Demecolcine-assisted enucleation for bovine cloning

The present study demonstrated that demecolcine treatment for at least 30 min produces a membrane protrusion in metaphase II-stage bovine oocytes. The maternal chromosome mass is condensed within the protrusion, which makes it easy to remove the maternal chromosomes for nuclear transfer (NT). Maturation promoting factor activity, but not mitogen-activated protein kinase activity, increased up to 30% in oocytes during demecolcine treatment. One normal healthy calf was obtained after transfer of four NT blastocysts produced following demecolcine treatment. Demecolcine treatment did not increase the potential of NT oocytes to develop into blastocysts. The present study demonstrated that chemically-assisted removal of chromosomes is effective for bovine cloning.     

In Vitro Phosphorylation of Proteins in IAA-Treated Mesocotyls in Zea mays

Five-mm sections of elongation zones of Zea mesocotyls wereincubated for designated periods with various concentrationsof IAA. In vitro protein phosphorylation in the soluble fraction(85,000 x g supernatant) prepared from the sections was analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The phosphorylation of proteins in the soluble fraction thathad been prepared from sections incubated for 20 min in thepresence of 10{small tilde}^s M IAA was greater than that inthe sections incubated for 20 min without IAA. The amount ofphosphorylation of proteins per protein became higher when higherconcentrations increased (10{small tilde}⁸—10{small tilde}⁵M).The growth of sections incubated in the presence of 10{smalltilde}⁸ M IAA or higher concentrations was greater than thatof sections incubated in the absence of IAA. The promotion ofgrowth by IAA was greater at higher concentrations of IAA. Proteinsin the soluble fraction, prepared from sections incubated for20 min in the presence of 10{small tilde}⁵ M IAA, were phosphorylatedin the presence of either 10 fM cAMP, 10 µM cGMP, 100µM W-7, 100 µM W-5, 20 µM H-7 or 20 µMHA1004. The calmodulin antagonist, W-7, and the inhibitor ofprotein kinase C, H-7, inhibited the phosphorylation of proteinsstimulated by incubation with IAA. These results suggest thatIAA promotes cell elongation via protein phosphorylation thatdepends on calmodulin-dependent protein kinase and protein kinaseC. (Received November 29, 1995; Accepted May 20, 1996)     

Cutting edge: tyk2 is required for the induction and nuclear translocation of Daxx which regulates IFN-alpha-induced suppression of B lymphocyte formation

IFN-alpha inhibits B lymphocyte development, and the nuclear protein Daxx has been reported to be essential for this biological activity. We show in this study that IFN-alpha inhibits the clonal proliferation of B lymphocyte progenitors in response to IL-7 in wild-type, but not in tyk2-deficient, mice. In addition, the IFN-alpha-induced up-regulation and nuclear translocation of Daxx are completely abrogated in the absence of tyk2. Therefore, tyk2 is directly involved in IFN-alpha signaling for the induction and translocation of Daxx, which may result in B lymphocyte growth arrest and/or apoptosis.     

CrkL directs ASAP1 to peripheral focal adhesions

Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express an ADP-ribosylation factor (Arf)-specific GTPase-activating protein (GAP), ASAP1, as a CrkL-binding protein. In spreading platelets, most endogenous ASAP1 is localized at peripheral focal adhesions. To determine the physiologic significance of the CrkL-ASAP1 association, we overexpressed CrkL, ASAP1, or both in combination in COS7 cells. Unlike endogenous ASAP1 in platelets, overexpressed ASAP1 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both endogenous and expressed ASAP1 accumulated at CrkL-induced focal adhesions. An SH2-mutated CrkL, which cannot localize at focal adhesions, failed to recruit ASAP1 into focal adhesions. Thus, CrkL appears to be a lynchpin between ASAP1 and peripheral focal adhesions.     

