Proteomic analysis of circulating human monocytes in coronary artery disease

Monocytes play an important role in inflammation and atherosclerosis; however, the molecular details underlying these diverse functions are not completely understood. Proteomic analysis of monocytes can provide new insights into their biological role in coronary artery disease (CAD). Twenty angiographically confirmed male, CAD patients (≥50% stenosis) attending cardiology clinic of Nehru Hospital, PGIMER, Chandigarh, and who were not receiving any lipid lowering therapy and 20 TMT negative subjects who served as controls were enrolled in the study. Circulating monocytes isolated from overnight fasting blood samples were analyzed by 2D gel electrophoresis (pH 4-7), and differentially expressed protein spots were subjected to mass spectrometry and identification of proteins. We observed 333 ± 40 protein spots in monocytes from patients and 312 ± 20 in controls; out of which 63 protein spots showed altered intensity in CAD patients. Thirteen spots showed fivefold increased and two protein spots showed fivefold decreased expression in CAD group as compared to control group, respectively. Two proteins showing decreased expression in monocytes from CAD patients were identified as: (i) glutathione transferase and (ii) heat shock protein 70 KDa. Proteins showing increased expression in CAD patients were identified as: (i) vimentin, (ii) mannose binding lectin receptor protein, and (iii) S100A8 calcium-binding protein. The results of our study offer identification of several proteins in monocytes which can provide new perspectives in role of monocytes in pathogenesis of atherosclerosis.     

Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) expression is epigenetically regulated by one-carbon metabolism in invasive duct cell carcinoma of breast

In view of recent studies highlighting the prognostic relevance of expression and CpG island methylator phenotype (CIMP) of Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) in invasive duct cell carcinoma (IDC), we hypothesized in this article that impaired one-carbon metabolism might influence CIMP phenotype of BNIP3. In order to substantiate the prognostic relevance of BNIP3, we explored its association with 8-oxo-2'deoxyguanosine (8-oxodG), a marker of oxidative stress with prognostic relevance. BNIP3 expression and CIMP phenotype were studied using semi-quantitative RT-PCR and combined bisulfite restriction analysis (COBRA), respectively, in 56 IDC tumors. Eight polymorphisms in one-carbon metabolism were studied using PCR-RFLP and PCR-AFLP approaches. 8-oxodG was measured using competitive ELISA kit. BNIP3 was found to be upregulated in IDC (cases vs. controls: 0.94 ± 0.05 vs. 0.18 ± 0.08, P < 0.0001). COBRA analysis confirmed hypomethylation of BNIP3 promoter CpG island in these cases. CIMP phenotype of BNIP3 showed positive association with tubule formation (P = 0.034) and methionine synthase reductase (MTRR) A66G (P = 0.002); inverse association with cytosolic serine hydroxyl methyltransferase (cSHMT) C1420T (P < 0.005) and 8-oxodG (<10% vs. >10% methylation: 7.24 ± 2.77 ng/ml vs. 4.42 ± 2.93 ng/ml, P < 0.0005); and no association with nuclear pleomorphism or mitotic index or ER, PR, and HER statuses. Synergistic effect of MTR A2756G and MTRR A66G variants on BNIP3 hypermethylator phenotype was clearly evident (P < 0.0007). MTRR A66G and cSHMT C1420T polymorphisms influence CIMP phenotype of BNIP3, thus epigenetically regulating BNIP3 in breast cancer. The linear association between BNIP3 and 8-oxodG substantiates the role of BNIP3 as redox sensor as well as prognostic marker in breast cancer.     

Dynamics of microRNAs in bull spermatozoa

Background

Degenerative effects of critical regulators of reproduction, the kisspeptin peptides, on cellular aspects of sexually immature male gonads are known but similar information on accessory sex glands remain elusive.

Methods

Prepubertal laboratory rats were injected kisspeptin-10 at three different dosage concentrations (10 pg, 1 ng and 1 microgram) for a period of continuous 12 days at the rate of two doses per day. Control rats were maintained in parallel. The day following the end of the experimental period, seminal vesicles were removed and processed for light and electron microscopic examination using the standard methods. DNA damage was estimated by DNA ladder assay and DNA fragmentation assay.

Results

The results demonstrated cellular degeneration. Epithelial cell height of seminal vesicles decreased significantly at all doses (P < 0.05). Marked decrease in epithelial folds was readily noticeable, while the lumen was dilated. Ultrastructural changes were characterized by dilatation of endoplasmic reticulum and Golgi complex, heterochromatization of nuclei, invagination of nuclear membranes and a decreased number of secretory granules. Percent DNA damage to the seminal vesicle was 19.54 +/- 1.98, 38.06 +/- 2.09 and 58.18 +/- 2.59 at 10 pg, 1 ng and 1 microgram doses respectively.

