Interferon-gamma augments hydrolysis of LTA4 to LTB4 by endothelial cells

LTB4 is a potent mediator of inflammation acting at local sites inflammation. LTB4 increases the lymphocyte binding to and penetration through the endothelium. In this paper we demonstrate that while endothelial cells were unable to metabolize LTB4 from arachidonic acid they were able to hydrolyse LTA4 into LTB4 in a granulocyte-endothelial co-culture assay. This hydrolysis is markedly increased if endothelial cells were pretreated with IFN-γ prior to the assay. The IFN-γ induced effect was shown to be time- and dose-dependent. The ability of endothelial cells to hydrolyse LTA4 to LTB4 may provide an answer how LTB4 can be produced in large quantities by non-heamatopoetic cells (i.e by endothelial cells) at sites of acute inflammation.     

Reagents For The Selective Immobilization Of Oligonucleotides On Solid Supports

New reagents (CPGs and phosphoramidites) for automatic solid phase synthesis of modified oligonucleotides were designed. Three oligonucleotides carrying fluorescent label at the 5′-terminus and an anchor group at the 3′-terminus were prepared and their immobilization in orthogonal conditions on solid supports was studied.     

Variations in enzyme activity in stomach and pancreatic tissue and digesta in piglets around weaning

A study was performed to investigate the effect of weaning at 4 weeks of age on the activity of digestive enzymes in the stomach and pancreatic tissue and in digesta from 3 days prior to weaning to 9 days postweaning in 64 piglets. In stomach tissue the activity of pepsin and gastric lipase was determined. Pepsin activity declined abruptly after weaning but 5 days postweaning the weaning level was regained and in the gastric contents no change in pepsin activity was observed. Weaning did not influence the activity of gastric lipase. The activity of eight enzymes and a cofactor was measured in pancreatic tissue. The effect of weaning on the enzyme activity was highly significant for all enzymes except elastase. The activity of all enzymes remained at the weaning level during day 1–2 postweaning followed by a reduction of the activity. The activity of trypsin, carboxypeptidase A, amylase and lipase exhibited minimum activity 5 days postweaning. Trypsin activity increased to the preweaning level on day 7–9 whereas the activity of the others increased but did not reach the preweaning level. The activity of chymotrypsin, carboxypeptidase B and carboxyl ester hydrolase decreased during the entire experimental period. In digesta no effect of weaning was observed on the activity of amylase and trypsin. The activity of chymotrypsin was reduced after weaning in the proximal third of the small intestine and lipase and carboxyl ester hydrolase activity was reduced in the middle and distal parts of the small intestine after weaning. The present study shows that the activities of the digestive enzymes in the pancreatic tissue are affected by weaning. Even though the pancreatic secretion cannot be judged from these results they show that the enzymes respond differently to weaning. In general the activity of the digestive enzymes in pancreatic tissue is low on day 5 postweaning which in interaction with other factors may increase the risk of developing postweaning diarrhoea.     

Delineation of the key amino acids involved in neutrophil inhibitory factor binding to the I-domain supports a mosaic model for the capacity of integrin alphaMbeta 2 to recognize multiple ligands

To gain insight into the mechanism by which the alpha(M)I-domain of integrin alpha(M)beta(2) interacts with multiple and unrelated ligands, the identity of the neutrophil inhibitory factor (NIF) recognition site was sought. A systematic strategy in which individual amino acid residues within three previously implicated segments were changed to those in the alpha(L)I-domain, which is structurally very similar but does not bind NIF, was implemented. The capacity of the resulting mutants, expressed as glutathione S-transferase fusion proteins, to recognize NIF was assessed. These analyses ultimately identified Asp(149), Arg(151), Gly(207), Tyr(252), and Glu(258) as critical for NIF binding. Cation binding, a function of the metal ion-dependent adhesion site (MIDAS) motif, was assessed by terbium luminescence to evaluate conformational perturbations induced by the mutations. All five mutants bound terbium with unaltered affinities. When the five residues were inserted into the alpha(L)I-domain, the chimera bound NIF with high affinity. Another ligand of alpha(M)beta(2), C3bi, which is known to use the same segments of the alpha(M)I-domain in engaging the receptor, failed to bind to the chimeric alpha(L)I-domain. Thus, the alpha(M)I-domain appears to present a mosaic of exposed amino acids within surface loops on its MIDAS face, and different ligands interact with different residues to attain high affinity binding.     

Perylene attached to DNA through stiff or flexible linker: duplex stability and FRET

Two fluorescent nucleosides, 5-(perylen-3-ylethynyl)-2'-deoxyuridine and 5-[(perylen-3-yl)methoxypropyn-1-yl]-2'-deoxyuridine, were incorporated into synthetic oligodeoxyribonucleotides and spectral properties of the conjugates and their duplexes were studied.     

