Structures of acidic O-linked polylactosaminoglycans on human skim milk mucins

O-Linked glycans were isolated from human skim milk mucins or mucin-derived high-molecular weight glycopeptides and fractionated by anion exchange chromatography into neutral and acidic alditols. Major oligosaccharides contained in the acidic fraction were purified by high performance liquid chromatography and structurally characterized by a combination of fast atom bombardment mass spectrometry, methylation analysis and 500 MHz 1H-nuclear magnetic resonance spectroscopy. The structural aspects exhibited by these major species in the acidic fraction resemble those established previously for the neutral oligosaccharides from human skim milk mucins: 1) the size of the alditols varies from tri- to decasaccharides, 2) the core structure is of the ubiquitous type 2, 3) the backbone sequences are of the poly-N-acetyllactosamine type with a particular preponderance of linearly extended GlcNAc beta(1-3)Gal (major) or GlcNAc beta(1-6)Gal units (minor).     

Karyotype analysis in Hyacinthella dalmatica (Hyacinthaceae) reveals vertebrate-type telomere repeats at the chromosome ends.

Chromosome analysis of three different populations of Hyacinthella dalmatica (Lallem.) Trinajsti?, an endemic species of the coastal region of southeastern Europe, showed a unique chromosome number, 2n = 2x = 20, and bimodal karyotype with one large and nine smaller pairs of chromosomes. Staining with fluorochromes CMA3 (chromomycin A3) and DAPI (4,6-diamidino-2-phenylindole) revealed heterochromatic regions associated with NORs, centromeres, and several interstitial heterochromatic bands on the longest chromosome pair. Double-target FISH with two ribosomal DNA probes revealed one locus of 5S rRNA genes in the pericentromeric region of chromosome pair 3 and one locus of 18S-5.8S-26S rRNA genes on the short arm of chromosome pair 4 in all plants and populations analyzed. Southern hybridization analysis and FISH experiments demonstrated that the distal ends of H. dalmatica chromosomes contain the vertebrate telomere (5'-TTAGGG-3') repeat type rather than the Arabidopsis (5'-TTTAGGG-3') heptamer, and so suggest that this Asparagales species along with Aloe and Othocallis contains the vertebrate-type telomere repeat.     

Hexokinase-coupled radioassays: Glyceraldehyde-3-phosphate dehydrogenase and enolase

A general method for the assay of enzymes which produce ATP, or are susceptible to be coupled to ATP-producing enzymes, is described. We have applied it to the assay of glyceraldehyde-3-phosphate dehydrogenase and enolase. For these enzymes, the product of the reaction, 1,3-bisphosphoglycerate or phosphoenolpyruvate, were coupled to ADP and either phosphoglycerate kinase or pyruvate kinase, respectively. The ATP formed in both cases is used by hexokinase plus labeled glucose to produce labeled glucose 6-phosphate which is quantitatively separated in small Dowex 1 columns and measured by liquid scintillation spectrometry. The conditions described permitted the detection of 0.1 mU of glyceraldehyde-3-phosphate dehydrogenase or enolase. As a further example of the sensitivity of the radioassay, effluents of a CM-cellulose column charged with an extract prepared from one single frog oocyte were analyzed and shown to contain a single enolase and two glyceraldehyde-3-phosphate dehydrogenase fractions.     

Genetic variability in sodium-glucose cotransporter 2 influences glycemic control and risk for diabetic retinopathy in type 2 diabetes patients

