Suppression of soluble adenylyl cyclase protects smooth muscle cells against oxidative stress-induced apoptosis

Apoptosis of vascular smooth muscle cells (VSMC) significantly contributes to the instability of advanced atherosclerotic plaques. Oxygen radicals are an important cause for VSMC death. However, the precise mechanism of oxidative stress-induced VSMC apoptosis is still poorly understood. Here, we aimed to analyse the role of soluble adenylyl cylclase (sAC). VSMC derived from rat aorta were treated with either H₂O₂ (300 µmol/L) or DMNQ (30 µmol/L) for 6 h. Oxidative stress-induced apoptosis was prevented either by treatment with 30 µmol/L KH7 (a specific inhibitor of sAC) or by stable sAC-knockdown (shRNA-transfection). A similar effect was found after inhibition of protein kinase A (PKA). Suppression of the sAC/PKA-axis led to a significant increase in phosphorylation of the p38 mitogen-activated protein kinase under oxidative stress accompanied by a p38-dependent phosphorylation/inactivation of the pro-apoptotic Bcl-2-family protein Bad. Pharmacological inhibition of p38 reversed these effects of sAC knockdown on apoptosis and Bad phosphorylation, suggesting p38 as a link between sAC and apoptosis. Analysis of the protein phosphatases 1 and 2A activities revealed an activation of phosphatase 1, but not phosphatase 2A, under oxidative stress in a sAC/PKA-dependent manner and its role in controlling the p38 phosphorylation. Inhibition of protein phosphatase 1, but not 2A, prevented the pro-apoptotic effect of oxidative stress. In conclusion, sAC/PKA-signaling plays a key role in the oxidative stress-induced apoptosis of VSMC. The cellular mechanism consists of the sAC-promoted and protein phosphatase 1-mediated suppression of p38 phosphorylation resulting to activation of the mitochondrial pathway of apoptosis.     

The kinase homology domain of receptor guanylyl cyclase C: ATP binding and identification of an adenine nucleotide sensitive site

The role of the kinase homology domain (KHD) in receptor guanylyl cyclases is to regulate the activity of the catalytic guanylyl cyclase domain. The KHD lacks many of the amino acids required for phosphotransfer activity and, therefore, is not expected to possess kinase activity. Guanylyl cyclase activity of the receptor guanylyl cyclase C (GC-C) is modulated by ATP, and computational modeling showed that the KHD can adopt a structure similar to protein kinases, suggesting that the KHD is the site for ATP interaction. A monoclonal antibody, GCC:4D7, raised to the KHD of GC-C, fails to react with GC-C in the presence of ATP and ATP analogues that regulate GC-C catalytic activity, indicating that a conformational change occurs in the KHD on ATP binding. Mapping of the epitope of the antibody through the use of recombinant protein constructs and phage display showed that the epitope for GC-C:4D7 lies immediately C-terminal to a critical lysine residue (Lys516 in GC-C), required for ATP interaction in protein kinases. By employing a novel approach utilizing ATP-agarose affinity chromatography, we demonstrate that the intracellular domain of GC-C and the KHD bind ATP. Mutation of Lys516 to Ala abolishes ATP binding. Thus, this report is the first to show direct ATP binding to the pseudokinase domain of receptor guanylyl cyclase C, as well as to identify dramatic conformational changes that occur in this domain on ATP binding, akin to those seen in catalytically active protein kinases.     

FBXL20 promotes breast cancer malignancy by inhibiting apoptosis through degradation of PUMA and BAX

Apoptosis is a programmed cell death that efficiently removes damaged cells to maintain tissue homeostasis. Defect in apoptotic machinery can lead to tumor development, progression, and resistance to chemotherapy. PUMA (p53 upregulated modulator of apoptosis) and BAX (BCL2-associated X protein) are among the most well-known inducers of apoptosis. It has been reported that expression levels of BAX and PUMA are controlled at the posttranslational level by phosphorylation. However, the posttranslational regulation of these proapoptotic proteins remains largely unexplored. In this study, using biochemical, molecular biology, flow cytometric, and immunohistochemistry techniques, we show that PUMA and BAX are the direct target of the F-box protein FBXL20, which restricts their cellular levels. FBXL20 directs the proteasomal degradation of PUMA and BAX in a protein kinase AKT1-dependent manner to promote cancer cell proliferation and tumor growth. Interestingly, inactivation of AKT1 results in activation of another protein kinase GSK3α/β, which facilitates the proteasomal degradation of FBXL20 by another F-box protein, FBXO31. Thus, a switch between two signaling kinases AKT1 and GSK3α/β modulates the functional activity of these proapoptotic regulators, thereby determining cell survival or death. RNAi-mediated ablation of FBXL20 results in increased levels of PUMA as well as BAX, which further enhances the sensitivity of cancer cells to chemotherapeutic drugs. We showed that high level expression of FBXL20 in cancer cells reduces therapeutic drug-induced apoptosis and promotes chemoresistance. Overall, this study highlights the importance of targeting FBXL20 in cancers in conjunction with chemotherapy and may represent a promising anticancer strategy to overcome chemoresistance.     

