Diagnosing benign and malignant lesions in breast tissue sections by using IR-microspectroscopy

The collection of IR spectra through microscope optics and the visualization of the IR data by IR imaging represent a visualization approach, which uses infrared spectral features as a native intrinsic contrast mechanism. To illustrate the potential of this spectroscopic methodology in breast cancer research, we have acquired IR-microspectroscopic data from benign and malignant lesions in breast tissue sections by point microscopy with spot sizes of 30-40 μm. Four classes of distinct breast tissue spectra were defined and stored in the data base: fibroadenoma (a total of 1175 spectra from 14 patients), ductal carcinoma in situ (a total of 1349 spectra from 8 patients), connective tissue (a total of 464 spectra), and adipose tissue (a total of 146 spectra). Artifical neural network analysis, a supervised pattern recognition method, was used to develop an automated classifier to separate the four classes. After training the artifical neural network classifier, infrared spectra of independent external validation data sets (“unknown spectra”) were analyzed. In this way, all spectra (a total of 386) taken from micro areas inside the epithelium of fibroadenomas from 4 patients were correctly classified. Out of the 421 spectra taken from micro areas of the in situ component of invasive ductal carcinomas of 3 patients, 93% were correctly identified. Based on these results, the potential of the IR-microspectroscopic approach for diagnosing breast tissue lesions is discussed.     

In silico pharmacogenetics of warfarin metabolism

Pharmacogenetic approaches can be instrumental for predicting individual differences in response to a therapeutic intervention. Here we used a recently developed murine haplotype-based computational method to identify a genetic factor regulating the metabolism of warfarin, a commonly prescribed anticoagulant with a narrow therapeutic index and a large variation in individual dosing. After quantification of warfarin and nine of its metabolites in plasma from 13 inbred mouse strains, we correlated strain-specific differences in 7-hydroxywarfarin accumulation with genetic variation within a chromosomal region encoding cytochrome P450 2C (Cyp2c) enzymes. This computational prediction was experimentally confirmed by showing that the rate-limiting step in biotransformation of warfarin to its 7-hydroxylated metabolite was inhibited by tolbutamide, a Cyp2c isoform-specific substrate, and that this transformation was mediated by expressed recombinant Cyp2c29. We show that genetic variants responsible for interindividual pharmacokinetic differences in drug metabolism can be identified by computational genetic analysis in mice.     

Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge

Lipase B from Candida antarctica (CalB) is a versatile biocatalyst for various bioconversions. In this study, the thermostability of CalB was improved through the introduction of a new disulfide bridge. Analysis of the B‐factors of residue pairs in CalB wild type (CalB‐WT) followed by simple flexibility analysis of residues in CalB‐WT and its designated mutants using FIRST server were newly proposed to enhance the selective power of two computational tools (MODIP and DbD v1.20) to predict the possible disulfide bonds in proteins for the enhancement of thermostability. Five residue pairs (A162‐K308, N169‐F304, Q156_‐L163, S50‐A273, and S239C‐D252C) were chosen and the respective amino acid residues were mutated to cysteine. In the results, CalB A162C‐K308C showed greatly improved thermostability while maintaining its catalytic efficiency compared to that of CalB‐WT. Remarkably, the temperature at which 50% of its activity remained after 60‐min incubation (T) of CalB A162C_K308C was increased by 8.5°C compared to that of CalB‐WT (55 and 46.5°C, respectively). Additionally, the half‐life at 50°C of CalB A162C‐K308C was 4.5‐fold higher than that of CalB‐WT (220 and 49 min, respectively). The improvement of thermostability of CalB A162C‐K308C was elucidated at the molecular level by molecular dynamics (MD) simulation. Biotechnol. Bioeng. 2012; 109:867–876. © 2011 Wiley Periodicals, Inc.     

Azemiopsin from Azemiops feae viper venom, a novel polypeptide ligand of nicotinic acetylcholine receptor

Azemiopsin, a novel polypeptide, was isolated from the Azemiops feae viper venom by combination of gel filtration and reverse-phase HPLC. Its amino acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and, unlike similar venom isolates, does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a β-structure. Peptide synthesis was used to verify the determined sequence and to prepare peptide in sufficient amounts to study its biological activity. Azemiopsin efficiently competed with α-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC(50) 0.18 ± 0.03 μm) and with lower efficiency to human α7 nAChR (IC(50) 22 ± 2 μm). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (α1β1εδ) than the fetal form (α1β1γδ), EC(50) being 0.44 ± 0.1 μm and 1.56 ± 0.37 μm, respectively. The peptide had no effect on GABA(A) (α1β3γ2 or α2β3γ2) receptors at a concentration up to 100 μm or on 5-HT(3) receptors at a concentration up to 10 μm. Ala scanning showed that amino acid residues at positions 3-6, 8-11, and 13-14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a disulfide-containing peptide from the Tropidechis wagleri venom, shares with it a homologous C-terminal hexapeptide, but is the first natural toxin that blocks nAChRs and does not possess disulfide bridges.     

