Synapsin I is a major endogenous substrate for protein L-isoaspartyl methyltransferase in mammalian brain

The accumulation of potentially deleterious L-isoaspartyl linkages in proteins is prevented by the action of protein L-isoaspartyl O-methyltransferase, a widely distributed enzyme that is particularly active in mammalian brain. Methyltransferase-deficient (knock-out) mice exhibit greatly increased levels of isoaspartate and typically succumb to fatal epileptic seizures at 4-10 weeks of age. The link between isoaspartate accumulation and the neurological abnormalities of these mice is poorly understood. Here, we demonstrate that synapsin I from knock-out mice contains 0.9 +/- 0.3 mol of isoaspartate/mol of synapsin, whereas the levels in wild-type and heterozygous mice are undetectable. Transgenic mice that selectively express methyltransferase only in neurons show reduced levels of synapsin damage, and the degree of reduction correlates with the phenotype of these mice. Isoaspartate levels in synapsin from the knock-out mice are five to seven times greater than those in the average protein from brain cytosol or from a synaptic vesicle-enriched fraction. The isoaspartyl sites in synapsin from knock-out mice are efficiently repaired in vitro by incubation with purified methyltransferase and S-adenosyl-L-methionine. These findings demonstrate that synapsin I is a major substrate for the isoaspartyl methyltransferase in neurons and suggest that isoaspartate-related alterations in the function of presynaptic proteins may contribute to the neurological abnormalities of mice deficient in this enzyme.     

Human monoclonal anti-La antibodies. The La protein resides on a subset of Ro particles

We have addressed the problem of anti-La autoimmune responses by defining the specific binding sites of human mAb to the La protein. Two human anti-La mAb were developed; one an IgM (kappa) (designated 8G3) and the second an IgG1 (kappa) (9A5) isotype. The mAb 8G3 immunoprecipitated the La RNA and La protein from crude human cell lysates; bound the 50-kDa La protein and a 28-kDa digestion fragment in immunoblots, and recognized a small defined internal segment from the cloned La protein. In contrast, the IgG isotype (9A5) failed to precipitate native La from cell lysates but bound the same segment of digested La protein and the same polypeptide of 131 amino acids in length from the cloned La protein. Immunoprecipitation experiments performed with these mAb demonstrated that the La protein is a component of a subset of Ro particles. The data suggest that the La protein is not present on the hY RNA in the absence of the Ro polypeptide. These observations may define functional subsets or maturation states of hY RNA based on their association with Ro or Ro and La polypeptides.     

Genetic diversity and relatedness within packs in an intensely hunted population of wolvesCanis lupus

A population of grey wolvesCanis lupus Linnaeus, 1758 inhabiting Bia?owie?a Primeval Forest (BPF) on the Polish-Belarussian border has recovered after near extermination in the 1970s. Currently, it is intensively hunted in the Belarussian part of BPF and protected in the Polish part. We used a combination of molecular analysis, radiotracking, and field observation to study genetic diversity of the population after natural recolonisation and the consequences of heavy hunting for the genetic composition and social structure of wolf packs. Both microsatellite and mtDNA analyses revealed high genetic diversity. For 29 individuals and 20 microsatellite loci, the mean expected heterozygosity was 0.733. Four mtDNA haplotypes were found. Three of them had earlier been described from Europe. Their geographic distribution suggests that wolves recolonising BPF immigrated mainly from the north-east, and less effectively from the east and south-east. We traced the composition of 6 packs for a total of 26 pack-years. Packs were family units (a breeding pair with offspring) with occasional adoption of unrelated adult males, which occurred more frequently in packs living in the Belarussian part of the BPF, due to heavy hunting and poaching. Breeding pairs were half-sibs or unrelated wolves. Pair-bonds in the breeding pair lasted from 1 to 4 years and usually broke by the death of one or both mates. Successors of breeding females were their daughters, while a successor of a breeding male could be either his son or an alien wolf. As is evident from Bia?owie?a’s wolves, high genetic diversity may result from immigration of outside individuals, which are easily recruited to a heavily exploited local population.     

