Microarray compound screening (microARCS) to identify inhibitors of HIV integrase

A novel high-throughput strand transfer assay has been developed, using Microarray Compound Screening (microARCS) technology, to identify inhibitors of human immunodeficiency virus (HIV) integrase. This technology utilizes agarose matrices to introduce a majority of the reagents throughout the assay. Integration of biotinylated donor DNA with fluorescein isothiocyanate (FITC)-labeled target DNA occurs on a SAM membrane in the presence of integrase. An anti-FITC antibody conjugated to alkaline phosphatase (AP) was used to do an enzyme-linked immunosorbent assay with the SAM. An agarose gel containing AttoPhos, a substrate of AP, was used for detection of the integrase reactions on the SAM. For detection, the AttoPhos gel was separated from the SAM after incubation and then the gel was imaged using an Eagle Eye II closed-circuit device camera system. Potential integrase inhibitors appear as dark spots on the gel image. A library of approximately 250,000 compounds was screened using this HIV integrase strand transfer assay in microARCS format. Compounds from different structural classes were identified in this assay as novel integrase inhibitors.     

Dissection of the structural determinants involved in formation of the dimeric form of D-amino acid oxidase from Rhodotorula gracilis: role of the size of the betaF5-betaF6 loop

The role of the long loop connecting beta-strands F5 and F6 (21 amino acids, Pro302-Leu-Asp-Arg-Thr-Lys-Ser-Pro-Leu-Ser-Leu-Gly-Arg-Gly-Ser-Ala-Arg-Ala-Ala-Lys-Glu322) present in Rhodotorula gracilis d-amino acid oxidase (RgDAAO) was investigated by site-directed mutagenesis. This loop was proposed to play an important role in the 'head-to-tail' monomer-monomer interaction of this dimeric flavoenzyme: in particular, by means of electrostatic interactions between positively charged residues of the betaF5-betaF6 loop of one monomer and negatively charged residues belonging to the alpha-helices I3' and I3" of the other monomer. We produced a mutant of RgDAAO (namely, DAAO-DeltaLOOP2), in which only minor structural perturbations were introduced (only five amino acids were deleted; new sequence of the betaF5-betaF6 loop is Pro302-Leu-Asp-Arg-Thr-Leu-Gly-Arg-Gly-Ser-Ala-Arg-Ala-Ala-Lys-Glu317), and the charge of the betaF5-betaF6 loop not modified. The DeltaLOOP2 mutant is monomeric, has a weaker binding with the FAD cofactor, a decrease of the kinetic efficiency, and slight modifications in its spectral properties. The short version of the loop does not allow a correct monomer-monomer interaction, and its presence in the monomeric DAAO is a destabilizing structural element since the DeltaLOOP2 mutant is highly susceptible to proteolysis. These results, confirming the role of this loop in the subunits interaction and thus in stabilization of the sole dimeric form of RgDAAO, put forward the evidence that even a short deletion of the loop generates a consistent variation of the enzyme structure-function properties.     

Direct selection of human genomic loci by microarray hybridization

We applied high-density microarrays to the enrichment of specific sequences from the human genome for high-throughput sequencing. After capture of 6,726 approximately 500-base 'exon' segments, and of 'locus-specific' regions ranging in size from 200 kb to 5 Mb, followed by sequencing on a 454 Life Sciences FLX sequencer, most sequence reads represented selection targets. These direct selection methods supersede multiplex PCR for the large-scale analysis of genomic regions.     

Adherence of human erythroleukemia cells inhibits proliferation without inducing differentiation.

To investigate the effect of extracellular matrix molecules in the megakaryocytic lineage, we studied the role of integrin engagement in the proliferation and differentiation of human erythroleukemia (HEL) cells. HEL cells grew in suspension, but their adherence depended upon the presence of matrix proteins or protein kinase C signaling. Adherence by itself did not trigger commitment of these cells but accelerated phorbol 12-myristate 13-acetate-induced differentiation. HEL cells adhered to fibronectin mainly through alpha5beta1, and this receptor acted synergetically with alpha4beta1. Integrin engagement induced cell growth arrest through mitogen-activated protein kinase inactivation. Such down-regulation of the mitogen-activated protein kinase pathway by integrin engagement was suggested as a megakaryocytic-platelet lineage specificity. This signaling was not restricted to a peculiar integrin but was proposed as a general mechanism in these cells.     

