C(6)-oxidation followed by C(5)-epimerization of guar gum studied by high field NMR

Guar gum, a beta-D-(1-->4)-linked D-mannan with alpha-D-galactopyranosyl units attached as side groups, was treated with alpha-galactosidase, an enzyme that splits off the alpha-D-galactosyl units to obtain a galactomannan with a low galactose content. The galactose-depleted polysaccharide was then selectively oxidized in C(6) position and epimerized using mannuronan C(5)-epimerases, namely AlgE1, AlgE4, AlgE6, and their mixtures, obtaining new pseudo-alginates. In this paper, we report a full high field 1D and 2D NMR study of guar gum as such and of the galactose-depleted, oxidized and epimerized compounds, respectively. From the 1H NMR spectra, the degree of epimerization, the distribution of mannuronic acid (M) and guluronic acid (G) residues and the average G-block length, N(G>1), were obtained. By means of NMR diffusion experiments, it was also shown that no significant degradation of the polysaccharide occurs as a consequence of the epimerization reactions.     

Xanthones from Polygala alpestris (Rchb.)

Bioactivity-guided fractionation of Polygala alpestris L. (Rchb.) extracts led to the identification of two new xanthones, 1,3,7-trihydroxy-2,6-dimethoxyxanthone (1) and 2,3-methylenedioxy-4,7-dihydroxyxanthone (2). In addition five known compounds 3,4-dimethoxy-1,7-dihydroxyxanthone (3), 1,3-dihydroxy-7-methoxyxanthone (4), 1,7-dihydroxy-2,3-dimethoxyxanthone (5), 3',6-O-disinapoyl sucrose (6) and 3',5'-dimethoxybiphenyl-4-olo (7) were isolated. The structures of the isolated compounds were established by means of high resolution mass spectrometry, mono- and bi-dimensional NMR spectroscopy. All isolated compounds were tested for cytotoxic activity against three tumor cell lines (LoVo, HL-60, K 562).     

Involvement of metalloprotease-2 in the development of human brain microvessels

The involvement of the metalloprotease-2 (MMP-2) in vessel development was investigated in the human telencephalon by double immunoreactions with antibodies to the enzyme, latent (proMMP-2) and active (aMMP-2) forms, and an antibody against collagen type IV, a constitutive component of the extracellular matrix (ECM) of the vessel basal lamina. MMP-2 is expressed in both 12- and 18-week telencephalic vessels, the proenzyme being mainly localised in endothelial cells and the active form prevailing in -actin-reactive periendothelial cells identified as pericytes. Endothelial cells intensely positive for aMMP-2 were revealed in some microvessels and appeared locally associated with discontinuities of the collagen basal lamina. No detectable expression of MMP-2 was observed in perivascular glial processes revealed by vimentin/glial fibrillary acidic protein immunostainings. Double immunoreactions performed to further investigate telencephalon angiogenesis have demonstrated that both the endothelial cells and pericytes strongly express vascular endothelial growth factor (VEGF). Taken together, the results indicate that MMP-2 is largely involved in human brain angiogenesis and suggest that endothelial cells and pericytes tightly interplay in both angiogenesis mechanisms, by ECM proteolysis, and angiogenesis regulation, by local (autocrine/juxtacrine) VEGF action.Francesco Girolamo and Daniela Virgintino contributed equally to this work     

Countercurrent distribution of two distinct SNARE complexes mediating transport within the Golgi stack

Genetic and biochemical evidence has established that a SNARE complex consisting of syntaxin 5 (Sed5)-mYkt6 (Ykt6)-GOS28 (Gos1)-GS15 (Sft1) is required for transport of proteins across the Golgi stack in animals (yeast). We have utilized quantitative immunogold labeling to establish the cis-trans distribution of the v-SNARE GS15 and the t-SNARE subunits GOS28 and syntaxin 5. Whereas the distribution of the t-SNARE is nearly even across the Golgi stack from the cis to the trans side, the v-SNARE GS15 is present in a gradient of increasing concentration toward the trans face of the stack. This contrasts with a second distinct SNARE complex, also required for intra-Golgi transport, consisting of syntaxin 5 (Sed5)-membrin (Bos1)-ERS24 (Sec22)-rBet1 (Bet1), whose v-(rBet1) and t-SNARE subunits (membrin and ERS24), progressively decrease in concentration toward the trans face. Transport within the stack therefore appears to utilize countercurrent gradients of two Golgi SNAREpins and may involve a mechanism akin to homotypic fusion.     

