Molecular characterization and solution properties of enzymatically tailored arabinoxylans

Two α-l-arabinofuranosidases with different substrate specificities were used to modify the arabinose-to-xylose ratio of cereal arabinoxylans: one enzyme (AXH-m) removed the l-arabinofuranosyl substituents from the monosubstituted xylopyranosyl residues and the other (AXH-d3) the (1 → 3)-linked l-arabinofuranosyl units from the disubstituted xylopyranosyl residue. In this study, we noticed that not only the arabinose-to-xylose ratio but also the position of the arabinofuranosyl substituents affects the water-solubility of arabinoxylans. The AXH-d3 treatment had no significant effect on the solution conformation of arabinoxylans, but the density of the arabinoxylan molecules decreased in DMSO solution after AXH-m modification. The possible heterogeneity of arabinoxylans complicated the interpretation of data describing the macromolecular properties of the enzymatically modified samples.     

Automated detection of remote homology

The classification of a newly identified protein as a member of a superfamily is important for focusing experiments on its most likely functions. Such classification, often performed by hand, has now been fully automated. This sophisticated new approach takes into account not only alignment scores but also a number of other computable attributes, such as functional sites deduced from sequence conservation patterns.     

Single bioreactor gastrointestinal tract simulator for study of survival of probiotic bacteria

The aim of the present study was to design an in vitro model system to evaluate the probiotic potential of food. A single bioreactor system-gastrointestinal tract simulator (GITS) was chosen for process simulation on account of its considerable simplicity compared to multi-vessel systems used in previous studies. The bioreactor was evaluated by studying the viability of four known probiotic bacteria (Lactobacillus acidophilus La-5, Lactobacillus johnsonii NCC 533, Lactobacillus casei strain Shirota, and Lactobacillus rhamnosus GG) as a function of their physiological state. L. acidophilus and L. johnsonii survived in GITS better when introduced at an early stationary or exponential phase compared to being previously stored for 2 weeks at 4 degrees C. These two species were more resistant to bile salts and survived better than L. casei and L. rhamnosus GG. The latter two species gave large losses (up to 6 log) in plate counts independent of growth state due to the bile. However, experiments with some commercial probiotic products containing Lb. GG bacteria showed much better survival compared with model food (modified deMan-Rogosa-Sharpe growth medium), thus demonstrating the influence of the food matrix on the viability of bacteria. The study demonstrated that GITS can be successfully used for evaluation of viability of probiotic bacteria and functionality of probiotic food.     

Translocation of phosphatidylthreonine and -serine to mitochondria diminishes exponentially with increasing molecular hydrophobicity

Some cultured cells contain significant amounts of a rarely recognized phospholipid, phosphatidylthreonine. Since phosphatidylthreonine is a structural analog of phosphatidylserine, the question rises whether it is transported to mitochondria and decarboxylated to phosphatidylisopropanolamine therein. We studied this issue with hamster kidney cell-line using a novel approach, i.e. electrospray mass-spectrometry and stable isotope-labeled precursors. Scanning for a neutral loss of 155, which is characteristic for phosphatidylisopropanolamine, indicated that this lipid is indeed present. The identity of phosphatidylisopropanolamine was supported by the following: (i) it co-chromatographed with phosphatidylethanolamine; (ii) its molecular species profile was similar to that of phosphatidylethanolamine; (iii) its head group was labeled from ¹³C-threonine; and (iv) its concentration increased in parallel with phosphatidylthreonine. Tests with solubilized decarboxylase and subcellular fractionation studies indicated that the low cellular content of phosphatidylisopropanolamine is due to inefficient decarboxylation, rather than poor translocation of phosphatidylthreonine to mitochondria. Importantly, the average hydrophobicity of phosphatidylisopropanolamine molecular species was significantly less than that of phosphatidylthreonine species, indicating that hydrophilic phosphatidylthreonine species translocate to mitochondria far more rapidly than hydrophobic ones. Parallel results were obtained for phosphatidylserine. These findings imply that efflux from the ER membrane could be the rate-limiting step in the phosphatidylthreonine and -serine translocation to mitochondria.     

Root fungal colonisations of the understory grass <Emphasis Type="Italic">Deschampsia flexuosa</Emphasis> after top-canopy harvesting

Aims

Root fungal relationships in forest understory may be affected by tree harvesting. Deschampsia flexuosa forms a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi functioning in nutrient uptake, and a more loose association with dark septate endophytic (DSE) fungi. We asked how harvesting affects fungal colonisations and whether DSE is more prone to change than AM.

Methods

Deschampsia flexuosa plants were sampled close to a control or a cut tree after top-canopy harvesting in a primary successional site. Colonisations were studied using light microscopy. Shoot N%, vegetation cover and soil nutrients were determined.

Results

Tree harvesting did not affect vegetation and soil parameters, except potassium (K⁺) increasing near cut trees. AM colonisation did not change, while DSE increased. Shoot N% increased with increasing DSE near cut trees. Hyaline septate (HSE) hyphae and soil K⁺ and magnesium (Mg²⁺) were positively correlated near control trees. Lichen cover and HSE correlated negatively.

Conclusions

DSE colonisation increased but AM did not change after harvesting. Positive correlation of DSE with shoot N% near cut trees may suggest a role for DSE in favouring plant nitrogen uptake after disturbance in an open microsite. HSE may play a role in K⁺ and Mg²⁺ uptake.

