Non-regulatory CD8+CD45RO+CD25+ T-lymphocytes may compensate for the loss of antigen-inexperienced CD8+CD45RA+ T-cells in old age

The age-related decline in immune system functions is responsible for the increased prevalence of infectious diseases and the low efficacy of vaccination in elderly individuals. In particular, the number of peripheral naive T-cells declines throughout life and they exhibit severe functional defects at advanced age. However, we have recently identified a non-regulatory CD8+CD45RO+ CD25+ T-cell subset that occurs in a subgroup of healthy elderly individuals, who still exhibit an intact humoral immune response following influenza vaccination. Here, we demonstrate that CD8+CD45RO+CD25+ T-cells share phenotypic and functional characteristics with naive CD8+CD45RA+CD28+ T-cells from young individuals, despite their expression of CD45RO. CD8+CD45RO+ CD25+ T-cells also have long telomeres and upon antigenic challenge, they efficiently expand in vitro and differentiate into functional effector cells. The expanded population also maintains a diverse T-cell receptor repertoire. In conclusion, CD8+CD45RO+CD25+ T-cells from elderly individuals compensate for the loss of functional naive T-cells and may therefore be used as a marker of immunological competence in old age.     

The Reductive Half-reaction of Xanthine Dehydrogenase from Rhodobacter capsulatus: THE ROLE OF GLU232 IN CATALYSIS*

The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu²³² in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that k_red, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu²³² being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both k_red and k_red/K_d from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu²³² of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pK_a of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme.     

Antisecretory factor modulates GABAergic transmission in the rat hippocampus

Antisecretory Factor (AF) is a protein that has been implicated in the suppression of intestinal hypersecretion and inflammation. Intestinal secretion and inflammation are partly under local and central neural control raising the possibility that AF might exert its action by modulating neural signaling. In the present study we have investigated whether AF can modulate central synaptic transmission. Evoked glutamatergic and GABAergic synaptic transmissions were investigated using extracellular recordings in the CA1 region of hippocampal slices from adult rats. AF (0.5 microg/ml) suppressed GABA(A)-mediated synaptic transmission by about 40% while having no effect on glutamatergic transmission. Per oral administration of cholera toxin as well as feeding of rats with a diet containing hydrothermally processed cereals, known to upregulate endogenous AF plasma activity, mimicked the effect of exogenously administered AF on hippocampal GABAergic transmission. Our results identify AF as a neuromodulator and further raise the possibility that the hippocampus and AF are involved in a gut-brain loop controlling intestinal secretion and inflammation.     

Sweet Substitute: a software tool for in silico fragmentation of peptide-linked N-glycans

A software tool, Sweet Substitute, is described, which assists tandem mass spectrometry (MS/MS)-based glycosylation characterization from within a tryptic digest. The algorithm creates a virtual nanoelectrospray-quadrupole time-of-flight style-MS/MS spectrum of any user-defined N-linked glycan structure. An empirical peak height modeling routine is implemented in the program. By comparing the theoretical MS/MS data with the deconvoluted and deisotoped experimental MS/MS data, the user is able to quickly assess whether a proposed candidate oligosaccharide structure is a plausible one.     

Fine-mapping and candidate gene analysis of bovine spinal muscular atrophy

Bovine spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disease, has been mapped at moderate resolution to the distal part of Chromosome 24. In this article we confirm this location and fine-map the SMA locus to an interval of approximately 0.8 cM at the very distal end of BTA24. Despite remarkable similarity to human SMA, the causative gene SMN can be excluded in bovine SMA. However, the interval where the disease now has been mapped contains BCL2, like SMN an antiapoptotic factor, and shown to bind to SMN. Moreover, knockout mice lacking the BCL2 gene show rapid motor neuron degeneration with early postnatal onset, as observed in bovine SMA. A comparative cattle/human map of the distal end of BTA24, based on the emerging bovine genome sequencing data, shows conserved synteny to HSA18 with hints of a segmental duplication and pericentic inversion just after the last available bovine marker DIK4971. This synteny lets us conclude that SMA is in immediate vicinity of the telomere. Candidate gene analysis of BCL2, however, excludes most of this gene, except its promoter region, and draws attention to the neighboring gene VPS4B, part of the endosomal protein-sorting machinery ESCRT-III which is involved in several neurodegenerative diseases. Stefan Krebs and Ivica Medugorac contributed equally to this work and agreed to be considered as first authors.     