Exclusion of golgi residents from transport vesicles budding from Golgi cisternae in intact cells

A central feature of cisternal progression/maturation models for anterograde transport across the Golgi stack is the requirement that the entire population of steady-state residents of this organelle be continuously transported backward to earlier cisternae to avoid loss of these residents as the membrane of the oldest (trans-most) cisterna departs the stack. For this to occur, resident proteins must be packaged into retrograde-directed transport vesicles, and to occur at the rate of anterograde transport, resident proteins must be present in vesicles at a higher concentration than in cisternal membranes. We have tested this prediction by localizing two steady-state residents of medial Golgi cisternae (mannosidase II and N-acetylglucosaminyl transferase I) at the electron microscopic level in intact cells. In both cases, these abundant cisternal constituents were strongly excluded from buds and vesicles. This result suggests that cisternal progression takes place substantially more slowly than most protein transport and therefore is unlikely to be the predominant mechanism of anterograde movement.     

Mitochondrial and ion channel gene alterations in autism

To evaluate the potential importance in autistic subjects of copy number variants (CNVs) that alter genes of relevance to bioenergetics, ionic metabolism, and synaptic function, we conducted a detailed microarray analysis of 69 autism probands and 35 parents, compared to 89 CEU HapMap controls. This revealed that the frequency CNVs of≥100kb and CNVs of≥10 Kb were markedly increased in probands over parents and in probands and parents over controls. Evaluation of CNVs≥1Mb by chromosomal FISH confirmed the molecular identity of a subset of the CNVs, some of which were associated with chromosomal rearrangements. In a number of the cases, CNVs were found to alter the copy number of genes that are important in mitochondrial oxidative phosphorylation (OXPHOS), ion and especially calcium transport, and synaptic structure. Hence, autism might result from alterations in multiple bioenergetic and metabolic genes required for mental function. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Life on the Half-Shell: Consequences of a Carapace in the Evolution of Armadillos (Xenarthra: Cingulata)

Without doubt, the possession of an armored carapace represents one of the most conspicuous morphological features of all cingulates. Here, we review some of the many ways in which the carapace may have influenced the evolution of other features of extant armadillos (Xenarthra: Cingulata). Effects range from physiological impacts on respiration and thermoregulation, to mechanical and other constraints on reproduction. Additionally, in mammals, armor has been linked with relatively slow plantigrade locomotion, which in turn may have promoted the low metabolic rate and exploitation of a low quality diet typically observed in armadillos. Finally, this network of relationships may help to explain the lack of any obvious kin-selected altruism in the polyembryonic armadillos, such as the nine-banded armadillo (Dasypus novemcinctus), because of time and energy constraints associated with a short active period devoted almost exclusively to feeding. In mammals, there has been growing interest in describing an ecological “lifestyle” as the particular way in which each species makes its living, and how this lifestyle constrains the evolution of other phenotypic traits. Based on our review, it appears the carapace has been a major determinant of the lifestyle of armadillos and has played a central role in shaping the evolution of many other features of these animals.     

Procalcitonin Predicts Real-Time PCR Results in Blood Samples from Patients with Suspected Sepsis

Background

Early diagnosis and rapid bacterial identification are of primary importance for outcome of septic patients. SeptiFast® (SF) real-time PCR assay is of potential utility in the etiological diagnosis of sepsis, but it cannot replace blood culture (BC) for routine use in clinical laboratory. Procalcitonin (PCT) is a marker of sepsis and can predict bacteremia in septic patients. The aim of the present study was to investigate whether PCT serum levels could predict SF results, and could help screening febrile patients in which a SF assay can improve the etiological diagnosis of sepsis.

