Structural study of Ac-Phe-[Orn-Pro-dCha-Trp-Arg], a potent C5a receptor antagonist, by NMR

The cyclic hexapeptide Ac(0)-Phe(1)-[Orn(2)-Pro(3)-dCha(4)-Trp(5)-Arg(6)] (the square brackets denote cyclization) is a potent antagonist against C5a (the a-fragment of complement protein C5) binding to C5a receptor (C5aR) and an excellent candidate to become a therapeutic agent against diseases that involve unregulated activation of the complement system. We present the solution structure determination of this cyclic C5aR peptide antagonist (cC5aR-pa), using nuclear magnetic resonance (NMR) data and restrained molecular dynamics-based simulated annealing in torsion angle space with NMR-derived distance and torsion angle restraints. The calculated NMR ensemble of structures demonstrates the presence of a predominant conformation of a distorted type II' beta-turn in the segment Pro(3)-dCha(4)-Trp(5)-Arg(6). We critically examine the calculated structure with measured NMR parameters, such as nuclear Overhauser enhancement (NOE) connectivity patterns and intensities characteristic of specific structures, (3)J(H(N)-H(alpha)) scalar coupling constants, temperature coefficients for NH groups, and differences between observed chemical shifts and their random coil values. The raw NMR data are consistent with the presence of the type II' beta-turn, but also indicate the presence of conformational inter-conversion. The calculated three-dimensional coordinates for cC5aR-pa will form the basis for further computational studies and for the development of pharmacophore models.     

Proteomic profiling of aging in the mouse heart: Altered expression of mitochondrial proteins

Using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry, we have used a systems biology approach to study the molecular basis of aging of the mouse heart. We have identified 8 protein spots whose expression is up-regulated due to aging and 36 protein spots whose expression is down-regulated due to aging (p0.05 as judged by Wilcoxon Rank Sum test). Among the up-regulated proteins, we have characterized 5 protein spots and 2 of them, containing 3 different enzymes, are mitochondrial proteins. Among the down-regulated proteins, we have characterized 27 protein spots and 16 of them are mitochondrial proteins. Mitochondrial damage is believed to be a key factor in the aging process. Our current study provides molecular evidence at the level of the proteome for the alteration of structural and functional parameters of the mitochondria that contribute to impaired activity of the mouse heart due to aging.     

Cytomorphological spectrum in scar endometriosis: a study of eight cases

OBJECTIVE: Endometriosis due to ectopic endometrial tissue that responds to hormonal stimulation and is extremely rare in a surgical scar appearing in 0.1% of women who have undergone Caesarean section. Fine needle aspiration cytology (FNAC) can be a valuable diagnostic aid in the evaluation of these subcutaneous abdominal masses. METHODS: We present the cytomorphological spectrum in eight cases of abdominal wall endometriosis diagnosed by FNAC over a 6-year period (June 1998-June 2004) in Mubarak Al-Kabeer Hospital (Kuwait). The patients ranged from 27 to 56 years of age. Seven had a prior Caesarean section and one had a hysterectomy for fibroid. They presented 3-8 years later with nodules in/near the abdominal scar. Five cases presented with a painful nodule, fluctuant with the menstrual cycle. RESULTS: Cytological findings comprised epithelial clusters and fusiform stromal cells with numerous haemosiderin-laden macrophages. Mild to moderate epithelial atypia was observed in three cases. A diagnosis of endometriosis was rendered in all the eight cases and tissue was available in four cases. CONCLUSIONS: Endometriotic nodules need to be differentiated from other benign/malignant masses and evaluated for possible malignant transformation. FNAC provides a safe and effective tool for diagnosis thereby obviating the need for other procedures.     

pH dependence of stability of the 10th human fibronectin type III domain: a computational study

We present detailed computational studies based on electrostatic calculations to evaluate the origins of pKa values and the pH dependence of stability for the 10th type III domain of human fibronectin (FNfn10). One of our goals is to validate the calculation protocols by comparison to experimental data (Koide, A.; Jordan, M. R.; Horner, S.; Batori, V.; Koide, S. Biochemistry 2001, 40, 10326-10333). Another goal is to evaluate the sensitivity of the calculated ionization free energies and apparent pKa values on local structural fluctuations, which do not alter the structural convergence to a particular architecture, by using a complete ensemble of solution NMR structures and the NMR average minimized structure of FNfn10 (Main, A. L.; Harvey, T. S.; Baron, M.; Boyd, J.; Campbell, I. D. Cell 1992, 71, 671-678). Our calculations demonstrate that, at high ionic strength, FNfn10 is more stable at low pH compared to neutral pH, in overall agreement with experimental data. This behavior is attributed to contributions from unfavorable Coulombic interactions in a surface patch for the pairs Asp7-Glu9 and Asp7-Asp23. The unfavorable interactions are decreased at low pH, where the acidic residues become neutral, and are further decreased at high ionic strength because of increased screening by salt ions. Elimination of the unfavorable interactions in the theoretical mutants Asp7Asn (D7N) and Asp7Lys (D7K) produce higher calculated stabilities at neutral pH and any ionic strength compared to the wild-type, in agreement with the experimental data. We also discuss subtleties in the calculated apparent pKa values and ionization free energies, which are not in agreement with the experimental data. This work demonstrates that comparative electrostatic calculations can provide rapid predictions of pH-dependent properties of proteins and can be significant aids in guiding the design of proteins with tailored properties.     

