An endogenous factor which interacts with synaptosomal membrane Na+, K+-ATPase activation by K+

In previous papers, the isolation of brain soluble fractions able to modify neuronal Na⁺, K⁺-ATPase activity has been described. One of those fractions-peak I-stimulates membrane Na⁺, K⁺-ATPase while another-peak II-inhibits this enzyme activity, and has other ouabain-like properties. In the present study, synaptosomal membrane Na⁺, K⁺-ATPase was analyzed under several experimental conditions, using ATP orp-nitrophenylphosphate (p-NPP) as substrate, in the absence and presence of cerebral cortex peak II. Peak II inhibited K⁺-p-NPPase activity in a concentration dependent manner. Double reciprocal plots indicated that peak II uncompetitively inhibits K⁺-p-NPPase activity regarding substrate, Mg²⁺ and K⁺ concentration. Peak II failed to block the known K⁺-p-NPPase stimulation caused by ATP plus Na⁺. At various K⁺ concentrations, percentage K⁺-p-NPPase inhibition by peak II was similar regardless of the ATP plus Na⁺ presence, indicating lack of correlation with enzyme phosphorylation. Na⁺, K⁺-ATPase activity was decreased by peak II depending on K⁺ concentration. It is postulated that the inhibitory factor(s) present in peak II interfere(s) with enzyme activation by K⁺.     

Managing Bone Health in Breast Cancer

ObjectiveOsteoporosis is a common condition that can be caused or exacerbated by estrogen deficiency.MethodsThis narrative review will discuss optimizing bone health in the setting of adjuvant endocrine treatments for hormone receptor–positive breast cancer and the current use of antiresorptive agents as adjuvant therapy and as bone modifying agents.ResultsAdjuvant endocrine treatments for hormone receptor–positive breast cancer (tamoxifen and aromatase inhibitors) affect bone health. The exact effect depends on the agent used and the menopausal state of the woman. Antiresorptive medications for osteoporosis, bisphosphonates and denosumab, lower the risk of bone loss from aromatase inhibitors. Use of bisphosphonates as adjuvant treatment in breast cancer, regardless of hormone receptor status, is increasing because of benefits seen to cancer relapse and survival.ConclusionOptimizing bone health in women with breast cancer during and after cancer treatment is informed by an understanding of breast cancer treatment and its skeletal effect.     

Crystal structure of human annexin I at 2.5 A resolution.

cDNA coding for N-terminally truncated human annexin I, a member of the family of Ca(2+)-dependent phospholipid binding proteins, has been cloned and expressed in Escherichia coli. The expressed protein is biologically active, and has been purified and crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 139.36 A, b = 67.50 A, and c = 42.11 A. The crystal structure has been determined by molecular replacement at 3.0 A resolution using the annexin V core structure as the search model. The average backbone deviation between these two structures is 2.34 A. The structure has been refined to an R-factor of 17.7% at 2.5 A resolution. Six calcium sites have been identified in the annexin I structure. Each is located in the loop region of the helix-loop-helix motif. Two of the six calcium sites in annexin I are not occupied in the annexin V structure. The superpositions of the corresponding loop regions in the four domains show that the calcium binding loops in annexin I can be divided into two classes: type II and type III. Both classes are different from the well-known EF-hand motif (type I).     

Refinement of the structure of human basic fibroblast growth factor at 1.6 A resolution and analysis of presumed heparin binding sites by selenate substitution.

