Structural studies of an exopolysaccharide produced by Streptococcus thermophilus THS

The structure of an extracellular polysaccharide (EPS) from Streptococcus thermophilus THS has been determined. A combination of component analysis, methylation analysis and NMR spectroscopy shows that the polysaccharide is composed of pentasaccharide repeating units. Sequential information was obtained by two-dimensional (1)H,(1)H-NOESY and (1)H,(13)C-HMBC NMR experiments. NMR data indicate different mobility within the EPS with a stiffer backbone and a more flexible side-chain.     

Quantitative detection of cell surface protein expression by time-resolved fluorimetry.

A method is introduced for quantitative detection of cell surface protein expression. The method is based on immunocytochemistry, the use of long decay time europium(III) chelate and platinum(II) porphyrin labels, and detection of photoluminescence emission from adhered cells by time-resolved fluorimetry. After immunocytochemistry, the assay wells are evaporated to dryness and measured in the dry state. This protocol allows repeated and postponed analysis and microscopy imaging. In order to investigate the performance of the method, we chose expression of intercellular adhesion molecule-1 (ICAM-1) of endothelial cell line EAhy926 as a research target. The expression of ICAM-1 on the cells was enhanced by introduction of a cytokine, tumour necrosis factor-alpha (TNFalpha). The method gave signal:background ratios (S:B) of 20 and 9 for europium and platinum labels, respectively, whereas prompt fluorescent FITC label gave a S:B of 3. Screening window coefficients (=Z'-factor) were >0.5 for all the three labels, thus indicating a score for an excellent screening assay. In conclusion, the method appears to be an appropriate choice for protein expression analysis, both in high-throughput screening applications, and for detailed sample investigation by fluorescent microscopy imaging.     

Reinnervation of arterial grafts by adrenergic nerves occurs in rats as indicated by increased levels of noradrenaline

To investigate the changes in noradrenaline concentrations in transplanted arterial grafts in rats, 31 female rats 4 to 6 weeks old of the AO/Ks:OC strain were operated on. Femoral arterial grafts were anastomosed to carotid arteries and compared with control femoral segments. Six rats were included in each follow-up group at 0, 1, 4, and 12 weeks, and there were seven rats in the 20-week follow-up group. High-performance liquid chromatography was used to determine the concentrations of noradrenaline. The operation itself decreased noradrenaline concentrations in the grafts to 76 percent of that in the control segments. One week after the operation, the noradrenaline concentration had fallen to 1.7 percent of the control values and started to recover thereafter. One month after the operation, it was 23 percent; at 3 months, it was 31 percent; and at 5 months, it was 43 percent of control values. The decrease from time 0 to 1 week was significant (p = 0.001), as was the increase from 1 week to 20 weeks (p = 0.004). Noradrenaline concentrations had fallen significantly 1 week after the operation and thereafter they increased to levels comparable to those seen in the immediate postoperative period.     

Evaluation of chemical pretreatment of contaminated soil for improved PAH bioremediation

The efficiency of several chemical treatments as potential enhancers of the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in contaminated soil was evaluated by analyzing the mineralization of ¹⁴C-labeled phenanthrene, pyrene, and benzo(a)pyrene. The effect of nonionic surfactants with Fenton oxidation and combinations of surfactants with the Fenton oxidation was evaluated in a microtiter plate assay. The surfactants selected for the study were Tween 80, Brij 35, Tergitol NP-10, and Triton X-100. The addition of Fentons reagent significantly enhanced the mineralization of pyrene at the two concentrations studied: 2.8 M H₂O₂ with 0.1 M FeSO₄ and 0.7 M H₂O₂ with 0.025 M FeSO₄. Phenanthrene mineralization was also positively induced by the Fenton treatments. However, none of the treatments had a significant effect on benzo(a)pyrene mineralization. Surfactant additions at concentrations of 20% and 80% of the aqueous critical micelle concentration did not significantly affect the mineralization rates. When surfactant addition was combined with the Fenton oxidation, reduced mineralization rates were obtained when compared with mineralization after Fentons treatment alone. The results indicate that the addition of Fentons reagent may enhance the mineralization of PAHs in contaminated soil, whereas the addition of surfactants has no significant beneficial effect. The efficiency of the Fenton oxidation may decrease when surfactants are added simultaneously with Fentons reagent to contaminated soil.     

A partial skeleton of Prodeinotherium bavaricum (Proboscidea, Mammalia) from the Middle Miocene of Unterzolling (Upper Freshwater Molasse, Germany)

Here, a partial skeleton of Prodeinotherium bavaricum from Unterzolling (Southern Germany) is documented. The following elements are preserved and described for the first time: cervical vertebrae 1-2 and 5-7, the first thoracic vertebra, one lumbar vertebra, trapezium, metacarpals 1-5, tibia, calcaneus, endo- and mesocuneiform, cuboid, the fourth metatarsal, and some phalanges. Comparisons with the skeletons of P. bavaricum from Franzensbad (Czech Republic) and Deinotherium giganteum from Eserovo (Bulgaria) show osteological differences that are described and discussed.     