Anti-biofilm activities of essential oils rich in carvacrol and thymol against Salmonella Enteritidis

The aim of the present study was to determine the bioactive compounds in four essential oils (EO’s) from Origanum heracleoticum, Origanum vulgare, Thymus vulgaris and Thymus serpyllum and to assess their antimicrobial and anti-biofilm activity against Salmonella Enteritidis. Strains were previously characterized depending on the expression of the extracellular matrix components cellulose and curli fimbriae as rdar (red, dry and rough) and bdar morphotype (brown, dry and rough). This study revealed that the EO’s and EOC’s (carvacrol and thymol) investigated showed inhibition of biofilm formation at sub-minimum inhibitory concentration. Comparing the efficacy of EO’s and EOC’s in the inhibition of biofilm formation between the strains with different morphotype (rdar and bdar) did not show a statistically significant difference. Results related to the effectiveness of EO’s and EOC’s (the essential oil components, carvacrol and thymol) on eradication of preformed 48?h old biofilms indicated that biofilm reduction occurred in a dose-dependent manner over time.     

Intracellular protein modification associated with altered T cell functions in autoimmunity

Posttranslational protein modifications influence a number of immunologic responses ranging from intracellular signaling to protein processing and presentation. One such modification, termed isoaspartyl (isoAsp), is the spontaneous nonenzymatic modification of aspartic acid residues occurring at physiologic pH and temperature. In this study, we have examined the intracellular levels of isoAsp residues in self-proteins from MRL(+/+), MRL/lpr, and NZB/W F(1) mouse strains compared with nonautoimmune B10.BR mice. In contrast to control B10.BR or NZB/W mice, the isoAsp content in MRL autoimmune mice increased and accumulated with age in erythrocytes, brain, kidney, and T lymphocytes. Moreover, T cells that hyperproliferate to antigenic stimulation in MRL mice also have elevated intracellular isoAsp protein content. Protein l-isoaspartate O-methyltransferase activity, a repair enzyme for isoAsp residues in vivo, remains stable with age in all strains of mice. These studies demonstrate a role for the accumulation of intracellular isoAsp proteins associated with T cell proliferative defects of MRL autoimmune mice.     

Fatty acid profiles of Trachinus radiatus Cuvier, 1829 (Perciformes‐Trachinoidei,Trachinidae)

The goal of this study was to analyse the fatty acid (FA) profiles of the streaked (starry) weever (Trachinus radiatus), a prized food fish in the countries of its distribution. Fish (N = 20) were sampled in July 2011. Location: 42.761019°N, 17.765090°W; Adriatic Sea, Elaphite Islands near Dubrovnik, Croatia, at 5–10 m depth using longline hooks; body length ranges: 24.1–47.2 cm, weight ranges: 120–960 g. Morphological species determination was genetically confirmed (Folmer region of COI gene). Biochemical analysis of T. radiatus muscular tissue (filet) revealed average ± SD dry matter of 252.3 ± 14.8 g/kg w.w.; moisture of 747.7 ± 14.8 g/kg w.w., and ash of 28.0 ± 6.9 g/kg w.w. Intramuscular crude protein content exceeded the total lipid (TL = 11.9 ± 4.0 g/kg w.w.) content approximately 17.7 fold. Unsaturated FA (UFA) was higher than saturated FA (SFA), with a predominance of polyunsaturated FA (PUFA). The ω3:ω6 ratio was 4.9:1, respectively. Among individually determined fatty acids, the PUFA 22:6n3 (DHA) was highly present (29.99 ± 2.75% TL) followed by a relatively high 20:5n3 (EPA) content. There was 25 fold higher EPA content than of substrate αLNA, and a 15.5 fold higher DHA content to DPAn3. Such ratios indicate that besides trophic ingestion, FA bioconversion elongase/desaturase synthesis pathways toward ω3 PUFA in Weevers could be highly efficient.     

B cells drive early T cell autoimmunity in vivo prior to dendritic cell-mediated autoantigen presentation

Both B cells and dendritic cells (DCs) have been implicated as autoantigen-presenting cells in the activation of self-reactive T cells. However, most self-proteins are ubiquitously and/or developmentally expressed, making it difficult to determine the source and the exposure of autoantigens to APCs in a controlled manner. In this study, we have used an Ig transgenic mouse model to examine the mechanisms by which B cells and other APCs acquire and present lupus autoantigens in vivo. Targeting a lupus autoantigen, the small nuclear ribonucleoprotein particle D protein, to the BCR activates autoreactive T cells in the periphery. Our in vivo studies demonstrate that autoantigen-specific B cells, when present in the repertoire, are the first subset of APCs to capture and present self-proteins for activating T cells. Thereafter, DCs acquire self-Ag and become effective APCs for stimulating the same subsets of autoreactive T cells. This mechanism provides one explanation of how early steps in autoimmunity can focus responses, via BCR, at a small group of self-proteins among the total milieu of intracellular self-proteins. Subsequently, DCs and other professional APCs may then amplify and perpetuate the autoimmune T cell response.