The complexity of mitochondrial outer membrane permeability and VDAC regulation by associated proteins

Previous studies have shown that class II β-tubulin plays a key role in the regulation of oxidative phosphorylation (OXPHOS) in some highly differentiated cells, but its role in malignant cells has remained unclear. To clarify these aspects, we compared the bioenergetic properties of HL-1 murine sarcoma cells, murine neuroblastoma cells (uN2a) and retinoic acid - differentiated N2a cells (dN2a). We examined the expression and possible co-localization of mitochondrial voltage dependent anion channel (VDAC) with hexokinase-2 (HK-2) and βII-tubulin, the role of depolymerized βII-tubuline and the effect of both proteins in the regulation of mitochondrial outer membrane (MOM) permeability. Our data demonstrate that neuroblastoma and sarcoma cells are prone to aerobic glycolysis, which is partially mediated by the presence of VDAC bound HK-2. Microtubule destabilizing (colchicine) and stabilizing (taxol) agents do not affect the MOM permeability for ADP in N2a and HL-1 cells. The obtained results show that βII-tubulin does not regulate the MOM permeability for adenine nucleotides in these cells. HL-1 and NB cells display comparable rates of ADP-activated respiration. It was also found that differentiation enhances the involvement of OXPHOS in N2a cells due to the rise in their mitochondrial reserve capacity. Our data support the view that the alteration of mitochondrial affinity for ADNs is one of the characteristic features of cancer cells. It can be concluded that the binding sites for tubulin and hexokinase within the large intermembrane protein supercomplex Mitochondrial Interactosome, could be different between muscle and cancer cells.     

Stereochemical aspects of cholinesterase catalysis

Proceeding from the sterochemical regularities of the nucleophilic substitution reaction at the carbonyl group and the assumption that the spatial structure of the active center of cholinesterases is complementary to the molecule of the ester substrates for these enzymes, some general features of the stereoselectivity phenomena in the reactions of acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) with organophosphorus inhibitors are discussed. For these enzymes the models of the active center are proposed in terms of different binding sites and the catalytic center. On the basis of this model, the stereochemical pecularities and the physicochemical background of the stereoselectivity effects on enzyme inhibition, reactivation, and “aging” reactions can be understood. Knowledge of the absolute configuration of several chiral organophosphorus inhibitors also makes it possible to determine the absolute spatial arrangement of the hydrophobic binding sites on the active surface of cholinesterases.     

Evidence for Genetic Heterogeneity in the Carbohydrate-Deficient Glycoprotein Syndrome Type I (CDG1)

We have analyzed a series of polymorphic markers on chromosome 16p13 in 17 families with carbohydrate-deficient glycoprotein syndrome type I (CDG1). First, linkage to the region between D16S406 and D16S500 is confirmed. The telomeric border of the candidate region is now definitively placed proximal to D16S406 by crossovers observed in 2 families. Second, in 1 family with 2 affected siblings, the disease is not linked to chromosome 16p. Genetic heterogeneity has not been previously reported for CDG1, and this observation has implications for prenatal diagnosis. Third, allelic associations suggest that the disease locus is localized close to D16S414/D16S497. This places the region of interest centromeric of its published localization.     

Hypoglycosylation of a brain glycoprotein ({beta}-trace protein) in CDG syndromes due to phosphomannomutase deficiency and N-acetylglucosaminyl-transferase II deficiency

Human β-trace protein is a major intrathecally synthesizedpolypeptide constituent of human cerebrospinal fluid. We havepreviously shown that this protein is almost quantitativelymodified with biantennary complex-type N-linked oli-gosaccharideswhich show "brain-type" glycosylation characteristics (Hoffmann,A.et al, J. Neurochenu, 63, pp. 2185-2191,1994). In the presentstudy human β-trace protein from the cerebrospinal fluid(CSF) of patients with carbohydrate-deficient glycoprotein syndrome(CDGS) due to phospho-mannomutase (PMM) deficiency and N-acetyl-glucosami-nyltransferaseII (GlcNAc-T II) deficiency as well as from control individualswas studied by Western blot analysis. The protein from pooledCSFs was purified by immunoaffinity chromatography. The proteinfrom the five patients with CDGS PMM deficiency showed threeprotein bands upon SDS-PAGE analysis corresponding to the di-,mono-, and unglycosylated polypeptide forms. Carbohydrate structuralanalysis of the enzymatically liberated N-glycans was performedapplying mapping by HPAEC-PAD, methylation analysis as wellas MALD/TOF-MS. Essentially identical oli-gosaccharide structureswere detected in β-TP from type I patients and controladult pooled CSF. The β-trace protein from two patientswith GlcNAc-T II deficiency showed a single di-N-glycosylatedprotein band with a significantly lower molecular weight thanthe di-glycosylated polypeptide from control patients and theβ-trace protein from pooled adult CSF. β-TP from GlcNAc-TII deficiency patients shared only three oligosaccharides outof the 13 observed in β-TP from controls or patients withPMM deficiency. The major oligosaccharide structures of theglycoprotein from patients with GlcNAc-T n deficiency were foundto be monoanten-nary asialo- or monosialylated lactosamine-typechains with proximal fucose and bisecting GlcNAc. "brain-type" N-glycosylation carbohydrate deficiency glycoprotein syndrome human β-trace protein phosphomannomutase deficiency GlcNAc-transferase II deficiency human cerebrospinal fluid     

Diversity and Stability of Lactic Acid Bacteria in Rye Sourdoughs of Four Bakeries with Different Propagation Parameters

