Gene transfer preferentially selects MHC class I positive tumour cells and enhances tumour immunogenicity

The modulated expression of MHC class I on tumour tissue is well documented. Although the effect of MHC class I expression on the tumorigenicity and immunogenicity of MHC class I negative tumour cell lines has been rigorously studied, less is known about the validity of gene transfer and selection in cell lines with a mixed MHC class I phenotype. To address this issue we identified a C26 cell subline that consists of distinct populations of MHC class I (H-2D/K) positive and negative cells. Transient transfection experiments using liposome-based transfer showed a lower transgene expression in MHC class I negative cells. In addition, MHC class I negative cells were more sensitive to antibiotic selection. This led to the generation of fully MHC class I positive cell lines. In contrast to C26 cells, all transfectants were rejected in vivo and induced protection against the parental tumour cells in rechallenge experiments. Tumour cell specificity of the immune response was demonstrated in in vitro cytokine secretion and cytotoxicity assays. Transfectants expressing CD40 ligand and hygromycin phosphotransferase were not more immunogenic than cells expressing hygromycin resistance alone. We suggest that the MHC class I positive phenotype of the C26 transfectants had a bearing on their immunogenicity, because selected MHC class I positive cells were more immunogenic than parental C26 cells and could induce specific anti-tumour immune responses. These data demonstrate that the generation of tumour cell transfectants can lead to the selection of subpopulations that show an altered phenotype compared to the parental cell line and display altered immunogenicity independent of selection marker genes or other immune modulatory genes. Our results show the importance of monitoring gene transfer in the whole tumour cell population, especially for the evaluation of in vivo therapies targeted to heterogeneous tumour cell populations.     

Glycomic strategy for efficient linkage analysis of di-, oligo- and polysialic acids

Sialic acid polymers of glycoproteins and glycolipids are characterized by a high diversity in nature and are involved in distinct biological processes depending inter alia on the glycosidic linkages between the present sialic acid residues. Though suitable protocols are available for chain length and sialic acid determination, sensitive methods for linkage analysis of di-, oligo-, and polysialic acids (di/oligo/polySia) are still pending. In this study, we have established a highly sensitive glycomic strategy for this purpose which is based on permethylation of di/oligo/polySia after tagging their reducing ends with the fluorescent dye 1,2-diamino-4,5-methylenedioxybenzene (DMB). Using DMB-labeled sialic acid di/oligo/polymers glycosidic linkages could be efficiently determined and, optionally, the established working procedure can be combined with HPLC for in depth characterization of distinct di/oligo/polySia chains. Moreover, the outlined approach can be directly applied to mammalian tissue samples and linkage analysis of sialic acid polymers present in biopsy samples of neuroblastoma tissue demonstrating the usefulness of the outlined work flow to screen, for example, cancer tissue for the presence of distinct variants of di/oligo/polySia as potentially novel biomarkers. Hence, the described strategy offers a highly sensitive and efficient strategy for identification of glycosidic linkages in sialic acid di/oligo/polymers of glycoproteins and glycolipids.     

Dual role of the Jak1 FERM and kinase domains in cytokine receptor binding and in stimulation-dependent Jak activation

Jak1 is a tyrosine kinase that noncovalently forms tight complexes with a variety of cytokine receptors and is critically involved in signal transduction via cytokines. Jaks are predicted to have a 4.1, ezrin, radixin, moesin (FERM) domain at their N terminus. FERM domains are composed of three structurally unrelated subdomains (F1, F2, and F3) which are in close contact to one another and form the clover-shaped FERM domain. We generated a model structure of the Jak1 FERM domain, based on solved FERM structures and the alignments with other FERM domains. To destabilize different subdomains and to uncover their exact function, we mutated specific hydrophobic residues conserved in FERM domains and involved in hydrophobic core interactions. In this study, we show that the structural integrity of the F2 subdomain of the FERM domain of Jak1 is necessary to bind the IFN-gammaRalpha. By mutagenesis of hydrophobic residues in the hydrophobic core between the three FERM subdomains, we find that the structural context of the FERM domain is necessary for the inhibition of Jak1 phosphorylation. Thus, FERM domain mutations can have repercussions on Jak1 function. Interestingly, a mutation in the kinase domain (Jak1-K907E), known to abolish the catalytic activity, also leads to an impaired binding to the IFN-gammaRalpha when this mutant is expressed at endogenous levels in U4C cells. Our data show that the structural integrity of both the FERM domain and of the kinase domain is essential for both receptor binding and catalytic function/autoinhibition.     