Organization and dynamics of tryptophan residues in tetrameric and monomeric soybean agglutinin: Studies by steady-state and time-resolved fluorescence,phosphorescence and chemical modification

We have investigated the organization and dynamics of tryptophan residues in tetrameric, monomeric and unfolded states of soybean agglutinin (SBA) by selective chemical modification, steady-state and time-resolved fluorescence, and phosphorescence. Oxidation with N-bromosuccinimide (NBS) modifies two tryptophans (Trp 60 and Trp 132) in tetramer, four (Trp 8, Trp 203 and previous two) in monomer, and all six (Trp 8, Trp 60, Trp 132, Trp 154, Trp 203 and Trp 226) in unfolded state. Utilizing wavelength-selective fluorescence approach, we have observed a red-edge excitation shift (REES) of 10 and 5 nm for tetramer and monomer, respectively. A more pronounced REES (21 nm) is observed after NBS oxidation. These results are supported by fluorescence anisotropy experiments. Acrylamide quenching shows the Stern–Volmer constant (K_SV) for tetramer, monomer and unfolded SBA being 2.2, 5.0 and 14.6 M⁻¹, respectively. Time-resolved fluorescence studies exhibit biexponential decay with the mean lifetime increasing along tetramer (1.0 ns) to monomer (1.9 ns) to unfolded (3.6 ns). Phosphorescence studies at 77 K give more structured spectra, with two (0,0) bands at 408.6 (weak) and 413.2 nm for tetramer. However, a single (0,0) band appears at 411.8 and 407.2 nm for monomer and unfolded SBA, respectively. The exposure of hydrophobic surface in SBA monomer has been examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, which shows ∼20-fold increase in ANS fluorescence compared to that for tetramer. The mean lifetime of ANS also shows a large increase (12.0 ns) upon binding to monomer. These results may provide important insight into the role of tryptophans in the folding and association of SBA, and oligomeric proteins in general.     

Relevance of weak flavin binding in human D‐amino acid oxidase

In the brain, the human flavoprotein D ‐amino acid oxidase (hDAAO) is involved in the degradation of the gliotransmitter D ‐serine, an important modulator of NMDA‐receptor‐mediated neurotransmission; an increase in hDAAO activity (that yields a decrease in D ‐serine concentration) was recently proposed to be among the molecular mechanisms leading to the onset of schizophrenia susceptibility. This human flavoenzyme is a stable homodimer (even in the apoprotein form) that distinguishes from known D ‐amino acid oxidases because it shows the weakest interaction with the flavin cofactor in the free form. Instead, cofactor binding is significantly tighter in the presence of an active site ligand. In order to understand how hDAAO activity is modulated, we investigated the FAD binding process to the apoprotein moiety and compared the folding and stability properties of the holoenzyme and the apoprotein forms. The apoprotein of hDAAO can be distinguished from the holoenzyme form by the more “open” tertiary structure, higher protein fluorescence, larger exposure of hydrophobic surfaces, and higher sensitivity to proteolysis. Interestingly, the FAD binding only slightly increases the stability of hDAAO to denaturation by urea or temperature. Taken together, these results indicate that the weak cofactor binding is not related to protein (de)stabilization or oligomerization (as instead observed for the homologous enzyme from yeast) but rather should represent a means of modulating the activity of hDAAO. We propose that the absence in vivo of an active site ligand/substrate weakens the cofactor binding, yielding the inactive apoprotein form and thus avoiding excessive D ‐serine degradation.     

Doubly linked,A-type proanthocyanidin trimer and other constituents of Ixora coccinea leaves and their antioxidant and antibacterial properties

Phytochemical investigation of the ethyl acetate fraction of the methanol extract of the leaves of Ixora coccinea led to the isolation and identification of an A-type trimeric proanthocyanidin epicatechin-(2β → O → 7, 4β → 8)–epicatechin-(5 → O → 2β, 6 → 4β)–epicatechin named ixoratannin A-2 along with seven known compounds, epicatechin, procyanidin A2, cinnamtannin B-1, and four flavon-3-ol rhamnosides viz: kaempferol-7-O-α-l-rhamnnoside, kaempferol-3-O-α-l-rhamnoside, quercetin-3-O-α-l-rhamnopyranoside, and kaempferol-3,7-O-α-l-dirhamnoside. The structures were elucidated by the application of IR, UV, MS, 1D-, and 2D-NMR spectroscopic analyses and by comparison with literature data. Antioxidant evaluation of isolated compounds revealed that ixoratannin A-2 and cinnamtannin B-1 were the most active compounds in DPPH, inhibition of lipid peroxidation and nitric oxide radical scavenging assays. Antibacterial activities were assessed by means of agar-diffusion assays using Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtilis. All tested compounds inhibited the growth of B. subtilis, while only epicatechin and quercetin-3-O-α-l-rhamnopyranoside inhibited the growth of E. coli.     

Production of recombinant cholesterol oxidase containing covalently bound FAD in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3)-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food) cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in Escherichia coli of type II cholesterol oxidase from Brevibacterium sterolicum (BCO).