闭时,栓质细胞中积累了大量的K⁺和H₂O₂,且栓质细胞中K⁺和H₂O₂的积累始终与副卫细胞中K⁺和H₂O₂的积累变化一致,而硅质细胞和长细胞没有K⁺和H₂O₂的积累。该研究确定了玉米叶表皮短细胞发生的时期;展示了其发育过程的形态学变化特征;发现栓质细胞中K⁺和H₂O₂的积累随气孔器开关呈周期性变化,且与副卫细胞中K⁺和H₂O₂的积累变化保持一致。     

μ- and μ4-η coordination of A2H2 (A = C, Si, Ge, Sn and Pb) ligands with transition metals

The heavier analogs of C₂H₂ have been studied at the B3LYP level for their μ and μ₄-η² coordination properties with the transition metals. Based on known alkyne compounds, transition metal fragments [W₂(μ-NH)(Cp)₂(Cl)₂] and [Fe₄(CO)₁₂] have been chosen. The SBKJC relativistic effective core potentials and their associated basis sets were used on W, Fe, Sn and Pb, and the 6-31G(d) basis set was used on all other elements. All the complexes of Si₂H₂, Ge₂H₂, Sn₂H₂ and Pb₂H₂ are found to be local minima. The trans-twist nature of the ligand A₂H₂ (A = Si-Pb) is large in μ-coordinated complexes of W, and it is very small in μ₄-η² coordinated complexes of Fe. The electronic structure of these complexes was investigated using fragment molecular orbital method (FMO).     

Increased bone turnover is associated with protein and energy metabolism in adolescents with sickle cell anemia

Contribution of bone turnover to the hypercatabolic state observed in sickle cell anemia is unknown. We examined the association between markers of bone turnover and basal rates of whole body protein turnover and energy expenditure in 28 adolescents with homozygous sickle cell anemia (HbSS) and in 26 matched controls with normal phenotype (HbAA). Whole body protein breakdown and synthesis were measured using a stable isotope of [15N]glycine, resting energy expenditure was measured by whole room indirect calorimetry, and the rate of pyridinoline cross-link (PYD) excretion in urine and fasting serum levels of the type I procollagen carboxy-terminal propeptide (PICP) were measured with commercial kits. Urinary PYD and serum PICP were significantly elevated in HbSS patients. The increase in procollagen synthesis, indicated by high levels of PICP, was significantly correlated with increased whole body protein synthesis. The increase in type I collagen degradation, indicated by high PYD excretion, was significantly correlated with increased protein breakdown. We conclude that increased rates of bone turnover contribute to the increased rates of protein turnover and energy expenditure observed in adolescents with homozygous sickle cell anemia.     

H2 oxidation,O2 uptake and CO2 fixation in hydrogen treated soils

In many legume nodules, the H₂ produced as a byproduct of N₂ fixation diffuses out of the nodule and is consumed by the soil. To study the fate of this H₂ in soil, a H₂ treatment system was developed that provided a 300 cm³ sample of a soil:silica sand (2:1) mixture with a H₂ exposure rate (147 nmol H₂ cm^–3hr^–1) similar to that calculated exist in soils located within 1–4 cm of nodules (30–254 nmol H₂ cm^–3hr^–1). After 3 weeks of H₂ pretreatment, the treated soils had a K_m and V_max for H₂ uptake (1028 ppm and 836 nmol cm^–3 hr^–1, respectively) much greater than that of control, air-treated soil (40.2 ppm and 4.35 nmol cm^–3 hr^–1, respectively). In the H₂ treated soils, O₂, CO₂ and H₂ exchange rates were measured simultaneously in the presence of various pH₂. With increasing pH₂, a 5-fold increase was observed in O₂ uptake, and CO₂ evolution declined such that net CO₂ fixation was observed in treatments of 680 ppm H₂ or more. At the H₂ exposure rate used to pretreat the soil, 60% of the electrons from H₂ were passed to O₂, and 40% were used to support CO₂ fixation. The effect of H₂ on the energy and C metabolism of soil may account for the well-known effect of legumes in promoting soil C deposition.     

