Protein phosphatase 2Cbeta association with the IkappaB kinase complex is involved in regulating NF-kappaB activity

The NF-kappaB pathway is important in the control of the immune and inflammatory response. One of the critical events in the activation of this pathway is the stimulation of the IkappaB kinases (IKKs) by cytokines such as tumor necrosis factor-alpha and interleukin-1. Although the mechanisms that modulate IKK activation have been studied in detail, much less is known about the processes that down-regulate its activity following cytokine treatment. In this study, we utilized biochemical fractionation and mass spectrometry to demonstrate that protein phosphatase 2Cbeta (PP2Cbeta) can associate with the IKK complex. PP2Cbeta association with the IKK complex led to the dephosphorylation of IKKbeta and decreased its kinase activity. The binding of PP2Cbeta to IKKbeta was decreased at early times post-tumor necrosis factor-alpha treatment and was restored at later times following treatment with this cytokine. Experiments utilizing siRNA directed against PP2Cbeta demonstrated an in vivo role for this phosphatase in decreasing IKK activity at late times following cytokine treatment. These studies are consistent with the ability of PP2Cbeta to down-regulate cytokine-induced NF-kappaB activation by altering IKK activity.     

Establishment and characterization of three immortal bovine muscular epithelial cell lines

We have established three immortal bovine muscular epithelial (BME) cell lines, one spontaneously immortalized (BMES), the second SV40LT-mediated (BMEV) and the third hTERT-mediated (BMET). The morphology of the three immortal cell lines was similar to that of early passage primary BME cells. Each of the immortal cell lines made cytokeratin, a typical epithelial marker. BMET grew faster than the other immortal lines and the BME cells, in 10% FBS-DMEM medium, whereas neither the primary cells nor the three immortal cell lines grew in 0.5% FBS-DMEM. The primary BME cells and the immortal cell lines, with the exception of BMES, made increasing amounts of p53 protein when treated with doxorubicin, a DNA damaging agent. On the other hand, almost half of the cells in populations of the three immortal cell lines may lack p16(INK4a) regulatory function, compared to primary BME cells that were growth arrested by enforced expression of p16(INK4a). In soft-agar assays, the primary cells and immortal cell lines proved to be less transformed in phenotype than HeLa cells. The three immortal epithelial-type cell lines reported here are the first cell lines established from muscle tissue of bovine or other species.     

Pioglitazone, a synthetic ligand for PPARγ, induces apoptosis in RB-deficient human colorectal cancer cells

No published data are available about the expression of peroxisome proliferator-activated receptor γ (PPARγ) and the role of PPARγ in retinoblastoma protein (RB)-deficient human colorectal cancer (CRC) cells (SNU-C4 and SNU-C2A). Our aim was to investigate whether PPARγ is expressed in SNU-C4 and SNU-C2A cells and to elucidate possible molecular mechanisms underlying the effect of pioglitazone, a synthetic ligand for PPARγ, on cell growth in these cell lines. RT-PCR and Western blot analysis showed that both human CRC cell lines expressed PPARγ mRNA and protein. Pioglitazone inhibited the cell growth of both cell lines through G2/M phase block and apoptosis. In addition, pioglitazone caused a down-regulation of the X chromosome-linked inhibitor of apoptosis (XIAP), Bcl-2, and cyclooxygenase-2 (COX-2) under conditions leading to PPARγ down-regulation. These results suggest that pioglitazone may have therapeutic relevance or significance in the treatment of human CRC, and the down-regulation of XIAP, Bcl-2, and COX-2 may contribute to pioglitazone-induced apoptosis in these and other RB-deficient cell lines and tumors.     

Immunosuppressants inhibit hormone-stimulated Mg2+ uptake in mouse distal convoluted tubule cells

