Identification of Novel ��-Synuclein Isoforms in Human Brain Tissue by using an Online NanoLC-ESI-FTICR-MS Method

Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are neurodegenerative diseases that are characterized by intra-neuronal inclusions of Lewy bodies in distinct brain regions. These inclusions consist mainly of aggregated α-synuclein (α-syn) protein. The present study used immunoprecipitation combined with nanoflow liquid chromatography (LC) coupled to high resolution electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) to determine known and novel isoforms of α-syn in brain tissue homogenates. N-terminally acetylated full-length α-syn (Ac-α-syn?????) and two N-terminally acetylated C-terminally truncated forms of α-syn (Ac-α-syn????? and Ac-α-syn?????) were found. The different forms of α-syn were further studied by Western blotting in brain tissue homogenates from the temporal cortex Brodmann area 36 (BA36) and the dorsolateral prefrontal cortex BA9 derived from controls, patients with DLB and PD with dementia (PDD). Quantification of α-syn in each brain tissue fraction was performed using a novel enzyme-linked immunosorbent assay (ELISA).     

Inbreeding-environment interactions for fitness: complex relationships between inbreeding depression and temperature stress in a seed-feeding beetle

It is commonly argued that inbred individuals should be more sensitive to environmental stress than are outbred individuals, presumably because stress increases the expression of deleterious recessive alleles. However, the degree to which inbreeding depression is dependent on environmental conditions is not clear. We use two populations of the seed-feeding beetle, Callosobruchus maculatus, to test the hypotheses that (a) inbreeding depression varies among rearing temperatures, (b) inbreeding depression is greatest at the more stressful rearing temperatures, (c) the degree to which high or low temperature is stressful for larval development varies with inbreeding level, and (d) inbreeding depression is positively correlated between different environments. Inbreeding depression (δ) on larval development varied among temperatures (i.e., there was a significant inbreeding-environment interaction). Positive correlations for degree of inbreeding depression were consistently found between all pairs of temperatures, suggesting that at least some loci affected inbreeding depression across all temperatures examined. Despite variation in inbreeding depression among temperatures, inbreeding depression did not increase consistently with our proxy for developmental stress. However, inbreeding changed which environments are benign versus stressful for beetles; although 20°C was not a stressful rearing temperature for outbred beetles, it became the most stressful environment for inbred larvae. The finding that inbreeding-environment interactions can cause normally benign environments to become stressful for inbred populations has important consequences for many areas of evolutionary genetics, artificial breeding (for conservation or food production), and conservation of natural populations.     

Effects of elevated carbon dioxide, ozone and water availability on spring wheat growth and yield

Spring wheat (Triticum aestivum L. cv. Dragon) was exposed to elevated carbon dioxide (CO₂), alone (1995) or in combination with two levels of increased ozone (O₃) (1994) or increased irrigation (1996) during three successive growing seasons as part of the EU ESPACE‐wheat programme and conducted in open‐top chambers (OTCs) and ambient air (AA) plots at Östad, 50 km north‐east of Göteborg, Sweden. Doubling the CO₂ concentration had a positive effect on grain yield in all 3 years (+21, +7 and +11%, respectively), although only statistically significant in 1994. That year was characterised by a warm and dry summer in comparison with 1995 and 1996, in which the summers were more humid and typical for south‐west Sweden. In 1994, the CO₂‐induced increase in grain yield was associated with an increase in the duration of the green leaf area, a positive effect on straw yield and on the number of ears per square metre and a negative effect (?13%) on grain protein concentration. Harvest index was unaffected by the elevated CO₂ concentration. The only statistically significant effect of elevated CO₂ in 1995 was a decrease in the grain protein concentration (?11% in both CO₂ concentrations), and in 1996 an increase (+21%) in the straw yield. In 1996 the soil water potential was less negative in elevated CO₂, which is likely to reflect a lower water consumption of these plants. Addition of extra O₃ significantly affected the grain yield (?6 and ?10%, respectively) and the 1 000‐grain weight negatively (?3 and ?6%). Statistically significant interactions between CO₂ and O₃ were obtained for the number of ears per unit area and for the 1 000‐grain weight. The 1 000‐grain weight was negatively affected by O₃ in low CO₂, but remained unaffected in the high CO₂ treatment. There was a significant decrease (?6%) in the grain protein concentration induced by elevated irrigation. The chambers, compared with AA plots, had a positive effect on plant development and on grain yield in all 3 years.     

Differential retrotranslocation of mitochondrial Bax and Bak

The Bcl-2 proteins Bax and Bak can permeabilize the outer mitochondrial membrane and commit cells to apoptosis. Pro-survival Bcl-2 proteins control Bax by constant retrotranslocation into the cytosol of healthy cells. The stabilization of cytosolic Bax raises the question whether the functionally redundant but largely mitochondrial Bak shares this level of regulation. Here we report that Bak is retrotranslocated from the mitochondria by pro-survival Bcl-2 proteins. Bak is present in the cytosol of human cells and tissues, but low shuttling rates cause predominant mitochondrial Bak localization. Interchanging the membrane anchors of Bax and Bak reverses their subcellular localization compared to the wild-type proteins. Strikingly, the reduction of Bax shuttling to the level of Bak retrotranslocation results in full Bax toxicity even in absence of apoptosis induction. Thus, fast Bax retrotranslocation is required to protect cells from commitment to programmed death.     

