离子色谱法测定无水磷酸氢二钠中阴离子及不同级别产品质量比较

摘 要 目的：建立离子色谱法同时测定无水磷酸氢二钠中的氟离子、氯离子、硝酸根和硫酸根离子的含量,并评价不同级别无水磷酸氢二钠的质量状况。 方法: 采用Dionex IonPac AS18(4 mm×250 mm)色谱柱,以10,40 mmol·L-1 氢氧化钾淋洗液梯度洗脱,流速为1.0 ml·min -1 ,进样量为25 μl。 结果: 氟离子、氯离子、硝酸根和硫酸根离子质量浓度分别在0.02～0.40,0.01~0.80,0.06~1.00和0.10~5.00 μg·ml-1 范围内与峰面积均呈良好线性关系,r分别为0.999 5,0.999 7,0.999 9和0.999 5,平均回收率分别为92.26%,93.17%,99.80%,91.81%,RSD分别为0.87%,2.32%,3.07%,1.49%(n=6),供试品溶液在30 h内稳定。 结论: 本方法准确度高、重复性好,可用于测定无水磷酸氢二钠中的氟离子、氯离子、硝酸根和硫酸根离子的含量,为建立无水磷酸氢二钠的质量标准提供参考。     

硫氢化钠对糖尿病心肌病大鼠心脏保护作用及对JNK/FoxO1/Bcl-2信号通路的影响

摘要： 目的 观察外源性硫化氢的供体硫氢化钠 （NaHS） 对糖尿病心肌病 （DCM） 大鼠模型心肌损伤的保护作用及相关机制。方法 运用糖尿病饮食及链脲佐菌素 （STZ） 腹腔注射法建立DCM大鼠模型; 选取DCM大鼠16只, 正常大鼠16只, 随机分为DCM+0.9%氯化钠组 （DCM+Saline组）、 DCM+硫氢化钠组 （DCM+NaHS组）、 正常大鼠+0.9%氯化钠组 （Control+Saline组）、 正常大鼠+硫氢化钠组 （Control+NaHS组）, 每组8只; 予以DCM+NaHS组、 Control+NaHS组腹腔注射NaHS溶液100 mmol/ （kg·d）（浓度12 mmol/L, 体积8.3 mL/kg）, DCM+Saline组、 Control+Saline组大鼠腹腔注射等量生理盐水, 持续12周。实验结束行心脏超声测定左室射血分数 （LVEF） 及左室缩短分数 （LVFS）, 随后取左室心肌组织, 采用HE染色及Masson染色观察心肌组织病理改变, Western blot实验检测B淋巴细胞瘤-2基因 （Bcl-2）、磷酸化氨基末端蛋白激酶 （p-JNK）、 磷酸化叉头蛋白家族1 （p-FoxO1） 蛋白的表达。结果 糖尿病及硫氢化钠对大鼠心功能指标、 p-JNK、 p-FoxO1及Bcl-2蛋白有影响, 除外Bcl-2蛋白均存在交互作用 （P＜0.05）; 其中, DCM大鼠 LVEF及LVFS下降、 心肌细胞肥大及心肌间质纤维化, Bcl-2、 p-FoxO1蛋白表达下降、 p-JNK蛋白表达增加 （P＜ 0.05）; NaHS可以使DCM大鼠心功能指标、 病理组织变化得到改善, Bcl-2、 p-FoxO1蛋白表达增加、 p-JNK蛋白表达减少 （P＜0.05）。结论 DCM心肌损伤与细胞凋亡相关, 硫氢化钠可以减轻DCM心肌损伤保护心脏功能, 可能通过作用于JNK/FoxO1/Bcl-2发挥作用。     

