1,4-Benzodiazepine-2,5-diones as small molecule antagonists of the HDM2-p53 interaction: discovery and SAR

A library of 1,4-benzodiazepine-2,5-diones was screened for binding to the p53-binding domain of HDM2 using Thermofluor, a miniaturized thermal denaturation assay. The hits obtained were shown to bind to HDM2 in the p53-binding pocket using a fluorescence polarization (FP) peptide displacement assay. The potency of the series was optimized, leading to sub-micromolar antagonists of the p53-HDM2 interaction.     

Nuclear targeting and cell cycle regulatory function of human BARD1

The BARD1 gene is mutated in a subset of breast and ovarian cancers, implicating BARD1 as a potential tumor suppressor. BARD1 gains a ubiquitin E3 ligase activity when heterodimerized with BRCA1, but the only known BRCA1-independent BARD1 function is a p53-dependent proapoptotic activity stimulated by nuclear export to the cytoplasm. We described previously the nuclear-cytoplasmic shuttling of BARD1, and in this study, we identify the transport sequences that target BARD1 to the nucleus and show that they are essential for BARD1 regulation of the cell cycle. We used deletion mapping and mutagenesis to define two active nuclear localization signals (NLSs) present in human BARD1 that are not conserved in rodent BARD1. Site-directed mutagenesis of the primary bipartite NLS abolished BARD1 nuclear import and caused its cytoplasmic accumulation. Using flow cytometry and 5-bromo-2-deoxyuridine incorporation assays, we discovered that transiently expressed BARD1 can elicit a p53-independent cell cycle arrest in G1 phase, and that this was abrogated by mutation of the BARD1 NLS but not by mutation of the nuclear export signal. Thus, BARD1 regulation of the cell cycle is a nuclear event and may be linked to its induced expression during mitosis and its possible involvement in the DNA damage checkpoint.     

Alanine-scanning mutations in domain 4 of anthrax toxin protective antigen reveal residues important for binding to the cellular receptor and to a neutralizing monoclonal antibody

A panel of variants with alanine substitutions in the small loop of anthrax toxin protective antigen domain 4 was created to determine individual amino acid residues critical for interactions with the cellular receptor and with a neutralizing monoclonal antibody, 14B7. Substituted protective antigen proteins were analyzed by cellular cytotoxicity assays, and their interactions with antibody were measured by plasmon surface resonance and analytical ultracentrifugation. Residue Asp683 was the most critical for cell binding and toxicity, causing an approximately 1000-fold reduction in toxicity, but was not a large factor for interactions with 14B7. Substitutions in residues Tyr681, Asn682, and Pro686 also reduced toxicity significantly, by 10-100-fold. Of these, only Asn682 and Pro686 were also critical for interactions with 14B7. However, residues Lys684, Leu685, Leu687, and Tyr688 were critical for 14B7 binding without greatly affecting toxicity. The K684A and L685A variants exhibited wild type levels of toxicity in cell culture assays; the L687A and Y688A variants were reduced only 1.5- and 5-fold, respectively.     

Effect of furosemide on hyperpnea-induced airway obstruction, injury, and microvascular leakage

Freed, Arthur N., Varsha Taskar, Brian Schofield, andChiharu Omori. Effect of furosemide on hyperpnea-induced airway obstruction, injury, and microvascular leakage. J. Appl. Physiol. 81(6): 2461-2467, 1996.Furosemideattenuates hyperpnea-induced airway obstruction (HIAO) in asthmaticsubjects via unknown mechanism(s). We studied the effect of furosemideon dry air-induced bronchoconstriction, mucosal injury, andbronchovascular hyperpermeability in a canine model of exercise-inducedasthma. Peripheral airway resistance (Rp) was recorded before and aftera 2-min dry-air challenge (DAC) at 2,000 ml/min. After pretreatmentwith aerosolized saline containing 0.75% dimethyl sulfoxide, DACincreased Rp 72 ± 11% (SE, n = 7) above baseline; aerosolized furosemide(10³ M) reduced thisresponse by ~50 ± 6% (P < 0.01). To assess bronchovascular permeability, colloidal carbon wasinjected (1 ml/kg iv) 1 min before DAC, and after 1 h, the vehicle- andfurosemide-treated airways were prepared for morphometric analysis.Light microscopy confirmed previous studies showing that DAC damagedthe airway epithelium and enhanced bronchovascular permeability.Furosemide did not inhibit dry air-induced mucosal injury orbronchovascular hyperpermeability and in fact tended to increase airwaydamage and vascular leakage. This positive trend toward enhancedbronchovascular permeability in DAC canine peripheral airways isconsistent with the hypothesis that furosemide inhibits HIAO in part byenhancing microvascular leakage and thus counterbalancing theevaporative water loss that occurs during hyperpnea.

