A novel human glycosyltransferase: primary structure and characterization of the gene and transcripts

We report the identification and primary structure of a novel human glycosyltransferase, B3GTL (beta3-glycosyltransferase-like). The 498 residue protein consists of a short cytoplasmic N-terminal "tail" (residues 1-4), a single transmembrane domain with type II topology (residues 5-28), a "stem" region (residues 29-260), and a catalytic domain (residues 261-498). The genomes of Anopheles gambiae, Drosophila melanogaster, and Caenorhabditis elegans encode potential orthologs which share 31-39% sequence identity with B3GTL, as well as the following features: a conserved catalytic domain containing a triple aspartate motif (DDD) at its core, a conserved pattern of cysteine residues, a C-terminal KDEL-like motif, and conserved residues and motifs that affiliate this novel group with a family of beta3-glycosyltransferases (GT31 in the CAZY classification). The B3GTL gene lacks canonical TATA and CAAT boxes and contains three functional polyadenylation sites. It is transcribed in a wide range of tissues and in TGF-beta-treated T84 epithelial cells.     

Differences in glycosylation and sperm-egg binding inhibition of pregnancy-related glycodelin

Glycodelin is a glycoprotein produced in many glands, particularly those of reproductive tissues. It appears as different glycoforms in amniotic fluid (glycodelin-A) and seminal plasma (glycodelin-S), but only glycodelin-A inhibits gamete adhesion. In the present study, glycodelin from secretory-phase endometrium, first-trimester pregnancy decidua, and midtrimester amniotic fluid was studied with respect to physicochemical properties, including glycosylation patterns and inhibitory activity of sperm-egg binding. Purified glycodelins from all these sources were similar in isoelectric focusing and in lectin immunoassays using lectins from Wisteria floribunda and Sambucus nigra. Likewise, the glycodelins inhibited sperm-egg binding in a dose-dependent manner, as measured by hemizona-binding assay. However, subtle quantitative physicochemical and biological differences were found between glycodelins from different sources as well as within the same tissue/fluid between different individuals. Differences were most pronounced between endometrial glycodelins from nonpregnancy and first-trimester pregnancy. The glycan structures studied by fast-atom bombardment mass spectrometry of individual amniotic fluid glycodelin-A samples also showed interindividual quantitative differences. In conclusion, glycodelins from different female reproductive tract tissues and amniotic fluid share substantial similarity, allowing all of them to be called glycodelin-A. However, these glycodelins exhibit quantitative physicochemical and functional differences between different sources and individuals.     

Enhancing the thermal stability of avidin. Introduction of disulfide bridges between subunit interfaces

In this study we showed that tetrameric chicken avidin can be stabilized by introducing intermonomeric disulfide bridges between its subunits. These covalent bonds had no major effects on the biotin binding properties of the respective mutants. Moreover, one of the mutants (Avd-ccci) maintained its tetrameric integrity even in denaturing conditions. The new avidin forms Avd-ci and Avd-ccci, which have native --> denatured transition midpoints (T(m)) of 98.6 and 94.7 degrees C, respectively, in the absence of biotin, will find use in applications where extreme stability or minimal leakage of subunits is required. Furthermore, we showed that the intramonomeric disulfide bridges found in the wild-type avidin affect its stability. The mutant Avd-nc, in which this bridge was removed, had a lower T(m) in the absence of biotin than the wild-type avidin but showed comparable stability in the presence of biotin.     