Enzyme-dependent variations in the polysialylation of the neural cell adhesion molecule (NCAM) in vivo

Polysialic acid (polySia), an alpha2,8-linked polymer of N-acetylneuraminic acid, represents an essential regulator of neural cell adhesion molecule (NCAM) functions. Two polysialyltransferases, ST8SiaII and ST8SiaIV, account for polySia synthesis, but their individual roles in vivo are still not fully understood. Previous in vitro studies defined differences between the two enzymes in their usage of the two NCAM N-glycosylation sites affected and suggested a synergistic effect. Using mutant mice, lacking either enzyme, we now assessed in vivo the contribution of ST8SiaII and ST8SiaIV to polysialylation of NCAM. PolySia-NCAM was isolated from mouse brains and trypsinized, and polysialylated glycopeptides as well as glycans were analyzed in detail. Our results revealed an identical glycosylation and almost complete polysialylation of N-glycosylation sites 5 and 6 in polySia-NCAM irrespective of the enzyme present. The same sets of glycans were substituted by identical numbers of polySia chains in vivo, the length distribution of which, however, differed with the enzyme setting. Expression of ST8SiaIV alone led to higher amounts of short polySia chains and gradual decrease with length, whereas exclusive action of ST8SiaII evoked a slight reduction in long polySia chains only. These variations were most pronounced at N-glycosylation site 5, whereas the polysialylation pattern at N-glycosylation site 6 did not differ between NCAM from wild-type and ST8SiaII- or ST8SiaIV-deficient mice. Thus, our fine structure analyses suggest a comparable quality of polysialylation by ST8SiaII and ST8SiaIV and a distinct synergistic action of the two enzymes in the synthesis of long polySia chains at N-glycosylation site 5 in vivo.     

A high density of X-linked genes for general cognitive ability: a run-away process shaping human evolution?

The incidence of mental disability is 30% higher in males than in females. We have examined entries in the OMIM database that are associated with mental disability and for several other common defects. Our findings indicate that compared with the autosomes, the X chromosome contains a significantly higher number of genes that, when mutated, cause mental impairment. We propose that these genes are involved in the development of cognitive abilities and thus exert a large X-chromosome effect on general intelligence in humans. We discuss these conclusions with regard to the conservation of the vertebrate X-chromosomal linkage group and to human evolution.     

Variable thermal stress tolerance of the reef-associated symbiont-bearing foraminifera Amphistegina linked to differences in symbiont type

Coral Reefs - Adaptation, acclimatization and symbiont diversity are known to regulate thermal tolerance in corals, but the role of these mechanisms remains poorly constrained in other...     

Effects of selenium deficiency on fatty acid metabolism in rats fed fish oil-enriched diets.

The hepatic fatty acid metabolism was investigated in rats stressed by selenium deficiency and enhanced fish oil intake. Changes in the composition of lipids, peroxides, and fatty acids were studied in the liver of rats fed either a Sedeficient (8 microg Se/kg) or a Se-adequate (300 microg Se/kg) diet, both rich in n-3 fatty acid-containing fish oil (100 g/kg diet) and vitamin E (146 mg alpha-tocopherol/kg diet). The two diets were identical except for their Se content. Se deficiency led to a decrease in hair coat density and quality as well as to changes in liver lipids, individual lipid fractions and phospholipid fatty acid composition of the liver. The low Se status did reduce total and reduced glutathione in the liver but did not affect the hepatic malondialdehyde level. In liver phospholipids (PL), Se deficiency significantly reduced levels of palmitic acid [16:0], fatty acids of the n-3 series such as DHA [22:6 n-3], and other long-chain polyunsaturates C-20-C-22, but increased n-6 fatty acids such as linoleic acid (LA) [18:2 n-6]. Thus, the conversion of LA to arachidonic acid was reduced and the ratio of n-6/n-3 fatty acids was increased. As in liver PL, an increase in the n-6/n-3 ratio was also observed in the mucosal total fatty acids of the small intestine. These results suggest that in rats with adequate vitamin E and enhanced fish oil intake, Se deficiency affects the lipid concentration and fatty acid composition in the liver. The changes may be related to the decreased levels of selenoenzymes with antioxidative functions. Possible effects of Se on absorption, storage and desaturation of fatty acids were also discussed.