Synthesis of (1,4)-naphthoquinono-[3,2-c]-1H-pyrazoles and their (1,4)-naphthohydroquinone derivatives as antifungal, antibacterial, and anticancer agents

A series of (1,4)-naphthoquinono [3,2-c]-1H-pyrazoles and their (1,4)-naphthohydroquinone derivatives 2-7 were synthesized and evaluated for antifungal, antibacterial, and anticancer activities. The structure-activity relationship of these compounds was studied and the results show that the compound 2b exhibited in vitro antifungal activity against Candida albicans and Cryptococcus neoformans, and also possessed antibacterial profile against Klebsiella pneumoniae and Escherichia coli whereas 1c showed anticancer activity against Walker 256 Carcinosarcoma in rats.     

Neural cell adhesion molecule-associated polysialic acid potentiates alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor currents

The highly negatively charged polysialic acid (PSA) is a carbohydrate predominantly carried by the neural cell adhesion molecule (NCAM) in mammals. NCAM and, in particular, PSA play important roles in cellular and synaptic plasticity. Here we investigated whether PSA modulates the activity of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptors (AMPA-Rs). Single channel recordings of affinity-purified AMPA-Rs reconstituted in lipid bilayers revealed that bacterially derived PSA, called colominic acid, prolonged the open channel time of AMPA-R-mediated currents by severalfold and altered the bursting pattern of the receptor channels but did not modify AMPA-R single channel conductance. This effect was reversible, concentration-dependent, and specific, since monomers of sialic acid and another negatively charged carbohydrate, chondroitin sulfate, did not potentiate single channel AMPA-R currents. Recombinant PSA-NCAM also potentiated currents mediated by reconstituted AMPA-Rs. In pyramidal neurons acutely isolated from the CA1 region of the early postnatal hippocampus, l-glutamate or AMPA (applied in the presence of antagonists blocking voltage-gated Na(+) and K(+) currents and N-methyl-d-aspartate and metabotropic glutamate receptors) induced inward currents, which were significantly increased by co-application of colominic acid. Chondroitin sulfate did not affect AMPA-R-mediated currents in CA1 neurons. The effect of colominic acid was age-dependent, since in pyramidal neurons from adult hippocampus, colominic acid failed to potentiate glutamate responses. Thus, our study demonstrates age-dependent potentiation of AMPA receptors by PSA via a mechanism probably involving direct PSA-AMPA-R interactions. This mechanism might amplify AMPA-R-mediated signaling in immature cells, thereby affecting their development.     

Identification of residues in the human guanylate-binding protein 1 critical for nucleotide binding and cooperative GTP hydrolysis

The guanylate-binding proteins (GBPs) form a group of interferon-gamma inducible GTP-binding proteins which belong to the family of dynamin-related proteins. Like other members of this family, human guanylate-binding protein 1 (hGBP1) shows nucleotide-dependent oligomerisation that stimulates the GTPase activity of the protein. A unique feature of the GBPs is their ability to hydrolyse GTP to GDP and GMP. In order to elucidate the relationship between these findings, we designed point mutants in the phosphate-binding loop (P-loop) as well as in the switch I and switch II regions of the protein based on the crystal structure of hGBP1. These mutant proteins were analysed for their interaction with guanine nucleotides labeled with a fluorescence dye and for their ability to hydrolyse GTP in a cooperative manner. We identified mutations of amino acid residues that decrease GTPase activity by orders of magnitude a part of which are conserved in GTP-binding proteins. In addition, mutants in the P-loop were characterized that strongly impair binding of nucleotide. In consequence, together with altered GTPase activity and given cellular nucleotide concentrations this results in hGBP1 mutants prevailingly resting in the nucleotide-free (K51A and S52N) or the GTP bound form (R48A), respectively. Using size-exclusion chromatography and analytical ultracentrifugation we addressed the impact on protein oligomerisation. In summary, mutants of hGBP1 were identified and biochemically characterized providing hGBP1 locked in defined states in order to investigate their functional role in future cell biology studies.     

Identification of aspirin and diclofenac binding proteins in the red complex pathogens

Red complex organisms are a group of organisms (Porphyromonas gingivalis ATCC 33277, Treponema denticola ATCC 35405, Tannerella forsythia ATCC 43037) that have been identified for the causation of periodontal diseases. Aspirin and diclofenac have been used as regular analgesics. Therefore, it is of interest to document the identification of aspirin and diclofenac binding proteins in the red complex pathogens using the STITCH v.5 pipeline. The virulence properties of these proteins were analyzed using VICMPred and VirulentPred software. Thus, we document 000 number of proteins having optimal binding features with the known analgesics.     

