Effects of Leghaemoglobin Components on Nitrogen Fixation and Oxygen Consumption

The effects of purified oxyleghaemoglobin components added toa suspension of bacteroids from soybean and pea root noduleprepared anaerobically were studied in terms of nitrogen fixationand oxygen consumption. Soybean leghaemoglobin components (Lba and Lb c) and pea leghaemoglobin components (Lb I and Lb IV)have different O₂-binding affinities. Lb a and Lb IV showedhigher O₂-binding affinities than Lb c and Lb I. When anaerobicallyprepared bacteroids were incubated with these leghaemoglobincomponents separately under low oxygen tension and in the presenceof a reduction system, Lb a and Lb IV were more effective forboth nitrogen fixation and oxygen consumption than Lb c andLb I. These results suggest that leghaemoglobin components participatein more effective nitrogen fixation by controlling oxygen transportto bacteroids. (Received July 7, 1981; Accepted November 2, 1981)     

Isolation of an Internal Deletion Mutant of the Arabidopsis thaliana ABI3 Gene

An Arabidopsis thaliana mutant that produces green seeds thatare highly insensitive to exogenous ABA, non-dormant and severelydesiccation intolerant was isolated from a population of fastneutron-irradiated seeds. Molecular and genetic analysis ofthis mutant shows that these phenotypes are caused by an internaldeletion of approximately one third of the ABI3 gene. Thereforeabi3 mutants with the above phenotypes are representative ofnull alleles at this locus. (Received December 3, 1993; Accepted January 22, 1994)     

Effect of volume of oocyte cytoplasm on embryo development after parthenogenetic activation, intracytoplasmic sperm injection, or somatic cell nuclear transfer

Animal cloning methods are now well described and are becoming routine. Yet, the frequency at which live cloned offspring are produced remains below 5%, irrespective of the nuclear donor species or cell type. One possible explanation is that the reprogramming factor(s) of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we have increased the oocyte volume by oocyte fusion and examined its subsequent development. We constructed oocytes with volumes two to nine times greater than the normal volume by the electrofusion or mechanical fusion of intact and enucleated oocytes. We examined their in vitro and in vivo developmental potential after parthenogenetic activation, intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). When the fused oocytes were activated parthenogenetically, most developed to morulae or blastocysts, regardless of their original size. Diploid fused oocytes were fertilized by ICSI and developed normally and after embryo transfer, we obtained 12 (4-15%) healthy and fertile offspring. However, enucleated fused oocytes could not support the development of mice cloned by SCNT. These results suggest that double fused oocytes have normal potential for development after fertilization, but oocytes with extra cytoplasm do not have enhanced reprogramming potential.     

Fucosylation of N-glycans regulates the secretion of hepatic glycoproteins into bile ducts

Fucosylated alpha-fetoprotein (AFP) is a highly specific tumor marker for hepatocellular carcinoma (HCC). However, the molecular mechanism by which serum level of fucosylated AFP increases in patients with HCC remains largely unknown. Here, we report that the fucosylation of glycoproteins could be a possible signal for secretion into bile ducts in the liver. We compared oligosaccharide structures on glycoproteins in human bile with those in serum by several types of lectin blot analyses. Enhanced binding of biliary glycoproteins to lectins that recognize a fucose residue was observed over a wide range of molecular weights compared with serum glycoproteins. A structural analysis of oligosaccharides by two-dimensional mapping high performance liquid chromatography and matrix-assisted laser desorption ionization time-of flight mass spectrometry confirmed the increases in the fucosylation of biliary glycoproteins. Purification followed by structural analysis on alpha1-antitrypsin, alpha1-acid glycoprotein and haptoglobin, which are synthesized in the liver, showed higher fucosylation in bile than in serum. To find direct evidence for fucosylation and sorting signal into bile ducts, we used alpha1-6 fucosyltransferase (Fut8)-deficient mice because fucosylation of glycoproteins produced in mouse liver was mainly an alpha1-6 linkage. Interestingly, the levels of alpha1-antitrypsin and alpha1-acid glycoprotein were quite low in bile of Fut8-deficient mice as compared with wild-type mice. An immunohistochemical study showed dramatic changes in the localization of these glycoproteins in the liver of Fut8-deficient mice. Taken together, these results suggest that fucosylation is a possible signal for the secretion of glycoproteins into bile ducts in the liver. A disruption in this system might involve an increase in fucosylated AFP in the serum of patients with HCC.     

