Differentiation-associated modulation of heparan sulfate structure and function in CaCo-2 colon carcinoma cells

Heparan sulfate species expressed by different cell and tissue types differ in their structural and functional properties. Limited information is available on differences in regulation of heparan sulfate biosynthesis within a single tissue or cell population under different conditions. We have approached this question by studying the effect of cell differentiation on the biosynthesis and function of heparan sulfate in human colon carcinoma cells (CaCo-2). These cells undergo spontaneous differentiation in culture when grown on semipermeable supports; the differentiated cells show phenotypic similarity to small intestine enterocytes. Metabolically labeled heparan sulfate was isolated from the apical and basolateral media from cultures of differentiated and undifferentiated cells. Compositional analysis of disaccharides, derived from the contiguous N-sulfated regions of heparan sulfate, indicated a greater proportion of 2-O- sulfated iduronic acid units and a smaller amount of 6-O-sulfated glucosamine units in differentiated than in undifferentiated cells. By contrast, the overall degree of sulfation, the chain length and the size distribution of the N-acetylated regions were similar regardless the differentiation status of the cells. The structural changes were found to affect the binding of heparan sulfate to the long isoform of platelet-derived growth factor A chain but not to fibroblast growth factor 2. These findings show that heparan sulfate structures change during cell differentiation and that heparan sulfate-growth factor interactions may be affected by such changes.      

Analysis of circulating regulatory T cells in patients with metastatic prostate cancer pre- versus post-vaccination

We have previously shown that the suppressive function of regulatory T cells (Tregs) from peripheral blood mononuclear cells (PBMCs) is enhanced in patients with prostate cancer when compared with healthy individuals. Two phase II studies using the PSA-TRICOM vaccine in patients with metastatic castration-resistant prostate cancer (mCRPC) showed evidence of patient benefit in terms of enhanced survival. The Halabi nomogram has been used to predict survival (HPS) of patients with mCRPC treated with conventional chemotherapy or second-line hormonal therapy. Tregs from PBMCs of patients (n = 23) with mCRPC were obtained pre- and post-three monthly vaccinations, and analyzed for number, phenotype, and suppressive function. Changes post- versus pre-vaccination in these parameters were compared with 3-year survival and HPS. No differences in Treg numbers were observed post- versus pre-vaccination. Trends (P = 0.029) were observed between overall survival (OS) and a decrease in Treg suppressive function post- versus pre-vaccination. Trends were also observed in analyzing effector:Treg (CD4⁺CD25⁺CD127⁻FoxP3⁺CTLA4⁺) ratio post- versus pre-vaccination with OS versus HPS. These data provide preliminary evidence for a possible association between improved OS and a decrease in Treg function when PBMCs are analyzed after three monthly vaccinations. Patients with an OS > HPS were more likely to have decreased Treg function following vaccine. Larger studies to confirm and extend these findings are warranted.     

Substitution bias, rapid saturation, and the use of mtDNA for nematode systematics

Only relatively recently have researchers turned to molecular methods for nematode phylogeny reconstruction. Thus, we lack the extensive literature on evolutionary patterns and phylogenetic usefulness of different DNA regions for nematodes that exists for other taxa. Here, we examine the usefulness of mtDNA for nematode phylogeny reconstruction and provide data that can be used for a priori character weighting or for parameter specification in models of sequence evolution. We estimated the substitution pattern for the mitochondrial ND4 gene from intraspecific comparisons in four species of parasitic nematodes from the family Trichostrongylidae (38-50 sequences per species). The resulting pattern suggests a strong mutational bias toward A and T, and a lower transition/transversion ratio than is typically observed in other taxa. We also present information on the relative rates of substitution at first, second, and third codon positions and on relative rates of saturation of different types of substitutions in comparisons ranging from intraspecific to interordinal. Silent sites saturate extremely quickly, presumably owing to the substitution bias and, perhaps, to an accelerated mutation rate. Results emphasize the importance of using only the most closely related sequences in order to infer patterns of substitution accurately for nematodes or for other taxa having strongly composition-biased DNA. ND4 also shows high amino acid polymorphism at both the intra- and interspecific levels, and in higher level comparisons, there is evidence of saturation at variable amino acid sites. In general, we recommend using mtDNA coding genes only for phylogenetics of relatively closely related nematode species and, even then, using only nonsynonymous substitutions and the more conserved mitochondrial genes (e.g., cytochrome oxidases). On the other hand, the high substitution rate in genes such as ND4 should make them excellent for population genetics studies, identifying cryptic species, and resolving relationships among closely related congeners when other markers show insufficient variation.      

