Design and synthesis of 2-substituted benzoxazoles as novel PTP1B inhibitors

A series of benzoxazole compounds containing oxamic acid were synthesized and screened for the PTP1B inhibition. Compound 31d showed best biochemical potency (K_i) of 6.7 μM. Structure–activity relationship were explained with the help of molecular modeling approach.     

Controlled release of nutrients to mammalian cells cultured in shake flasks

Though cell culture-based protein production processes are rarely carried out under batch mode of operation, cell line and initial process development operations are usually carried out in batch mode due to simplicity of operation in widely used scale down platforms like shake flasks. Nutrient feeding, if performed, is achieved by bolus addition of concentrated feed solution at different intervals, which leads to large transient increases in nutrient concentrations. One negative consequence is increased waste metabolite production. We have developed a hydrogel-based nutrient delivery system for continuous feeding of nutrients in scale down models like shake flasks without the need for manual feed additions or any additional infrastructure. Continuous delivery also enables maintaining nutrient concentrations at low levels, if desired. The authors demonstrate the use of these systems for continuous feeding of glucose and protein hydrolysate to a suspension Chinese Hamster Ovary (CHO) culture in a shake flask. Glucose feeding achieved using the glucose-loaded hydrogel resulted in a 23% higher integral viable cell density and an 89% lower lactate concentration at the end of the culture when compared with a bolus-feed of glucose.     

Effect of Different Interferonα2 Preparations on IP10 and ET-1 Release from Human Lung Cells

Background

Alfa-interferons (IFNα2a, IFNα2b, 40KDa-PEGIFNα2a and 12KDa-PEGIFNα2b) are effective treatments for chronic hepatitis C infection. However, their usage has been associated with a variety of adverse events, including interstitial pneumonitis and pulmonary arterial hypertension. Although rare, these adverse events can be severe and potentially life-threatening, emphasizing the need for simple biomarkers of IFN-induced lung toxicity.

Methods

Human lung microvascular endothelial cells (HLMVEC), human pulmonary artery smooth muscle (HPASM) cells and A549 cells were grown under standard conditions and plated into 96- or 6-well plates. Cells were stimulated with various concentrations of different IFNs in hydrocortisone-free medium. After 24 and 48 hours, IP10 and ET-1 were measured by ELISA in conditioned medium. In a second set of experiments, cells were pre-treated with tumour necrosis factor-α (TNF-α) (10 ng/mL).

Results

IFNα2a, IFNα2b, 40KDa-PEGIFNα2a and 12KDa-PEGIFNα2b, but not IFNλ, induced IP10 (CXCL10) release and increased IP10 gene induction in HLMVEC. In addition, all four IFNα preparations induced IP10 release from HPASM cells and A549 cells pre-treated with TNFα. In each of these cell types, 40KDa-PEGIFNα2a was significantly less active than the native forms of IFNα2a, IFNα2b or 12KDa-PEGIFNα2b. Similarly, IFNα2a, IFNα2b and 12KDa-PEGIFNα2b, but not 40KDa-PEGIFNα2a, induced endothelin (ET)-1 release from HPASM cells.

Conclusions

Consistent with other interstitial pulmonary diseases, both IP10 and ET1 may serve as markers to monitor IFN-induced lung toxicity in patients. In addition, both markers may also serve to help characterize the risk associated with IFNα preparations to induce lung toxicity.     

Viral Toll Like Receptor activation of pulmonary vascular smooth muscle cells results in endothelin-1 generation; relevance to pathogenesis of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare but fatal condition in which raised pulmonary vascular resistance leads to right heart failure and death. Endothelin-1 is a potent endogenous vasoconstrictor, which is considered to be central to many of the events that lead to PAH, and is an important therapeutic target in the treatment of the condition. In many cases of PAH, the aetiology is unknown but inflammation is increasingly thought to play an important role and viruses have been implicated in the development of disease. The Toll Like Receptors (TLRs) play a key role in innate immune responses by initiating specific anti-bacterial and anti-viral defences in recognition of signature molecular motifs on the surface of invading pathogens. In this study, we set out to examine the expression of bacterial and viral TLRs in human pulmonary artery smooth muscle cells and to establish whether their activation could be relevant to PAH. We found that the viral TLR3 and bacterial TLRs 4 and 6 were most abundantly expressed in human pulmonary artery smooth muscle cells. Using specific TLR ligands, we found that activation of TLRs 3 and 4 resulted in IL-8 release by human pulmonary artery smooth muscle cells but that only TLR3 stimulation resulted in IP10 and endothelin-1 release. These data suggest that human pulmonary artery smooth muscle cells express significant levels of viral TLR3 and respond to its activation by releasing endothelin-1. This may have importance in understanding the association between viruses and the development of PAH.     