Structural elucidation of the viscous exopolysaccharide produced by Lactobacillus helveticus Lb161

A viscous extracellular polysaccharide produced by Lactobacillus helveticus Lb161 isolated from raw milk has been investigated. Sugar and methylation analysis, and 1H and 13C NMR spectroscopy revealed that the polysaccharide is composed of a heptasaccharide repeating unit. The sequence of sugar residues was determined by use of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear multiple bond connectivity experiments. The structure of the repeating unit of the exopolysaccharide from L. helveticus Lb161 is as follows: carbohydrate structure [see text]. The polysaccharide contains approximately 0.6 equivalents of O-acetyl group per repeating unit (not located).     

Structural analysis of the exopolysaccharide produced by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> ST1 solely by NMR spectroscopy

The use of lactic acid bacteria in fermentation of milk results in favorable physical and rheological properties due to in situ exopolysaccharide (EPS) production. The EPS from S. thermophilus ST1 produces highly viscous aqueous solutions and its structure has been investigated by NMR spectroscopy. Notably, all aspects of the elucidation of its primary structure including component analysis and absolute configuration of the constituent monosaccharides were carried out by NMR spectroscopy. An array of techniques was utilized including, inter alia, PANSY and NOESY-HSQC TILT experiments. The EPS is composed of hexasaccharide repeating units with the following structure: → 3)[α-d-Glcp-(1 → 4)]-β-d-Galp-(1 → 4)-β-d-Glcp-(1 → 4)[β-d-Galf-(1 → 6)]-β-d-Glcp-(1 → 6)-β-d-Glcp-(1 →, in which the residues in square brackets are terminal groups substituting backbone sugar residues that consequently are branch-points in the repeating unit of the polymer. Thus, the EPS consists of a backbone of four sugar residues with two terminal sugar residues making up two side-chains of the repeating unit. The molecular mass of the polymer was determined using translational diffusion experiments which resulted in M_w = 62 kDa, corresponding to 64 repeating units in the EPS.     

Mitochondrial DNA haplogroups in early-onset Alzheimer's disease and frontotemporal lobar degeneration

Background

Alleles of apolipoprotein E (APOE) are the major genetic risk factor for late onset Alzheimer's Disease (LOAD). Recently, an APOE splice variant that retains intron 3 (APOE-I3) was identified. To gain insight into the possible role of this isoform in LOAD, we quantified its expression in a cohort of 56 human brain specimens by using quantitative RT-PCR.

Results

We found that APOE-I3 generally represents a low percentage (< 0.5%) of overall APOE expression. However, in one specimen, the proportion of APOE-I3 was increased about ~13 fold. This specimen was unique in the cohort for possessing the minor allele of an intron 3 single nucleotide polymorphism (SNP), rs12982192. Additionally, an allelic expression imbalance study indicated that the rs12982192 minor allele was associated with increased APOE-I3 expression.

Conclusions

Overall, we interpret our results as suggesting that APOE-I3 represents a minor portion of APOE expression and that rs12982192 is associated with APOE intron 3 retention. Since the minor allele of this SNP is on the same haplotype as the minor allele of rs429358, which defines the APOE4 allele, we speculate that rs12982192 may reflect a modest loss of mRNA encoding functional APOE4.     

Reddish spring colouring of deciduous leaves: a sign of ecotype?

Several deciduous woody plant species produce anthocyanins during leaf development in spring and again during leaf senescence in autumn. The leaves of Betula pendula Roth (silver birch) commonly exhibit transient reddening in juvenile leaves under northern growing conditions, with the intensity of the red colour varying among individual trees. The objective of our study was to test the hypothesis that the accumulation of foliar anthocyanins during spring in leaves of B. pendula is an ecotypic response. Chlorophyll fluorescence ratio (Fv/Fm), leaf reflectance and anthocyanin concentrations were measured, in relation to phenology in spring, summer and autumn from birches used for landscaping with either red or green-emergent leaves. The results suggest that (1) the trees with green- or red-emergent juvenile leaves represent different populations, and (2) that the red-emergent leaves senesced earlier, indicating that (3) trees with red-emerging leaves belong to a more northern ecotype than the trees with green-emerging leaves. The role of anthocyanin synthesis in a northern radiation environment is discussed.     

Inhibition of the cellular function of perforin by 1-amino-2,4-dicyanopyrido[1,2-a]benzimidazoles

A high throughput screen showed the ability of a 1-amino-2,4-dicyanopyrido[1,2-a]benzimidazole analogue to directly inhibit the lytic activity of the pore-forming protein perforin. A series of analogues were prepared to study structure-activity relationships (SAR) for the this activity, either directly added to cells or released in situ by KHYG-1 NK cells, at non-toxic concentrations. These studies showed that the pyridobenzimidazole moiety was required for effective activity, with strongly basic centres disfavoured. This class of compounds was relatively unaffected by the addition of serum, which was not the case for a previous class of direct inhibitors.