Microdetermination of galactose and galactose 1-phosphate in dried blood spots

Newborn screening for galactosemia (galactose-1-phosphate uridyltransferase deficiency), as well as for other defects in galactose metabolism (galactokinase deficiency and uridine diphosphogalactose 4-epimerase deficiency), requires a method of determining both galactose and galactose 1-phosphate in dried blood. We have developed a sequential quantitative method for the microdetermination of galactose and galactose 1-phosphate that can be applied to 3-mm-diameter disks of dried blood and that can be used with a Technion Autoanalyser II equipped with a fluorometer.Galactose is determined by the fluorescence of NADH following treatment with β-galactose dehydrogenase and with the consequent reduction of NAD. The complete system includes alkaline phosphatase for the hydrolysis of galactose 1-phosphate, so that the total amounts of a galactose and galactose 1-phosphate are determined. For the measurement of galactose alone, alkaline phosphate is omitted from the system. The difference in fluorescence between that from the complete system and that from the alkaline phosphatase-omitted system yields the concentration of galactose 1-phosphate.     

Suramin sensitizing cells to ionizing radiation by inactivating DNA-dependent protein kinase

Here we report that suramin sensitizes LM217, MDA-MB-468, T98G and A431 cells to ionizing radiation. Suramin sensitized cells to X radiation in a dose-dependent fashion, and longer exposure to suramin before X irradiation resulted in more efficient sensitization. The dose-modifying factors calculated from the survival curves were 1.18 in LM217 cells and 1.37 in MDA-MB-468 cells. Suramin did not sensitize Scid cells that had no DNA-dependent protein kinase activity. Suramin inhibited DNA-dependent protein kinase activity in vitro and in vivo. The concentration of suramin resulting in 50% inhibition in vitro was 1.7 microM in LM217 cells and 2.4 microM in MDA-MB-468 cells. Exposure of LM217 and MDA-MB-468 cells to suramin did not affect the level of Ku70 (G22P1) or Ku80 (XRCC5), but it increased the level of DNA-PKcs(PRKDC). Suramin did not sensitize LM217 or MDA-MB-468 cells to UV radiation. Suramin's effects were not caused by accumulation of cells in a specific phase of the cell cycle. These results suggest that suramin sensitizes cells to ionizing radiation by inhibiting DNA-dependent protein kinase activity.     

Variation in gene expression and aberrantly regulated chromosome regions in cloned mice

DNA microarray analysis was used to determine the precise genome-wide gene expression profiles of somatic cloned mice derived from Sertoli and cumulus cells. It demonstrated unexpectedly large epigenetic diversity in neonatal cloned mice, despite their normal appearance and genetic identity. In three neonatal tissues of the cloned mice, the expression of 9-40% of the genes examined was more than two times higher or lower in donor cell-dependent or -independent manners compared with normal controls. Relatively few (0.4-4%) of the genes exhibited up- or downregulation in the same manner in both types of clone. A cluster analysis of the variation in gene expression led to the identification of several chromosome regions in which gene expression was aberrantly controlled in the somatic clones. These results provide a more complete understanding of how somatic clones differ from each other and from normal individuals produced by sexual reproduction and indicate the significant difficulties that face the application of somatic cloning in regenerative medicine.     

Stretch-induced morphological changes of human endothelial cells depend on the intracellular level of Ca2+ rather than of cAMP