Functional and Structural Characterization of Melanin from Brevibacillus invocatus Strain IBA

Doklady Biological Sciences - Melanin is a polyphenol or indolic dark brown to black pigment of macromolecules that has a variety of biological functions including UV defence, desiccation, and...     

Unique Drought Resistance Functions of the Highly ABA-Induced Clade A Protein Phosphatase 2Cs

Six Arabidopsis (Arabidopsis thaliana) clade A protein phosphatase 2Cs (PP2Cs) have established abscisic acid (ABA) signaling roles; however, phenotypic roles of the remaining three "HAI" PP2Cs, Highly ABA-Induced1 (HAI1), AKT1-Interacting PP2C1/HAI2, and HAI3, have remained unclear. HAI PP2C mutants had enhanced proline and osmoregulatory solute accumulation at low water potential, while mutants of other clade A PP2Cs had no or lesser effect on these drought resistance traits. hai1-2 also had increased expression of abiotic stress-associated genes, including dehydrins and late embryogenesis abundant proteins, but decreased expression of several defense-related genes. Conversely, the HAI PP2Cs had relatively less impact on several ABA sensitivity phenotypes. HAI PP2C single mutants were unaffected in ABA sensitivity, while double and triple mutants were moderately hypersensitive in postgermination ABA response but ABA insensitive in germination. The HAI PP2Cs interacted most strongly with PYL5 and PYL7 to -10 of the PYL/RCAR ABA receptor family, with PYL7 to -10 interactions being relatively little affected by ABA in yeast two-hybrid assays. HAI1 had especially limited PYL interaction. Reduced expression of the main HAI1-interacting PYLs at low water potential when HAI1 expression was strongly induced also suggests limited PYL regulation and a role of HAI1 activity in negatively regulating specific drought resistance phenotypes. Overall, the HAI PP2Cs had greatest effect on ABA-independent low water potential phenotypes and lesser effect on classical ABA sensitivity phenotypes. Both this and their distinct PYL interaction demonstrate a new level of functional differentiation among the clade A PP2Cs and a point of cross talk between ABA-dependent and ABA-independent drought-associated signaling.     

The flip side of phospho‐signalling: Regulation of protein dephosphorylation and the protein phosphatase 2Cs

Protein phosphorylation is a key signalling mechanism and has myriad effects on protein function. Phosphorylation by protein kinases can be reversed by protein phosphatases, thus allowing dynamic control of protein phosphorylation. Although this may suggest a straightforward kinase–phosphatase relationship, plant genomes contain five times more kinases than phosphatases. Here, we examine phospho‐signalling from a protein phosphatase centred perspective and ask how relatively few phosphatases regulate many phosphorylation sites. The most abundant class of plant phosphatases, the protein phosphatase 2Cs (PP2Cs), is surrounded by a web of regulation including inhibitor and activator proteins as well as posttranslational modifications that regulate phosphatase activity, control phosphatase stability, or determine the subcellular locations where the phosphatase is present and active. These mechanisms are best established for the Clade A PP2Cs, which are key components of stress and abscisic acid signalling. We also describe other PP2C clades and illustrate how these phosphatases are highly regulated and involved in a wide range of physiological functions. Together, these examples of multiple layers of phosphatase regulation help explain the unbalanced kinase–phosphatase ratio. Continued use of phosphoproteomics to examine phosphatase targets and phosphatase–kinase relationships will be important for deeper understanding of phosphoproteome regulation.     

Transformations in plant-available forms of iron and manganese as influenced by manuring and cropping under irrigation

Summary Vertisols from field plots fertilized continuously with P, K, FYM alone and in combination at the rate of 60 kg P₂O₅, 30 kg K₂O and 1.5 ton FYM per ha were tested for different forms of Fe and Mn as a consequence of continuous cropping with sorghum and wheat. The transformations that occurred in different forms of plant-available iron were greater than those of Mn. Exchangeable and easily reducible Fe contents were markedly influenced by treatments. In manganese, transformations of easily reducible to exchangeable forms were conspicuous.     

