Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex

Cytochrome bc(1) is an integral membrane protein complex essential for cellular respiration and photosynthesis; it couples electron transfer from quinol to cytochrome c to proton translocation across the membrane. Specific bc(1) inhibitors have not only played crucial roles in elucidating the mechanism of bc(1) function but have also provided leads for the development of novel antibiotics. Crystal structures of bovine bc(1) in complex with the specific Q(o) site inhibitors azoxystrobin, MOAS, myxothiazol, stigmatellin and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole were determined. Interactions, conformational changes and possible mechanisms of resistance, specific to each inhibitor, were defined. Residues and secondary structure elements that are capable of discriminating different classes of Q(o) site inhibitors were identified for the cytochrome b subunit. Directions in the displacement of the cd1 helix of cytochrome b subunit in response to various Q(o) site inhibitors were correlated to the binary conformational switch of the extrinsic domain of the iron-sulfur protein subunit. The new structural information, together with structures previously determined, provide a basis that, combined with biophysical and mutational data, suggest a modification to the existing classification of bc(1) inhibitors. bc(1) inhibitors are grouped into three classes: class P inhibitors bind to the Q(o) site, class N inhibitors bind to the Q(i) site and the class PN inhibitors target both sites. Class P contains two subgroups, Pm and Pf, that are distinct by their ability to induce mobile or fixed conformation of iron-sulfur protein.     

Assessment of Sarcocystis neurona sporocyst viability and differentiation between viable and nonviable sporocysts using propidium iodide stain

Sarcocystis neurona has become recognized as the major causative agent of equine protozoal myeloencephalitis (EPM) in the Americas. At least 3 pathogenic species of Sarcocystis, including S. neurona, can be isolated from opossums. Methods are needed to ascertain whether these isolates are viable and capable of causing infections. In this study, the nuclear stain propidium iodide (PI) was used to differentiate between live (viable) and heat-killed (nonviable) S. neurona sporocysts. PI was excluded by live sporocysts but penetrated compromised sporocyst membrane and stained sporozoite nuclei of dead sporocysts. After live and dead sporocysts were mixed at various ratios, the number of unstained sporocysts detected after the staining procedure correlated significantly (r2 = 0.9978) with the expected numbers of live sporocysts. Sporocyst mixtures were also assayed for in vitro excystation and development in tissue cultures. The correlation between the percentage of plaques formed in tissue cultures and the percentage of expected infectious (live) sporocysts in each mixture was r2 = 0.6712. By analysis of variance, no statistically significant difference was measured between the percentage of viable sporocysts and the percentage of infectious sporocysts (P = 0.3902) in each mixture. In addition, there was evidence of a relation between PI impermeability of sporocysts and animal infectivity. These results suggest that the PI dye-exclusion technique can be a useful tool in identifying viability and potential infectivity of S. neurona sporocysts and in differentiating between viable and nonviable sporocysts.     

CXC chemokine ligand 10 controls viral infection in the central nervous system: evidence for a role in innate immune response through recruitment and activation of natural killer cells

How chemokines shape the immune response to viral infection of the central nervous system (CNS) has largely been considered within the context of recruitment and activation of antigen-specific lymphocytes. However, chemokines are expressed early following viral infection, suggesting an important role in coordinating innate immune responses. Herein, we evaluated the contributions of CXC chemokine ligand 10 (CXCL10) in promoting innate defense mechanisms following coronavirus infection of the CNS. Intracerebral infection of RAG1(-/-) mice with a recombinant CXCL10-expressing murine coronavirus (mouse hepatitis virus) resulted in protection from disease and increased survival that correlated with a significant increase in recruitment and activation of natural killer (NK) cells within the CNS. Accumulation of NK cells resulted in a reduction in viral titers that was dependent on gamma interferon secretion. These results indicate that CXCL10 expression plays a pivotal role in defense following coronavirus infection of the CNS by enhancing innate immune responses.     

Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture

Efficient replication of hepatitis C virus (HCV) subgenomic RNA in cell culture requires the introduction of adaptive mutations. In this report we describe a system which enables efficient replication of the Con1 subgenomic replicon in Huh7 cells without the introduction of adaptive mutations. The starting hypothesis was that high amounts of the NS5A hyperphosphorylated form, p58, inhibit replication and that reduction of p58 by inhibition of specific kinase(s) below a certain threshold enables HCV replication. Upon screening of a panel of kinase inhibitors, we selected three compounds which inhibited NS5A phosphorylation in vitro and the formation of NS5A p58 in cell culture. Cells, transfected with the HCV Con1 wild-type sequence, support HCV RNA replication upon addition of any of the three compounds. The effect of the kinase inhibitors was found to be synergistic with coadaptive mutations in NS3. This is the first direct demonstration that the presence of high amounts of NS5A-p58 causes inhibition of HCV RNA replication in cell culture and that this inhibition can be relieved by kinase inhibitors.     

Gender differences in human immunodeficiency virus type 1-specific CD8 responses in the reproductive tract and colon following nasal peptide priming and modified vaccinia virus Ankara boosting

Induction of mucosal anti-human immunodeficiency virus type 1 (HIV-1) T-cell responses in males and females will be important for the development of a successful HIV-1 vaccine. An HIV-1 envelope peptide, DNA plasmid, and recombinant modified vaccinia virus Ankara (rMVA) expressing the H-2D(d)-restricted cytotoxic T lymphocyte P18 epitope were used as immunogens to test for their ability to prime and boost anti-HIV-1 T-cell responses at mucosal and systemic sites in BALB/c mice. We found of all prime-boost combinations tested, an HIV-1 Env peptide subunit mucosal prime followed by systemic (intradermal) boosting with rMVA yielded the maximal induction of gamma interferon (IFN-gamma) spot-forming cells in the female genital tract and colon. However, this mucosal prime-systemic rMVA boost regimen was minimally immunogenic for the induction of genital, colon, or lung anti-HIV-1 T-cell responses in male mice. We determined that a mucosal Env subunit immunization could optimally prime an rMVA boost in female but not male mice, as determined by the magnitude of antigen-specific IFN-gamma responses in the reproductive tracts, colon, and lung. Defective mucosal priming in male mice could not be overcome by multiple mucosal immunizations. However, rMVA priming followed by an rMVA boost was the optimal prime-boost strategy for male mice as determined by the magnitude of antigen-specific IFN-gamma responses in the reproductive tract and lung. Thus, prime-boost immunization strategies able to induce mucosal antigen-specific IFN-gamma responses were identified for male and female mice. Understanding the cellular and molecular basis of gender-determined immune responses will be important for optimizing induction of anti-HIV-1 mucosal immune responses in both males and females.     

The severe acute respiratory syndrome coronavirus Nsp15 protein is an endoribonuclease that prefers manganese as a cofactor

Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has endoribonuclease activity that preferentially cleaved 5' of uridylates of RNAs. Blocking either the 5' or 3' terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn(2+) at 2 to 10 mM was needed for optimal endoribonuclease activity, but Mg(2+) and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn(2+) needed for endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus endoribonuclease.     

cDNA cloning, expression, purification, distribution, and characterization of biologically active canine alanine aminotransferase-1