BackgroundGluconeogenesis and renal glucose excretion in kidneys both play an important role in glucose homeostasis. Sodium-glucose cotransporter (SGLT2), coded by the SLC5A2 gene is responsible for reabsorption up to 99% of the filtered glucose in proximal tubules. SLC5A2 genetic polymorphisms were suggested to influence glucose homeostasis. We investigated if common SLC5A2 rs9934336 polymorphism influences glycemic control and risk for macro or microvascular complications in Slovenian type 2 diabetes (T2D) patients.MethodsAll 181 clinically well characterized T2D patients were genotyped for SLC5A2 rs9934336 G>A polymorphism. Associations with glycemic control and T2D complications were assessed with nonparametric tests and logistic regression.ResultsSLC5A2 rs9934336 was significantly associated with increased fasting blood glucose levels (P<0.001) and HbA1c levels under the dominant genetic model (P=0.030). After adjustment for T2D duration, significantly higher risk for diabetic retinopathy was present in carriers of at least one polymorphic SLC5A2 rs9934336 A allele compared to non-carriers (OR=7.62; 95%CI=1.65-35.28; P=0.009).ConclusionsOur pilot study suggests an important role of SLC5A2 polymorphisms in the physiologic process of glucose reabsorption in kidneys in T2D patients. This is also the first report on the association between SLC5A2 polymorphism and diabetic retinopathy.     

Identification of stool proteins in C57BL/6J mice by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry

Gastrointestinal disease is a major cause of mortality in humans and animals, and the detection of disease-associated protein in stool is an established diagnostic method in this context. Yet, no data currently exists about the protein composition of mammalian faeces. Using a newly developed two-dimensional (2D) gel method, 28 of the most abundant proteins in murine faeces were identified. Mammalian faeces contains protein from multiple species (from the individual, from gastrointestinal bacteria, from food, etc.). Yet, it was found that the majority of mouse stool proteins were of mouse origin, with a minority of proteins being derived from food (in particular soybean glycinin and conglycinin) and bacteria (flagellin). Most mouse proteins were proteases and saccharidases derived from the exocrine pancreas. In addition, two unexpected mouse proteins were identified: one was a newly described mucin-like protein from intestinal goblet cells (FcγBP); the other was the secreted form of carbonic anhydrase (type VI) from salivary gland. The data suggest that 2D analysis of faecal protein is likely to provide meaningful information about the physiological stage of the gastrointestinal tract. Compared with studies based on biopsies, faecal protein analysis may reduce the number of laboratory animals, and might also allow quicker bridging from animal studies to humans, where biopsy material is more difficult to obtain and is less relevant for general practice use.     

Establishment of the 1st European Tissue Bank and a Vision of the Future of Tissue Banking

The paper offers a short history of the oldest European tissue bank, in cooperation with important international organizations and eduction authorities. The final part details the organization of development in research and clinical use by the bank in the field of human embryonal cells.     

Mechanisms of hydrazine toxicity in rat liver investigated by proteomics and multivariate data analysis

A proteomics approach combined with multivariate data analysis was used to examine the hepatotoxic effect of hydrazine in 30 male Sprague Dawley rats, assigned to four treatment groups and two control groups. Liver samples from the individual animals were resolved by two-dimensional differential gel electrophoresis (2-D DIGE) and protein patterns from the 2-D gels were analyzed by principal component analysis (PCA) and partial least squares regression (PLSR). The PCA plot was able to describe the variation in the protein expression related to dose and time, by separation or clustering of different animal groups. PLSR followed by variable selection (Jack-knifing) was used to select proteins that varied significantly in relation to the dose related response of the hydrazine treatment. The 10 up-regulated and 10 down-regulated proteins with highest rank in the PLSR model were identified by mass spectrometry. Hydrazine treatment induced altered expression of proteins related to lipid metabolism, Ca(2+) homeostasis, thyroid hormone pathways and stress response. Several of the identified proteins have not previously been implicated in hydrazine toxicity and may thus be regarded as new potential biomarkers of induced liver toxicity.     

