Application of HN(C)N to rapid estimation of 1J(N-C(alpha)) coupling constants correlated to psi torsion angles in proteins: implication to structural genomics

We recently described a triple resonance experiment, HN(C)N, for sequential correlation of H(N) and 15N atoms in (15N, 13C) labeled proteins [J. Biomol. NMR. 20 (2001) 135]. Here, we describe an approach based on this experiment for estimation of one bond N-C(alpha) J-couplings in medium size labeled proteins, which seem to show good correlations with psi torsion angles along the protein backbone. The approach uses the ratio of the intensities of the sequential and diagonal peaks in the F(2)-F(3) planes of the HN(C)N spectrum. The reliability of the approach has been demonstrated using a short peptide wherein the coupling constants have been measured by the present method and also independently from peak splittings in HSQC spectra. The two results agree within 10%. The applicability of the procedure to proteins has been demonstrated using doubly labeled FK506 binding protein (FKBP, molecular mass approximately 12 kDa). Coupling constant estimates have been obtained for 62 out of 100 non-proline residues and they show a correlation with psi torsion angles, as has been reported before. This semi-quantitative application of HN(C)N extends the significance of the experiment especially, in the context of structural genomics, since the single experiment, not only provides a great enhancement in the speed of resonance assignment, but also provides quantitative structural information.     

A suppressive effect of the adenovirus 5 protein E1B 55K on apoptosis induced by IL-3 deprivation and gamma-irradiation

The murine IL-3-dependent myeloid cell line 32D undergoes a rapid death when deprived of interleukin-3 (IL-3), a process that is suppressed or delayed by the constitutive expression of Bcl-2 or the Bcl-2-related Bcl-xL survival protein. The adenovirus type 5 E1B region encodes an E1B 55K protein, that has been reported to bind and inactivate the p53 protein that plays an important role in the induction of apoptosis. In order to explore the potential effect of the E1B 55K protein on IL-3 deprival-induced cell death, we have established 32D cell lines overexpressing the adenovirus E1B 55K protein and compared its ability to modulate the cell death with that of the human Bcl-2 protein. We observed that E1B 55K, as Bcl-2, delays the cell death caused by either IL-3-deprivation or DNA damage induced by gamma-irradiation. Cell-cycle analysis after IL-3 deprivation indicated that surviving Bcl-2 transfectants accumulate predominantly in the G0/G1 phase of the cell cycle, while E1B 55K transfectants survive in both G0/G1 and the S and G2/M phases of the cell cycle. zVAD-fmk, a broad caspase inhibitor, prevented chromatin condensation and fragmentation, but not cell death, suggesting that IL-3 deprivation induces a cell death program in which the caspases are dispensable. In contrast, both E1B 55K and Bcl-2 allowed cell survival and prevented the typical features of programmed cell death, such as phosphatidyl-serine exposure, loss of mitochondrial membrane potential, and chromatin condensation and fragmentation. Our findings indicate that the adenovirus 5 E1B 55K protein has the capability to act as a survival factor, and suggest that E1B 55K exerts its effect upstream of the activation of effector caspases, by preventing the loss of mitochondrial membrane potential induced by IL-3 deprivation.     

Methyl 3β-(4-[125I]Iodophenyl)Tropane-2β-Carboxylate In Vitro Binding to Dopamine and Serotonin Transporters Under "Physiological" Conditions

Abstract: Methyl 3β-(4-[¹²⁵I]iodophenyl)tropane-2β-carboxylate ([¹²³I]β-CIT) is a single photon emission computed tomographic radiotracer for in vivo labeling of dopamine (DA) and serotonin (5-HT) transporters. Single photon emission computed tomographic experiments in nonhuman primates showed that [¹²³I]β-CIT in vivo binding to DA transporters had a much slower washout than binding to 5-HT transporters. This observation was not predicted from previously published in vitro studies. These studies, performed at 22°C in nonphysiological buffer, reported similar affinity of [¹²⁵I]β-CIT for DA and 5-HT transporters. We now report [¹²⁵I]β-CIT binding parameters to fresh rat membranes at 22°C and 37°C, in a buffer mimicking the composition of cerebrospinal fluid. At both temperatures, binding to DA transporters was best fit by a twosite model, whereas binding to 5-HT transporters was compatible with one population of sites. At 22°C, [¹²⁵I]β-CIT showed similar affinity to high-affinity DA (0.39 n M ) and 5-HT transporter sites (0.47 n M ). Increasing the incubation temperature from 22°C to 37°C reduced binding to DA transporters by 60%, whereas binding to 5-HT transporters was only marginally affected. In vitro kinetic experiments failed to detect significant differences in on or off rates that could explain the observed in vivo kinetics. These experiments thus failed to explain [¹²³ I]β-CIT in vivo uptake kinetics, suggesting the existence of specific factors affecting the in vivo situation.     