The three-dimensional structure of human basic fibroblast growth factor has been refined to a crystallographic residual of 16.1% at 1.6 A resolution. The structure has a Kunitz-type fold and is composed of 12 antiparallel beta-strands, 6 of which form a beta-barrel. One bound sulfate ion has been identified in the model, hydrogen bonded to the side chains of Asn 27, Arg 120, and Lys 125. The side chain of Arg 120 has two conformations, both of which permit hydrogen bonds to the sulfate. This sulfate binding site has been suggested as the binding site for heparin (Eriksson, A.E., Cousens, L.S., Weaver, L.H., & Matthews, B.W., 1991, Proc. Natl. Acad. Sci. USA 88, 3441-3445). Two beta-mercaptoethanol (BME) molecules are also included in the model, each forming a disulfide bond to the S gamma atoms of Cys 69 and Cys 92, respectively. The side chain of Cys 92 has two conformations of which only one can bind BME. Therefore the BME molecule is half occupied at this site. The locations of possible sulfate binding sites on the protein were examined by replacing the ammonium sulfate in the crystallization medium with ammonium selenate. Diffraction data were measured to 2.2 A resolution and the structure refined to an R-factor of 13.8%. The binding of the more electron-dense selenate ion was identified at two positions. One position was identical to the sulfate binding site identified previously. The second selenate binding site, which is of lower occupancy, is situated 5.6 A from the first. This ion is hydrogen bonded by the side chain of Lys 135 and Arg 120. Thus the side chain of Arg 120 binds two selenate ions simultaneously. It is suggested that the observed second selenate binding site should also be considered as a possible binding site for heparin, or that both selenate binding sites might simultaneously contribute to the binding of heparin.     

Higher-order interactions of Bcr-Abl can broaden chronic myeloid leukemia (CML) drug repertoire

Bcr-Abl, a nonreceptor tyrosine kinase, is associated with leukemias, especially chronic myeloid leukemia (CML). Deletion of Abl's N-terminal region, to which myristoyl is linked, renders the Bcr-Abl fusion oncoprotein constitutively active. The substitution of Abl's N-terminal region by Bcr enables Bcr-Abl oligomerization. Oligomerization is critical: it promotes clustering on the membrane, which is essential for potent MAPK signaling and cell proliferation. Here we decipher the Bcr-Abl specific, step-by-step oligomerization process, identify a specific packing surface, determine exactly how the process is structured and identify its key elements. Bcr's coiled coil (CC) domain at the N-terminal controls Bcr-Abl oligomerization. Crystallography validated oligomerization via Bcr-Abl dimerization between two Bcr CC domains, with tetramerization via tight packing between two binary assemblies. However, the structural principles guiding Bcr CC domain oligomerization are unknown, hindering mechanistic understanding and drugs exploiting it. Using molecular dynamics (MD) simulations, we determine that the binary complex of the Bcr CC domain serves as a basic unit in the quaternary complex providing a specific surface for dimer–dimer packing and higher-order oligomerization. We discover that the small α1-helix is the key. In the binary assembly, the helix forms interchain aromatic dimeric packing, and in the quaternary assembly, it contributes to the specific dimer–dimer packing. Our mechanism is supported by the experimental literature. It offers the key elements controlling this process which can expand the drug discovery strategy, including by Bcr CC-derived peptides, and candidate residues for small covalent drugs, toward quenching oligomerization, supplementing competitive and allosteric tyrosine kinase inhibitors.     

Crystallization and preliminary X-ray analysis of nonglycosylated tissue inhibitor of metalloproteinases-1, N30QN78Q TIMP-1

A nonglycosylated (N₃₀QN₇₈Q) form of the human tissue inhibitor of metalloproteinases, TIMP-1, has been prepared and crystallized in a form suitable for X-ray diffraction analysis. Small single crystals have been grown using sodium tartrate as a precipitant. The crystals are in space group P2₁, with cell dimensions a = 35.28, b = 53.95, c = 48.56, and β = 96.0°. There is a single molecule of TIMP-1 in the asymmetric unit. The crystals diffract to at least 2.3 Å resolution. Complete data have been collected to 2.9 Å and a search for heavymetal derivatives is in progress. © 1993 Wiley-Liss, Inc.     

