Iron(III) and aluminum(III) complexes with hydroxypyrone ligands aimed to design kojic acid derivatives with new perspectives

With the aim to design new chelators for the clinical treatment of different diseases involving the trivalent metal ions Fe(III) and Al(III), we present the equilibria of kojic acid and its derivative 6-[5-hydroxy-2-hydroxymethyl-pyran-4-one]-5-hydroxy-2-hydroxymethyl-pyran-4-one with these two metal ions. Potentiometric and spectrophotometric techniques for iron, and potentiometry and ¹H NMR for aluminum were used, supported by X-ray, electrospray ionization-mass spectrometry (ESI-MS), calorimetry and quantum chemical calculations. In this work, evidence is given on the formation of MeL, MeL₂, and MeL₃ complexes of both metal ions with kojic acid, confirmed by the X-ray structure of the FeL₃ complex, and of variously protonated Me₂L₂ and MeL₂ complexes of 6-[5-hydroxy-2-hydroxymethyl-pyran-4-one]-5-hydroxy-2-hydroxymethyl-pyran-4-one. The extremely good pFe value for this second ligand gives confidence to, and opens perspectives for, the search of new kojic acid derivatives.     

Modification of the clozapine structure by parallel synthesis

A structure-activity study based on the core structure of clozapine 1b was accomplished by utilizing high-throughput synthesis. Several focused libraries were designed and synthesized to quickly develop SAR. The results indicate that by varying different regions of clozapine, both D(1)-selective and D(2)-selective compounds can be obtained.     

SCH 57790: a novel M2 receptor selective antagonist

As a decrease in cholinergic neurons has been observed in Alzheimer's Disease (AD), therapeutic approaches to AD include inhibition of acetylcholinesterase to increase acetylcholine levels. Evidence suggests that acetylcholine release in the CNS is modulated by negative feedback via presynaptic M2 receptors, blockade of which should provide another means of increasing acetylcholine release. Structure-activity studies of [4-(phenylsulfonyl)phenyl]methylpiperazines led to the synthesis of 4-cyclohexyl-alpha-[4-[[4-methoxyphenyl]sulfinyl]-phenyl]-1-piperazin eacetonitrile. This compound, SCH 57790, binds to cloned human M2 receptors expressed in CHO cells with an affinity of 2.78 nM; the affinity at M1 receptors is 40-fold lower. SCH 57790 is an antagonist at M2 receptors expressed in CHO cells, as the compound blocks the inhibition of adenylyl cyclase activity mediated by the muscarinic agonist oxotremorine. This compound should be useful in assessing the potential of M2 receptor blockade for enhancement of cognition.     

The effects of the main body cations on the peptidase(s) activity against pyroGlu6[125I-Tyr8]SP6-11 in the nuclear and synaptosomal fraction of the cortex and hippocampus of rat brain

1. Peptidase(s) activity of nuclear and synaptosomal fraction from cortex and hippocampus of rat brain against pyroGlu6[125I-Tyr8]SP6-11 was evaluated in different concentration of Ca2+, Mg2+, K+ and Na+ in about "isotonic" conditions. 2. The effects of studied ions on the peptidase activities forming N-terminal and C-terminal fragments are different especially in synaptosomes of both areas. 3. The differences of ionic requirements for N- and C-forming activities are particularly relevant for Ca2+ at the cortex and K+ at the hippocampus. 4. Ca2+ activate forming of N-terminal fragments in the nuclear fraction whereas inhibit it in synaptosomes from both areas. 5. The ionic requirements for C-terminal fragments' formation in synaptosomes of both areas are contradictory.     

Prostate‐specific membrane antigen expression in the vasculature of primary lung carcinomas associates with faster metastatic dissemination to the brain

Glioblastomas and brain metastases (BM) of solid tumours are the most common central nervous system neoplasms associated with very unfavourable prognosis. In this study, we report the association of prostate‐specific membrane antigen (PSMA) with various clinical parameters in a large cohort of primary and secondary brain tumours. A tissue microarray containing 371 cases of ascending grades of gliomas pertaining to astrocytic origin and samples of 52 cases of primary lung carcinomas with matching BM with follow‐up time accounting to 10.4 years was evaluated for PSMA expression using immunohistochemistry. In addition, PSMA expression was studied in BM arising from melanomas and breast carcinomas. Neovascular expression of PSMA was evident alongside with high expression in the proliferating microvasculature of glioblastomas when compared to the tumour cell expression. This result correlated with the results obtained from the in silico (cancer genome databases) analyses. In gliomas, only the vascular expression of PSMA associated with poor overall survival but not the tumour cell expression. In the matched primary lung cancers and their BM (n = 52), vascular PSMA expression in primary tumours associated with significantly accelerated metastatic dissemination to the brain with a tendency towards poor overall survival. Taken together, we report that the vascular expression of PSMA in the primary and secondary brain tumours globally associates with the malignant progression and poor outcome of the patients.     