Conclusion

The study reveals that continuous administration of kisspeptin does not lead to an early maturation but instead severe degeneration of sexually immature seminal vesicles.     

A linear ablating system in the left and right atrium: feasibility, catheter performance and clinical results

IntroductionWe describe the use of a ablating system to compartmentalise and regionally isolate the atria in paroxysmal and persistent atrial fibrillation (AF).Methods40 patients were studied, 25 paroxysmal AF and 14 persistent AF. One patient enrolled was later found to be in left atrial flutter and was excluded. The Cardima Revelation® TX catheter system with Intellitemp® Radiofrequency (RF) energy control device and a Medtronic Atakar® RF generator were used to place wide area circumferential ablations to achieve conduction block into the left and right sided pulmonary veins. Roof lines and mitral isthmus lines were also performed. In patients with persistent AF and in repeat procedures, right atrial compartmentalisation was performed with an anterior superior vena cava (SVC) to inferior vena cava (IVC) line and a septal SVC to IVC line.ResultsAt 6 months, 18 of the 39 patients were asymptomatic, 10 had improved symptoms and 22 were in sinus rhythm. In the paroxysmal group, 11 were asymptomatic, 7 had improved symptoms and 16 (64%) were in sinus rhythm. In the persistent group, 7 were asymptomatic, 3 had improved symptoms and 6 (43%) were in sinus rhythm. The total group AF burden was 37.8 ± 5.4 hrs pre-procedure and 23.1 ± 5.1 hrs at 6 months post procedure. Mean temperature, impedance and power recorded at each pole demonstrated effective power delivery at all poles. No catheter charring was observed, complication rates were comparable to standard AF ablation technique.ConclusionLinear ablation in the left and right atria to mimic Cox’s Maze is feasible and safe using this ablating system.     

Brain size at birth throughout human evolution: a new method for estimating neonatal brain size in hominins

An increase in brain size is a hallmark of human evolution. Questions regarding the evolution of brain development and obstetric constraints in the human lineage can be addressed with accurate estimates of the size of the brain at birth in hominins. Previous estimates of brain size at birth in fossil hominins have been calculated from regressions of neonatal body or brain mass to adult body mass, but this approach is problematic for two reasons: modern humans are outliers for these regressions, and hominin adult body masses are difficult to estimate. To accurately estimate the brain size at birth in extinct human ancestors, an equation is needed for which modern humans fit the anthropoid regression and one in which the hominin variable entered into the regression equation has limited error. Using phylogenetically sensitive statistics, a resampling approach, and brain-mass data from the literature and from National Primate Research Centers on 362 neonates and 2802 adults from eight different anthropoid species, we found that the size of the adult brain can strongly predict the size of the neonatal brain (r² = 0.97). This regression predicts human brain size, indicating that humans have precisely the brain size expected as an adult given the size of the brain at birth. We estimated the size of the neonatal brain in fossil hominins from a reduced major axis regression equation using published cranial capacities of 89 adult fossil crania. We suggest that australopiths gave birth to infants with cranial capacities that were on average 180 cc (95% CI: 158–205 cc), slightly larger than the average neonatal brain size of chimpanzees. Neonatal brain size increased in early Homo to 225 cc (95% CI: 198–257 cc) and in Homo erectus to approximately 270 cc (95% CI: 237–310 cc). These results have implications for interpreting the evolution of the birth process and brain development in all hominins from the australopiths and early Homo, through H. erectus, to Homo sapiens.     

恶性疟原虫abstrakt同源基因的克隆及DEAD盒家族蛋白的序列分析

DEAD盒蛋白家族的ATP依赖性的RNA解旋酶类参与细胞内几乎所有的RNA代谢过程 ,在几乎所有生物的细胞生长发育过程中扮演着众多不可或缺的角色。在本实验中 ,通过PCR和探针杂交相结合的筛选方法 ,筛选恶性疟原虫 (Plasmodiumfalciparum)的基因组文库 ,克隆了FH1F———abstrakt同源基因的完整序列。通过搜索已经完成测序的恶性疟原虫基因组数据库 ,推测FH1F序列定位在第 5条染色体上。FH1F全长2 80 4bp,包含一个 1 1 6 1bp的完整阅读框 ,编码一个由 386个氨基酸组成的蛋白。对FH1F蛋白序列用BlastP进行搜索和分析以及用DNAStar与许多典型的DEAD盒蛋白序列进行比对分析 ,结果均提示FH1F蛋白应该是DEAD盒家族的一个Abstrakt蛋白。另一方面 ,用DNAStar对已知所有完整的DEAD盒蛋白进行详细的序列分析以及用Mega对这些序列进行系统发育研究的结果都显示 :DEAD盒家族的蛋白聚类成为若干不同的亚群 ;与DEAD盒蛋白的一般保守序列相比 ,Abstrakt,eIF 4A ,Vasa ,P6 8等不同亚群的DEAD盒蛋白在保守区具有各自不同的结构特征。本文对不同的DEAD盒蛋白的结构特征进行了总结并试图给出不同亚群分类上的结构标准 ,对Abstrakt蛋白在本应高度保守的位点上异常于其它DEAD盒蛋白的氨基酸残基的取代也进行了相关的初步分析     

