Tezosentan increases nitric oxide signaling via enhanced hydrogen peroxide generation in lambs with surgically induced acute increases in pulmonary blood flow

We have previously shown that acute increases in pulmonary blood flow (PBF) are limited by a compensatory increase in pulmonary vascular resistance (PVR) via an endothelin‐1 (ET‐1) dependent decrease in nitric oxide synthase (NOS) activity. The mechanisms underlying the reduction in NO signaling are unresolved. Thus, the purpose of this study was to elucidate mechanisms of this ET‐1–NO interaction. Pulmonary arterial endothelial cells were acutely exposed to shear stress in the presence or absence of tezosentan, a combined ET_A/ET_B receptor antagonist. Shear increased NO_x, eNOS phospho‐Ser1177, and H₂O₂ and decreased catalase activity; tezosentan enhanced, while ET‐1 attenuated all of these changes. In addition, ET‐1 increased eNOS phospho‐Thr495 levels. In lambs, 4 h of increased PBF decreased H₂O₂, eNOS phospho‐Ser1177, and NO_X levels, and increased eNOS phospho‐Thr495, phospho‐catalase, and catalase activity. These changes were reversed by tezosentan. PEG‐catalase reversed the positive effects of tezosentan on NO signaling. In all groups, opening the shunt resulted in a rapid increase in PBF by 30 min. In vehicle‐ and tezosentan/PEG‐catalase lambs, PBF did not change further over the 4 h study period. PVR fell by 30 min in vehicle‐ and tezosentan‐treated lambs, and by 60 min in tezosentan/PEG‐catalase‐treated lambs. In vehicle‐ and tezosentan/PEG‐catalase lambs, PVR did not change further over the 4 h study period. In tezosentan‐treated lambs, PBF continued to increase and LPVR to decrease over the 4 h study period. We conclude that acute increases in PBF are limited by an ET‐1 dependent decrease in NO production via alterations in catalase activity, H₂O₂ levels, and eNOS phosphorylation. J. Cell. Biochem. 114: 435–447, 2013. © 2012 Wiley Periodicals, Inc.     

Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell

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Prediction of Radiation Esophagitis in Non–Small Cell Lung Cancer Using Clinical Factors,Dosimetric Parameters,and Pretreatment Cytokine Levels

Radiation esophagitis (RE) is a common adverse event associated with radiotherapy for non–small cell lung cancer (NSCLC). While plasma cytokine levels have been correlated with other forms of radiation-induced toxicity, their association with RE has been less well studied. We analyzed data from 126 patients treated on 4 prospective clinical trials. Logistic regression models based on combinations of dosimetric factors [maximum dose to 2 cubic cm (D2cc) and generalized equivalent uniform dose (gEUD)], clinical variables, and pretreatment plasma levels of 30 cytokines were developed. Cross-validated estimates of area under the receiver operating characteristic curve (AUC) and log likelihood were used to assess prediction accuracy. Dose-only models predicted grade 3 RE with AUC values of 0.750 (D2cc) and 0.727 (gEUD). Combining clinical factors with D2cc increased the AUC to 0.779. Incorporating pretreatment cytokine measurements, modeled as direct associations with RE and as potential interactions with the dose-esophagitis association, produced AUC values of 0.758 and 0.773, respectively. D2cc and gEUD correlated with grade 3 RE with odds ratios (ORs) of 1.094/Gy and 1.096/Gy, respectively. Female gender was associated with a higher risk of RE, with ORs of 1.09 and 1.112 in the D2cc and gEUD models, respectively. Older age was associated with decreased risk of RE, with ORs of 0.992/year and 0.991/year in the D2cc and gEUD models, respectively. Combining clinical with dosimetric factors but not pretreatment cytokine levels yielded improved prediction of grade 3 RE compared to prediction by dose alone. Such multifactorial modeling may prove useful in directing radiation treatment planning.     

Biodegradation of chlorpropham and its major products by <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NKC-1

Chlorpropham [isopropyl N-(3-chlorophenyl) carbamate] (CIPC), an important phenyl carbamate herbicide, has been used as a plant growth regulator and potato sprout suppressant (Solanum tuberosum L) during long-term storage. A bacterium capable of utilizing the residual herbicide CIPC as a sole source of carbon and energy was isolated from herbicide-contaminated soil samples employing selective enrichment method. The isolated bacterial strain was identified as Bacillus licheniformis NKC-1 on the basis of its morphological, cultural, biochemical characteristics and also by phylogenetic analysis based on 16S rRNA gene sequences. The organism degraded CIPC through its initial hydrolysis by CIPC hydrolase enzyme to yield 3-chloroaniline (3-CA) as a major metabolic product. An inducible 3-CA dioxygenase not only catalyzes the incorporation of molecular oxygen but also removes the amino group by the deamination yielding a monochlorinated catechol. Further, degradation of 4-chlorocatechol proceeded via ortho- ring cleavage through the maleylacetate process. 3-Chloroaniline and 4-chlorocatechol are the intermediates in the CIPC degradation which suggested that dechlorination had occurred after the aromatic ring cleavage. The presence of these metabolites has been confirmed by using ultra-violet (UV), high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), Fourier transmission-infrared (FT-IR), proton nuclear magnetic resonance (¹H NMR) and gas chromatography-mass (GC-MS) spectral analysis. Enzyme activities of CIPC hydrolase, 3-CA dioxygenase and chlorocatechol 1, 2-dioxygenase were detected in the cell-free-extract of the CIPC culture and are induced by cells of NKC-1 strain. These results demonstrate the biodegradation pathways of herbicide CIPC and promote the potential use of NKC-1 strain to bioremediate CIPC-contaminated environment with subsequent release of ammonia, chloride ions and carbon dioxide.     

