Brain alpha-dystroglycan displays unique glycoepitopes and preferential binding to laminin-10/11

alpha-Dystroglycan was quantitatively enriched from mammalian brain based on its uniform reactivity with Vicia villosa agglutinin and resolved into sub-populations possessing or lacking the sulfated glucuronic acid epitope recognized by monoclonal antibody HNK-1. We generated a new monoclonal antibody specific for a glycoepitope on brain alpha-dystroglycan but absent from alpha-dystroglycan expressed in all other tissues examined. Finally, we found that laminin-10/11 preferentially bound to brain alpha-dystroglycan compared to skeletal muscle alpha-dystroglycan. Our results suggest that tissue-specific glycosylation modifies the laminin binding specificity of alpha-dystroglycan.     

Bcr/Abl interferes with the Fanconi anemia/BRCA pathway: implications in the chromosomal instability of chronic myeloid leukemia cells

Chronic myeloid leukemia (CML) is a malignant clonal disorder of the hematopoietic system caused by the expression of the BCR/ABL fusion oncogene. Although it is well known that CML cells are genetically unstable, the mechanisms accounting for this genomic instability are still poorly understood. Because the Fanconi anemia (FA) pathway is believed to control several mechanisms of DNA repair, we investigated whether this pathway was disrupted in CML cells. Our data show that CML cells have a defective capacity to generate FANCD2 nuclear foci, either in dividing cells or after DNA damage. Similarly, human cord blood CD34(+) cells transduced with BCR/ABL retroviral vectors showed impaired FANCD2 foci formation, whereas FANCD2 monoubiquitination in these cells was unaffected. Soon after the transduction of CD34(+) cells with BCR/ABL retroviral vectors a high proportion of cells with supernumerary centrosomes was observed. Similarly, BCR/ABL induced a high proportion of chromosomal abnormalities, while mediated a cell survival advantage after exposure to DNA cross-linking agents. Significantly, both the impaired formation of FANCD2 nuclear foci, and also the predisposition of BCR/ABL cells to develop centrosomal and chromosomal aberrations were reverted by the ectopic expression of BRCA1. Taken together, our data show for the first time a disruption of the FA/BRCA pathway in BCR/ABL cells, suggesting that this defective pathway should play an important role in the genomic instability of CML by the co-occurrence of centrosomal amplification and DNA repair deficiencies.     

Detailed lipid analysis of yolk platelets of amphibian (Bufo arenarum) oocytes

Yolk platelets, the principal components of amphibian oocytes, have been generally considered as material reservoirs. Their biochemical composition and function during oogenesis and early development have not been fully elucidated. The aim of this study was to carry out a lipidic characterization of yolk platelets from full-grown Bufo arenarum oocytes. Ovarian oocytes were manually obtained and the subcellular fraction was isolated by centrifugation at low velocity. Lipids were separated by thin-layer chromatography. For compositional analysis, they were derived by methanolysis, being identified and quantified in a gas-liquid chromatograph. Phospholipid content indicates that phosphatidylcholine and phosphatidylethanolamine are the main phospholipids followed by phosphatidylinositol, sphingomyelin, phosphatidylserine, and phosphatidic acid. Phospholipidic profile is similar to that in whole oocytes except for the absence of diphosphatidylglycerol in yolk platelets. Oleic, palmitic, and linoleic acids are the main fatty acids in phosphatidylcholine, and oleic acid is the principal one in phosphatidylethanolamine. In phosphatidic acid, palmitic, estearic, palmitoleic, and oleic acids represent 68 mol% of the total acyl groups. Phosphatidylinositol, enriched in arachidonic acid, is the most unsaturated phospholipid while sphingomyelin shows the lowest unsaturation index. The acyl group distribution in triacylglycerols is similar when yolk platelets and whole oocytes are compared. Polar and neutral lipids of yolk platelets determine the lipidic profile of the whole oocyte. The presence of unusual fatty acids as 14:0, 15:0, 15:1, 17:0, and 17:1 in phospholipids and triacylglycerols may indicate an oxidation mechanism different from beta-oxidation in yolk platelets and/or a structural and functional relation with mitochondria. Given that yolk platelets in amphibian oocytes may act in a dynamic fashion in development, their role should be reconsidered.     