18O Labeling of Chlorophyll d in Acaryochloris marina Reveals That Chlorophyll a and Molecular Oxygen Are Precursors

The cyanobacterium Acaryochloris marina was cultured in the presence of either H₂¹⁸O or ¹⁸O₂, and the newly synthesized chlorophylls (Chl a and Chl d) were isolated using high performance liquid chromatography and analyzed by mass spectroscopy. In the presence of H₂¹⁸O, newly synthesized Chl a and d, both incorporated up to four isotopic ¹⁸O atoms. Time course H₂¹⁸O labeling experiments showed incorporation of isotopic ¹⁸O atoms originating from H₂¹⁸O into Chl a, with over 90% of Chl a ¹⁸O-labeled at 48 h. The incorporation of isotopic ¹⁸O atoms into Chl d upon incubation in H₂¹⁸O was slower compared with Chl a with ∼50% ¹⁸O-labeled Chl d at 115 h. The rapid turnover of newly synthesized Chl a suggested that Chl a is the direct biosynthetic precursor of Chl d. In the presence of ¹⁸O₂ gas, one isotopic ¹⁸O atom was incorporated into Chl a with approximately the same kinetic incorporation rate observed in the H₂¹⁸O labeling experiment, reaching over 90% labeling intensity at 48 h. The incorporation of two isotopic ¹⁸O atoms derived from molecular oxygen (¹⁸O₂) was observed in the extracted Chl d, and the percentage of double isotopic ¹⁸O-labeled Chl d increased in parallel with the decrease of non-isotopic-labeled Chl d. This clearly indicated that the oxygen atom in the C3¹-formyl group of Chl d is derived from dioxygen via an oxygenase-type reaction mechanism.     

Hydrogen peroxide is required for abscisic acid-induced NH4+ accumulation in rice leaves

The role of H₂O₂ in abscisic acid (ABA)-induced NH₄⁺ accumulation in rice leaves was investigated. ABA treatment resulted in an accumulation of NH₄⁺ in rice leaves, which was preceded by a decrease in the activity of glutamine synthetase (GS) and an increase in the specific activities of protease and phenylalanine ammonia-lyase (PAL). GS, PAL, and protease seem to be the enzymes responsible for the accumulation of NH₄⁺ in ABA-treated rice leaves. Dimethylthiourea (DMTU), a chemical trap for H₂O₂, was observed to be effective in inhibiting ABA-induced accumulation of NH₄⁺ in rice leaves. Inhibitors of NADPH oxidase, diphenyleneiodonium chloride (DPI) and imidazole (IMD), and nitric oxide donor (N-tert-butyl-α-phenylnitrone, PBN), which have previously been shown to prevent ABA-induced increase in H₂O₂ contents in rice leaves, inhibited ABA-induced increase in the content of NH₄⁺. Similarly, the changes of enzymes responsible for NH₄⁺ accumulation induced by ABA were observed to be inhibited by DMTU, DPI, IMD, and PBN. Exogenous application of H₂O₂ was found to increase NH₄⁺ content, decrease GS activity, and increase protease and PAL-specific activities in rice leaves. Our results suggest that H₂O₂ is involved in ABA-induced NH₄⁺ accumulation in rice leaves.     

Components and fractions for differently bound water molecules of dipalmitoylphosphatidylcholine-water system as studied by DSC and H-NMR spectroscopy

Differently bound water molecules of dipalmitoylphosphatidylcholine (DPPC)-H₂O system were investigated with differential scanning calorimetry (DSC). According to a method previously reported by us, the ice-melting DSC curves of the DPPC-H₂O samples of varying water contents were deconvoluted into multiple components, and the ice-melting enthalpies for the individual deconvoluted components were used to estimate average molar ice-melting enthalpies for freezable interlamellar and bulk waters, respectively. With these average molar ice-melting enthalpies, the numbers of differently bound water molecules of the DPPC-H₂O system were calculated at varying water contents and were used to construct a water distribution diagram of this system. Furthermore, to evaluate the reliability of the present DSC deconvolution method, ²H-NMR T₁ measurements of DPPC-²H₂O system were carried out at 5 °C of the gel phase temperature, and components and fractions for differently bound water (²H₂O) molecules were estimated from the analysis of nonexponential magnetization recovery curves.     

Determination of muscle protein synthesis rates in fish using H2O and H NMR analysis of alanine

Following administration of deuterated water (²H₂O), the fractional synthetic rate (FSR) of a given endogenous protein can be estimated by ²H-enrichment quantification of its alanine residues. Currently, this is measured by mass spectrometry following a derivatization procedure. Muscle FSR was measured by ¹H/²H NMR analysis of alanine from seabass kept for 6 days in 5% ²H-enriched saltwater, following acid hydrolysis and amino acid isolation by cation-exchange chromatography of muscle tissue. The analysis is simple and robust, and provides precise measurements of excess alanine ²H-enrichment in the 0.1–0.4% range from 50 mmol of alanine recovered from muscle protein.