     

Protection against vaccinia virus challenge by CD8 memory T cells resolved by molecular mimicry

Live vaccinia virus (VV) vaccination has been highly successful in eradicating smallpox. However, the mechanisms of immunity involved in mediating this protective effect are still poorly understood, and the roles of CD8 T-cell responses in primary and secondary VV infections are not clearly identified. By applying the concept of molecular mimicry to identify potential CD8 T-cell epitopes that stimulate cross-reactive T cells specific to lymphocytic choriomeningitis virus (LCMV) and VV, we identified after screening only 115 peptides two VV-specific immunogenic epitopes that mediated protective immunity against VV. An immunodominant epitope, VV-e7r130, did not generate cross-reactive T-cell responses to LCMV, and a subdominant epitope, VV-a11r198, did generate cross-reactive responses to LCMV. Infection with VV induced strong epitope-specific responses which were stable into long-term memory and peaked at the time virus was cleared, consistent with CD8 T cells assisting in the control of VV. Two different approaches, direct adoptive transfer of VV-e7r-specific CD8 T cells and prior immunization with a VV-e7r-expressing ubiquitinated minigene, demonstrated that memory CD8 T cells alone could play a significant role in protective immunity against VV. These studies suggest that exploiting cross-reactive responses between viruses may be a useful tool to complement existing technology in predicting immunogenic epitopes to large viruses, such as VV, leading to a better understanding of the role CD8 T cells play during these viral infections.     

Integrin CD11c/CD18 α-Chain Phosphorylation Is Functionally Important

CD11c/CD18 (α_Xβ₂, p150/95, or complement receptor 4, CR4) is a monocyte/macrophage-enriched integrin that has been reported to bind to a variety of ligands. These include cell surface proteins, extracellular matrix proteins, and soluble ligands. The regulation of ligand binding to CD11c/CD18 has remained poorly understood. Previous work has shown that both α-chain and β-chain phosphorylations of CD11a/CD18 and CD11b/CD18 are needed for activity, but no corresponding studies on CD11c/CD18 have been performed. In this study, we have identified the phosphorylation site of CD11c as Ser-1158 and show that it is pivotal for adherence and phagocytosis.     

Premature birth and circadian preference in young adulthood: evidence from two birth cohorts

A preference for eveningness (being a “night owl”) and preterm birth (<37 weeks of gestation) are associated with similar adversities, such as elevated blood pressure, impaired glucose regulation, poorer physical fitness, and lower mood. Yet, it remains unclear if and how preterm birth is associated with circadian preference. The aim of this study was to assess this association across the whole gestation range, using both objective and subjective measurements of circadian preference.

Circadian preference was measured among 594 young adults (mean age 24.3 years, SD 1.3) from two cohorts: the ESTER study and the Arvo Ylppö Longitudinal Study. We compared 83 participants born early preterm (<34 weeks) and 165 late preterm (34 to <37 weeks) with those born at term (≥37 weeks, n = 346). We also compared very low birth weight (VLBW, <1500 g) participants with term-born controls. We obtained objective sleep data with actigraphs that were worn for a mean period of 6.8 (SD 1.4) nights. Our primary outcome was sleep midpoint during weekdays and weekend. The sleep midpoint is the half-way time between falling asleep and waking up, and it represents sleep timing. We also investigated subjective chronotype with the Morningness–Eveningness Questionnaire (MEQ) in 688 (n = 138/221/329) ESTER participants. The MEQ consists of 19 questions, which estimates the respondent to be of a “morning”, “evening,” or “intermediate” chronotype, based on the Morningness–Eveningness Score (MES). We analyzed the data from the actigraphs and the MES with three linear regression models, and analyzed distribution of the chronotype class with Pearson χ2.

There were no consistent differences across the study groups in sleep midpoint. As compared with those born at term, the mean differences in minutes:seconds and 95% confidence intervals for the sleep midpoint were: early preterm weekdays 11:47 (?8:34 to 32:08), early preterm weekend 4:14 (?19:45 to 28:13), late preterm weekdays ?10:28 (?26:16 to 5:21), and late preterm weekend ?1:29 (?20:36 to 17:37). There was no difference in sleep timing between VLBW-participants and controls either. The distribution of chronotype in the MEQ among all participants was 12.4% morningness, 65.4% intermediate, and 22.2% eveningness. The distribution of the subjective chronotype class did not differ between the three gestational age groups (p = 0.98). The linear regression models did not show any influence of gestational age group or VLBW status on the MES (all p > 0.5).

We found no consistent differences between adults born early or late preterm and those born at term in circadian preference. The earlier circadian preference previously observed in those born smallest is unlikely to extend across the whole range of preterm birth.     

Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution

The multispanning membrane protein vacuole membrane protein 1 (VMP1) marks and regulates endoplasmic reticulum (ER)‐domains associated with diverse ER‐organelle membrane contact sites. A proportion of these domains associate with endosomes during their maturation and remodeling. We found that these VMP1 domains are enriched in choline/ethanolamine phosphotransferase and phosphatidylinositol synthase (PIS1), 2 ER enzymes required for the synthesis of various phospholipids. Interestingly, the lack of VMP1 impairs the formation of PIS1‐enriched ER domains, suggesting a role in the distribution of phosphoinositides. In fact, depletion of VMP1 alters the distribution of PtdIns4P and proteins involved in the trafficking of PtdIns4P. Consistently, in these conditions, defects were observed in endosome trafficking and maturation as well as in Golgi morphology. We propose that VMP1 regulates the formation of ER domains enriched in lipid synthesizing enzymes. These domains might be necessary for efficient distribution of PtdIns4P and perhaps other lipid species. These findings, along with previous reports that involved VMP1 in regulating PtdIns3P during autophagy, expand the role of VMP1 in lipid trafficking and explain the pleiotropic effects observed in VMP1‐deficient mammalian cells and other model systems.