Alanine aminotransferase (ALT) is a pyridoxal enzyme found mainly in the liver and kidney, but also in small amounts in the heart, muscle, fat, and brain. Serum aminotransferase activities have been used broadly as surrogate markers for tissue injury and disease in human and veterinary clinical settings and in safety assessment of chemicals and pharmaceuticals. Because of its relative abundance in liver, increased serum ALT activity is generally considered indicative of liver damage. Two ALT isoenzymes, ALT1 and ALT2, are known and have been cloned and sequenced from human, rat, and mouse. In this study, we have cloned the complementary DNA encoding the canine orthologue of ALT1 (cALT1). The complete cDNA sequence comprised 1852 bases and contained a 1485-base open reading frame, which encodes a polypeptide of 494 amino acid residues. Canine ALT1 shares 87.7, 87.2, and 87.0% amino acid identity to its human, mouse, and rat orthologues, respectively. The cDNA was expressed in Escherichia coli, with a N-terminal His (6x) tag, and the recombinant enzyme was purified using immobilized metal-affinity chromatography. The final yield of the purified recombinant cALT1 was greater than 5mg/L culture. The alanine transaminase activity of purified cALT1 was 229.81U/mg protein, which is approximately 38-fold higher than that of total soluble recombinant E. coli cell lysate, confirming that the enzyme is a functional ALT. Evaluation of various canine tissues by RT-PCR revealed that the level of ALT1 expression is in the order of: heart>liver>fat approximately brain approximately gastrocnemius>kidney. The purified cALT1 will be helpful to develop isoenzyme-specific anti-bodies, which could further improve the diagnostic resolution of current ALT assays in drug safety studies.     

A perikinetochoric ring defined by MCAK and Aurora-B as a novel centromere domain

Mitotic Centromere-Associated Kinesin (MCAK) is a member of the kinesin-13 subfamily of kinesin-related proteins. In mitosis, this microtubule-depolymerising kinesin seems to be implicated in chromosome segregation and in the correction of improper kinetochore-microtubule interactions, and its activity is regulated by the Aurora-B kinase. However, there are no published data on its behaviour and function during mammalian meiosis. We have analysed by immunofluorescence in squashed mouse spermatocytes, the distribution and possible function of MCAK, together with Aurora-B, during both meiotic divisions. Our results demonstrate that MCAK and Aurora-B colocalise at the inner domain of metaphase I centromeres. Thus, MCAK shows a “cone”-like three-dimensional distribution beneath and surrounding the closely associated sister kinetochores. During the second meiotic division, MCAK and Aurora-B also colocalise at the inner centromere domain as a band that joins sister kinetochores, but only during prometaphase II in unattached chromosomes. During chromosome congression to the metaphase II plate, MCAK relocalises and appears as a ring below each sister kinetochore. Aurora-B also relocalises to appear as a ring surrounding and beneath kinetochores but during late metaphase II. Our results demonstrate that the redistribution of MCAK at prometaphase II/metaphase II centromeres depends on tension across the centromere and/or on the interaction of microtubules with kinetochores. We propose that the perikinetochoric rings of MCAK and Aurora-B define a novel transient centromere domain at least in mouse chromosomes during meiosis. We discuss the possible functions of MCAK at the inner centromere domain and at the perikinetochoric ring during both meiotic divisions.     

Forensic visualization of foreign matter in human tissue by near-infrared spectral imaging: methodology and data mining strategies.

BACKGROUND: Rapidity of data acquisition, high image fidelity and large field of view are of tremendous value when looking for chemical contaminants or for the proverbial "needle in the haystack" - in this case foreign inclusions in histologic sections of biopsy or autopsy tissues. Near infrared chemical imaging is one of three chemical imaging techniques (NIR, MIR and Raman) based on vibrational spectroscopy, and provides distinct technical advantages for this application. METHODS: We have chosen to utilize and evaluate near infrared (NIR) imaging for studies of foreign materials in tissue because the experimental configuration is relatively simple, data collection is rapid, and large sample areas can be screened with high image fidelity and spatial resolution. RESULTS: We have shown that NIR imaging can readily find and identify silicone gel inclusions in biological tissue samples. Additionally, preliminary results indicate that spectral signatures in the data set are also potentially sensitive to structural changes in the surrounding tissue that may be induced by the foreign body. CONCLUSIONS: NIR chemical imaging is a powerful, non-destructive tool for localization and identifying foreign contaminants in biological tissue. Preliminary results indicate that NIR imaging is also sensitive enough to differentiate tissue types (perhaps based on collagen structural differences), and provide data on the spatial localization of these components.