Utilization of horticultural waste (Apple Pomace) for multiple carbohydrase production from Rhizopus delemar F2 under solid state fermentation

The brown rot fungus Rhizopus delemar F₂ was shown to produce extracellular thermostable and multiple carbohydrase enzymes. The potential of Rhizopus delemar F2 in utilizing apple pomace under solid state fermentation (SSF) is the purpose of the study. Solid state fermentation (SSF) is a very effective technique opposed to submerged fermentation in various aspects. Enhanced production of multiple carbohydrases 18.20?U?g^?1 of cellulose, 158.30?U?g^?1 of xylanase, 61.50?U?g^?1 of pectinase and amylase 21.03?U?g^?1 was released by microwave pretreatment of apple pomace at 450?W for 1?min and then by incubation the culture thus obtained at 30?°C for 6 days with moisture content of 1:4.5. Apple pomace can serve as a potential source of raw material for the production of multiple carbohydrases. Besides, it can find great commercial significance in production of bioethanol and various industries like textile, fruit juice, paper and pulp industry.     

Skeletal abnormalities in doubly heterozygous Bmp4 and Bmp7 mice

Analysis of the skeletal phenotypes caused by the genetic inactivation of individual Bmps, along with the study of their expression patterns, suggest possible functional redundancy of these molecules. To investigate the effect on skeleton development of the combined absence of some Bmp genes expressed in the same areas, we have intercrossed heterozygous Bmp7 mice with Bmp2^+/−, Bmp4^+/−, or Bmp5^+/− animals. Bmp2/7 and Bmp5/7 double heterozygous animals do not present with any abnormalities. In contrast, Bmp4/7 double heterozygotes develop minor defects in two restricted areas of the skeleton, the rib cage, and the distal part of the limbs. In the ribs, Bmp4 and Bmp7 seem to act in the same pathway to assure proper guidance of mesenchymal condensations of the ribs extending toward the sternum. In the limbs, these molecules appear to play a similar role in controlling digit number, possibly through induction of apoptosis in the interdigital and anterior mesenchyme. Dev. Genet. 22:340–348, 1998. © 1998 Wiley-Liss, Inc.     

Insights into the crystal structure of BRD2-BD2 – phenanthridinone complex and theoretical studies on phenanthridinone analogs

Bromodomain and extra-terminal family proteins recognize the acetylated histone code on chromatin and participate in downstream processes like DNA replication, modification, and repair. As part of epigenetic approaches, BRD2 and BRD4 were identified as putative targets, for the management of chronic diseases. We have recently reported the discovery of a new scaffold of the phenanthridinone-based inhibitor (L10) of the second bromodomain of BRD2 (BRD2-BD2). Here, we present the crystal structure of the BRD2-BD2, refined to 1.4 Å resolution, in complex with β-mercaptoethanol (a component of the protein buffer). The β-mercaptoethanol covalently links to C425 of BD2 in the acetyl-lysine binding pocket, to form a modified cysteine mercaptoethanol (CME). The CME modification significantly hinders the entry of ligands into the BD2 binding pocket, suggesting that β-mercaptoethanol should be removed during protein production process. Next, to confirm whether phenanthridionone scaffold is a new inhibitor family of BRD2-BD2, we have determined the crystal structure of BD2 in complex with 6(5H)-Phenanthridinone (a core moiety of L10), refined to 1.28 Å resolution. It confirmed that the phenanthridinone molecule, unambiguously, binds to BD2. Moreover, we performed molecular docking and molecular dynamic studies on selected phenanthridinone analogs. The predicted L10 analogs are stable with essential hydrophobic and hydrophilic interactions with BD2 during molecular dynamic simulations. We propose that the predicted phenanthridinone analogs may be potential molecules for inhibiting the BD2 function of acetylated histone recognition.     

Purification and characterisation of prolactin from sheep and buffalo pituitaries

A study of the problem of structural variants of proteins and their relative contribution to the expressed immunological and biological activity has been initiated using sheep and buffalo prolactins as models. The feasibility of obtaining immunologically and biologically active prolactin in high yields from the discarded ’acid pellet’ of sheep and buffalo pituitaries has been demonstrated. This permits use of the same batch of glands for purifying lutropin, follitropin and prolactin as side fractions. The major component in preparations of buffalo prolactin has a molecular size of 24 kDa. The preparations were active in a radioligand binding inhibition assay and in a rat liver based radioreceptor assay. Charge and size isomers of sheep prolactin and buffalo prolactin have been observed. The reference sheep prolactin did not, in preliminary work, give any indication of being glycosylated. However radioactive sulphate was found to be incorporated into prolactin-rich fractions of sheep and buffalo pituitariesin vitro. By physico-chemical and immunochemical criteria the [³⁵S]-labelled material was similar to standard reference prolactin. The structural implications of sulphation have been probed. Presented at the 3rd National Symposium on Bioorganic Chemistry, 1987, Hyderabad.