Microbial diversity of the Soldhar hot spring,India, assessed by analyzing 16S rRNA and protein-coding genes

Bacterial diversity of the Soldhar hot spring, located in the Chamoli district of Uttarakhand, India, was investigated using a clone library, denaturing gradient gel electrophoresis (DGGE) and functional genes. Physicochemical analysis of sediment samples indicated an oligotrophic environment with very low sulfur content. Based on the 16S rRNA gene studies Proteobacteria was the most predominant group in all the three approaches. Other dominant phyla were Deinococcus-Thermus and Aquificae. Pyrobaculum was the only archaeal genus detected by DGGE. In the functional gene analysis, the nifH library showed a single operational taxonomic unit (OTU) related to the genus Paenibacillus whereas the aoxB library showed three OTUs related to Acidovorax, Aminobacter and Agrobacterium. Our results demonstrate for the first time both the bacterial and archaeal diversity in the Soldhar hot spring by culture-independent techniques, thereby providing important information that will increase our understanding of the microbial ecology of the Soldhar hot spring.     

Fermentative production of rhamnolipid and purification by adsorption chromatography

Glycolipids are one of the major classes of biosurfactants in which the rhamnolipids are best studied. The present work investigates the optimization of inoculum age and batch time for maximizing the yield of rhamnolipid from Pseudomonas aeruginosa (MTCC 2453). The yield and titer of rhamnolipids were maximum in the fermentation batch with an inoculum age of 24?hr. Batch time studies were performed on biomass production, rhamnolipid production, and sunflower oil utilization. The maximum yield of rhamnolipid was achieved at 96?hr when the culture cells were in the late exponential/early stationary phase. At optimum substrate concentration, maximum yield of 10.8?g/L was achieved. Further, downstream processing of crude rhamnolipid from broth using organic solvent extraction and subsequent purification using adsorption chromatography was done. In this study, chromatographic method was developed for purification of rhamnolipid by adsorption phenomena with more than 88.7% purity and 86.5% recovery. The present study provides new perspective on concepts involving separation by adsorption. Further antimicrobial properties and surfactant properties were studied for rhamnolipid production.     

Size exclusion chromatography of zein and xanthophylls: Optimization of operating parameters

Ethanol-soluble components of corn (zein and xanthophylls) were separated and purified by size exclusion chromatography. Aqueous ethanol was used as the solvent for the entire process from extraction through chromatography. The effect of operating and design parameters, such as temperature, flow rate, loading mass, loading volume, column height and diameter, on productivity and resolution of the components was studied. Using a one-variable-at-a-time (OVAT) approach with 1 cm × 60 cm columns, optimum conditions were determined to be 40 °C temperature, 0.25 mL/min flow rate, volume loading of 22 mL corn extract, mass loading at 70 g/L of corn extract. All components could be resolved with base line separation with a 240 cm column length. Column diameter did not affect separation, implying linear scalability with constant flow distribution.     

Porohyperelastic finite element modeling of abdominal aortic aneurysms

Abdominal aortic aneurysm (AAA) is the gradual weakening and dilation of the infrarenal aorta. This disease is progressive, asymptomatic, and can eventually lead to rupture--a catastrophic event leading to massive internal bleeding and possibly death. The mechanical environment present in AAA is currently thought to be important in disease initiation, progression, and diagnosis. In this study, we utilize porohyperelastic (PHE) finite element models (FEMs) to investigate how such modeling can be used to better understand the local biomechanical environment in AAA. A 3D hypothetical AAA was constructed with a preferential anterior bulge assuming both the intraluminal thrombus (ILT) and the AAA wall act as porous materials. A parametric study was performed to investigate how physiologically meaningful variations in AAA wall and ILT hydraulic permeabilities affect luminal interstitial fluid velocities and wall stresses within an AAA. A corresponding hyperelastic (HE) simulation was also run in order to be able to compare stress values between PHE and HE simulations. The effect of AAA size on local interstitial fluid velocity was also investigated by simulating maximum diameters (5.5 cm, 4.5 cm, and 3.5 cm) at the baseline values of ILT and AAA wall permeability. Finally, a cyclic PHE simulation was utilized to study the variation in local fluid velocities as a result of a physiologic pulsatile blood pressure. While the ILT hydraulic permeability was found to have minimal affect on interstitial velocities, our simulations demonstrated a 28% increase and a 20% decrease in luminal interstitial fluid velocity as a result of a 1 standard deviation increase and decrease in AAA wall hydraulic permeability, respectively. Peak interstitial velocities in all simulations occurred on the luminal surface adjacent to the region of maximum diameter. These values increased with increasing AAA size. PHE simulations resulted in 19.4%, 40.1%, and 81.0% increases in peak maximum principal wall stresses in comparison to HE simulations for maximum diameters of 35 mm, 45 mm, and 55 mm, respectively. The pulsatile AAA PHE FEM demonstrated a complex interstitial fluid velocity field the direction of which alternated in to and out of the luminal layer of the ILT. The biomechanical environment within both the aneurysmal wall and the ILT is involved in AAA pathogenesis and rupture. Assuming these tissues to be porohyperelastic materials may provide additional insight into the complex solid and fluid forces acting on the cells responsible for aneurysmal remodeling and weakening.