The Functional Interrelationship between Gap Junctions and Fenestrae in Endothelial Cells of the Liver Organoid

Functional intact liver organoid can be reconstructed in a radial-flow bioreactor when human hepatocellular carcinoma (FLC-5), mouse immortalized sinusoidal endothelial M1 (SEC) and A7 (HSC) hepatic stellate cell lines are cocultured. The structural and functional characteristics of the reconstructed organoid closely resemble the in vivo liver situation. Previous liver organoid studies indicated that cell-to-cell communications might be an important factor for the functional and structural integrity of the reconstructed organoid, including the expression of fenestrae. Therefore, we examined the possible relationship between functional intact gap junctional intercellular communication (GJIC) and fenestrae dynamics in M1-SEC cells. The fine morphology of liver organoid was studied in the presence of (1) irsogladine maleate (IM), (2) oleamide and (3) oleamide followed by IM treatment. Fine ultrastructural changes were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and compared with control liver organoid data. TEM revealed that oleamide affected the integrity of cell-to-cell contacts predominantly in FLC-5 hepatocytes. SEM observation showed the presence of fenestrae on M1-SEC cells; however, oleamide inhibited fenestrae expression on the surface of endothelial cells. Interestingly, fenestrae reappeared when IM was added after initial oleamide exposure. GJIC mediates the number of fenestrae in endothelial cells of the liver organoid.     

Inulavosin and its benzo‐derivatives,melanogenesis inhibitors,target the copper loading mechanism to the active site of tyrosinase

Tyrosinase, a melanosomal membrane protein containing copper, is a key enzyme for melanin synthesis in melanocytes. Inulavosin inhibits melanogenesis by enhancing a degradation of tyrosinase in lysosomes. However, the mechanism by which inulavosin redirects tyrosinase to lysosomes is yet unknown. The analyses of structure–activity relationship of inulavosin and its benzo‐derivatives reveal that the hydroxyl and the methyl groups play a critical role in their inhibitory activity. Intriguingly, the docking studies to tyrosinase suggest that the compounds showing inhibitory activity bind through hydrophobic interactions to the cavity of tyrosinase below which the copper‐binding sites are located. This cavity is proposed to be required for the association with a chaperon that assists in copper loading to tyrosinase in Streptomyces antibioticus. Inulavosin and its benzo‐derivatives may compete with the copper chaperon and result in a lysosomal mistargeting of apo‐tyrosinase that has a conformational defect.     

Formaldehyde Fixation Contributes to Detoxification for Growth of a Nonmethylotroph, Burkholderia cepacia TM1, on Vanillic Acid

During bacterial degradation of methoxylated lignin monomers, such as vanillin and vanillic acid, formaldehyde is released through the reaction catalyzed by vanillic acid demethylase. When Burkholderia cepacia TM1 was grown on vanillin or vanillic acid as the sole carbon source, the enzymes 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) were induced. These enzymes were also expressed during growth on Luria-Bertani medium containing formaldehyde. To understand the roles of these enzymes, the hps and phi genes from a methylotrophic bacterium, Methylomonas aminofaciens 77a, were introduced into B. cepacia TM1. The transformant strain constitutively expressed the genes for HPS and PHI, and these activities were two- or threefold higher than the activities in the wild strain. Incorporation of [¹⁴C]formaldehyde into the cell constituents was increased by overexpression of the genes. Furthermore, the degradation of vanillic acid and the growth yield were significantly improved at a high concentration of vanillic acid (60 mM) in the transformant strain. These results suggest that HPS and PHI play significant roles in the detoxification and assimilation of formaldehyde. This is the first report that enhancement of the HPS/PHI pathway could improve the degradation of vanillic acid in nonmethylotrophic bacteria.     

Cytologic features based on the expression of E-cadherin and catenins in lung adenocarcinoma.

OBJECTIVE: To investigate the relationship between E-cadherin-associated cell-to-cell adhesion and cytologic features in preoperative cytologic lung adenocarcinoma specimens. STUDY DESIGN: Evaluation of the relationship between cell-to-cell adhesion, formation of cellular clusters and frequency of single cells in 31 cases of primary lung adenocarcinoma, collected by brush and needle cytology preoperatively. RESULTS: Most cases with remarkable overlapping of cells in compact cellular clusters and a few solitary cells maintained cell-to-cell adhesion. Cellular clusters that had a slight tendency to overlap, a small cell-to-cell adhesion area and a high frequency of solitary cells tended to lack E-cadherin-associated cell-to-cell adhesion. CONCLUSION: Formation of cellular clusters and the appearance of solitary cancer cells are closely related to E-cadherin-associated cell-to-cell adhesion. Therefore, it is highly likely that cytologic features may indicate malignant behavior, such as local invasion and lymph node metastasis, in primary lung adenocarcinoma.