Effect of nitric oxide on the growth of Chlamydophila pneumoniae

Chlamydophila pneumoniae is an important human intracellular pathogen; however, the pathogenesis of C. pneumoniae infection is poorly understood and the immune control mechanism versus host cells is not completely known. The role of the nitric oxide (NO) synthase pathway in inhibiting the ability of C. pneumoniae to infect macrophage J774 cells and the ability of NO to damage isolated C. pneumoniae were investigated. Exposure of infected cultures to recombinant murine gamma interferon (MurIFN-gamma) resulted in increased production of NO and reduced viability. Addition of 2-(N,N-diethylamino)-diazenolase-2-oxide before infection of J774 cells or during chlamydial cultivation released NO, both resulting in a reduction in the viability of C. pneumoniae in a dose-dependent way. These results indicate that immune control of chlamydial growth in murine macrophage cells may trigger a mechanism that includes NO release with effects on the multiplication of the microorganism, thus suggesting that NO may play a role in preventing the systemic spread of Chlamydia.     

Phosphorylation modulates the alpha-helical structure and polymerization of a peptide from the third tau microtubule-binding repeat

Paired helical filaments (PHFs) isolated from patients with Alzheimer's disease (AD) mainly consist of the microtubule-associated protein tau in a hyperphosphorylated form. It has been found that PHFs are the first example of pathological protein aggregation associated with formation of alpha-helices [Biochemistry (2002) 41, 7150-5]. In an effort to investigate the interplay between phosphorylation and the putative role of short regions of alpha-helix in the polymerization of tau, we have focused on the region of tau encompassing residues 317 to 335. This region is able to form protein fibrils in vitro and has two serines that are often found phosphorylated in PHFs. Using trifluoroethanol as an indicator of the alpha-helix, we find that the stability of the alpha-helix conformation is enhanced by phosphorylation. Circular dichroism data show that the phosphorylated peptide in water presents a content in alpha-helix similar to the unphosphorylated peptide at 40% of trifluoroethanol. Phosphorylation also stimulates the effect of juglone in promoting the in vitro polymerization. Furthermore, Fourier transformed infrared spectroscopy of samples of phosphorylated peptide polymerized with juglone renders a spectrum with maxima at approximately 1665 and approximately 1675 cm(-1), which are suggestive of a mixture of turns and alpha-helix conformations. Our results provide a direct mechanistic connection between phosphorylation and polymerization in tau. The connection between phosphorylation and polymerization appears to involve formation of alpha-helix structure.     

Multiple scattering x-ray absorption studies of Zn2+ binding sites in bacterial photosynthetic reaction centers

Binding of transition metal ions to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides has been previously shown to slow light-induced electron and proton transfer to the secondary quinone acceptor molecule, Q(B). On the basis of x-ray diffraction at 2.5 angstroms resolution a site, formed by AspH124, HisH126, and HisH128, has been identified at the protein surface which binds Cd(2+) or Zn(2+). Using Zn K-edge x-ray absorption fine structure spectroscopy we report here on the local structure of Zn(2+) ions bound to purified RC complexes embedded into polyvinyl alcohol films. X-ray absorption fine structure data were analyzed by combining ab initio simulations and multiparameter fitting; structural contributions up to the fourth coordination shell and multiple scattering paths (involving three atoms) have been included. Results for complexes characterized by a Zn to RC stoichiometry close to one indicate that Zn(2+) binds two O and two N atoms in the first coordination shell. Higher shell contributions are consistent with a binding cluster formed by two His, one Asp residue, and a water molecule. Analysis of complexes characterized by approximately 2 Zn ions per RC reveals a second structurally distinct binding site, involving one O and three N atoms, not belonging to a His residue. The local structure obtained for the higher affinity site nicely fits the coordination geometry proposed on the basis of x-ray diffraction data, but detects a significant contraction of the first shell. Two possible locations of the second new binding site at the cytoplasmic surface of the RC are proposed.     

Inhomogeneous alginate gel spheres: an assessment of the polymer gradients by synchrotron radiation-induced X-ray emission, magnetic resonance microimaging, and mathematical modeling

It has been previously demonstrated that calcium alginate gels prepared by dialysis often exhibit a concentration inhomogeneity being the polymer concentration considerably lower in the center of the gel than at the edges. Inhomogeneity may be a preferred structure in microcapsules due to low porosity and higher stability so that it is interesting to evaluate the polymer gradient in spherically symmetrical small alginate beads (1.0-0.7 mm diameter) obtained in different conditions. In this paper, two complementary techniques have been used to investigate this aspect. The concentration gradient of alginate has been analyzed by measuring both the spatial distribution of calcium ions in sections of alginate gel spheres, by means of x-ray fluorescence spectroscopy, and the T2 relaxation behavior on intact gel beads using magnetic resonance microimaging. The experimentally determined gradients from three-dimensional gels provide data to reevaluate the parameter estimates in the recently reported mathematical model for alginate gel formation (A. Mikkaelsen and A. Elgsaeter, Biopolymers, 1995, Vol. 36, pp. 17-41). The model may account for the gels being less inhomogeneous when nongelling sodium or magnesium ions are added during gelation.     

cis expression of the F12 human immunodeficiency virus (HIV) Nef allele transforms the highly productive NL4-3 HIV type 1 to a replication-defective strain: involvement of both Env gp41 and CD4 intracytoplasmic tails