乳腺癌改良根治术患者术后复发转移的危险因素及血清CA125、COX-2、sTNFR-P55的预测价值研究

摘要 目的：探讨乳腺癌改良根治术患者术后复发转移的危险因素及血清糖类抗原125（CA125）、环加氧酶-2（COX-2）、可溶性肿瘤坏死因子受体P55（sTNFR-P55）的预测价值。方法：对2014年1月至2016年12月新疆医科大学第一附属医院收治的109例行乳腺癌改良根治术的乳腺癌患者进行前瞻性研究,所有患者术后均随访5年,其中2例失访,107例完成随访。根据5年内患者复发转移情况将其分为复发转移组（n=31）和未复发转移组（n=76）。收集患者入院时的临床病理资料,采用电化学发光法检测术前血清CA125,采用酶联免疫吸附法检测术前血清COX-2、sTNFR-P55。采用logistic回归模型分析患者术后复发转移的影响因素,绘制受试者工作特征（ROC）曲线评估血清CA125、COX-2、sTNFR-P55对术后复发转移的预测价值。结果：复发转移组肿瘤直径＞5 cm、浸润性非特殊癌、脉管癌栓、雌激素受体（ER）/孕激素受体（PR）阴性、无内分泌治疗构成比、TNM分期IIIA期、腋窝淋巴结转移数量4~9个构成比高于未复发转移组（P<0.05）。复发转移组血清CA125、COX-2、sTNFR-P55水平高于未复发转移组（P<0.05）。多因素logistic回归分析结果显示,肿瘤直径＞5 cm、浸润性非特殊癌、TNM分期IIIA期、脉管癌栓、腋窝淋巴结转移数量4~9个、CA125升高、COX-2升高、sTNFR-P55升高是乳腺癌改良根治术患者术后5年内复发转移的独立危险因素（OR=1.318、1.213、1.223、1.137、1.257、1.241、1.313、1.351,P<0.05）。血清CA125、COX-2、sTNFR-P55均可有效预测乳腺癌术后复发转移,曲线下面积（AUC）分别为0.803、0.749、0.761,三指标联合预测术后复发转移的AUC为0.915,灵敏度和特异度分别为0.94、0.83。结论：肿瘤直径、浸润性非特殊癌、TNM分期、脉管癌栓、腋窝淋巴结转移数量以及术前血清CA125、COX-2、sTNFR-P55异常升高是乳腺癌改良根治术患者术后5年内复发转移的危险因素,术前血清CA125、COX-2、sTNFR-P55联合检测可预测乳腺癌改良根治术后的复发转移风险。     

Immunohistochemical characterisation of differentiated CAD cells: expression of peptides and chromogranins

The CNS-derived cell line, CAD cell line, when grown in a protein free medium (PFM), differentiates to neuron-like cells with very long processes. It was previously studied biochemically and found to express TH activity, some neurospecific proteins, but no glial proteins. We have now further studied the CAD cells and focused on the expression of various neuropeptides, GAP-43 and GFAP. All peptides studied were present, including TH, but also GFAP, in contrast to earlier studies. A different kind of processes, short, slender and distributed like a “fringe” around cell body and along processes was observed, NESP55 but not other chromogranins was present in these “fringes”, GAP43 showed some degree of overlapping with NESP55. The results show that even after differentiation in PFM, the CAD cells express a palette of neuropeptides and chromogranins, catecholaminergic markers as well as the glia-specific GFAP. Our efforts to induce exocytosis/endocytosis from the peptide granules by high K⁺ were, however, unsuccessful. Due to long processes, the CAD cells may represent a good model for studying intracellular transport, and, since the cells express both neuronal and glial characteristics, it may be useful for investigating the influence of different trophic/growth factors on the expression of various neuronal characteristics.     

Changes in cell wall polysaccharides in developing barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) coleoptiles

Cell wall polysaccharides in developing barley coleoptiles were examined using acetic acid–nitric acid extraction, alditol acetate and methylation analyses and enzymatic digestion. The coleoptile cell wall from imbibed grain was rich in pectic polysaccharides (30 mol%), arabinoxylan (25 mol%), cellulose (25 mol%) and xyloglucan (6 mol%), but contained only low levels of (13,14)--D-glucan (1 mol%). During 5 days of coleoptile growth, pectic polysaccharides decreased steadily to about 9 mol%, while (13,14)--D-glucan increased to 10 mol%. Following the cessation of growth of the coleoptiles at about 5 days, (13,14)--D-glucan content rapidly decreased to 1 mol%. The cellulose content of the walls remained at about 35–40 mol% throughout coleoptile growth. Similarly, arabinoxylan content remained essentially constant at 25–30 mol% during growth, although the ratio of substituted to unsubstituted 4-linked xylosyl units decreased from about 4:1 to 1:1. Xyloglucan content ranged from 6 mol% to 10 mol% and the oligosaccharide profile determined using a xyloglucan-specific endoglucanase and MALDI-TOF mass spectrometry indicated that the oligosaccharides XXGG and XXGGG were the principal components, with one and two acetyl groups, respectively, Thus, dramatic changes in wall composition were detected during the growth of barley coleoptiles, both with respect to the relative abundance of individual wall constituents and to the fine structure of the arabinoxylans.     

