Pigment epithelium-derived factor in ulcerative colitis: possible relationship with disease activity

OBJECTIVES: Pigment epithelium-derived factor (PEDF) is an endogenous most potential angiogenic inhibitor and increased expression of PEDF in intestinal mucosa specimens was shown in the course of ulcerative colitis (UC). The aim of the present study was to evaluate serum concentration of pigment epithelium-derived growth factor, a potent anti-angiogenic factor and its possible association with vascular endothelial growth factor (VEGF) levels and disease activity. METHODS: Concentrations of PEDF and VEGF were measured in sera of 33 patients (13 females and 20 males) with active UC. RESULTS: There was significant increase of serum PEDF (32.3+/-2.9 vs. 20.6+/-4.7 ng/mL, P<0.05) as well as VEGF (326.4+/-58.1 vs. 110.9+/-15.7 pg/mL, P<0.05) in UC patients compared to healthy controls. Serum PEDF showed strong, positive correlation with endoscopic score (r=0.622, P<0.001), while such association was absent in respect to VEGF (r=0.05, P=0.77). In contrast serum VEGF decreased in severe UC comparing to patients with a mild course of disease, however the difference was not significant (274.9+/-64.9 vs. 360.4+/-103.4 pg/mL, P=0.53). CONCLUSIONS: Increase in serum PEDF during UC, especially in severe forms of disease suggests its involvement in UC pathogenesis.     

Increased level of fibrinogen chains in the proteome of blood platelets in secondary progressive multiple sclerosis patients

Epidemiological studies indicate a high risk of stroke, heart failure and myocardial infarction in patients with multiple sclerosis, especially in its secondary progressive (SPMS) phase. Some ischaemic events are directly associated with abnormal platelet functions and their prothrombotic activity. Recent reports, including this study, confirm the increased activation of circulating platelets in SPMS, and also show increased platelet reactivity, among other responses, as well as strong aggregation. In this current study, we conducted a comparative analysis of the platelet proteome in SPMS patients and in healthy controls, to demonstrate the quantitative and qualitative differences likely to affect functional changes observed in SPMS. During densitometry evaluation of 2‐D fluorescence difference gel electrophoresis, we observed differences between the electrophoretic patterns of SPMS platelets and the control samples. To determine a detailed characterisation of the proteome changes in the SPMS patients’ blood platelets, in the next stage, we performed mass spectrometry of selected spots and indicated the increased presence of four proteins (fibrinogen, α‐2 macroglobulin, septin‐14 and tubulin β‐1 chain). The most important of these is the increased amount of prothrombotic protein, fibrinogen, which seems to confirm the accuracy of the imaging and potentially explains the increased risk of platelet‐origin thrombotic events. This study provides new knowledge of the potential existence of the molecular mechanisms responsible for the acceleration of the platelet pro‐coagulant function in SPMS. This can help to identify new targets for therapy, which can then be used not only in the second stage of the disease.     

Molecular discrimination of type-I over type-II methionyl aminopeptidases

Two residues that are conserved in type-I methionyl aminopeptidases (MetAPs) but are absent in all type-II MetAPs are the cysteine residues (Escherichia coli MetAP-I: C59 and C70) that reside at the back of the substrate recognition pocket. These Cys residues are 4.4 A apart and do not form a disulfide bond. Since bacteria and fungi contain only type-I MetAPs while all human cells contain both type-I and type-II MetAPs, type-I MetAPs represent a novel antibiotic/antifungal target if type-I MetAPs can be specifically targeted over type-II. Based on reaction of the thiol-specific binding reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) with the type-I MetAP from E. coli and the type-II MetAP from Pyrococcus furiosus, the type-I MetAP can be selectively inhibited. Verification that DTNB covalently binds to C59 in EcMetAP-I was obtained by mass spectrometry (MS) from reaction of DTNB with the C59A and C70A mutant EcMetAP-I enzymes. In addition, two inhibitors of EcMetAP-I, 5-iodopentaphosphonic acid (1) and 6-phosphonohexanoic acid (2), were designed and synthesized. The first was designed as a selective-C59 binding reagent while the second was designed as a simple competitive inhibitor of EcMetAP. Indeed, inhibitor 1 forms a covalent interaction with C59 based on activity assays and MS measurements, while 2 does not. These data indicate that type-I MetAPs can be selectively targeted over type-II MetAPs, suggesting that type-I MetAPs represent a new enzymatic target for antibacterial or antifungal agents.     

Socio-economic position and type 2 diabetes risk factors: patterns in UK children of South Asian, black African-Caribbean and white European origin

Background

Socio-economic position (SEP) and ethnicity influence type 2 diabetes mellitus (T2DM) risk in adults. However, the influence of SEP on emerging T2DM risks in different ethnic groups and the contribution of SEP to ethnic differences in T2DM risk in young people have been little studied. We examined the relationships between SEP and T2DM risk factors in UK children of South Asian, black African-Caribbean and white European origin, using the official UK National Statistics Socio-economic Classification (NS-SEC) and assessed the extent to which NS-SEC explained ethnic differences in T2DM risk factors.

Methods and Findings

Cross-sectional school-based study of 4,804 UK children aged 9–10 years, including anthropometry and fasting blood analytes (response rates 70%, 68% and 58% for schools, individuals and blood measurements). Assessment of SEP was based on parental occupation defined using NS-SEC and ethnicity on parental self-report. Associations between NS-SEC and adiposity, insulin resistance (IR) and triglyceride differed between ethnic groups. In white Europeans, lower NS-SEC status was related to higher ponderal index (PI), fat mass index, IR and triglyceride (increases per NS-SEC decrement [95%CI] were 1.71% [0.75, 2.68], 4.32% [1.24, 7.48], 5.69% [2.01, 9.51] and 3.17% [0.96, 5.42], respectively). In black African-Caribbeans, lower NS-SEC was associated with lower PI (−1.12%; [−2.01, −0.21]), IR and triglyceride, while in South Asians there were no consistent associations between NS-SEC and T2DM risk factors. Adjustment for NS-SEC did not appear to explain ethnic differences in T2DM risk factors, which were particularly marked in high NS-SEC groups.