The negligible chondritic contribution in the lunar soils water

Recent data from Apollo samples demonstrate the presence of water in the lunar interior and at the surface, challenging previous assumption that the Moon was free of water. However, the source(s) of this water remains enigmatic. The external flux of particles and solid materials that reach the surface of the airless Moon constitute a hydrogen (H) surface reservoir that can be converted to water (or OH) during proton implantation in rocks or remobilization during magmatic events. Our original goal was thus to quantify the relative contributions to this H surface reservoir. To this end, we report NanoSIMS measurements of D/H and ⁷Li/⁶Li ratios on agglutinates, volcanic glasses, and plagioclase grains from the Apollo sample collection. Clear correlations emerge between cosmogenic D and ⁶Li revealing that almost all D is produced by spallation reactions both on the surface and in the interior of the grains. In grain interiors, no evidence of chondritic water has been found. This observation allows us to constrain the H isotopic ratio of hypothetical juvenile lunar water to δD ≤ −550‰. On the grain surface, the hydroxyl concentrations are significant and the D/H ratios indicate that they originate from solar wind implantation. The scattering distribution of the data around the theoretical D vs. ⁶Li spallation correlation is compatible with a chondritic contribution <15%. In conclusion, (i) solar wind implantation is the major mechanism responsible for hydroxyls on the lunar surface, and (ii) the postulated chondritic lunar water is not retained in the regolith.Three types of sources could contribute to lunar superficial and mantle water, namely: (i) a primordial indigenous source identified in apatites (1–4), volcanic glasses (5, 6), and plagioclase phases (7) supporting a common origin of water for the Earth−Moon system (8–10); (ii) an addition of H₂O-rich material via impacts of carbonaceous chondrites (CCs) and cometary materials (11, 12); and (iii) a proton implantation by the solar wind (SW) (13–18). Because magmatic water was incorporated in apatites, i.e., in the last minerals crystallized from lunar melts, the D/H ratio of these minerals was used to identify the source of this water. Indeed, all inner solar system objects (Earth, Moon, CCs) show an average water D/H ratio around 150 × 10⁻⁶ with variations lying between 125 × 10⁻⁶ and 220 × 10⁻⁶. However, in lunar materials, a variety of processes may have altered this D/H ratio, namely: isotopic fractionation during the outgassing of the melt under vacuum, the reduction of water into H₂ by the highly reduced lunar melts, or the contribution of D from spallation reactions. The possible oxidation of SW H into water during silicate melting could also be considered as a possible source for this mantellic water. Indeed, production of water by SW implantation is now considered as a ubiquitous process in the solar system (13, 19) and one of the possible mechanisms for bringing water to the Moon’s surface. However, its contribution relative to chondritic or cometary sources is still debated (20).The D/H ratio (reported here in δD units) is commonly used to identify water sources. However, the Moon being an airless body unprotected by a planetary magnetic field, space weathering (21) modifies the δD of implanted H or of water adsorbed on grains, complicating the identification of the sources. Several types of space contributions can be distinguished: (i) water vapor deposition (22) resulting from carbonaceous chondrite or comet impacts; (ii) low-energy SW particles (∼1 keV/u) that are implanted in silicate grains (23), yielding a 200-nm-thick rim; and (iii) high-energy solar (SCR; 0.5–1.0 MeV/u) and galactic (GCR, 0.1–10 GeV/u) cosmic rays that penetrate the rocks down to a few centimeters to a few meters, respectively. These high-energy particles are responsible for the production of cosmogenic D and ⁶Li via the so-called spallation reactions (24, 25). As a consequence, the D/H ratios of the rim and of the interior of grains do not record the same information: (i) The rim contains SW H, cosmogenic elements, and water redeposited after the impacts of water-rich bodies, whereas (ii) the interior of grains contains the cosmogenic elements and lunar volatiles trapped in the melt.To estimate the relative proportions of SW and cosmogenic D in the hydrogen budget of grains in soils, we use the ⁷Li/⁶Li ratio as a record of the average concentration of spallation products (26). This approach offers two advantages: (i) The amount of cosmogenic D is considered as a free parameter and does not rely on the usual assumptions of theoretical calculations of spallation yields (5, 8, 9, 13), and (ii) in addition to a small isotope fractionation restricted to 6‰ (27), departure of the ⁷Li/⁶Li ratio toward low values can be unambiguously attributed to the contribution of the cosmogenic ⁶Li (28) [the cosmogenic ⁷Li/⁶Li ratio lies between 1.4 and 2.0 (29) while the lunar ratio is 12.15].     

The nature of protein folding pathways

How do proteins fold, and why do they fold in that way? This Perspective integrates earlier and more recent advances over the 50-y history of the protein folding problem, emphasizing unambiguously clear structural information. Experimental results show that, contrary to prior belief, proteins are multistate rather than two-state objects. They are composed of separately cooperative foldon building blocks that can be seen to repeatedly unfold and refold as units even under native conditions. Similarly, foldons are lost as units when proteins are destabilized to produce partially unfolded equilibrium molten globules. In kinetic folding, the inherently cooperative nature of foldons predisposes the thermally driven amino acid-level search to form an initial foldon and subsequent foldons in later assisted searches. The small size of foldon units, ∼20 residues, resolves the Levinthal time-scale search problem. These microscopic-level search processes can be identified with the disordered multitrack search envisioned in the “new view” model for protein folding. Emergent macroscopic foldon–foldon interactions then collectively provide the structural guidance and free energy bias for the ordered addition of foldons in a stepwise pathway that sequentially builds the native protein. These conclusions reconcile the seemingly opposed new view and defined pathway models; the two models account for different stages of the protein folding process. Additionally, these observations answer the “how” and the “why” questions. The protein folding pathway depends on the same foldon units and foldon–foldon interactions that construct the native structure.     