     

The human loci DNF15S2 and D3S94 have a high degree of sequence similarity to acyl-peptide hydrolase and are located at 3p21.3.

The short arm of chromosome 3 undergoes genetic loss in most small-cell lung cancers and renal cell carcinomas. The most frequently deleted region includes the DNF15S2 locus (mapped to 3p21), suggesting that a putative recessive tumor-suppressor gene might be located nearby. A cosmid clone, cA476, contains the D3S94 locus and two HTF islands and detects a PstI RFLP. We have isolated cDNAs homologous to conserved fragments within cA476; and these cDNAs have 96% sequence similarity to a cDNA derived from the DNF15S2 locus. Sequence information from cDNAs derived from both the rat and pig acyl-peptide hydrolase (E.C.3.4.19.1) gene show that they have a high degree of sequence similarity to cDNAs derived from D3S94 and DNF15S2, suggesting that they are all the same locus. Cosmid cA476 (DNF15S2) has been mapped, by fluorescent in situ hybridization, to chromosome 3p21.3. D3S94 and DNF15S2 are quite distinct from aminoacylase 1 (ACY1), which has been physically linked to D3S2, D3S92, and D3S93, all localized within 3p21.1.     

Induction of tannin acyl hydrolase (EC 3.1.1.20) activity in some members of fungi imperfecti

The potential of gallotannin, methyl gallate, gallic acid, and pyrogallol to induce tannin acyl hydrolase (EC 3.1.1.20) activity in Aspergillus niger, Aspergillus fischerii, Fusarium solani, and Trichoderma viride has been investigated. The maximum induction ratios recorded were A. fischerii (26.7), F. solani (26.1), and T. viride (40.7) when the fungi were induced with gallotannin, gallic acid, and methyl gallate, respectively. A. niger was devoid of basal enzyme activity. The gallotannin tolerance limits for A. niger, A. fischerii, F. solani, and T. viride were determined by progressively increasing the gallotannin concentration and were found to be 20, 4, 3, and 3%, respectively. F. solanii induced with 3% gallotannin has been recommended for the production of tannase enzyme.     

Phosphatidylserine externalization and procoagulant activation of erythrocytes induced by Pseudomonas aeruginosa virulence factor pyocyanin

The opportunistic pathogen Pseudomonas aeruginosa causes a wide range of infections in multiple hosts by releasing an arsenal of virulence factors such as pyocyanin. Despite numerous reports on the pleiotropic cellular targets of pyocyanin toxicity in vivo, its impact on erythrocytes remains elusive. Erythrocytes undergo an apoptosis‐like cell death called eryptosis which is characterized by cell shrinkage and phosphatidylserine (PS) externalization; this process confers a procoagulant phenotype on erythrocytes as well as fosters their phagocytosis and subsequent clearance from the circulation. Herein, we demonstrate that P. aeruginosa pyocyanin‐elicited PS exposure and cell shrinkage in erythrocyte while preserving the membrane integrity. Mechanistically, exposure of erythrocytes to pyocyanin showed increased cytosolic Ca²⁺ activity as well as Ca²⁺‐dependent proteolytic processing of μ‐calpain. Pyocyanin further up‐regulated erythrocyte ceramide abundance and triggered the production of reactive oxygen species. Pyocyanin‐induced increased PS externalization in erythrocytes translated into enhanced prothrombin activation and fibrin generation in plasma. As judged by carboxyfluorescein succinimidyl‐ester labelling, pyocyanin‐treated erythrocytes were cleared faster from the murine circulation as compared to untreated erythrocytes. Furthermore, erythrocytes incubated in plasma from patients with P. aeruginosa sepsis showed increased PS exposure as compared to erythrocytes incubated in plasma from healthy donors. In conclusion, the present study discloses the eryptosis‐inducing effect of the virulence factor pyocyanin, thereby shedding light on a potentially important mechanism in the systemic complications of P. aeruginosa infection.     

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of beta2-adrenergic receptors

Phosphatidylinositol 3-kinase inhibitors have been shown to affect endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated beta(2)-adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate beta(2)-adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the beta-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of beta(2)-adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of beta(2)-adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of beta(2)-adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes.     

Green Synthesized Silver Nanoparticles Using Lactobacillus Acidophilus as an Antioxidant,Antimicrobial, and Antibiofilm Agent Against Multi-drug Resistant Enteroaggregative Escherichia Coli

Probiotics and Antimicrobial Proteins - The present study was envisaged to employ the green synthesis and characterization of silver nanoparticles (AgNPs) using the potential probiotic strain...