When exposed to a uni-axial cyclic stretch, cultured human umbilical vein endothelial cells (HUVECs) align and elongate perpendicular to the stretch axis. Previous studies showed that forskolin inhibited stretch-induced orientation of endothelial cells, suggesting that adenosine 3:5-cyclic monophosphate (cAMP) plays an important role in the shape change. However, we have recently shown that stretch-induced shape changes in cultured HUVECs are due to increased [Ca2+]i. In the present study, we examined the possible role of cAMP in stretch-induced shape changes in cultured HUVECs. Application of uni-axial cyclic stretch induced a gradual rise in cAMP reaching a peak level at 60 min after the onset of stretch. The adenylate cyclase activator, forskolin, increased the basal level of cAMP but inhibited the rise in [Ca2+]i resulting in no cell shape changes. In contrast, N 6,2-dibutyryladenosine 3:5-cyclic monophosphate (dbcAMP) enhanced the stretch-induced increase in cAMP and [Ca2+]i and resulted in cell shape changes. On the other hand, 2'5'-dideoxyadenosine (DDA), an adenylate cyclase inhibitor, inhibited stretch-induced increases in cAMP and [Ca2+]i resulting in no cell shape changes. In summary, our data showed that cell shape changes were consistently dependent on [Ca2+]i rather than cAMP levels. We conclude that the primary second messenger in the stretch-induced shape changes in HUVECs is intracellular Ca2+ rather than cAMP.     

Molecular cloning and linkage analysis of complement C3 and C4 genes of the Japanese medaka fish

 The thioester-containing complement components, C3 and C4, are believed to have arisen by gene duplication from a common ancestor, and the mammalian C4 gene resides in the vicinity of the C2 and B genes within the major histocompatibility complex (MHC) class III region. To analyze the evolution of both the complement system and the MHC, we determined the complete primary structures of two C3 genes, termed Orla C3-1 and Orla C3-2, and one C4 gene, termed Orla C4, of a teleost, Japanese medaka fish (Oryzias latipes), by analyzing cDNA clones isolated from a liver library constructed using the inbred AA2 strain. The deduced basic structures of Orla C3-1, C3-2, and C4, such as the subunit chain structure, the thioester site, and the proteolytic activation site, are similar to their mammalian counterparts. However, the catalytic His residue which greatly increases the rate of thioester reaction, is replaced by Ala in Orla C3-2, implying functional differentiation between two C3 molecules. Mapping analysis revealed a close linkage between the C3-1 and C3-2 genes, indicating that they arose by a local duplication rather than by a genome-wide tetraploidization. The C4 gene belongs to a different linkage group, and no linkage was observed among the C3, C4, Bf/C2, MHC class I, and MHC class II loci. These results suggest that the MHC class III complement region was established in the tetrapod lineage, or lost in the teleost lineage. Received: 15 July 1999 / Revised: 3 September 1999     

Analysis of the sequence variations in the Mhc DRB1-like gene of the endangered Humboldt penguin (Spheniscus humboldti)

The Major Histocompatibility Complex (Mhc) genomic region of many vertebrates is known to contain at least one highly polymorphic class II gene that is homologous in sequence to one or other of the human Mhc DRB1 class II genes. The diversity of the avian Mhc class II gene sequences have been extensively studied in chickens, quails, and some songbirds, but have been largely ignored in the oceanic birds, including the flightless penguins. We have previously reported that several penguin species have a high degree of polymorphism on exon 2 of the Mhc class II DRB1-like gene. In this study, we present for the first time the complete nucleotide sequences of exon 2, intron 2, and exon 3 of the DRB1-like gene of 20 Humboldt penguins, a species that is presently vulnerable to the dangers of extinction. The Humboldt DRB1-like nucleotide and amino acid sequences reveal at least eight unique alleles. Phylogenetic analysis of all the available avian DRB-like sequences showed that, of five penguin species and nine other bird species, the sequences of the Humboldt penguins grouped most closely to the Little penguin and the mallard, respectively. The present analysis confirms that the sequence variations of the Mhc class II gene, DRB1, are useful for discriminating among individuals within the same penguin population as well those within different penguin population groups and species.The nucleotide sequence and amino acid sequence data reported in this paper have been submitted to the DDBJ database and have been assigned the accession numbers AB088371–AB088374, AB089199, AB154393–AB154399, and AB162144.     

