Myogenic differentiation of p53- and Rb-deficient immortalized and transformed bovine fibroblasts in response to MyoD

We have established in culture a spontaneously immortalized bovine embryonic fibroblast (BEF) cell line that has lost p53 and p16(INK4a) functions. MyoD is a muscle-specific regulator capable of inducing myogenesis in a number of cell types. When the BEF cells were transduced with MyoD they differentiated efficiently to desmin-positive myofibers in the presence of 2% horse serum and 1.7 nM insulin. The myogenic differentiation of this cell line was more rapid and obvious than that of C2C12 cells, as judged by morphological changes and expression of various muscle regulatory factors. To confirm that lack of the p53 and p16(INK4a) pathway does not prevent MyoD-mediated myogenesis, we established a cell line transformed with SV40LT (BEFV) and introduced MyoD into it. In the presence of 2% horse serum and 1.7 nM insulin, the MyoD-transduced BEFV cells differentiated like the MyoD-transduced BEFS cells, and displayed a similar pattern of expression of muscle regulatory proteins. Taken together, our results indicate that MyoD overexpression overcomes the defect in muscle differentiation associated with immortalization and cell transformation caused by the loss of p53 and Rb functions.     

Establishment of immortal swine kidney epithelial cells

Using normal swine kidney epithelial (SKE) cells that were shown to be senescent at passages 12 to 14, we have established one lifespan-extended cell line and two lifespan-extended cell lines by exogenous introduction of the human catalytic subunit of telomerase (hTERT) and simian virus 40 large T-antigen (SV40LT), all of which maintain epithelial morphology and express cytokeratin, a marker of epithelial cells. SV40LT- and hTERT-transduced immortal cell lines appeared to be smaller and exhibited more uniform morphology relative to primary and spontaneously immortalized SKE cells. We determined the in vitro lifespan of primary SKE cells using a standard 3T6 protocol. There were two steps of the proliferation barrier at 12 and 20, in which a majority of primary SKE cells appeared enlarged, flattened, vacuolated, and ss-galactosidase-positive, all phenotypical characteristics of senescent cells. Lifespan-extended SKE cells were eventually established from most of the cellular foci, which is indicative of spontaneous cellular conversion at passage 23. Beyond passage 25, the rate of population doubling of the established cells gradually increased. At passage 30, immortal cell lines grew faster than primary counterpart cells in 10% FBS-DMEM culture conditions, and only SV40LT-transduced immortal cells grew faster than primary and other SKE immortal cells in 0.5% FBS-DMEM. These lifespan-extended SKE cell lines failed to grow in an anchorage-independent manner in soft-agar dishes. Hence, three immortalized swine kidney epithelial cells that are not transformed would be valuable biological tools for virus propagation and basic kidney epithelial cell research.     

Effects of the grafting on the fluorescence properties of CdTe nanocrystals

In this paper, we systematically investigated the influence of graft reagents having an amino or a carboxyl terminus with different chain lengths on the fluorescence properties of water‐soluble thioglycolic acid‐stabilized CdTe nanocrystals (TGA–CdTe). Strong enhancement effects of the grafting on the fluorescence intensity of TGA–CdTe were observed. The experiment results demonstrated that short‐chain‐length grafting can increase the fluorescence intensity of CdTe nanocrystals (NCs) better than long‐chain‐length grafting, and the grafting did not influence the emission wavelength of the CdTe NCs. The fluorescence intensity of the carboxyl‐grafted TGA–CdTe was more stable than that of the amino‐grafted TGA–CdTe at wide pH ranges (pH 5.1–10.0). Copyright © 2009 John Wiley & Sons, Ltd.     