Performance of monolayered cervical smears in a gynecology outpatient setting in Kuwait

OBJECTIVE: To compare ThinPrep (TP) Papanicolaou smears (Cytyc Corp., Box-borough, Massachusetts, U.S.A.) with matching conventional Papanicolaou (CP) smears for specimen adequacy, cytologic quality, diagnostic accuracy and screening time. STUDY DESIGN: In this prospective study of 1,024 women a split-sample, matched-pair design in favor of CP slides based on single-blind criteria was followed with a smear on a glass slide for CP and the remaining material collected in Preserv-Cyt solution (Cytyc) for a TP smear. A Cytobrush (Medscand, Hollywood, Florida, U.S.A.) was used for smear preparation for CP. TP smears were processed in ThinPrep 2000 (Cytyc). Smears were stained with Papanicolaou stain and were interpreted according to the Bethesda system. RESULTS: The number of satisfactory but limited (SBL) cases with TP were 77 (7.5%) as compared to 127 (12.4%) with the CP method. This reduction in SBL smears with the TP method and consequent increase in satisfactory smears were highly significant (P < .001) by McNemar's test. As regards unsatisfactory smears in discordant pairs, although the number of unsatisfactory smears was higher with TP (41 cases) as against CP (27 cases), the difference was not statistically significant (P < .05). The split-sample method showed a high correlation between the CP and TP diagnoses. TP smears had a significant advantage over CP smears in the reduction in the number of ASCUS and AGUS cases (14 vs. 29) (P < .05) and increased the pickup rate of LSIL, 6 vs. 1. Time taken to screen the TP smears was half that of CP smears. No cases of LSIL or HSIL were missed on TP smears. CONCLUSION: The liquid-based processor significantly improved the adequacy and quality of smears, resulting in fewer recall cases for SBL smears, leading to more definitive diagnoses in atypical cases, increasing the pickup rate of LSILs and reducing the screening time. A machine handling multiple specimens automatically would decrease cost and be an asset to a cytopathology laboratory.     

Purification of recombinant human carbonic anhydrase-II by metal affinity chromatography without incorporating histidine tags

Due to their involvement in diverse pathological conditions, carbonic anhydrases have been the targets of drug developments for the treatments of glaucoma, epilepsy, high altitude sickness, as well as cancer. Of about 14 isozymes of carbonic anhydrases, carbonic anhydrase-II (hCA-II) has been most extensively investigated from the structural, functional, and inhibitor design point of view. We discovered that hCA-II preferentially binds to the Sepharose-iminodiacetate (IDA)-Zn(2+) column, and such binding does not require incorporation of either N- or C-terminal histidine tags in the protein structure. By using the Sepharose-IDA-Zn(2+) affinity column, we purified the Escherichia coli expressed hCA-II with an overall recovery of 76%. The purified enzyme showed a single band on the SDS-PAGE. Due to ease in preparing the Sepharose-IDA-Zn(2+) column, and purifying hCA-II just in one step, the overall protocol will be ideal for producing bulk quantities of the enzyme for high throughput screening of inhibitors.     

Genetic diversity and population structure of invasive and native populations of Erigeron canadensis L.

小蓬草入侵地和原产地种群的遗传多样性和种群结构 外来入侵植物对全球生物多样性造成了危害。小蓬草(Erigeron canadensis L.)是危害最为严重的外来农业杂草之一,代表了洲际入侵的典型例子。本研究利用10个多态性SSR位点,分别对采自中国江苏和浙江省的入侵地和采自美国阿拉巴马州的原产地各5个种群、共计312个植株的基因型进行了遗传多样性和遗传结构分析。结果表明,江苏省和浙江省的入侵种群显示出与阿拉巴马州原产地种群相似的遗传多样性,表明入侵期间没有严重的遗传瓶颈。利用STRUCTURE对种群结构的分析结果显示,种群之间分化较低,在原产地和入侵范围内均只仅检测到两个基因群。在入侵种群中观察到的遗传多样性较高,表明在入侵初期可能存在多次引入或引入了遗传背景不同的繁殖体。上述研究为阐明小蓬草这一全球有害杂草在中国东部的入侵动态提供了新的证据。在防除实践中,应注意防范小蓬草入侵种群和本地种群之间的种子基因流传播,阻止除草剂抗性植株的引入和扩散。