Structural insight into the molecular mechanism of allosteric activation of human cystathionine β-synthase by S-adenosylmethionine

Cystathionine β-synthase (CBS) is a heme-dependent and pyridoxal-5′-phosphate–dependent protein that controls the flux of sulfur from methionine to cysteine, a precursor of glutathione, taurine, and H₂S. Deficiency of CBS activity causes homocystinuria, the most frequent disorder of sulfur amino acid metabolism. In contrast to CBSs from lower organisms, human CBS (hCBS) is allosterically activated by S-adenosylmethionine (AdoMet), which binds to the regulatory domain and triggers a conformational change that allows the protein to progress from the basal toward the activated state. The structural basis of the underlying molecular mechanism has remained elusive so far. Here, we present the structure of hCBS with bound AdoMet, revealing the activated conformation of the human enzyme. Binding of AdoMet triggers a conformational change in the Bateman module of the regulatory domain that favors its association with a Bateman module of the complementary subunit to form an antiparallel CBS module. Such an arrangement is very similar to that found in the constitutively activated insect CBS. In the presence of AdoMet, the autoinhibition exerted by the regulatory region is eliminated, allowing for improved access of substrates to the catalytic pocket. Based on the availability of both the basal and the activated structures, we discuss the mechanism of hCBS activation by AdoMet and the properties of the AdoMet binding site, as well as the responsiveness of the enzyme to its allosteric regulator. The structure described herein paves the way for the rational design of compounds modulating hCBS activity and thus transsulfuration, redox status, and H₂S biogenesis.Cystathionine β-synthase (CBS; EC 4.2.1.22) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes the β-replacement of the hydroxyl group of l-serine (Ser) by l-homocysteine (Hcy), yielding cystathionine (Cth) (1). A deficient activity of human CBS (hCBS) is the cause of classical homocystinuria [CBS-deficient homocystinuria (CBSDH); Online Mendelian Inheritance in Man (OMIM) no. 236200], an autosomal, recessive inborn error of sulfur amino acid metabolism, characterized by increased levels of Hcy in plasma and urine. CBSDH manifests as a combination of connective tissue defects, skeletal deformities, vascular thrombosis, and mental retardation (2).The hCBS is a homotetrameric enzyme whose subunits are organized into three structural domains. The N-terminal region binds heme and is thought to function in redox sensing and/or enzyme folding (3, 4). The central catalytic core shows the fold of the type II family PLP-dependent enzymes (5, 6). Finally, the C-terminal region consists of a tandem pair of CBS motifs (7–9) that bind S-adenosylmethionine (AdoMet) and lead to an increase in catalytic activity by up to fivefold (10, 11). The CBS motif pair, commonly known as a “Bateman module” (12, 13), is responsible for CBS subunit tetramerization (14, 15). The presence of pathogenic missense mutations in this region often does not impair enzyme activity but typically interferes with binding of AdoMet and/or the enzyme’s activation by AdoMet (15–17). Removal of the regulatory region leads to a dimer with much increased activity (14, 15). Recently, we showed that removal of residues 516–525, forming a flexible loop of the CBS2 motif of hCBS, yields dimeric species (hCBSΔ516–525) with intact AdoMet binding capacity and activity responsiveness to AdoMet similar to a native hCBS WT (18).hCBS is regulated by a complex molecular mechanism that remains poorly understood. More than a decade ago, we and others hypothesized that hCBS might exist in two different conformations: a “basal” state with low activity, where the C-terminal regulatory domain would restrict the access of substrates into the catalytic site, and an AdoMet-bound “activated” state, where the AdoMet-induced conformational change would allow for enzyme activation (16, 19). Recently, we have unveiled the relative orientations of the regulatory and catalytic domains in hCBS (18), which were in a striking contrast to those of both the previous in silico models (20, 21) and the Drosophila melanogaster (dCBS) structure (22). Our data showed that, although the pairing mode and the orientation of catalytic cores are similar in both insect dCBS and hCBS, the position of their regulatory domains is markedly different (18). In the basal state, the Bateman modules from each hCBS unit are far apart and do not interact with each other, being placed just above the entrance of the catalytic site of the complementary subunit, thus hampering the access of substrates into this cavity. Our hCBSΔ516–525 structure additionally revealed the presence of two major cavities in the Bateman module, S1 and S2, one of which (S2) is solvent-exposed and probably represents the primary binding site for AdoMet (18). These findings are in agreement with the much higher basal activity of dCBS and its inability to bind or to be regulated by AdoMet (23, 24) and suggest that the structural basis underlying the regulation of the human enzyme markedly differs from CBS regulation in insects or yeast (24). Taken together, the available data indicate that binding of AdoMet to the Bateman module weakens the interaction between the regulatory domain and the catalytic core although the mechanism and the magnitude of the underlying structural effect are still under debate (16, 19, 25–27).To solve the molecular mechanism of hCBS regulation by AdoMet, we have analyzed the crystals of an engineered hCBSΔ516–525 protein that bears the mutation E201S, which potentially weakens and/or disrupts the interaction between the Bateman module and the catalytic core (Fig. 1A), thus favoring the activation of the enzyme. The data presented here fill a long-sought structural gap by unraveling the crystal structure of AdoMet-bound hCBS, thus providing the overall fold of the enzyme in its activated conformation and the identity of the AdoMet binding sites. Comparison with the structures of hCBS in basal conformation and constitutively activated dCBS was instrumental in the understanding of the regulatory role played by the C-terminal domain as well as the effect of some of the pathogenic mutations in the activation and/or inhibition of this key molecule of transsulfuration.Open in a separate windowFig. 1.Interactions between protein domains in basal hCBS. (A) In hCBSΔ516–525, residues Y484, N463, and S466 anchor the Bateman module (blue) to the protein core (gray) through H-bonds with the residues E201 and D198 from the loop L191–202, thus occluding the entrance to the catalytic pocket. (B) The CBS-specific activity of selected hCBS variants in the absence (blue bars) and the presence (red bars) of 300 µM AdoMet. hCBS enzyme species marked with “Δ” lack residues 516–525 and form dimers.     

