Definition of microsatellite size variants forTnfa andHsp70 in autoimmune and nonautoimmune mouse strains

We have cloned and sequenced the upstream regulatory region of tumor necrosis factor (Tnfa) gene in 12 different mouse strains and identified an allelic polymorphism in the upstream regulatory region of the mouseTnfa gene. TheTNF allele found in the NZW strain is distinct from those of all otherH-2 haplotypes, supporting our previous suggestion that this allele may be associated with a regulatory or structural defect. In addition, simple tandem repeat sequences (microsatellites) within the promoter region of theTnfa gene and the 3 untranslated region of one of the members of the HSP70 family (Hsp68c clone) were utilized as genetic markers. Ten TNF size variants and twelve HSP70 variants were identified in over forty mouse strains. Using these markers in a set of congenic mice, we mapped this member of the HSP70 family to the central portion of theH-2 complex, centromeric to theTnfa gene. The NOD and NZW strains carry uniqueHSP70 alleles based on the variability in the length of this marker. These findings raise the possibility that this protein may play a role in the association of the major histocompatibility complex with these autoimmune diseases.     

Measurement of stagnancy in a pipe line using a radioactive tracer and labelled bacteria

Summary A stainless steel T joint was used to simulate stagnant volume in a pipe-line. Residence time distribution in the lines was measured using a radiotracer (Cr⁵¹) which emitted radiation. The radiation was measured externally to the pipe line. From the experimental data the dead volume of the system was calculated. The effect of the depth of T and flow velocity on dead volume was evaluated. Labelled bacteria were used in some experiments instead of chromium. No significant difference in behavior between ionic chromium and bacteria labeled with chromium was found.     

Structure of a novel class II phospholipase D: catalytic cleft is modified by a disulphide bridge

Phospholipases D (PLDs) are principally responsible for the local and systemic effects of Loxosceles envenomation including dermonecrosis and hemolysis. Despite their clinical relevance in loxoscelism, to date, only the SMase I from Loxosceles laeta, a class I member, has been structurally characterized. The crystal structure of a class II member from Loxosceles intermedia venom has been determined at 1.7 Å resolution. Structural comparison to the class I member showed that the presence of an additional disulphide bridge which links the catalytic loop to the flexible loop significantly changes the volume and shape of the catalytic cleft. An examination of the crystal structures of PLD homologues in the presence of low molecular weight compounds at their active sites suggests the existence of a ligand-dependent rotamer conformation of the highly conserved residue Trp230 (equivalent to Trp192 in the glycerophosphodiester phosphodiesterase from Thermus thermophofilus, PDB code: 1VD6) indicating its role in substrate binding in both enzymes. Sequence and structural analyses suggest that the reduced sphingomyelinase activity observed in some class IIb PLDs is probably due to point mutations which lead to a different substrate preference.     

Multimodal Magnetic Resonance Imaging Study of Treatment-Na?ve Adults with Attention-Deficit/Hyperactivity Disorder

Background

Attention-Deficit/Hiperactivity Disorder (ADHD) is a prevalent disorder, but its neuroanatomical circuitry is still relatively understudied, especially in the adult population. The few morphometric magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) studies available to date have found heterogeneous results. This may be at least partly attributable to some well-known technical limitations of the conventional voxel-based methods usually employed to analyze such neuroimaging data. Moreover, there is a great paucity of imaging studies of adult ADHD to date that have excluded patients with history of use of stimulant medication.

Methods

A newly validated method named optimally-discriminative voxel-based analysis (ODVBA) was applied to multimodal (structural and DTI) MRI data acquired from 22 treatment-naïve ADHD adults and 19 age- and gender-matched healthy controls (HC).

Results

Regarding DTI data, we found higher fractional anisotropy in ADHD relative to HC encompassing the white matter (WM) of the bilateral superior frontal gyrus, right middle frontal left gyrus, left postcentral gyrus, bilateral cingulate gyrus, bilateral middle temporal gyrus and right superior temporal gyrus; reductions in trace (a measure of diffusivity) in ADHD relative to HC were also found in fronto-striatal-parieto-occipital circuits, including the right superior frontal gyrus and bilateral middle frontal gyrus, right precentral gyrus, left middle occipital gyrus and bilateral cingulate gyrus, as well as the left body and right splenium of the corpus callosum, right superior corona radiata, and right superior longitudinal and fronto-occipital fasciculi. Volumetric abnormalities in ADHD subjects were found only at a trend level of significance, including reduced gray matter (GM) in the right angular gyrus, and increased GM in the right supplementary motor area and superior frontal gyrus.

