Chromosomal localization of the major histocompatibility complex (MHC) in the rhesus monkey and chimpanzee by fluorescence in situ hybridization.

Genes for the major histocompatibility complex (MHC) were localized by fluorescence in situ hybridization to the long arm of rhesus monkey chromosome 5. This localization contradicts previous reports, based on genetic investigation of somatic cell hybrids, that placed the MHC on chromosome 2 of this species. In the chimpanzee, the MHC loci were localized to 5p21.3, corresponding precisely to their location on human chromosome 6p21.3.     

Mesenchymal Stromal Cell Secretome Up-Regulates 47 kDa CXCR4 Expression,and Induce Invasiveness in Neuroblastoma Cell Lines

Neuroblastoma accounts for 15% of childhood cancer deaths and presents with metastatic disease of the bone and the bone marrow at diagnosis in 70% of the cases. Previous studies have shown that the Mesenchymal Stromal Cell (MSC) secretome, triggers metastases in several cancer types such as breast and prostate cancer, but the specific role of the MSC factors in neuroblastoma metastasis is unclear. To better understand the effect of MSC secretome on chemokine receptors in neuroblastoma, and its role in metastasis, we studied a panel of 20 neuroblastoma cell lines, and compared their invasive potential towards MSC-conditioned-RPMI (mRPMI) and their cytokine receptor expression profiles. Western blot analysis revealed the expression of multiple CXCR4 isoforms in neuroblastoma cells. Among the five major isoforms, the expression of the 47 kDa isoform showed significant correlation with high invasiveness. Pretreatment with mRPMI up-regulated the expression of the 47 kDa CXCR4 isoform and also increased MMP-9 secretion, expression of integrin α3 and integrin β1, and the invasive potential of the cell; while blocking CXCR4 either with AMD 3100, a CXCR4 antagonist, or with an anti-47 kDa CXCR4 neutralizing antibody decreased the secretion of MMP-9, the expression of integrin α3 and integrin β1, and the invasive potential of the cell. Pretreatment with mRPMI also protected the 47 kDa CXCR4 isoform from ubiquitination and subsequent degradation. Our data suggest a modulatory role of the MSC secretome on the expression of the 47 kDa CXCR4 isoform and invasion potential of the neuroblastoma cells to the bone marrow.     

Fragile X expression in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids under low and high thymidylate stress conditions

Expression of the fragile X site fra(X)(q27.3) was studied in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids. In these cells, low thymidylate stress, achieved by 5-fluoro-2'-deoxyuridine (FdU) treatment and by limiting the exogenous supply of thymidine (dT), induced fragile X expression. High thymidylate stress, produced by supplying excess amounts of dT, was also effective in inducing fragile X expression, even in a hybrid clone that retained a fragile X chromosome as the only human chromosome; addition of deoxycytidine (dC) completely abolished this effect. In contrast, 5-bromo-2'-deoxyuridine (BrdU) did not induce fragile X expression. Cell-cycle analysis of BrdU-deprived thymidine-auxotrophic hybrid cells indicated that one round of DNA replication under thymidylate stress conditions is sufficient for fragile X expression. Our results suggest that the expression is an intrinsic property of the fragile site itself, which is believed to be composed of replicon clusters with pyrimidine-rich DNA sequence(s).     

Expression level of enzymes related to in situ estrogen synthesis and clinicopathological parameters in breast cancer patients

In order to evaluate the importance of estrogen production in tumor and surrounding tissues, we measured mRNA expression levels of 5 enzymes participating to estrogen synthesis in situ and 4 breast cancer-related proteins in 27 pairs of tumor and non-malignant tissues. Steroid sulfatase (STS) mRNA was more frequently detected in tumor tissues rather than in their non-malignant counterparts. Estrogen sulfotransferase (EST) was constantly expressed with high level not only in tumor tissues but also in their surrounding non-malignant counterparts. In contrast, mRNA expression levels of aromatase, and 17β-hydroxysteroid dehydrogenase type I and II were relatively low and detected only in small proportion of the patients. We also measured the mRNA expression levels of the same nine genes in tumor tissues of 197 breast cancer patients, and analyzed relationship between the mRNA expression level and the clinicopathological parameters. The mRNA expression levels of STS, aromatase and erbB2 in tumor tissues increased as breast cancer progressed. The tumoral mRNA expression levels of STS, estrogen receptor β, and erbB2 in patients with recurrence were higher than those in patients without recurrence. Upregulation of STS expression plays an important role in tumor progression of human breast cancer and is considered to be responsible for estrogen production in tumor and surrounding tissues.     

Electron paramagnetic resonance study of ferrous cytochrome P-450scc-nitric oxide complexes: effects of cholesterol and its analogues

Electron paramagnetic resonance (EPR) spectra of nitric oxide (NO) complexes of ferrous cytochrome P-450scc were measured at 77 K for the first time without using the rapid-mixing and freeze-quenching technique. Without substrate the EPR spectra were very similar to those of cytochrome P-450cam (from Pseudomonas putida) and cytochrome P-450LM (from rat liver microsomes) with rhombic symmetry; gx = 2.071, gz = 2.001, gy = 1.962, and Az = 2.2 mT for 14NO complexes. Upon addition of substrates [such as cholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, 25-hydroxycholesterol, and 22-ketocholesterol], the EPR spectra exhibited many variations having rhombic symmetry in the major component and an additional minor component with less rhombic symmetry. Furthermore, addition of 20(S)-hydroxycholesterol caused a striking change in the EPR spectrum. The component with rhombic symmetry disappeared completely, and the component with less rhombic symmetry dominated (gx = 2.027, gz = 2.007, gy = 1.984, and Az = 1.76 mT for 14NO complexes). These observations suggest the existence of the following physiologically important natures: (1) the conformational flexibility of the active site of the enzyme due to the steric interaction between the substrate and the heme-bound ligand molecule and (2) the importance of the hydroxylation of the cholesterol side chain at the 20S position to proceed the side-chain cleavage reaction in cytochrome P-450scc.     

On the relationship between water-soluble protein and calcium in the egg ofOryzias latipes

Summary Quantitative analysis of total and bound calcium in the egg ofOryzias latipes was performed during; the course of early development. The amount of total calcium is nearly constant. 95% of total calcium is bound with the basic substance before fertilization, but 17% becomes free within 10 minutes after fertilization. The rate of bound calcium to total calcium recovers the original value by the blastula stage. The water-soluble protein patterns obtained by paper electrophoresis consist of five anion bands. The protein of band IV increases during the early embryogenesis, while that of band I decreases relatively. There are no remarkable changes in the amount of protein in bands II, III, and V. Among three bands of thin layer chromatogram with Sephadex gel (G-200), the amount of protein of band A (more than 160,000 in molecular weight) reduces gradually after fertilization, while no marked change is observed in bands B (about 100,000 in molecular weight) and C (about 30,000 in molecular weight).The greater part of incorporated⁴⁵Ca is detected in low molecular substance (less than 30,000 in molecular weight), and the rest in bands A, B, and C, respectively.