Conclusions

SEP is associated with T2DM risk factors in children but patterns of association differ by ethnic groups. Consequently, ethnic differences (which tend to be largest in affluent socio-economic groups) are not explained by NS-SEC. This suggests that strategies aimed at reducing social inequalities in T2DM risk are unlikely to reduce emerging ethnic differences in T2DM risk.     

Human cathepsin D

A literature survey was performed of human cathepsin D gene, cathepsin D biosynthesis, posttranslatory modifications, transport within the cell, substrate specificity and catalytic effect. Methods used to determine the activity and level of this proteinase as well as its role in the biochemistry and pathobiochemistry of cells, tissues and organs were considered.     

A Non-Synonymous HMGA2 Variant Decreases Height in Shetland Ponies and Other Small Horses

The identification of quantitative trait loci (QTL) such as height and their underlying causative variants is still challenging and often requires large sample sizes. In humans hundreds of loci with small effects control the heritable portion of height variability. In domestic animals, typically only a few loci with comparatively large effects explain a major fraction of the heritability. We investigated height at withers in Shetland ponies and mapped a QTL to ECA 6 by genome-wide association (GWAS) using a small cohort of only 48 animals and the Illumina equine SNP70 BeadChip. Fine-mapping revealed a shared haplotype block of 793 kb in small Shetland ponies. The HMGA2 gene, known to be associated with height in horses and many other species, was located in the associated haplotype. After closing a gap in the equine reference genome we identified a non-synonymous variant in the first exon of HMGA2 in small Shetland ponies. The variant was predicted to affect the functionally important first AT-hook DNA binding domain of the HMGA2 protein (c.83G>A; p.G28E). We assessed the functional impact and found impaired DNA binding of a peptide with the mutant sequence in an electrophoretic mobility shift assay. This suggests that the HMGA2 variant also affects DNA binding in vivo and thus leads to reduced growth and a smaller stature in Shetland ponies. The identified HMGA2 variant also segregates in several other pony breeds but was not found in regular-sized horse breeds. We therefore conclude that we identified a quantitative trait nucleotide for height in horses.     

Immunochemical and biological studies of antigens isolated from a strain of Bacteroides fragilis

Phenol/water-extracted lipopolysaccharide and a fraction HM, extracted with acetate buffer pH 2.0, from Bacteroides fragilis strain 62/73 are antigenically different as shown by immunodiffusion, passive haemagglutination, haemagglutination inhibition and preliminary chemical investigations. Biological activity, assessed with the local Shwartzmann reaction, was demonstrated for the lipopolysaccharide whereas antigen HM was almost inactive in this test. HM is immunogenic in rabbits. Antibodies against HM were detected in seven out of ten sera of healthy humans.     

Positively charged ceramide is a potent inducer of mitochondrial permeabilization

Ceramide-induced cell death is thought to be mediated by change in mitochondrial function, although the precise mechanism is unclear. Proposed models suggest that ceramide induces cell death through interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability, as an intermediate step in ceramide signal propagation. To investigate these models, we developed a new generation of positively charged ceramides that readily accumulate in isolated and in situ mitochondria. Accumulated, positively charged ceramides increased inner membrane permeability and triggered release of mitochondrial cytochrome c. Furthermore, the positively charged ceramide-induced permeability increase was suppressed by cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide (90%). These observations suggest that the inner membrane permeability increase is due to activation of specific ion transporters, not the generalized loss of lipid bilayer barrier functions. The difference in sensitivity of ceramide-induced ion fluxes to inhibitors of mitochondrial transporters suggests activation of at least two transport systems: the permeability transition pore and the electrogenic H(+) channel. Our results indicate the presence of specific ceramide targets in the mitochondrial matrix, the occupation of which triggers permeability alterations of the inner and outer mitochondrial membranes. These findings also suggest a novel therapeutic role for positively charged ceramides.     

The influence of intracellular idarubicin and daunorubicin levels on drug cytotoxicity in childhood acute leukemia

Uptake and efflux of two anthracyclines, idarubicin (IDA) and daunorubicin (DNR), was studied in childhood acute leukemia samples. A comparison of IDA and DNR transport phenomena in relation to drug cytotoxicity and expression of P-glycoprotein (PGP) was made. Intracellular content of IDA/DNR was determined by flow cytometry using the fluorescent properties of the drugs. In vitro drug cytotoxicity was measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. PGP expression was analysed by flow cytometry. The uptake and efflux rates were non-significantly higher for IDA than DNR. There were no differences between three types of leukemia with respect to drug content during accumulation and retention. After correction for the cell volume, intracellular concentration of both drugs in each moment of uptake and efflux was significantly lower in relapsed ALL and AML samples in comparison with initial ALL cells. Efflux, but not uptake, of both drugs was inversely correlated with PGP expression and IDA, but not DNR, cytotoxicity. The cytotoxicity was correlated with drug accumulation for both drugs and with drug retention for IDA. In conclusion, it seems that (1) intracellular content was related to the lipophilic properties of the drugs rather than to the type of leukemia, (2) decreased intracellular concentration of both drugs might have an impact on compromised therapy results in AML and relapsed ALL children, (3) IDA presents higher cytotoxicity, which possibly might be decreased by the presence of PGP. These results might have a practical impact on the rational design of new chemotherapy protocols.