Human umbilical cord blood-derived cells differentiate into hepatocyte-like cells in the Fas-mediated liver injury model

Human umbilical cord blood (HUCB) contains stem/progenitor cells, which can differentiate into a variety of cell types. In this study, we investigated whether HUCB cells differentiate into hepatocytes in vitro and in vivo. We also examined whether CD34 could be the selection marker of stem cells for hepatocytes. HUCB cells were obtained from normal full-term deliveries, and CD34(+/-) cells were further separated. For in vitro study, HUCB cells were cultured for 4 wk, and expressions of liver-specific genes were examined. For the in vivo study, nonobese diabetic/severe combined immunodeficient mice were subjected to liver injury by a Fas ligand-carried adenoviral vector or only radiated. Mice were treated simultaneously with or without cell transplantation of HUCB, CD34(+), or CD34(-) cells. After 4 wk, human-specific gene/protein expression was examined. In the in vitro study, human liver-specific genes were positive after 7 days of culture. The immunofluorescent study showed positive staining of alpha-fetoprotein, cytokeratin 19, and albumin in round-shaped cells. In the in vivo study, immunohistochemical analysis showed human albumin-positive, hepatocyte-specific antigen-positive cells in mouse livers of the Fas ligand/transplantation group. Fluorescence in situ hybridization analysis using the human Y chromosome also showed positive signals. However, no difference between transplanted cell types was detected. In contrast, immunopositive cells were not detected in the irradiated/transplantation group. The RT-PCR result also showed human hepatocyte-specific gene expressions only in the Fas ligand/transplantation group. HUCB cells differentiated into hepatocyte-like cells in the mouse liver, and liver injury was essential during this process. The differences between CD34(+) and CD34(-) cells were not observed in human hepatocyte-specific expression.     

Establishing apoptosis resistant cell lines for improving protein productivity of cell culture

The authors established apoptosis resistant COS–1, myeloma, hybridoma, and Friend leukemia cell lines by genetically engineering cells, aiming at more efficient protein production by cell culture. COS–1 cells, which are most widely used for eukariotic gene expression, were transfected with human bcl–2 gene. Both bcl–2 and mock transfected COS–1 cells were cultured at low (0.2%) serum concentration for 9 days. The final viable cell number of the bcl–2 transfected cells was ninefold of that of the mock transfectants. Both bcl–2 and mock transfectants were further transfected with the vector pcDNA- containing SV40 ori and immunoglobulin gene for transiently expressing protein. The bcl–2 expressing COS–1 cells produced more protein than the mock transfected COS–1 cells after 4 days posttransfection.Mouse myeloma p3-X63-Ag.8.653 cells, which are widely used as the partner for preparing hybridoma, and hybridoma 2E3 cells were transfected with human bcl–2 gene. Both bcl–2 transfected myeloma and hybridoma survived longer than the corresponding original cells in batch culture. The bcl–2 transfected 2E3 cells survived 2 to 4 four days longer in culture, producing 1.5- to 4-fold amount of antibody in comparison with the mock transfectants.Coexpression of bag–1 with bcl–2 improved survival of hybridoma 2E3 cells more than bcl–2 expression alone. The bag–1 and bcl–2 coexpressing cells produced more IgG than the the cells expressing bcl–2 alone.Apoptosis of Friend murine erythroleukemia(F-MEL) cells was suppressed with antisense c-jun expression. The antisense c-jun expressing cells survived 16 days at non-growth state.     

Endothelial connexin32 enhances angiogenesis by positively regulating tube formation and cell migration

The gap junction proteins connexin32 (Cx32), Cx37, Cx40, and Cx43 are expressed in endothelial cells, and regulate vascular functions involving inflammation, vasculogenesis and vascular remodeling. Aberrant Cxs expression promotes the development of atherosclerosis which is modulated by angiogenesis; however the role played by endothelial Cxs in angiogenesis remains unclear. In this study, we determined the effects of endothelial Cxs, particularly Cx32, on angiogenesis. EA.hy926 cells that had been transfected to overexpress Cx32 significantly increased capillary length and the number on branches compared to Cx-transfectant cells over-expressing Cx37, Cx40, and Cx43 or mock-treated cells. Treatment via intracellular transfer of anti-Cx32 antibody suppressed tube formation of human umbilical vein endothelial cells (HUVECs) compared to controls. In vitro wound healing assays revealed that Cx32-transfectant cells significantly increased the repaired area while anti-Cx32 antibody-treated HUVECs reduced it. Ex vivo aorta ring assays and in vivo matrigel plaque assays showed that Cx32-deficient mice impaired both vascular sprouting from the aorta and cell migration into the implanted matrigel. Therefore endothelial Cx32 facilitates tube formation, wound healing, vascular sprouting, and cell migration. Our results suggest that endothelial Cx32 positively regulates angiogenesis by enhancing endothelial cell tube formation and cell migration.