EPSA: A Novel Supercritical Fluid Chromatography Technique Enabling the Design of Permeable Cyclic Peptides

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Application of Hydrogen Peroxide to the Control of Eutrophic Lake Systems in Laboratory Assays

We exposed water samples from a recreational lake dominated by the cyanobacterium Planktothrix agardhii to different concentrations of hydrogen peroxide (H₂O₂). An addition of 0.33 mg·L⁻¹ of H₂O₂ was the lowest effective dose for the decay of chlorophyll-a concentration to half of the original in 14 h with light and 17 h in experiments without light. With 3.33 mg·L⁻¹ of H₂O₂, the values of the chemical oxygen demand (COD) decreased to half at 36 and 126 h in experiments performed with and without light, respectively. With increasing H₂O₂, there is a decrease in the total and faecal coliform, and this effect was made more pronounced by light. Total and faecal coliform were inhibited completely 48 h after addition of 3.33 mg·L⁻¹ H₂O₂. Although the densities of cyanobacterial cells exposed to H₂O₂ did not decrease, transmission electron microscope observation of the trichomes showed several stages of degeneration, and the cells were collapsed after 48 h of 3.33 mg·L⁻¹ of H₂O₂ addition in the presence of light. Our results demonstrate that H₂O₂ could be potentially used in hypertrophic systems because it not only collapses cyanobacterial cells and coliform bacteria but may also reduce chlorophyll-a content and chemical oxygen demand.     

Young porcine endocrine pancreatic islets cultured in fibrin show improved resistance toward hydrogen peroxide

Aim: To study the protective effect of a fibrin scaffold toward embedded young porcine endocrine pancreatic islets from hydrogen peroxide within the context of islet encapsulation in transplantation. Methods: After isolation and in vitro maturation, groups of 200 young porcine islet equivalents (IEQ) were embedded in a 200 µL fibrin gel and exposed to 2 concentrations (10 and 100 µM) of hydrogen peroxide (H₂O₂) to investigate the ability of fibrin to protect islets against apoptotic stimuli. As a control, young porcine islets were seeded in tissue culture polystyrene (TCPS) well plates and exposed to the same H₂O₂ concentrations. Islet integrity, viability and function were then investigated. Results: Morphologically, the integrity of islets embedded in fibrin gels was better preserved compared with that of islets cultured in TCPS plates, when exposed to H₂O₂. Immunofluorescence staining showed that insulin and glucagon expression was higher in islets cultured in fibrin. Overall, H₂O₂ incubation led to decreased insulin and glucagon expression. A TUNEL assay revealed elevated numbers of apoptotic cells for islets cultured in TCPS plates when compared with those embedded in fibrin. Islets cultured in TCPS plates and exposed to H₂O₂ had diminished ability to secrete insulin in response to glucose stimulation, whereas islets embedded in fibrin maintained their glucose responsiveness. Insulin trapped in fibrin was extracted and quantified, revealing insulin in the extract. Conclusions/Interpretation: Fibrin has a protective effect on young porcine endocrine pancreatic islets exposed to hydrogen peroxide.