Parathyroid hormone rapidly stimulates hyaluronan synthesis by periosteal osteoblasts in the tibial diaphysis of the growing rat

Short term treatment (3-24 h) with parathyroid hormone (PTH) stimulated the synthesis and accumulation of hyaluronan (HyA) in explant cultures of tibial diaphyses from young rats. PTH increased the overall HyA content of periosteum 5-fold, with the basal cambium layer exhibiting the greatest enhancement ( approximately 8-fold). PTH increased the HyA content of cortical bone by 2-fold while not affecting the HyA content of bone marrow. PTH treatment greatly enhanced HyA staining throughout all layers of the periosteum, although its most dramatic effect occurred in the basal cambium layer. Here, unlike in the control tissue sections, nearly all cambium-lining osteoblasts stained intensely positive for HyA. PTH treatment enhanced the HyA staining of osteocytes in cortical bone tissue sections to the extent that the lacunocanalicular system became visualized. Three significant findings were revealed in this study. First, mature periosteal osteoblasts, under natural conditions, do not contain much HyA in their surrounding extracellular matrix but dramatically enhance their matrix HyA content when treated with PTH. Second, pre-osteocytes and osteocytes contain more HyA in their natural matrix than mature lining osteoblasts, and they appear to have functional PTH receptors because they responded to PTH treatment with an enhancement of HyA content. Finally, it was observed that the lining cells along the endosteal surface of the diaphysis did not stain strongly positive for HyA either naturally or when exposed to PTH treatment. This indicates that periosteal and endosteal osteoblastic cell populations exhibit metabolic differences in their extracellular matrix responses to PTH.     

QSAR studies applied to the prediction of antigen-antibody interaction kinetics as measured by BIACORE

The objective of this work was to investigate the potential of the quantitative structure-activity relationships (QSAR) approach for predictive modulation of molecular interaction kinetics. A multivariate QSAR approach involving modifications in peptide sequence and buffer composition was recently used in an attempt to predict the kinetics of peptide-antibody interactions as measured by BIACORE. Quantitative buffer-kinetics relationships (QBKR) and quantitative sequence-kinetics relationships (QSKR) models were developed. Their predictive capacity was investigated in this study by comparing predicted and observed kinetic dissociation parameters (k(d)) for new antigenic peptides, or in new buffers. The range of experimentally measured k(d) variations was small (300-fold), limiting the practical value of the approach for this particular interaction. However, the models were validated from a statistical point of view. In QSKR, the leave-one-out cross validation gave Q(2) = 0.71 for 24 peptides (all but one outlier), compared to 0.81 for 17 training peptides. A more precise model (Q(2) = 0.92) could be developed when removing sets of peptides sharing distinctive structural features, suggesting that different peptides use slightly different binding modes. All models share the most important factor and are informative for structure-kinetics relationships. In QBKR, the measured effect on k(d) of individual additives in the buffers was consistent with the effect predicted from multivariate buffers. Our results open new perspectives for the predictive optimization of interaction kinetics, with important implications in pharmacology and biotechnology.     

Cellular distribution and contribution of cyclooxygenase COX-2 to diabetogenesis in NOD mouse

Unlike most other mammalian cells, beta-cells of Langerhans constitutively express cyclooxygenase (COX)-2 rather than COX-1. COX-2 is also constitutively expressed in type 1 diabetes (T1D) patients' periphery blood monocytes and macrophage. To understand the role of COX-2 in the beta-cell, we investigated COX-2 expression in beta-cells and islet infiltrates of NOD and BALB/c mice using fluorescence immunohistochemistry and cytochemical confocal microscopy and Western blotting. Immunostaining showed that COX-2 is expressed in islet-infiltrating macrophages, and that the expression of insulin and COX-2 disappeared concomitantly from the beta-cells when NOD mice progressed toward overt diabetes. Also cultured INS-1E cells coexpressed insulin and COX-2 but clearly in different subcellular compartments. Treatment with celecoxib increased insulin release from these cells in a dose-dependent manner in glucose concentrations ranging from 5 to 17 mM. Excessive COX-2 expression by the islet-infiltrating macrophages may contribute to the beta-cell death during insulitis. The effects of celecoxib on INS-1E cells suggest that PGE(2) and other downstream products of COX-2 may contribute to the regulation of insulin release from the beta-cells.     