Myosin 1G (Myo1G) is a haematopoietic specific myosin that localises to the plasma membrane and regulates cell elasticity

Immune cells navigate through different environments where they experience different mechanical forces. Responses to external forces are determined by the mechanical properties of a cell and they depend to a large extent on the actin-rich cell cortex. We report here that Myo1G, a previously uncharacterised member of class I myosins, is expressed specifically in haematopoietic tissues and cells. It is associated with the plasma membrane. This association is dependent on a conserved PH-domain-like myosin I tail homology motif and the head domain. However, the head domain does not need to be a functional motor. Knockdown of Myo1G in Jurkat cells decreased cell elasticity significantly. We propose that Myo1G regulates cell elasticity by deformations of the actin network at the cell cortex.

Structured summary

MINT-7307273: MYO1G (uniprotkb:B0I1T2) and Actin (uniprotkb:P60709) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-7307283: TfR (uniprotkb:P02786) and MYO1G (uniprotkb:B0I1T2) colocalize (MI:0403) by cosedimentation through density gradients (MI:0029)     

Application of fluorescent probes to study structural changes in Aspergillus fumigatus exposed to amphotericin B, itraconazole, and voriconazole

The broad objective of this study was to document patterns of structural changes following antifungal treatment, and to determine any relationship with minimum inhibitory concentration (MIC) of an antifungal. Three clinical isolates of Aspergillus fumigatus, with high, intermediate, and low amphotericin B (AB), itraconazole (IZ), and voriconazole (VZ) MICs were studied in 24-well plates with cover slips. The fluorescent probes used were Calcofluor White (cell wall), propidium iodide (nucleus), and MitoTracker Green FM (mitochondria). Fluorescent microscopy as early as 3-h after exposure revealed that AB treated hyphae had intact cell wall with deformed mitochondria and nuclei while IZ and VZ treated hyphae revealed no intact cell wall, and deformation of mitochondria and nuclei. At 48 h, AB treated cells revealed rupture of hyphae and disintegration of mitochondria, and nuclei, IZ treated hyphae were swollen with disintegration of mitochondria, and nuclei while VZ treated hyphae showed rupture and disintegration of mitochondria and nuclei. The structural changes for the three strains studied were similar in fluorescent microscopy as long as the incubation time and their respective MICs were used. Thus, AB, IZ, and VZ induced gross organelle defects in A. fumigatus nuclei, mitochondria, and cell wall, which were consistent with respective MICs of antifungals used.     

The human beta-defensin-3, an antibacterial peptide with multiple biological functions

A group of interesting molecules called defensins exhibit multiple functions but have been primarily recognized to possess a broad spectrum of antimicrobial activities. Studies have reported two different types of defensins (α and β) from human and animals, a cyclic θ defensin from rhesus, and several defensin-like peptides from plants. There is no amino acid sequence homology between these peptides, but they all contain three Cys-Cys disulfide linkages while the connectivities are different. Human β-defensin-3 (HβD-3) is the most recently discovered member of the host-defense peptide family that has attracted much attention. This molecule is expressed either constitutively or induced upon a challenge, and a growing evidence indicates the involvement of such molecules in adaptive immunity as well. It has been shown to exhibit antibacterial activities towards Gram-negative and Gram-positive bacteria as well as an ability to act as a chemo-attractant. Analysis of NMR structural data suggested a symmetrical dimeric form of this peptide in solution, which consists of three β strands and a short helix in the N-terminal region. While the disulfide linkages are known to provide the structural stability and stability against proteases, the biological relevance of this dimeric form was contradicted by another biological study. Since there is considerable current interest in developing HβD-3 for possible pharmaceutical applications, studies to further our understanding on the determinants of antibacterial activities and immunomodulatory function of HβD-3 are considered to be highly significant. The knowledge of its biosynthetic regulation will also help in understanding the role of HβD-3 in immunity. This article presents an overview of the expression and regulation of HβD-3 in humans, and the structure-function correlations among HβD-3 and its modified peptides are discussed emphasizing the functional importance. The future scope for studies on HβD-3 and design of short potent antimicrobial peptides, based on the native HβD-3 molecule, that do not interfere in the immunomodulatory function is also outlined.     

Kinase activation of ClC-3 accelerates cytoplasmic condensation during mitotic cell rounding

"Mitotic cell rounding" describes the rounding of mammalian cells before dividing into two daughter cells. This shape change requires coordinated cytoskeletal contraction and changes in osmotic pressure. While considerable research has been devoted to understanding mechanisms underlying cytoskeletal contraction, little is known about how osmotic gradients are involved in cell division. Here we describe cytoplasmic condensation preceding cell division, termed "premitotic condensation" (PMC), which involves cells extruding osmotically active Cl(-) via ClC-3, a voltage-gated channel/transporter. This leads to a decrease in cytoplasmic volume during mitotic cell rounding and cell division. Using a combination of time-lapse microscopy and biophysical measurements, we demonstrate that PMC involves the activation of ClC-3 by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in human glioma cells. Knockdown of endogenous ClC-3 protein expression eliminated CaMKII-dependent Cl(-) currents in dividing cells and impeded PMC. Thus, kinase-dependent changes in Cl(-) conductance contribute to an outward osmotic pressure in dividing cells, which facilitates cytoplasmic condensation preceding cell division.