Anticrotalic and antitumoral activities of gel filtration fractions of aqueous extract from Tabernaemontana catharinensis (Apocynaceae)

The high mortality caused by Crotalus durissus terrificus snake venom is mainly due to crotoxin, which acts on the neuromuscular junction inhibiting the mechanism mediating acetylcholine release, thus leading to motor and respiratory paralysis and subsequently to animal death. We recently demonstrated that the aqueous extract (AE) of Tabernaemontana catharinensis can inhibit the lethal activity of C. d. terrificus venom. Eight fractions, PI to PVIII, were obtained by gel filtration of the extract on Sephadex G-10, and assayed for lethality and cytotoxicity. Fraction PVII [2.0 mg/100 g rat/50 microl saline solution (ss)] injected intramuscularly (i.m.) 20 s after the venom (240 microg) or crotoxin (200 microg/50 microl ss) neutralized the lethal activity of 2 LD50 of both. Fractions PI, PVI and PVIII (5.0 mg/100 g rat/50 microl ss) presented potent antitumoral activity in vitro against cells from human breast carcinoma (SK-BR-3) after 24 h incubation, as measured by Mosmann colorimetric method. Fraction PVII contains 12-methoxy-4-methylvoachalotine as its major component. These results demonstrate that the antivenom and antitumoral activities of the AE of T. catharinensis are exerted by different substances present in fraction PVII and fractions PI, PVI and PVIII, respectively, whose characteristics are distinct in terms of staining and Rf when analyzed by thin layer chromatography. The results also show that a preliminary fractionation by Sephadex G-10 gel filtration is a good option as a first step for isolation of biologically active substances from T. catharinensis.     

The effect of post-translational modifications on the hemorrhagic activity of snake venom metalloproteinases

Metalloproteinases (MPs) are Zn(+)-dependent endoproteolytic enzymes, abundant in crotalid and viperid snake venoms. Most snake venom metalloproteinases (svMPs) are active on extracellular matrix components and this effect is thought to result in bleeding as a consequence of the basement membrane disruption in capillaries. Jararhagin and ACLH are hemorrhagic svMPs from Bothrops jararaca and Agkistrodon contortrix laticinctus venom, respectively. Both enzymes demonstrate proteolytic activity on fibrinogen and fibronectin and jararhagin inhibits collagen-induced platelet aggregation in vitro. This work describes the expression, purification and successful refolding of the recombinant ACLH zymogen (rPRO-ACLH) as well as the catalytic domain of jararhagin (rCDJARA). The heterologous proteins were produced in E. coli, an in vivo expression system that does not make post-translational modifications. The recombinant refolded proteins did not show any hemorrhagic activity in mice skin, as well as the native deglycosylated jararhagin and ACLH. However, they preserved their proteolytic activity on fibrinogen and fibronectin. It seems that the hemorrhagic properties of these hemorrhagins are dependent on post-translational modifications, whereas their proteolytic activity is not dependent on such modifications.     

Control of yeast fed-batch process through regulation of extracellular ethanol concentration

At high growth rates, the biomass yield of bakers yeast (Saccharomyces cerevisiae) decreases due to the production of ethanol. For this reason, it is standard industrial practice to use a fed-batch process whereby the specific growth rate, , is fixed at a level below the point of ethanol production, i.e., _crit. Optimally, growth should be maintained at _crit, but in practice, this is difficult because _crit is dependent upon strain and culture conditions. In this work, growth was maintained at a point just above _crit by regulating ethanol concentration in the bioreactor. The models used for control design are shown, as are the experimental results obtained when this strategy was implemented. This technique should be applicable to all microorganisms that exhibit an overflow type metabolism.     

淹水胁迫对I-69/55杨蒸腾作用的影响

对Ｉ－６９／５５杨在人为控制条件下进行淹水胁迫试验,对照和淹水下分别测定其叶片的蒸腾速率（Ｔｒ）和净光合速率（Ｐｎ）,结果表明,在胁迫条件下净光合速率、蒸腾速率和气孔导度都有所下降,但在测定的时间期限内净光合速率基本不变,而蒸腾速率和气孔导度则随淹水时间的延长而下降;对照条件下光合速率和蒸腾速率都具有明显的日变化双峰曲线和季节变化,夏季净光合速率和蒸腾速率最高;水分利用率的的季节变化明显,秋季最高