The receptor-CheW binding interface in bacterial chemotaxis

The basic structural unit of the signaling complex in bacterial chemotaxis consists of the chemotaxis kinase CheA, the coupling protein CheW, and chemoreceptors. These complexes play an important role in regulating the kinase activity of CheA and in turn controlling the rotational bias of the flagellar motor. Although individual three-dimensional structures of CheA, CheW, and chemoreceptors have been determined, the interaction between chemoreceptor and CheW is still unclear. We used nuclear magnetic resonance to characterize the interaction modes of chemoreceptor and CheW from Thermotoga maritima. We find that chemoreceptor binding surface is located near the highly conserved tip region of the N-terminal helix of the receptor, whereas the binding interface of CheW is placed between the β-strand 8 of domain 1 and the β-strands 1 and 3 of domain 2. The receptor-CheW complex shares a similar binding interface to that found in the "trimer-of-dimers" oligomer interface seen in the crystal structure of cytoplasmic domains of chemoreceptors from Escherichia coli. Based on the association constants inferred from fast exchange chemical shifts associated with receptor-CheW titrations, we estimate that CheW binds about four times tighter to its first binding site of the receptor dimer than to its second binding site. This apparent anticooperativity in binding may reflect the close proximity of the two CheW binding surfaces near the receptor tip or further, complicating the events at this highly conserved region of the receptor. This work describes the first direct observation of the interaction between chemoreceptor and CheW.     

Correlates of Protective Cellular Immunity Revealed by Analysis of Population-Level Immune Escape Pathways in HIV-1

HLA class I-associated polymorphisms identified at the population level mark viral sites under immune pressure by individual HLA alleles. As such, analysis of their distribution, frequency, location, statistical strength, sequence conservation, and other properties offers a unique perspective from which to identify correlates of protective cellular immunity. We analyzed HLA-associated HIV-1 subtype B polymorphisms in 1,888 treatment-naïve, chronically infected individuals using phylogenetically informed methods and identified characteristics of HLA-associated immune pressures that differentiate protective and nonprotective alleles. Over 2,100 HLA-associated HIV-1 polymorphisms were identified, approximately one-third of which occurred inside or within 3 residues of an optimally defined cytotoxic T-lymphocyte (CTL) epitope. Differential CTL escape patterns between closely related HLA alleles were common and increased with greater evolutionary distance between allele group members. Among 9-mer epitopes, mutations at HLA-specific anchor residues represented the most frequently detected escape type: these occurred nearly 2-fold more frequently than expected by chance and were computationally predicted to reduce peptide-HLA binding nearly 10-fold on average. Characteristics associated with protective HLA alleles (defined using hazard ratios for progression to AIDS from natural history cohorts) included the potential to mount broad immune selection pressures across all HIV-1 proteins except Nef, the tendency to drive multisite and/or anchor residue escape mutations within known CTL epitopes, and the ability to strongly select mutations in conserved regions within HIV''s structural and functional proteins. Thus, the factors defining protective cellular immune responses may be more complex than simply targeting conserved viral regions. The results provide new information to guide vaccine design and immunogenicity studies.     

Nitroxyl (HNO) stimulates soluble guanylyl cyclase to suppress cardiomyocyte hypertrophy and superoxide generation

Background

New therapeutic targets for cardiac hypertrophy, an independent risk factor for heart failure and death, are essential. HNO is a novel redox sibling of NO• attracting considerable attention for the treatment of cardiovascular disorders, eliciting cGMP-dependent vasodilatation yet cGMP-independent positive inotropy. The impact of HNO on cardiac hypertrophy (which is negatively regulated by cGMP) however has not been investigated.

Methods

Neonatal rat cardiomyocytes were incubated with angiotensin II (Ang II) in the presence and absence of the HNO donor Angeli''s salt (sodium trioxodinitrate) or B-type natriuretic peptide, BNP (all 1 µmol/L). Hypertrophic responses and its triggers, as well as cGMP signaling, were determined.

Results

We now demonstrate that Angeli''s salt inhibits Ang II-induced hypertrophic responses in cardiomyocytes, including increases in cardiomyocyte size, de novo protein synthesis and β-myosin heavy chain expression. Angeli''s salt also suppresses Ang II induction of key triggers of the cardiomyocyte hypertrophic response, including NADPH oxidase (on both Nox2 expression and superoxide generation), as well as p38 mitogen-activated protein kinase (p38MAPK). The antihypertrophic, superoxide-suppressing and cGMP-elevating effects of Angeli''s salt were mimicked by BNP. We also demonstrate that the effects of Angeli''s salt are specifically mediated by HNO (with no role for NO• or nitrite), with subsequent activation of cardiomyocyte soluble guanylyl cyclase (sGC) and cGMP signaling (on both cGMP-dependent protein kinase, cGK-I and phosphorylation of vasodilator-stimulated phosphoprotein, VASP).

Conclusions

Our results demonstrate that HNO prevents cardiomyocyte hypertrophy, and that cGMP-dependent NADPH oxidase suppression contributes to these antihypertrophic actions. HNO donors may thus represent innovative pharmacotherapy for cardiac hypertrophy.     

Bis-semiquinone (bi-radical) formation by photoinduced proton coupled electron transfer in covalently linked catechol-quinone systems: Aviram's hemiquinones revisited

The structure and reactivity of a covalently linked catechol-ortho-benzoquinone (hemiquinone) is studied by UV-Vis and IR absorption spectroscopy. Nanosecond transient absorption spectroscopy of the hemiquinone reveals the formation of bi-radical state consisting of two semiquinone units. It is a long-lived state resulting from proton coupled electron transfer (PCET).