Conclusions

Our results suggest that adult ADHD is associated with neuroanatomical abnormalities mainly affecting the WM microstructure in fronto-parieto-temporal circuits that have been implicated in cognitive, emotional and visuomotor processes.     

Novel DNA polymorphism in the mouse tumor necrosis factor receptors type 1 and type 2

The introduction of the polymerase chain reaction (PCR) provides an entirely new means of analyzing DNA polymorphism and makes practical the analysis of length variation in simple-sequence tandem repeats of dinucleotides. In the process of cloning and sequencing the mouse genomic DNA for tumor necrosis factor (TNF) receptors type 1 and type 2, we identified two simple dinucleotide repeats within the noncoding regions of TNF receptor type 1 and three such sequences within TNF receptor type 2. PCR analysis of these sequences, using genomic DNA from 21 different inbred and wild mouse strains, as demonstrated by running the amplified products on sequencing gels, showed that the repeats are highly polymorphic. We identified seven alleles of TNF receptor type 2 and five alleles of TNF receptor type 1. Using these polymorphic markers in two sets of recombinant inbred strains of mice, the chromosomal localization of Tnfr-1 was mapped to mouse chromosome 6 and Tnfr-2 was located to the distal portion of mouse chromosome 4.     

Novel method for the synthesis of urea backbone cyclic peptides using new Alloc‐protected glycine building units

Cyclization of bioactive peptides, utilizing functional groups serving as natural pharmacophors, is often accompanied with loss of activity. The backbone cyclization approach was developed to overcome this limitation and enhance pharmacological properties. Backbone cyclic peptides are prepared by the incorporation of special building units, capable of forming amide, disulfide and coordinative bonds. Urea bridge is often used for the preparation of cyclic peptides by connecting two amine functionalized side chains. Here we present urea backbone cyclization as an additional method for the preparation of backbone cyclic peptide libraries. A straightforward method for the synthesis of crystalline Fmoc‐N^α [ω‐amino(Alloc)‐alkyl] glycine building units is presented. A set of urea backbone cyclic Glycogen Synthase Kinase 3 analogs was prepared and assessed for protein kinase B inhibition as anticancer leads. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Oxygen-mediated cold-acclimation in cucumber (Cucumis sativus) seedlings

Cold acclimation of etiolated cucumber seedlings, consisting of cooling at 12°C for 48 h followed by a warming period at 25°C, led to tolerance to subsequent chilling at 2°C. Tolerance, as evidenced by freedom from chilling injury and continued growth, developed during the warming period in a time-course manner for 12 h but decreased with prolonged warming. A similar increase and subsequent decrease was also observed in the content of palmitic, linoleic and linolenic acids in total lipid fraction from cucumber hypocotyl tissue. During the warming period supra-ambient oxygen stimulated, whereas subambient oxygen inhibited, the increase in fatty acid content as well as development of chilling tolerance. A strong correlation between oxygen-mediated changes in fatty acid content and associated development of cold tolerance suggests that both these processes are interrelated. Cold acclimation, but not cold stress, led to an increase followed by a decrease in CO₂ evolution suggesting that a respiratory upsurge is yet another feature of cold acclimation in cucumbers.     

A peptide DNA surrogate accelerates autoimmune manifestations and nephritis in lupus-prone mice

Lupus-associated anti-DNA Abs display features of Ag selection, yet the triggering Ag in the disease is unknown. We previously demonstrated that the peptide DWEYSVWLSN is bound by a pathogenic anti-DNA Ab, and that immunization of nonautoimmune mice with this peptide induces autoantibodies and renal Ig deposition. To elucidate differences in the induced B cell responses in mice genetically predisposed to autoimmunity, young (NZB x NZW)F(1) mice were immunized with this peptide DNA mimetope. DWEYSVWLSN-immunized mice had significantly increased IgG anti-dsDNA, anti-laminin, and anti-cardiolipin Ab titers compared with controls. In addition, glomerular histopathology in the form of endocapillary disease and crescent formation was markedly more severe in DWEYSVWLSN-immunized mice. Analysis of mAbs from DWEYSVWLSN-immunized mice revealed that anti-peptide Abs were often cross-reactive with DNA. Genetic elements used in the Ab response in immunized mice were homologous to those used in the spontaneous anti-DNA response in (NZB x NZW)F(1) mice, as well as in other, experimentally induced anti-DNA Abs. Our results indicate that peptide immunization can induce a molecular genetic response common to a variety of stimuli that break tolerance to mammalian dsDNA. Based on the similarity between spontaneously arising anti-DNA Abs and several types of induced anti-DNA Abs, we suggest that there may be more than a single Ag that can trigger systemic lupus erythematosus.