Microfilament bundles in cultured cells : Correlation with anchorage independence and tumorigenicity in nude mice

The distribution of actin microfilament bundles in cell lines 3T3B, CHO, HeLa and CLID extracted with 0.1% Triton X-100 was examined by indirect immunofluorescence using human actin antibodies and by electron microscopy of whole cells grown directly on support grids. Anchorage dependence as determined by growth in soft agar and tumorigenicity in nude mice was also investigated. Immunofluorescent staining showed that CHO and HeLa cells have normal numbers and distributions of actin microfilament bundles as compared with similarly spread control 3T3B cells. A significant fraction of the mouse CLID cells showed comparable numbers of microfilament bundles as 3T3B cells but their distribution was markedly different. In many cases the bundles radiated from a region close to the cell's centre or near its projections and usually penetrated the projections. The presence of diffuse staining in 4% of the cell population also indicated the existence in these cells of disorganized actin. Electron microscope studies of well spread regions of negatively-stained, Triton-extracted cells corroborated the observations made with the immunofluorescence technique. In 3T3B, CHO and CLID cells abundant microtubules were found, colinearly arranged with actin filaments in the thin cytoplasmic extensions. While CLID, CHO and HeLa cells showed the capacity to grow in soft agar, only CLID and HeLa cells produced tumours in athymic nude mice. The observations suggest that a reduction or disorganisation of the actin microfilament bundles may not in itself be essential at least for the non-virally transformed cells studied to show anchorage independence or to produce tumours in nude mice.     

Non-HLA genes PTPN22, CDK6 and PADI4 are associated with specific autoantibodies in HLA-defined subgroups of rheumatoid arthritis

Introduction

Genetic susceptibility to complex diseases has been intensively studied during the last decade, yet only signals with small effect have been found leaving open the possibility that subgroups within complex traits show stronger association signals. In rheumatoid arthritis (RA), autoantibody production serves as a helpful discriminator in genetic studies and today anti-citrullinated cyclic peptide (anti-CCP) antibody positivity is employed for diagnosis of disease. The HLA-DRB1 locus is known as the most important genetic contributor for the risk of RA, but is not sufficient to drive autoimmunity and additional genetic and environmental factors are involved. Hence, we addressed the association of previously discovered RA loci with disease-specific autoantibody responses in RA patients stratified by HLA-DRB1*04.

Methods

We investigated 2178 patients from three RA cohorts from Sweden and Spain for 41 genetic variants and four autoantibodies, including the generic anti-CCP as well as specific responses towards citrullinated peptides from vimentin, alpha-enolase and type II collagen.

Results

Our data demonstrated different genetic associations of autoantibody-positive disease subgroups in relation to the presence of DRB1*04. Two specific subgroups of autoantibody-positive RA were identified. The SNP in PTPN22 was associated with presence of anti-citrullinated enolase peptide antibodies in carriers of HLA-DRB1*04 (Cochran-Mantel-Haenszel test P = 0.0001, P_corrected <0.05), whereas SNPs in CDK6 and PADI4 were associated with anti-CCP status in DRB1*04 negative patients (Cochran-Mantel-Haenszel test P = 0.0004, P_corrected <0.05 for both markers). Additionally we see allelic correlation with autoantibody titers for PTPN22 SNP rs2476601 and anti-citrullinated enolase peptide antibodies in carriers of HLA-DRB1*04 (Mann Whitney test P = 0.02) and between CDK6 SNP rs42041 and anti-CCP in non-carriers of HLA-DRB1*04 (Mann Whitney test P = 0.02).

Conclusion

These data point to alternative pathways for disease development in clinically similar RA subgroups and suggest an approach for study of genetic complexity of disease with strong contribution of HLA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0414-3) contains supplementary material, which is available to authorized users.     

Cervical cancer in Croatia: state of the art and possibilities for prevention

In Croatia, there are about 355 incident cases and about 100 deaths from cervical cancer every year. The aim of this study is to present the trends of cervical cancer incidence and mortality and to propose preventive strategies for cervical cancer in Croatia. Age-standardised and age-specific cervical cancer incidence rates were calculated for the period 1985-2004. For cervical cancer mortality data, the WHO Mortality Database was used. After an early decrease of cervical cancer incidence and mortality following the introduction of opportunistic screening in Croatia, no further decrease has been observed since the 1990s. An increase in incidence over the last 20 years was observed in the age-groups 40-44 and 45-49 years. To reduce cervical cancer rates, an organised cervical cancer screening programme is essential. In addition, HPV vaccination should be introduced in the school vaccination programme to achieve further reductions in cervical cancer incidence in the future.