EGFR Signaling Promotes β-Cell Proliferation and Survivin Expression during Pregnancy

Placental lactogen (PL) induced serotonergic signaling is essential for gestational β-cell mass expansion. We have previously shown that intact Epidermal growth factor –receptor (EGFR) function is a crucial component of this pathway. We now explored more specifically the link between EGFR and pregnancy-induced β-cell mass compensation. Islets were isolated from wild-type and β-cell-specific EGFR-dominant negative mice (E1-DN), stimulated with PL and analyzed for β-cell proliferation and expression of genes involved in gestational β-cell growth. β-cell mass dynamics were analyzed both with traditional morphometrical methods and three-dimensional optical projection tomography (OPT) of whole-mount insulin-stained pancreata. Insulin-positive volume analyzed with OPT increased 1.4-fold at gestational day 18.5 (GD18.5) when compared to non-pregnant mice. Number of islets peaked by GD13.5 (680 vs 1134 islets per pancreas, non-pregnant vs. GD13.5). PL stimulated beta cell proliferation in the wild-type islets, whereas the proliferative response was absent in the E1-DN mouse islets. Serotonin synthesizing enzymes were upregulated similarly in both the wild-type and E1-DN mice. However, while survivin (Birc5) mRNA was upregulated 5.5-fold during pregnancy in the wild-type islets, no change was seen in the E1-DN pregnant islets. PL induced survivin expression also in isolated islets and this was blocked by EGFR inhibitor gefitinib, mTOR inhibitor rapamycin and MEK inhibitor PD0325901. Our 3D-volumetric analysis of β-cell mass expansion during murine pregnancy revealed that islet number increases during pregnancy. In addition, our results suggest that EGFR signaling is required for lactogen-induced survivin expression via MAPK and mTOR pathways.     

The ITGAV rs3738919 variant and susceptibility to rheumatoid arthritis in four Caucasian sample sets

Introduction

Angiogenesis is an important process in the development of destructive synovial pannus in rheumatoid arthritis (RA). The ITGAV +gene encodes a cell cycle-associated antigen, integrin ανβ 3, which plays a role in RA angiogenesis. Previously, two independent studies identified an association between the major allele of the ITGAV single-nucleotide polymorphism (SNP) rs3738919 and RA. We therefore tested this association in an independent study using New Zealand (NZ) and Oxford (UK) RA case control samples.

Methods

We compared genotype frequencies in 740 NZ Caucasian RA patients and 553 controls genotyped for rs3738919, using a polymerase chain reaction-restriction fragment length polymorphism assay. A TaqMan genotyping SNP assay was used to type 713 Caucasian RA patients and 515 control samples from Oxford for the rs3738919 variant. Association of rs3738919 with RA was tested in these two sample sets using the chi-square goodness-of-fit test. The Mantel-Haenszel test was used to perform a meta-analysis, combining the genetic results from four independent Caucasian case control cohorts, consisting of 3,527 cases and 4,126 controls. Haplotype analysis was also performed using SNPs rs3911238, rs10174098 and rs3738919 in the Wellcome Trust Case Control Consortium, NZ and Oxford case control samples.

Results

We found no evidence for association between ITGAV and RA in either the NZ or Oxford sample set (odds ratio [OR] = 0.88, P_allelic = 0.11 and OR = 1.18, P_allelic = 0.07, respectively). Inclusion of these data in a meta-analysis (random effects) of four independent cohorts (3,527 cases and 4,126 controls) weakens support for the hypothesis that rs3738919 plays a role in the development of RA (OR_combined = 0.92, 95% confidence interval 0.80 to 1.07; P = 0.29). No consistent haplotype associations were evident.

Conclusions

Association of ITGAV SNP rs7378919 with RA was not replicated in NZ or Oxford case control sample sets. Meta-analysis of these and previously published data lends limited support for a role for the ITGAV in RA in Caucasians of European ancestry.     

Interferon-gamma augments hydrolysis of LTA4 to LTB4 by endothelial cells

LTB4 is a potent mediator of inflammation acting at local sites of inflammation. LTB4 increases the lymphocyte binding to and penetration through the endothelium. In this paper we demonstrate that while endothelial cells were unable to metabolize LTB4 from arachidonic acid they were able to hydrolyse LTA4 into LTB4 in a granulocyte-endothelial co-culture assay. This hydrolysis is markedly increased if endothelial cells were pretreated with IFN-gamma prior to the assay. The IFN-gamma induced effect was shown to be time- and dose-dependent. The ability of endothelial cells to hydrolyse LTA4 to LTB4 may provide an answer how LTB4 can be produced in large quantities by nonheamatopoetic cells (i.e. by endothelial cells) at sites of acute inflammation.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.