Stretch-induced cell proliferation is mediated by FAK-MAPK pathway

Previously we reported that a uni-axial cyclic stretch treatment of rat 3Y1 fibroblasts induced focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation (Wang et al., 2001) [Wang, J.G., Miyazu, M., Matsushita, E., Sokabe, M., Naruse, K., 2001. Uni-axial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation. Biochem. Biophys. Res. Comm. 288, 356-361]. In the present study, we investigated whether stretch-induced MAPK activation leads to proliferation of fibroblasts. 3Y1 fibroblasts were subjected to a uni-axial cyclic stretch treatment (1 Hz, 120% in length) and the bromodeoxyuridine (BrdU) incorporation was measured to access cell proliferation. BrdU incorporation increased in a time-dependent manner and became significant within 6 hours. To investigate the involvement of FAK, we transiently expressed FAK mutants that lacked tyrosine phosphorylation site (s) (F397Y, F925Y, F397/925Y). Transient expression of wild-type FAK or mock vector did not inhibit the stretch-induced BrdU incorporation, however, the FAK mutants significantly blocked BrdU incorporation. Treatment of the cells with MAPK inhibitors, PD98059 or SB203580, blocked extracellular signal-regulated kinase (ERK) phosphorylation and p38 MAPK phosphorylation, respectively, and also blocked stretch-induced BrdU incorporation. These results suggest that the stretch-induced FAK activation followed by MAPK activation plays an important role in the stretch-induced proliferation of 3Y1 fibroblasts.     

Molecular identification and immunohistochemical localization of atrial natriuretic peptide in the heart of the dromedary camel (Camelus dromedarius)

Atrial and B-type natriuretic peptide (ANP and BNP) are cardiac hormones synthesized and secreted by the myoendocrine cells of the heart. They exert potent actions on body fluid balance. Since various body organs including the heart are under high physiological stress during water and food deprivation in the desert nomads, we intended to perform molecular biological and histological studies of ANP in the heart of the dromedary camel Camelus dromedarius. Initially, we isolated cDNAs encoding ANP from the atrium and BNP from the atrium and ventricle of the dromedary camel. Putative mature ANP, deduced from the cDNA sequence, was identical to that of human and pig ANP, but the putative mature BNP was more diverse and was most similar to pig BNP (94% identity). Thus, we used antisera raised against human ANP that did not cross-react with pig BNP in the subsequent immunohistochemical studies. The ANP-expressing myoendocrine cells are most concentrated in the right atrium, to a lesser extent in the left atrium, and almost absent in the left ventricle. The immuno-positive cells are scattered uniformly in each region and are characterized by the presence of immunoreactive granular deposits around the nucleus. The left atrium comprises some ramifications of conductive cells (Purkinje fibers), some of which also contained ANP-immunoreactive granules. At the electron microscopic level, myoendocrine cells possessed secretory granules primarily in the perinuclear zone and a well-developed Golgi apparatus. The present study is the first comprehensive report dealing with the molecular cloning and immunohistochemical localization of ANP in the heart of a desert dwelling mammal.     

Expression and function of NJ-1 surface antigen in megakaryopoiesis

Immunostaining with NJ-1 monoclonal antibody (MoAb) revealed that NJ-1 is expressed on megakaryocytes (MKs). NJ-1-positive and lineage-negative progenitor cells have a higher potency to proliferate and differentiate into MKs. MKs were divided into NJ-1(+)MKs and NJ-1(-)MKs. NJ-1(+)MKs are immature MKs because of their low potential to generate pro-platelets. When cultured CD41-positive MK cells were analyzed with RT-PCR, we found that the expression of NJ-1 is down-regulated. NJ-1(+)MKs have a high adherent potential to endothelial cells comparing with NJ-1(-)MKs, and this binding ability could be inhibited by the NJ-1-Fc fusion protein. We hypothesize that NJ-1(+)MKs are immature MKs and the NJ-1 molecule is involved in MK adhesion to endothelial cells.