We identified the lactic acid bacteria within rye sourdoughs and starters from four bakeries with different propagation parameters and tracked their dynamics for between 5–28 months after renewal. Evaluation of bacterial communities was performed using plating, denaturing gradient gel electrophoresis, and pyrosequencing of 16S rRNA gene amplicons. Lactobacillus amylovorus and Lactobacillus frumenti or Lactobacillus helveticus, Lactobacillus pontis and Lactobacillus panis prevailed in sourdoughs propagated at higher temperature, while ambient temperature combined with a short fermentation cycle selected for Lactobacillus sanfranciscensis, Lactobacillus pontis, and Lactobacillus zymae or Lactobacillus helveticus, Lactobacillus pontis and Lactobacillus zymae. The ratio of species in bakeries employing room-temperature propagation displayed a seasonal dependence. Introduction of different and controlled propagation parameters at one bakery (higher fermentation temperature, reduced inoculum size, and extended fermentation time) resulted in stabilization of the microbial community with an increased proportion of L. helveticus and L. pontis. Despite these new propagation parameters no new species were detected.     

The influence of anthropometric characteristics to the handgrip and pinch strength in 6-10-year old children

The aim of the study was to investigate the relationships between handgrip and pinch strength values with basic body (body height, body mass, BMI) and with specific hand anthropometric parameters (fingers spans, lengths and perimeters) in prepubertal children. Body height, body mass and five fingers spans, lengths and perimeters were measured in 461 6-10 year old Estonian children according to Visnapuu & Jürim?e (2007). BMI was calculated (kg/m2). The maximal handgrip strength of the right and left hand was measured with hand dynamometer. The right and left key and tip pinch were measured with a pinch gauge. Body height, as a rule, in combination with BMI, was the strongest predictor of handgrip strength, especially in older children (about 40-60 % of the total variance, R2 x 100). From the hand anthropometry, the most important span parameter was FS2 (see explanations in the methods), which explained about 10-50% (R2 x 100) of the total variance. In older groups, the FS1 and FS3 were added to the models. From the length parameters, the most important was IFL, which in younger groups together with MFL explained 10-30% of the total variance, and in older groups the addition of RFL increased the influence to 45% (R2 x 100). From the perimeters, the most important one was P2 which explained the variability of the handgrip strength in younger groups by 15-30% (R2 x 100) and in older groups together with P3 and P4 even 30-40%. In children, the basic anthropometric parameters (body height and BMI) contribute more to the prediction of handgrip strength than the specific anthropometric parameters. With increasing age the contribution of basic and specific hand anthropometry increases and the relation between anthropometry and handgrip strength is stronger in boys compared with girls. The relation of basic and hand anthropometry to the tip and key pinch strength is relatively low.     

Multiplexed glycoproteomic analysis of glycosylation disorders by sequential yolk immunoglobulins immunoseparation and MALDI-TOF MS

This study applied yolk immunoglobulins immunoaffinity separation and MALDI-TOF MS for clinical proteomics of congenital disorders of glycosylation (CDG) and secondary glycosylation disorders [galactosemia and hereditary fructose intolerance (HFI)]. Serum transferrin (Tf) and alpha1-antitrypsin (AAT) that are markers for CDG, were purified sequentially to obtain high-quality MALDI mass spectra to differentiate single glycoforms of the native intact glycoproteins. The procedure was found feasible for the investigation of protein macroheterogeneity due to glycosylation site underoccupancy then ensuing the characterization of patients with CDG group I (N-glycan assembly disorders). Following PNGase F digestion of the purified glycoprotein, the characterization of protein microheterogeneity by N-glycan MS analysis was performed in a patient with CDG group II (processing disorders). CDG-Ia patients showed a typical profile of underglycosylation where the fully glycosylated glycoforms are always the most abundant present in plasma with lesser amounts of partially and unglycosylated glycoforms in this order. Galactosemia and HFI are potentially fatal diseases, which benefit from early diagnosis and prompt therapeutic intervention. In symptomatic patients with galactosemia and in those with HFI, MALDI MS of Tf and AAT depicts a hypoglycosylation profile with a significant increase of underglycosylated glycoforms that reverses by dietary treatment, representing a clue for diagnosis and treatment monitoring.     

High-Throughput Sequencing Approach Uncovers the miRNome of Peritoneal Endometriotic Lesions and Adjacent Healthy Tissues

Accumulating data have shown the involvement of microRNAs (miRNAs) in endometriosis pathogenesis. In this study, we used a novel approach to determine the endometriotic lesion-specific miRNAs by high-throughput small RNA sequencing of paired samples of peritoneal endometriotic lesions and matched healthy surrounding tissues together with eutopic endometria of the same patients. We found five miRNAs specific to epithelial cells – miR-34c, miR-449a, miR-200a, miR-200b and miR-141 showing significantly higher expression in peritoneal endometriotic lesions compared to healthy peritoneal tissues. We also determined the expression levels of miR-200 family target genes E-cadherin, ZEB1 and ZEB2 and found that the expression level of E-cadherin was significantly higher in endometriotic lesions compared to healthy tissues. Further evaluation verified that studied miRNAs could be used as diagnostic markers for confirming the presence of endometrial cells in endometriotic lesion biopsy samples. Furthermore, we demonstrated that the miRNA profile of peritoneal endometriotic lesion biopsies is largely masked by the surrounding peritoneal tissue, challenging the discovery of an accurate lesion-specific miRNA profile. Taken together, our findings indicate that only particular miRNAs with a significantly higher expression in endometriotic cells can be detected from lesion biopsies, and can serve as diagnostic markers for endometriosis.