Hypothalamic response to leptin changes during a hormonally induced estrous cycle in rats

The leptin system regulates body fat mass through a feedback loop between adipose tissue and the hypothalamus. To test if leptin responsiveness may be regulated we assayed hypothalamic response to leptin during the estrous cycle; when changes in food intake are known to occur. Immature rats were treated with pregnant mare’s serum gonadotropin (PMSG) to induce synchronized follicular development, ovulation and corpus luteum formation. Leptin response was estimated by measuring the in vitro induction of tis11, a primary response gene activated by STAT3-dependent cytokines in hypothalamic explants after leptin stimulation. In addition, mRNA levels of the suppressor cytokine signaling-3 (SOCS-3), a possible mediator of leptin resistance, were analyzed. Serum leptin levels did not change between days 2 and day 3 (corresponding to proestrus and estrus, respectively) but the response to leptin was higher on day 2 than on day 3 (p=0.05). Food intake displayed a tendency towards downregulation between day 1 and day 2 (p=0.057), and a tendency towards upregulation between day 2 and day 3 (p=0.072), although the body weight increased on day of the study (p<0.0001). There was no significant difference in hypothalamic expression of SOCS-3 between day 2 and day 3. In conclusion, we have shown that leptin responsiveness changes during a hormonally induced estrous cycle in rats. Our data suggest that a change in the hypothalamic response to leptin may cause the cyclic feeding behavior seen in rats.     

The Drosophila Ret gene is transcribed in multiple alternatively spliced forms

We have cloned and sequenced the 5' and 3' ends of the Drosophila homolog of the vertebrate c-ret gene, Ret, and have derived from it the predicted protein sequence of Ret. The extracellular domain of Ret is very widely diverged from that of its vertebrate counterparts but the cadherin motif present in vertebrate c-ret proteins can also be discerned in Ret. As with the vertebrate gene, multiple splice variants were detected at the 5'-end of Ret, one of which inserts an exon with a protein-terminating frameshift into the cDNA. In contrast to human c-ret, which may vary its signalling specificity by using splicing-derived, alternative C-terminal sequences, Ret cDNAs showed no variation at their 3'-ends.     

Fetal microchimerism in kidney biopsies of lupus nephritis patients may be associated with a beneficial effect

IntroductionMicrochimeric male fetal cells (MFCs) have been associated with systemic lupus erythematosus, and published studies have further correlated MFC with lupus nephritis (LN). In the present study, we evaluated the frequency of MFC in the renal tissue of patients with LN.MethodsTwenty-seven renal biopsies were evaluated: Fourteen were from women with clinical and laboratory findings of LN, and thirteen were from controls. Genomic DNA was extracted from kidney biopsies, and the male fetal DNA was quantified using real-time quantitative polymerase chain reactions for the detection of specific Y chromosome sequences.ResultsMFCs were detected in 9 (64%) of 14 of patients with LN, whereas no MFCs were found in the control group (P = 0.0006). No differences in pregnancy history were found between patients with LN and the control group. Significantly higher amounts of MFCs were found in patients with LN with serum creatinine ≤1.5 mg/dl. Furthermore, women with MFCs had significantly better renal function at the time of biopsy (P = 0.03). In contrast, patients with LN without MFCs presented with more severe forms of glomerulonephritis (World Health Organization class IV = 60% and class V = 40%).ConclusionsOur data indicate a high prevalence of MFCs in renal biopsy specimens from women with LN, suggesting a role for MFCs in the etiology of LN. The present report also provides some evidence that MFCs could have a beneficial effect in this disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0615-4) contains supplementary material, which is available to authorized users.     

Chitooligosaccharide inhibits RANKL-induced osteoclastogenesis and ligation-induced periodontitis by suppressing MAPK/ c-fos/NFATC1 signaling

Considering the high rate of osteoclast-related diseases worldwide, research targeting osteoclast formation/function is crucial. In vitro, we demonstrated that chitooligosaccharide (CS) dramatically inhibited osteoclastogenesis as well as osteoclast function dose-dependently. CS suppressed osteoclast-specific genes expression during osteoclastogenesis. Furthermore, we found that CS attenuated receptor activator of nuclear factor kappa B ligand (RANKL)-mediated mitogen-activated protein kinase (MAPK) pathway involving p38, erk1/2, and jnk, leading to the reduced expression of c-fos and nuclear factor of activated T cells c1 (NFATc1) during osteoclast differentiation. In vivo, we found CS protected rats from periodontitis-induced alveolar bone loss by micro-computerized tomography and histological analysis. Overall, CS inhibited RANKL-induced osteoclastogenesis and ligature-induced rat periodontitis model, probably by suppressing the MAPK/c-fos/NFATc1 signaling pathway. Therefore, CS may be a safe and promising treatment for osteoclast-related diseases.     

Ulysses - an application for the projection of molecular interactions across species

We developed Ulysses as a user-oriented system that uses a process called Interolog Analysis for the parallel analysis and display of protein interactions detected in various species. Ulysses was designed to perform such Interolog Analysis by the projection of model organism interaction data onto homologous human proteins, and thus serves as an accelerator for the analysis of uncharacterized human proteins. The relevance of projections was assessed and validated against published reference collections. All source code is freely available, and the Ulysses system can be accessed via a web interface .