Intraabdominal hydatid cyst: a case report

BACKGROUND: Hydatid disease is caused by Echinococcus granulosus, endemic in cattle and sheep-raising regions of the world such as Central Europe, South America, Australia, New Zealand and South Africa. Although hydatid disease is more common in liver and lung, it also affects brain, kidney, spleen and muscle. We present a case of intraabdominal hydatid cyst, diagnosed by fine needle aspiration cytology, producing an indentation of the liver, which is uncommon. CASE: A male patient presented with right side abdominal pain. On ultrasonography an intraabdominal solid mass (right hypochondrial) was revealed, and subsequently FNA was done. Smears were diagnostic of hydatid cyst. CONCLUSION: FNAC is a sensitive and rapid technique in diagnosis of hydatid cysts. The present case is unusual, owing to its presentation as a solid abdominal mass seeding over the liver and mimicking malignancy radiologically.     

Identification of peptide antagonists to glycoprotein Ibalpha that selectively inhibit von Willebrand factor dependent platelet aggregation

GPIbalpha is an integral membrane protein of the GPIb-IX-V complex found on the platelet surface that interacts with the A1 domain of von Willebrand factor (vWF-A1). The interaction of GPIbalpha with vWF-A1 under conditions of high shear stress is the first step in platelet-driven thrombus formation. Phage display was used to identify peptide antagonists of the GPIbalpha-vWF-A1 interaction. Two nine amino acid cysteine-constrained phage display libraries were screened against GPIbalpha revealing peptides that formed a consensus sequence. A peptide with sequence most representative of the consensus, designated PS-4, was used as the basis for an optimized library. The optimized selection identified additional GPIbalpha binding peptides with sequences nearly identical to the parent peptide. Surface plasmon resonance of the PS-4 parent and two optimized synthetic peptides, OS-1 and OS-2, determined their equilibrium dissociation GPIbalpha binding constants ( K Ds) of 64, 0.74, and 31 nM, respectively. Isothermal calorimetry corroborated the K D of peptide PS-4 with a resulting affinity value of 68 nM. An ELISA demonstrated that peptides PS-4, OS-1, and OS-2 competitively inhibited the interaction between the vWF-A1 domain and GPIbalpha-Fc in a concentration-dependent manner. All three peptides inhibited GPIbalpha-vWF-mediated platelet aggregation induced under high shear conditions using the platelet function analyzer (PFA-100) with full blockade observed at 150 nM for OS-1. In addition, OS-1 blocked ristocetin-induced platelet agglutination of human platelets in plasma with no influence on platelet aggregation induced by several agonists of alternative platelet aggregation pathways, demonstrating that this peptide specifically disrupted the GPIbalpha-vWF-A1 interaction.     

Biophysical analyses of human resistin: oligomer formation suggests novel biological function

Resistin, a small secreted peptide initially identified as a link between obesity and diabetes in mice, was shown to be involved in mediating inflammation in humans. We had shown earlier that recombinant human resistin has a tendency to form aggregates by formation of inter/intramolecular disulfide linkages and that it undergoes a concentration-dependent conformational change in secondary structure from alpha-helical to beta-sheet form. Here we report that this change in secondary structural conformation is due to the increase in the oligomeric form of human resistin as a function of protein concentration. Gel filtration analysis under different conditions further demonstrated that recombinant human resistin exists as a mixture of oligomer and trimer but is converted to a mixture of monomer and oligomer in the presence of 100 mM NaCl. We show that while the trimeric form of human resistin is stable to urea-induced denaturation, it is highly susceptible to NaCl and NaF, indicating the importance of ionic interactions in stabilization of trimer. In addition, urea was able to destabilize the oligomers indicating the involvement of hydrophobic interactions in oligomerization. Ionic as well as hydrophobic interactions stabilize the monomeric human resistin. Our data suggest that human resistin exists predominantly as oligomer and trimer in vitro. The oligomeric form of human resistin shows more potent effect on stimulation of proinflammatory cytokines. Therefore, it is very tempting to propose that the structural conformation of resistin may be involved in maintaining the very fine balance in regulation of macrophage function for successful response to a variety of pathological conditions.