Cyano-bridged Fe(II)-Cu(II) bimetallic assemblies: honeycomb-like and pentanuclear structures

A 2D honeycomb-like compound [Fe(CN)₆{Cu(apn)}₃]_n(ClO₄)_2n(H₂O)_4n (1) (apn=N-(3-aminopropyl)-1,3-propanediamine) and a pentanuclear compound [Fe(CN)₆{Cu(dmen)₂}₄](ClO₄)₄ (2) (dmen=N,N-dimethylethylenediamine) have been prepared and characterized. In the synthesis, the use of ferricyanide or ferrocyanide yielded identical products due to reduction of Fe(III) ion to Fe(II) in water. For 1, all cyanide groups of ferrocyanide are bonded to six Cu(II) ions of which two symmetry-related Cu atoms are linked to nitrogen atoms of cyanide ligands bound to the neighboring Fe(II) center, resulting in the honeycomb structure. The variations of the geometries around Cu(II) centers are between ideal trigonal bipyramidal and square pyramidal structures, which may arise from the relative structural arrangements of flexible apn ligands. For 2, all the Cu(II) ions can be seen as square pyramidal geometries composed of basal least-squares planes from four dmen nitrogen atoms and apical nitrogen atoms from cyanide bridge. The Cu-NC angle around Cu centers in 2 is 127.9(7)°, much acuter than that of 1, which is presumably associated with steric interactions between the bulky methyl groups of the dmen ligands on the neighboring Cu ions. Both compounds exhibit very weak antiferromagnetic interactions in the low temperature range.     

Role of glycosylation in the organic anion transporter OAT1

Organic anion transporters (OAT) play essential roles in the body disposition of clinically important anionic drugs, including antiviral drugs, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We reported previously (Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519-1524) that tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited organic anion transport in COS-7 cells expressing a mouse organic anion transporter (mOAT1), suggesting an important role of glycosylation in mOAT1 function. In the present study, we investigated the effect of disrupting putative glycosylation sites in mOAT1 as well as its human counterpart, hOAT1, by mutating asparagine to glutamine and assessing mutant transporters in HeLa cells. We showed that the putative glycosylation site Asp-39 in mOAT1 was not glycosylated but the corresponding site (Asp-39) in hOAT1 was glycosylated. Disrupting Asp-39 resulted in a complete loss of transport activity in both mOAT1 and hOAT1 without affecting their cell surface expression, suggesting that the loss of function is not because of deglycosylation of Asp-39 per se but rather is likely because of the change of this important amino acid critically involved in the substrate binding. Single replacement of asparagines at other sites had no effect on transport activity indicating that glycosylation at individual sites is not essential for OAT function. In contrast, a simultaneous replacement of all asparagines in both mOAT1 and hOAT1 impaired the trafficking of the transporters to the plasma membrane. In summary, we provided the evidence that 1) Asp-39 is crucially involved in substrate recognition of OAT1, 2) glycosylation at individual sites is not required for OAT1 function, and 3) glycosylation plays an important role in the targeting of OAT1 onto the plasma membrane. This study is the first molecular identification and characterization of glycosylation of OAT1 and may provide important insights into the structure-function relationships of the organic anion transporter family.     

AHNAK-mediated activation of phospholipase C-gamma1 through protein kinase C

We have recently shown that phospholipase C-gamma (PLC-gamma) is activated by the central repeated units (CRUs) of the AHNAK protein in the presence of arachidonic acid. Here we demonstrate that four central repeated units (4 CRUs) of AHNAK act as a scaffolding motif networking PLC-gamma and PKC-alpha. Specifically, 4 CRUs of AHNAK bind and activate PKC-alpha, which in turn stimulates the release of arachidonic acid near where PLC-gamma1 is localized. Moreover, 4 CRUs of AHNAK interacted with PLC-gamma and the concerted action of 4 CRUs with arachidonic acid stimulated PLC-gamma activity. Stimulation of NIH3T3 cells expressing 4 CRUs of AHNAK with phorbol 12-myristate 13-acetate resulted in the increased generation of total inositol phosphates (IP(T)) and mobilization of the intracellular calcium. Phorbol 12-myristate 13-acetate-dependent generation of IP(T) was completely blocked in NIH3T3 cells depleted of PLC-gamma1 by RNA interference. Furthermore, bradykinin, which normally stimulated the PLC-beta isozyme resulting in the generation of a monophasic IP(T) within 30 s in NIH3T3 cells, led to a biphasic pattern for generation of IP(T) in NIH3T3 cells expressing 4 CRUs of AHNAK. The secondary activation of PLC is likely because of the scaffolding activity of AHNAK, which is consistent with the role of 4 CRUs as a molecular linker between PLC-gamma and PKC-alpha.