Complex contribution of cyclophilin D to Ca2+-induced permeability transition in brain mitochondria, with relation to the bioenergetic state

Cyclophilin D (cypD)-deficient mice exhibit resistance to focal cerebral ischemia and to necrotic but not apoptotic stimuli. To address this disparity, we investigated isolated brain and in situ neuronal and astrocytic mitochondria from cypD-deficient and wild-type mice. Isolated mitochondria were challenged by high Ca(2+), and the effects of substrates and respiratory chain inhibitors were evaluated on permeability transition pore opening by light scatter. In situ neuronal and astrocytic mitochondria were visualized by mito-DsRed2 targeting and challenged by calcimycin, and the effects of glucose, NaCN, and an uncoupler were evaluated by measuring mitochondrial volume. In isolated mitochondria, Ca(2+) caused a large cypD-dependent change in light scatter in the absence of substrates that was insensitive to Ruthenium red or Ru360. Uniporter inhibitors only partially affected the entry of free Ca(2+) in the matrix. Inhibition of complex III/IV negated the effect of substrates, but inhibition of complex I was protective. Mitochondria within neurons and astrocytes exhibited cypD-independent swelling that was dramatically hastened when NaCN and 2-deoxyglucose were present in a glucose-free medium during calcimycin treatment. In the presence of an uncoupler, cypD-deficient astrocytic mitochondria performed better than wild-type mitochondria, whereas the opposite was observed in neurons. Neuronal mitochondria were examined further during glutamate-induced delayed Ca(2+) deregulation. CypD-knock-out mitochondria exhibited an absence or a delay in the onset of mitochondrial swelling after glutamate application. Apparently, some conditions involving deenergization render cypD an important modulator of PTP in the brain. These findings could explain why absence of cypD protects against necrotic (deenergized mitochondria), but not apoptotic (energized mitochondria) stimuli.     

A new sterol sulfate,Sch 572423, from a marine sponge,Topsentia sp

Bioassay-guided fractionation of an active fraction from a marine sponge Topsentia sp. in our marine fraction library (MFL) led to the isolation and identification of halistanol sulfate (1) and a new sterol sulfate Sch 572423 (2). Compounds 1 and 2 were identified as P2Y(12) inhibitors with IC(50) of 0.48 and 2.2 microM, respectively. The general method of purification for the MFL library and the structure elucidation of compound 2 are described.     

Digestion protocol for small protein amounts for nano-HPLC-MS(MS) analysis

A miniaturized tryptic digestion protocol for protein analysis has been developed, which works well for small amounts of proteins using small volume of reagents. The protocol starts from 10μL sample volume with total protein content in the low pmol or fmol range (alternatively expressed, in the low ng range). After adding various reagents the total volume of the tryptic digest will increase to 15μL only. This is especially advantageous for nano-HPLC-MS or MALDI analysis which requires (and allows) analysis of few μL aliquots only. Efficiency of the protocol was tested using nano-HLPC-MS(MS). The results show that the developed miniaturized digestion protocol performs at least as well, possibly even better, than conventional protocols using large sample amounts; and is far superior to digestion performed in larger volumes followed by solvent evaporation/resolvation. This is reflected both in signal intensities in MS and in the number of proteins identified by MS/MS.     

RabGEF1/Rabex-5 Regulates TrkA-Mediated Neurite Outgrowth and NMDA-Induced Signaling Activation in NGF-Differentiated PC12 Cells

Nerve growth factor (NGF) binds to its cognate receptor TrkA and induces neuronal differentiation by activating distinct downstream signal transduction events. RabGEF1 (also known as Rabex-5) is a guanine nucleotide exchange factor for Rab5, which regulates early endosome fusion and vesicular trafficking in endocytic pathways. Here, we used the antisense (AS) expression approach to induce an NGF-dependent sustained knockdown of RabGEF1 protein expression in stable PC12 transfectants. We show that RabGEF1 is a negative regulator of NGF-induced neurite outgrowth and modulates other cellular and signaling processes that are activated by the interaction of NGF with TrkA receptors, such as cell cycle progression, cessation of proliferation, and activation of NGF-mediated downstream signaling responses. Moreover, RabGEF1 can bind to Rac1, and the activation of Rac1 upon NGF treatment is significantly enhanced in AS transfectants, suggesting that RabGEF1 is a negative regulator of NGF-induced Rac1 activation in PC12 cells. Furthermore, we show that RabGEF1 can also interact with NMDA receptors by binding to the NR2B subunit and its associated binding partner SynGAP, and negatively regulates activation of nitric oxide synthase activity induced by NMDA receptor stimulation in NGF-differentiated PC12 cells. Our data suggest that RabGEF1 is a negative regulator of TrkA-dependent neuronal differentiation and of NMDA receptor-mediated signaling activation in NGF-differentiated PC12 cells.     

Mesenchymal precursor cells in the blood of normal individuals

Mesenchymal precursor cells found in the blood (BMPCs) of normal persons adhere to plastic and glass and proliferate logarithmically in DMEM-20% fetal calf serum (FCS) without growth factors. They form cells with fibroblast-like and stromal morphology, which is not affected by eliminating CD34, CD3, or CD14 cells. Osteogenic supplements (dexamethasone, ascorbic acid, and β-glycerophosphate) added to the culture inhibited fibroblast formation, and BMPCs assumed the cuboidal shape of osteoblasts. After 5 days in supplemented medium, the elutriated cells displayed alkaline phosphatase (AP), and the addition of bone morphogenetic protein (BMP)2 (1 ng) doubled AP production (P < 0.04). Two weeks later, 30% of the cells were very large and reacted with anti-osteocalcin antibody. The same cultures also contained sudanophlic adipocytes and multinucleated giant cells that stained for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptors. Cultured BMPCs immunostain with antibodies to vimentin, type I collagen, and BMP receptors, heterodimeric structures expressed on mesenchymal lineage cells. In addition, BMPCs stain with anti-CD105 (endoglin), a putative marker for bone-marrow mesenchymal stem cells (MSCs).