Mre11 deficiency in Arabidopsis is associated with chromosomal instability in somatic cells and Spo11-dependent genome fragmentation during meiosis

The Mre11/Rad50/Nbs1 complex is involved in many aspects of chromosome metabolism. Aberrant function of the complex is associated with defects in the DNA checkpoint, double-strand break repair, meiosis, and telomere maintenance. In this article, we report the consequences of Mre11 dysfunction for the stability of mitotic and meiotic chromosomes in Arabidopsis thaliana. Although plants homozygous for a T-DNA insertion in a conserved region of the MRE11 gene are viable, they exhibit growth defects and are infertile. Analysis of mitotic chromosomes prepared from the mutant plants revealed abundant dicentric chromosomes and chromosomal fragments. Fluorescence in situ hybridization showed that anaphase bridges are often formed by homologous chromosome arms. The frequency of chromosome fusions was not reduced in mre11 ku70 double mutants, suggesting that plants possess DNA end-joining activities independent of the Ku70/80 and Mre11 complexes. Cytogenetic examination of pollen mother cells revealed massive chromosome fragmentation and the absence of synapsis in the initial stages of meiosis. The fragmentation was substantially suppressed in mre11 spo11-1 double mutants, indicating that Mre11 is required for repair but not for the induction of Spo11-dependent meiotic DNA breaks in Arabidopsis.     

Toxic metals in freshwater fish from the Zagreb area as indicators of environmental pollution

The aim of this investigation was to determine the levels of heavy metals and metalloids in freshwater fish from the Zagreb area. A total of 216 freshwater fish samples from 5 sites were examined: Sava river upstream from Zagreb, Sava river at Zagreb, Sava river downstream from Zagreb, Jarun Lake, and 5 "ecologic" fishponds from the Zagreb surroundings. The metals lead, cadmium, mercury and the metalloid arsenic were determined by the method of atomic absorption spectrometry. The mean pooled levels of lead, cadmium and arsenic in all fish samples were 112.3 +/- 95 micrograms/kg, 8.5 +/- 11 micrograms/kg and 23.5 +/- 36 micrograms/kg, and did not exceed the allowed levels of 1,000 mg/kg, 100 micrograms/kg and 200 micrograms/kg, respectively. In 4 fish samples, the levels of mercury exceeded the allowed limit of 500 micrograms/kg (509, 596, 605 and 788 micrograms/kg), however, the pooled mean level of mercury was 127.8 +/- 90 micrograms/kg. There was no major difference in the levels of heavy metals between the two fish families observed, although the levels of lead, cadmium and mercury were higher in the family Ictaluridae (144 vs. 107 micrograms/kg, 10.4 vs. 8.2 micrograms/kg, and 153 vs. 124 micrograms/kg, respectively), whereas the level of arsenic was higher in the family Cyprinidae (23.8 vs. 21.8 micrograms/kg). Although the Sava river at Zagreb is the main recipient of sewage and wastewater in the Republic of Croatia, the levels of heavy metals were within the allowed limits in all groups of freshwater fish samples, with the exception of 4 samples that contained moderately elevated levels of mercury. Study results suggest that only mercury could be considered a good indicator of environmental pollution, because higher levels of mercury were measured in the fish from the Sava river than in the fish from the Jarun Lake and fishponds from the Zagreb surroundings, considering both pooleded and fish family specified data.     

Gender-specific growth patterns for stature,sitting height and limbs length in Croatian children and youth (3 to 18 years of age)

In a cross-sectional study of growth, 5,155 children (2,591 females, 2,564 males) from the town of Zagreb (Croatia) were measured. Four traits of linear dimensionality (stature, sitting height, arm and leg lengths) were studied in the age span of 3 to 18 years. A significant average annual increase of all four anthropometric parameters were observed up to 14 and 15 years of age in girls and 16 years of age in boys, showing that girls had a shorter growing period. In the prepubertal period until 9 years of age, gender differences were negligible. At the age of 10, boys were overgrown by girls in all parameters due to the earlier onset of puberty in girls. The growth gains for girls, when compared with those for boys, show a different pattern across variables. The female growth advantage remained in a two years period for the limbs length, but in a three year period for stature and the longest, for 4 years, for sitting height. The male predominance in size had an onset at the age of 13 for the limbs and in the age of 14 for stature and sitting height. The patterns of sexual dimorphism in stature and sitting height during growing years are similar to those observed in other populations of Europe. Growth of Croatian children and youth is very similar to that of the tallest European populations.