Methods

From 1009 febrile patients with suspected sepsis, 1009 samples for BC, SF real-time PCR, and PCT determination were obtained simultaneously, and results were compared and statistically analysed. Receiver operating characteristic (ROC) curves were generated to determine the area under the curve and to identify which cut-off of PCT value produced the best sensitivity to detect SF results.

Results

Mean PCT values of sera drawn simultaneously with samples SF positive (35.42±61.03 ng/ml) or BC positive (23.14±51.56 ng/ml) for a pathogen were statistically higher than those drawn simultaneously with SF negative (0.84±1.67 ng/ml) or BC negative (2.79±16.64 ng/ml) samples (p<0.0001). For SF, ROC analysis showed an area under the curve of 0.927 (95% confidence interval: 0.899–0.955, p<0.0001). The PCT cut-off value of 0.37 ng/ml showed a negative predictive value of 99%, reducing the number of SF assays of 53.9%, still identifying the 96.4% of the pathogens.

Conclusion

PCT can be used in febrile patients with suspected sepsis to predict SF positive or negative results. A cut-off value of 0.37 ng/ml can be considered for optimal sensitivity, so that, in the routine laboratory activity, SF assay should not be used for diagnosis of sepsis in an unselected patient population with a PCT value <0.37 ng/ml.     

Selective action of human sera differing in fatty acids and cholesterol content on in vitro gene expression

Serum constituents might directly affect metabolic diseases pathogenesis and are commonly used as diagnostic tool. The aim of this study was to investigate the human serum effect on in vitro gene expression, related to nutrients action and involved in lipid metabolism. In detail, 40 human sera were firstly analyzed in fatty acids profile by gas-chromatography. Then samples were tested through direct addition within culture medium on Hep G2 human hepatoma cells, comparing samples from hypercholesterolemic (average 273 mg/dl) versus normocholesterolemic male subjects (average 155 mg/dl), since this condition is a relevant disease risk factor and is typically consequent to nutritional style. Hypercholesterolemic sera produced a 0.4-fold reduction of sterol regulatory element binding protein 1c (SREBP-1c) mRNA (P < 0.05) and a 1.5-fold increase of UDP-glucuronosyltransferase 1A1 (UGT1A1) mRNA (P < 0.01). Samples with higher concentrations of n-6 fatty acids produced a higher expression of UGT1A1 mRNA. Total fatty acids [docosahexaenoic, eicosopentanoic, arachidonic, linolenic, and linoleic acid (DHA, EPA, AA, LNA, and LA, respectively)] in each serum resulted roughly inverse with trend of SREBP-1c mRNA expression. Serum AA, LA, and trans fatty acids were more abundant in hypercholesterolemic subjects (P < 0.01) while DHA as quota of detected fatty acids was significantly higher in normocholesterolemic subjects (P < 0.05). While it is not possible to indicate which component was responsible for the observed gene modulations, our data indicate that sera differing in lipid profiles, mainly associated with dietary behavior, differentially affect gene expression known to be involved in metabolic and nutritional related conditions.     

Defective autoimmune regulator-dependent central tolerance to myelin protein zero is linked to autoimmune peripheral neuropathy

Chronic inflammatory demyelinating polyneuropathy is a debilitating autoimmune disease characterized by peripheral nerve demyelination and dysfunction. How the autoimmune response is initiated, identity of provoking Ags, and pathogenic effector mechanisms are not well defined. The autoimmune regulator (Aire) plays a critical role in central tolerance by promoting thymic expression of self-Ags and deletion of self-reactive T cells. In this study, we used mice with hypomorphic Aire function and two patients with Aire mutations to define how Aire deficiency results in spontaneous autoimmune peripheral neuropathy. Autoimmunity against peripheral nerves in both mice and humans targets myelin protein zero, an Ag for which expression is Aire-regulated in the thymus. Consistent with a defect in thymic tolerance, CD4(+) T cells are sufficient to transfer disease in mice and produce IFN-γ in infiltrated peripheral nerves. Our findings suggest that defective Aire-mediated central tolerance to myelin protein zero initiates an autoimmune Th1 effector response toward peripheral nerves.