Synergistic Induction of Endothelin-1 by Tumor Necrosis Factor �� and Interferon �� Is due to Enhanced NF-��B Binding and Histone Acetylation at Specific ��B Sites

Endothelin-1 (ET-1) is a potent vasoconstrictor and co-mitogen for vascular smooth muscle and is implicated in pulmonary vascular remodeling and the development of pulmonary arterial hypertension. Vascular smooth muscle is an important source of ET-1. Here we demonstrate synergistic induction of preproET-1 message RNA and release of mature peptide by a combination of tumor necrosis factor α (TNFα) and interferon γ (IFNγ) in primary human pulmonary artery smooth muscle cells. This induction was prevented by pretreatment with the histone acetyltransferase inhibitor anacardic acid. TNFα induced a rapid and prolonged pattern of nuclear factor (NF)-κB p65 subunit activation and binding to the native preproET-1 promoter. In contrast, IFNγ induced a delayed activation of interferon regulatory factor-1 without any effect on NF-κB p65 nuclear localization or consensus DNA binding. However, we found cooperative p65 binding and histone H4 acetylation at distinct κB sites in the preproET-1 promoter after stimulation with both TNFα and IFNγ. This was associated with enhanced recruitment of RNA polymerase II to the ATG start site and read-through of the ET-1 coding region. Understanding such mechanisms is crucial in determining the key control points in ET-1 release. This has particular relevance to developing novel treatments targeted at the inflammatory component of pulmonary vascular remodeling.Endothelin-1 is a 21-amino acid peptide which is known to be both a potent vasoconstrictor and mitogen for vascular smooth muscle (1, 2). It is released as a 38-amino acid precursor (Big ET-1²) before cleavage to the mature ET-1 form. As such it has been implicated in the pathogenesis of vascular disease and is particularly associated with pulmonary arterial hypertension (3). Indeed, several endothelin receptor antagonists are now approved for the treatment of pulmonary arterial hypertension (4). However, endothelin receptor antagonists as a class are associated with potentially serious side effects (4), making new treatments aimed at blocking ET-1 synthesis an attractive alternative.Although endothelial cells are thought to be the main source of ET-1 release, several groups including our own have shown that ET-1 can be released from the more numerous vascular smooth muscle cells (5–10). The vascular pathology observed in pulmonary arterial hypertension is propagated by inflammation, and circulating levels of cytokines including tumor necrosis factor α (TNFα) are elevated in patients with pulmonary arterial hypertension (11–15). In many cell types cytokines mediate their biological effects at least in part by the activation of the nuclear factor κB (NF-κB) pathway (16), and a role for NF-κB in pulmonary arterial hypertension has been proposed (17). In addition, we have shown previously that a combination of TNFα and interferon γ (IFNγ) stimulates human pulmonary artery smooth muscle (HPASM) cells to release ET-1 (18). However, the mechanisms underlying this effect are unknown.The preproET-1 promoter region has been shown experimentally to possess binding sites for nuclear factor (NF)-1 and phorbol ester-sensitive c-Fos and c-Jun complexes (19), acute phase reactant regulatory proteins, and binding sites for AP-1 and GATA-2 (20–22). In addition, binding sites for interferon regulatory factor-1 (IRF-1) and NF-κB are predicted by Transfac analysis (23). The close proximity of the IRF-1 site and one of the NF-κB sites is characteristic of genes that are regulated by the synergistic action of TNFα and IFNγ, such as interleukin-6 (IL-6) and intercellular adhesion molecule-1 (24, 25), although ET-1 has not previously been recognized in this group.Our aims were, therefore, to investigate the role of NF-κB in ET-1 release by primary HPASM cells. In addition, we were interested in the role of histone acetylation in the epigenetic control of the ET-1 production. Understanding these novel mechanisms will allow a greater understanding of the pathogenesis of vascular remodeling in pulmonary vessels and aid in the development of new treatment strategies aimed at blocking synthesis of ET-1.     

Molecular docking and simulation analysis of the FimH protein with secondary metabolites from the Garcinia species

It is of interest to document the molecular docking and simulation analysis of the FimH protein with 10 secondary metabolites from Garcinia species in the context of drug discovery for Urinary tract infections (UTIs). We report the optimal binding features of flavonoids with the FimH protein for further consideration in drug discovery.