F12 human immunodeficiency virus type 1 (HIV-1) nef is a naturally occurring nef mutant cloned from the provirus of a nonproductive, nondefective, and interfering HIV-1 variant (F12-HIV). We have already shown that cells stably transfected with a vector expressing the F12-HIV nef allele do not downregulate CD4 receptors and, more peculiarly, become resistant to the replication of wild type (wt) HIV. In order to investigate the mechanism of action of such an HIV inhibition, the F12-HIV nef gene was expressed in the context of the NL4-3 HIV-1 infectious molecular clone by replacing the wt nef gene (NL4-3/chi). Through this experimental approach we established the following. First, NL4-3/chi and nef-defective (Deltanef) NL4-3 viral particles behave very similarly in terms of viral entry and HIV protein production during the first replicative cycle. Second, no viral particles were produced from cells infected with NL4-3/chi virions, whatever the multiplicity of infection used. The viral inhibition apparently occurs at level of viral assembling and/or release. Third, this block could not be relieved by in-trans expression of wt nef. Finally, NL4-3/chi reverts to a producer HIV strain when F12-HIV Nef is deprived of its myristoyl residue. Through a CD4 downregulation competition assay, we demonstrated that F12-HIV Nef protein potently inhibits the CD4 downregulation induced by wt Nef. Moreover, we observed a redistribution of CD4 receptors at the cell margin induced by F12-HIV Nef. These observations strongly suggest that F12-HIV Nef maintains the ability to interact with the intracytoplasmic tail of the CD4 receptor molecule. Remarkably, we distinguished the intracytoplasmic tails of Env gp41 and CD4 as, respectively, viral and cellular targets of the F12-HIV Nef-induced viral retention. For the first time, the inhibition of the viral life cycle by means of in-cis expression of a Nef mutant is here reported. Delineation of the F12-HIV Nef mechanism of action may offer additional approaches to interference with the propagation of HIV infection.     

High frequency of homozygosity of the HLA region in melanoma cell lines reveals a pattern compatible with extensive loss of heterozygosity

Malignant transformation of cells is frequently associated with abnormalities in human leukocyte antigen (HLA) expression. MHC class I loss or down-regulation in cancer cells is a major immune escape route used by a large variety of human tumours to evade antitumour immune responses mediated by cytotoxic T lymphocytes. The goal of our study was to explore HLA genotyping and phenotyping in a variety of melanoma tumour cell lines. A total of 91 melanoma cell lines were characterised for HLA class I and II genotype. In addition, 61 out of the 91 cell lines were also analysed for HLA class I and II cell surface molecule expression by flow cytometry. Unexpectedly, we found that 19.7% of the melanoma cell lines were homozygous for HLA class I genotypes, sometimes associated with HLA class II homozygosity (8.79%) and sometimes not (10.98%). The frequency of homozygosity was significantly higher compared with the control groups (1.6%). To identify the reasons underlying the high frequency of HLA homozygosity we searched for genomic deletions using eight pairs of highly polymorphic microsatellite markers covering the entire extended HLA complex on the short arm of chromosome 6. Our results were compatible with hemizygous deletions and suggest that loss of heterozygosity on chromosome arm 6p is a common feature in melanoma cell lines. In fact, although autologous normal DNA from the patients was not available and could not be tested, the retention in some cases of heterozygosity for a number of microsatellite markers would indicate a hemizygous deletion. In the rest of the cases, markers at 6p and 6q showed a single allele pattern indicating the probable loss of part or the whole of chromosome 6. These results led us to conclude that loss of heterozygosity in chromosome 6 is nonrandom and is possibly an immunologically relevant event in human malignant melanoma. Other well-established altered HLA class I phenotypes were also detected by flow cytometry that correspond to HLA class I total loss and HLA-ABC and/or specific HLA-B locus down-regulation.     

Physical map of the Azoarcus sp. strain BH72 genome based on a bacterial artificial chromosome library as a platform for genome sequencing and functional analysis

Azoarcus sp. strain BH72 is a Gram-negative proteobacterium of the beta subclass; it is a diazotrophic endophyte of graminaceous plants and can provide significant amounts of fixed nitrogen to its host plant Kallar grass. We aimed to obtain a physical map of the Azoarcus sp. strain BH72 chromosome to be directly used in functional analysis and as a part of an Azoarcus sp. BH72 genome project. A bacterial artificial chromosome (BAC) library was constructed and analysed. A representative physical map with a high density of marker genes was developed in which 64 aligned BAC clones covered almost the entire genome.     

Soil microbial community responses to additions of organic carbon substrates and heavy metals (Pb and Cr)

Microcosm experiments were conducted with soils contaminated with heavy metals (Pb and Cr) and aromatic hydrocarbons to determine the effects of each upon microbial community structure and function. Organic substrates were added as a driving force for change in the microbial community. Glucose represented an energy source used by a broad variety of bacteria, whereas fewer soil species were expected to use xylene. The metal amendments were chosen to inhibit the acute rate of organic mineralization by either 50% or 90%, and lower mineralization rates persisted over the entire 31-day incubation period. Significant biomass increases were abolished when metals were added in addition to organic carbon. The addition of organic carbon alone had the most significant impact on community composition and led to the proliferation of a few dominant phylotypes, as detected by PCR-denaturing gradient gel electrophoresis of bacterial 16S rRNA genes. However, the community-wide effects of heavy metal addition differed between the two carbon sources. For glucose, either Pb or Cr produced large changes and replacement with new phylotypes. In contrast, many phylotypes selected by xylene treatment were retained when either metal was added. Members of the Actinomycetales were very prevalent in microcosms with xylene and Cr(VI); gene copy numbers of biphenyl dioxygenase and phenol hydroxylase (but not other oxygenases) were elevated in these microcosms, as determined by real-time PCR. Much lower metal concentrations were needed to inhibit the catabolism of xylene than of glucose. Cr(VI) appeared to be reduced during the 31-day incubations, but in the case of glucose there was substantial microbial activity when much of the Cr(VI) remained. In the case of xylene, this was less clear.