A new locus for coeliac disease mapped to chromosome 15 in a population isolate

Coeliac disease is a common multifactorial disease with a strong genetic component, which is not entirely explained by the HLA association. Four previous whole-genome screens have produced somewhat inconsistent results suggesting genetic heterogeneity. We attempted to overcome this problem by performing a genome-wide scan in a Finnish sub-population, expected to be more homogeneous than the general population of Finland. The families in our study originate from the northeastern part of Finland, the Koilliskaira region, which has been relatively isolated since its founding in the 16th century. Genealogical studies have confirmed that the families share a common ancestor in the 16th century. Nine families with altogether 23 patients were genotyped for 399 microsatellite markers and the data were analysed with parametric linkage analysis using two dominant and one recessive model. A region on chromosome 15q11-q13 was implicated with a LOD score of 3.14 using a highly penetrant dominant model. Addition of more markers and one more sib-pair increased the LOD score to 3.74. This result gives preliminary evidence for existence of a susceptibility factor in this chromosomal region.     

Phylogenetic analysis of intestinal microflora indicates a novel Mycoplasma phylotype in farmed and wild salmon

All studies of the microbial community of the gastrointestinal tract of salmon to date have employed culture-based approaches, typically on pond- or tank-raised, freshwater animals. We present a phylogenetic survey of the bacterial populations present in the distal intestine of salmon from three different marine locations in Europe. This was accomplished through PCR amplification, cloning, and sequencing of partial 16S rDNA genes from microbial community DNA isolated from the contents of the GI tract distal to the pyloric ceca. Using this approach, the intestinal microbial communities of wild salmon from Scotland and pen-raised salmon from Scotland and Norway were compared. The predominating bacterial populations detected were Acinetobacter junii and a novel Mycoplasma phylotype. This Mycoplasma phylotype apparently comprised ~96% of the total microbes in the distal intestine of wild salmon. Substantial differences in intestinal microbial community composition and diversity were observed between the two groups of pen-raised salmon, which, in addition to geographical separation, were raised on different feeds. The microbial profiles found in this study were substantially different from those indicated in earlier culture-based studies for several species of fish, presumably because of the culture-independent techniques employed. Further, analysis of short-chain fatty acids in the digestive tract indicated that the decreasing redox gradient from proximal to distal reaches common to homeothermic animals was absent in salmon, and that the bacterial fermentation levels were much lower than are reported in homeothermic animals.     

Costs of herbivore resistance in clonal saplings of <Emphasis Type="Italic">Betula pendula</Emphasis>

Several studies have found genetic variation in plant resistance to herbivory. One of the explanations suggested for the observed intermediate levels of resistance are the costs of resistance, i.e., negative genetic correlations between resistance and other fitness components that may constrain the evolution of resistance. We studied the cost of herbivore resistance by investigating the genetic correlations between resistance traits and plant growth traits, and between resistance to insect and mammalian herbivores in cloned saplings of silver birch, Betula pendula. We used the performance of a geometrid moth, Epirrita autumnata, as an indicator of insect resistance. The numbers of resin droplets at the base and at the tip of the saplings correlate with mammalian resistance, and were thus used here as indicators of vole and hare resistance, respectively. We have previously observed genetic variation in these resistance traits. Further, we examined the correlations between several groups of secondary chemicals and plant growth traits. Finally, to reveal the effect of environmental factors on the trade-offs mentioned above, we investigated the correlations in saplings that were grown at two nutrient levels. We found significant negative correlations between indices of constitutive insect resistance and relative height growth in non-fertilized saplings, indicating cost of constitutive insect resistance. The two groups of secondary chemicals that have been shown to correlate strongly with constitutive insect resistance, i.e., condensed tannins and flavonol glycosides (especially myricetin glycosides), had different genetic correlations with plant traits; the concentration of condensed tannins did not correlate negatively with any of the plant traits, whereas the concentration of flavonol glycosides correlated negatively with plant height. Insect and mammalian resistance did not correlate negatively, indicating no ecological trade-offs.