Identification of the amino acid residues rendering TI-VAMP insensitive toward botulinum neurotoxin B

Botulinum neurotoxins types B, D, F, and G, and tetanus neurotoxin inhibit vesicular fusion via proteolytic cleavage of VAMP/Synaptobrevin, a core component of the membrane fusion machinery. Thus, these neurotoxins became widely used tools for investigating vesicular trafficking routes. Except for VAMP-1, VAMP-2, and Cellubrevin, no other member of the VAMP family represents a substrate for these neurotoxins. The molecular basis for this discrepancy is not known. A 34 amino acid residue segment of VAMP-2 was previously suggested to mediate the interaction with botulinum neurotoxin B, but the validity of the data was later questioned. To check whether this segment alone controls the susceptibility toward botulinum neurotoxin B, it was used to replace the corresponding segment in TI-VAMP. The resulting VAMP hybrid and VAMP-2 were hydrolysed at virtually identical rates. Resetting the VAMP-2 portion in the hybrid from either end to TI-VAMP residues gradually reduced the cleavability. A hybrid encompassing merely the VAMP-2 segment 71-80 around the Gln76/Phe77 scissile bond was still hydrolysed, albeit at a approximately tenfold lower cleavage rate. The contribution of each non-conserved amino acid of the whole 34-mer segment to the interaction was investigated employing VAMP-2. We find that the eight non-conserved residues of the 71-80 segment are all necessary for efficient cleavage. Mutation of an additional six residues located upstream and downstream of this segment affects substrate hydrolysis as well. Vice versa, a readily cleavable TI-VAMP molecule requires at the least the replacement of Ile158, Thr161, and the section 165-174 by Asp64, Ala67, and the 71-80 segment of VAMP-2, respectively. However, the insensitivity of TI-VAMP to botulinum neurotoxin B relies on at least 12 amino acid changes versus VAMP-2. These are scattered along an interface of 22 amino acid residues in length.     

Chromatin remodeling by ISW2 and SWI/SNF requires DNA translocation inside the nucleosome

Chromatin-remodeling complexes regulate access to nucleosomal DNA by mobilizing nucleosomes in an ATP-dependent manner. In this study, we find that chromatin remodeling by SWI/SNF and ISW2 involves DNA translocation inside nucleosomes two helical turns from the dyad axis at superhelical location-2. DNA translocation at this internal position does not require the propagation of a DNA twist from the site of translocation to the entry/exit sites for nucleosome movement. Nucleosomes are moved in 9- to 11- or approximately 50-base-pair increments by ISW2 or SWI/SNF, respectively, presumably through the formation of DNA loops on the nucleosome surface. Remodeling by ISW2 but not SWI/SNF requires DNA torsional strain near the site of translocation, which may work in conjunction with conformational changes of ISW2 to promote nucleosome movement on DNA. The difference in step size of nucleosome movement by SWI/SNF and ISW2 demonstrates how SWI/SNF may be more disruptive to nucleosome structure than ISW2.     

Structural analysis of the PP2C phosphatase tPphA from Thermosynechococcus elongatus: a flexible flap subdomain controls access to the catalytic site

The homologue of the phosphoprotein PII phosphatase PphA from Thermosynechococcus elongatus, termed tPphA, was identified and its structure was resolved in two different space groups, C222₁ and P4₁2₁2, at a resolution of 1.28 and 3.05 Å, respectively. tPphA belongs to a large and widely distributed subfamily of Mg²⁺/Mn²⁺-dependent phosphatases of the PPM superfamily characterized by the lack of catalytic and regulatory domains. The core structure of tPphA shows a high degree of similarity to the two PPM structures identified so far. In contrast to human PP2C, but similar to Mycobacterium tuberculosis phosphatase PstP, the catalytic centre exhibits a third metal ion in addition to the dinuclear metal centre universally conserved in all PPM members. The fact that the third metal is only liganded by amino acids, which are universally conserved in all PPM members, implies that the third metal could be general for all members of this family. As a specific feature of tPphA, a flexible subdomain, previously recognized as a flap domain, could be revealed. Comparison of different structural isomers of tPphA as well as site-specific mutagenesis implied that the flap domain is involved in substrate binding and catalytic activity. The structural arrangement of the flap domain was accompanied by a large side-chain movement of an Arg residue (Arg169) at the basis of the flap. Mutation of this residue strongly impaired protein stability as well as catalytic activity, emphasizing the importance of this amino acid for the regional polysterism of the flap subdomain and confirming the assumption that flap domain flexibility is involved in catalysis.