New Isopropyl Chromone and Flavanone Glucoside Compounds from the Leaves of Syzygium cerasiforme (Blume) Merr. & L.M.Perry and Their Inhibition of Nitric Oxide Production

A new isopropyl chromone ( 1 ) and a new flavanone glucoside ( 2 ) together with eleven known compounds ( 3–13 ) were isolated from the leaves of Syzygium cerasiforme (Blume) Merr. & L.M.Perry. Their structures were elucidated as 5,7-dihydroxy-2-isopropyl-6,8-dimethyl-4H-chromen-4-one ( 1 ), 5,7-dihydroxyflavanone 7-O-β-D-(6′′-O-galloylglucopyranoside) ( 2 ), strobopinin ( 3 ), demethoxymatteucinol ( 4 ), pinocembrin-7-O-β-D-glucopyranoside ( 5 ), (2S)-hydroxynaringenin-7-O-β-D-glucopyranoside ( 6 ), afzelin ( 7 ), quercetin ( 8 ), kaplanin ( 9 ), endoperoxide G3 ( 10 ), grasshopper ( 11 ), vomifoliol ( 12 ), litseagermacrane ( 13 ) by the analysis of HR-ESI-MS, NMR, and CD spectral data. Compounds 1 , 2 , 5 , 6 and 10 inhibited NO production on LPS-activated RAW264.7 cells with IC₅₀ values of 12.28±1.15, 8.52±1.62, 7.68±0.87, 9.67±0.57, and 6.69±0.34 μM, respectively, while the IC₅₀ values of the other compounds ranging from 33.38±0.78 to 86.51±2.98 μM, compared to that of the positive control, N^G-monomethyl-L-arginine acetate (L-NMMA) with an IC₅₀ value of 32.50±1.00 μM.     

Design,Synthesis, and Biological Evaluation of Novel Indanone Derivatives as Cholinesterase Inhibitors for Potential Use in Alzheimer's Disease

Indanone derivatives containing meta/para-substituted aminopropoxy benzyl/benzylidene moieties were designed based on the structures of donepezil and ebselen analogs as the cholinesterase inhibitors. The designed compounds were synthesized and their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities were measured. Inhibitory potencies (IC₅₀ values) for the synthesized compounds ranged from 0.12 to 11.92 μM and 0.04 to 24.36 μM against AChE and BChE, respectively. Compound 5 c showed the highest AChE inhibitory potency with IC₅₀ value of 0.12 μM, whereas the highest BChE inhibition was achieved by structure 7 b (IC₅₀=0.04 μM). Structure-activity relationship (SAR) analysis revealed that there is no significant difference between meta and para-substituted derivatives in AChE and BChE inhibition. However, the most potent AChE inhibitor 5 c belongs to meta-substituted compounds, while the most active BChE inhibitor is para-substituted derivative 7 b . The order of enzyme inhibition potency based on the substituted amine group is dimethyl amine>piperidine>morpholine. Compounds containing C=C linkage are more potent AChE inhibitors than the corresponding saturated structures. Molecular docking studies indicated that 5 c interacts with AChE in a very similar way to that observed experimentally for donepezil. The introduced indanone-aminopropoxy benzylidenes could be used in drug-discovery against Alzheimer's disease.     

FISCHERELLIN,A NEW ALLELOCHEMICAL FROM THE FRESHWATER CYANOBACTERIUM FISCHERELLA MUSCICOLA1

The benthic cyanobacterium Fischerella muscicola (Thur.) Gom. UTEX 1829 produces a secondary metabolite, fischerellin, that strongly inhibits other cyanobacteria and to a lesser extent members of the Chlorophyceae. Eubacteria are not affected. The major active compound is lipophilic and exhibits a molecular ion at m/z 408. It is heat- and acid-stable but decomposes in 1 M sodium hydroxide (80° C. 1 h). Fischerellin inhibits the photosynthetic but not the respiratory electron transport of cyanobacteria and chlorophytes. Its site of action is located in PS II. Two other species of Fischerella also produce fischerellin, indicating that the synthesis of such allelochemicals might be characteristic of the genus.