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) S18Y polymorphism in Alzheimer's disease

Alzheimer's disease (AD) is characterized by protein aggregates, i.e. senile plaques and neurofibrillary tangles. The ubiquitin-proteasome system has been proposed a role in proteolytic removal of these protein aggregates. Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme with important functions in recycling of ubiquitin. The S18Y polymorphism of the UCHL1 gene confers protection against Parkinson's disease. In this study, the genotype and allele frequencies of the UCHL1 S18Y polymorphism were investigated in 452 AD patients and 234 control subjects, recruited from four memory clinics in Sweden. Using a binary logistic regression model including UCHL1 allele A and APOE ε4 allele positivity, age and sex as covariates with AD diagnosis as dependent variable, an adjusted OR of 0.82 ([95% CI 0.55-1.24], P = 0.35) was obtained for a positive UCHL1 allele A carrier status. The present study thus do not support a protective effect of the UCHL1 S18Y polymorphism against AD.     

Purification of branched chain aminotransferase from rat heart mitochondria

This paper presents the first purification of the branched chain aminotransferase (EC 2.6.1.42) from rat heart mitochondria. The enzyme has been purified from the 100,000 x g supernatant obtained after sonication and ultracentrifugation of rat heart mitochondria. A combination of open column chromatography, high pressure liquid chromatography (HPLC), and discontinuous polyacrylamide disc gel electrophoresis was used. The key step in the procedure was hydrophobic interaction chromatography on HPLC. The final purification step was polyacrylamide disc gel electrophoresis where the enzyme appeared as a doublet. When electroeluted from the gel, each of these bands had the same specific activity demonstrating that there are two forms of the purified enzyme which differ slightly in electrical charge. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these two enzyme forms appeared as a single band with a molecular mass of 43 kDa. Size exclusion chromatography on Sephacryl S-100 identified the enzyme as a 50-kDa protein. These experiments argue against the existence of a dimeric form of this enzyme. The ratio of enzyme activity with leucine (0.84), valine (0.88), or glutamate (0.66) as amino acid substrate versus isoleucine remained constant throughout the purification procedure. Specific activity of the final preparation was 66 units/mg of enzyme protein. Polyclonal antibodies against the purified enzyme were raised in rabbits. On an immunoblot the antiserum recognized a 43-kDa protein in the 100,000 x g supernatant from a rat heart mitochondrial sonicate but did not recognize any proteins in rat brain cytosol. Quantitative immunodot assay resulted in an estimated enzyme content of about 100 micrograms of branched chain aminotransferase protein/g of heart, wet weight. Finally, 97% of the heart branched chain aminotransferase activity could be neutralized by the antiserum, but the antiserum would not neutralize aminotransferase activity in brain cytosol. These data suggest that close sequence homology does not exist between the two proteins.     

Different HLA DR-DQ associations in subgroups of idiopathic myasthenia gravis

We have investigated theHLA-DRB and -DQB gene polymorphism in 131 myasthenia gravis (MG) patients. TheHLA genotypes in these patients were assigned by means of restriction fragment length polymorphism (RFLP)-definedDR-DQ haplotypes, correlating to serologic HLA class II typing. Using this technique we could, among randomly selected non-thymomatous (NT)-MG patients, confirm the strong association to DR3, and 70% of the patients were found to carry a specific DR3-positiveDR-DQ haplotype,T-3.1. Furthermore, an analysis of T-3.1^– NT-MG patients revealed that 59 % were T-4.1^÷ (DR4, DQw8). Thymic hyperplasia was found in approximately 85 % of the T-3.1⁺ , as well as of the T-4.1⁺ /3.1^– patients. As previously observed, we found a clear dominance of females among the T-3.1⁺ NT-MG patients. However, among T-4.1⁺/3.1- patients, males were as common as females. Furthermore, the T-4.1⁺ patients were significantly older at the onset of disease than those who were T-3.1⁺. In female MG patients, the DRwl5-Dw2-positive haplotypeT-2.1 was strongly correlated with the presence of thymoma (T-MG). These data indicate that the HLA associations in early vs late onset of NT-MG are different, and that female patients with and without thymoma differ from each other with regard to HLA markers. Thus, at least three different HLA DR-DQ associations are found in subgroups of idiopathic MG.     

Interaction of estramustine phosphate with microtubule-associated proteins

We have reported [(1984) Cancer Res., in press] that estramustine phosphate inhibits microtubule assembly and disassembled preformed microtubules. We now present evidence that estramustine phosphate inhibits microtubule assembly by binding to the microtubule-associated proteins. We have found that: additional microtubule-associated proteins relieved the inhibition of assembly by estramustine phosphate; 3H-labelled estramustine phosphate bound predominantly to the microtubule-associated proteins; and the content of the microtubule-associated proteins was reduced in taxol reversed estramustine phosphate-inhibited microtubules.     

Improved yield of apple leaf protoplasts from in vitro cultured shoots by using very young leaves and adding L-methionine to the shoot medium

Protoplast isolation from apple leaf tissue of in-vitro cultured shoots is possible if very young leaves from buds are used. Digestible leaves are found in apple shoots of the subculture kerö 14 days after transferring the shoot and of M26 some days later.The yield of protoplasts is considerably improved if the apple shoots are cultured in a medium containing L-methionine (0.5 mM).