Immunosuppressants such as cyclosporinA and FK506 (tacrolimus) are widely prescribed to treat numerous disorders and to treat organ transplant recipients. However, cyclosporine A and FK506 are both known to produce hypomagnesaemia. The mechanism of this effect is still unclear. The present study determined the effects of immunosuppressant treatment on the parathyroid hormone (PTH) mediated Mg(2+) uptake and the mitogen-activated protein kinase (MAPK) activation in mouse distal convoluted tubule (MDCT) cells. The intracellular Ca(2+) and Mg(2+) concentrations in a single MDCT cell were measured by using the fluorescentdye Fura-2 AM and Mag-fura-2 AM, respectively. Cyclosporine A and FK506 illicited a transient increase of intracellular Ca(2+) from a basal level of 99 +/- 16 nM to 685 +/- 105 and 608 +/- 96 nM, respectively. In order to determine the Mg(2+) transport, the MDCT cells were Mg(2+)-depleted by culturing them in Mg(2+)-free media for 16 h, and the Mg(2+) uptake was measured by microfluorescence after placing the depleted cells in 1.5mM MgCl(2). The mean rate of Mg(2+) uptake, d([Mg(2+)](i))/dt, was 140 +/- 16 nM/s in the control MDCT cells. PTH increased the Mg(2+) uptake more than 2 times in this cell. Cyclosporine A (10 microM) and FK506 (0.1 microM) did not affect the basal Mg(2+)uptake (140 +/- 16 and 142 +/- 14 nM/s, respectively), but they inhibited the PTH-stimulated Mg(2+) entry, decreasing it from 248+/-12 to 147 +/- 7 and 148 +/- 14 nM/s, respectively. These effects were inhibited by L685818, which is a potent competitive antagonist of FK506. PTH stimulated the extracellular signal-regulated kinase1/2 (ERK1/2) protein synthesis. This PTH-stimulated ERK1/2 activation was inhibited by cyclosporine A and FK506. In the present study, the role of ERK1/2 activation on the PTH-dependent magnesium uptake was examined in MDCT cells, and we showed that immunosuppressants inhibit the hormone-stimulated Mg(2+) uptake into the MDCT cells by inhibiting the MAPK pathway.     

miR-24 triggers epidermal differentiation by controlling actin adhesion and cell migration

During keratinocyte differentiation and stratification, cells undergo extensive remodeling of their actin cytoskeleton, which is important to control cell mobility and to coordinate and stabilize adhesive structures necessary for functional epithelia. Limited knowledge exists on how the actin cytoskeleton is remodeled in epithelial stratification and whether cell shape is a key determinant to trigger terminal differentiation. In this paper, using human keratinocytes and mouse epidermis as models, we implicate miR-24 in actin adhesion dynamics and demonstrate that miR-24 directly controls actin cable formation and cell mobility. miR-24 overexpression in proliferating cells was sufficient to trigger keratinocyte differentiation both in vitro and in vivo and directly repressed cytoskeletal modulators (PAK4, Tks5, and ArhGAP19). Silencing of these targets recapitulated the effects of miR-24 overexpression. Our results uncover a new regulatory pathway involving a differentiation-promoting microribonucleic acid that regulates actin adhesion dynamics in human and mouse epidermis.     

Hepatic expression of cytochrome P450 in type 2 diabetic Goto-Kakizaki rats

Although hepatic expression of cytochrome P450 (CYP) changes markedly in diabetes, the role of ketone bodies in the regulation of CYP in diabetes is controversial. The present study was performed to determine the expression and activity of CYP in non-obese type II diabetic Goto-Kakizaki (GK) rats with normal levels of ketone bodies. In the present study, basal serum glucose levels increased 1.95-fold in GK rats, but acetoacetate and β-hydroxybutyrate levels were not significantly different. Hepatic expression of CYP reductase and CYP3A2 was up-regulated in the GK rats, and consequently, activities of CYP reductase and midazolam 4-hydroxylase, mainly catalyzed by CYP3A2, increased. In contrast, hepatic expression of CYP1A2 and CYP3A1 was down-regulated and the activities of 7-ethoxyresorufin-O-deethylase and 7-methoxyresorufin-O-demethylase, mainly catalyzed by CYP1A, also decreased in GK rats. Hepatic levels of microsomal protein and total CYP and hepatic expression of cytochrome b(5), CYP1B1, CYP2B1 and CYP2C11 were not significantly different between the GK rats and normal Wistar rats. Moreover, the expression and activity of CYP2E1, reported to be up-regulated in diabetes with hyperketonemia, were not significantly different between GK rats and control rats, suggesting that elevation of ketone bodies plays a critical role in the up-regulation of hepatic CYP2E1 in diabetic rats. Our results showed that the expression of hepatic CYP is regulated in an isoform-specific manner. The present results also show that the GK rat is a useful animal model for the pathophysiological study of non-obese type II diabetes with normal ketone body levels.     

Synthesis of norlignans and in vitro inhibitory activity of antigen-induced degranulation

The synthesis and biological evaluation of a series of novel norlignans are described. Norlignans were evaluated for their inhibitory activity on the release of β-hexosaminidase, a marker of degranulation, from RBL-2H3 cells induced by the IgE-antigen complex. The results showed that norlignans 4c and 4e potently inhibited degranulation, with IC(50) values of 18.3 and 17.9 μM, respectively.     

Roles of four Arabidopsis u-box e3 ubiquitin ligases in negative regulation of abscisic Acid-mediated drought stress responses

AtPUB18 and AtPUB19 are homologous U-box E3 ubiquitin ligases in Arabidopsis (Arabidopsis thaliana). AtPUB19 is a negative regulator of abscisic acid (ABA)-mediated drought responses, whereas the role of AtPUB18 in drought responses is unknown. Here, loss-of-function and overexpression tests identified AtPUB18 as a negative regulator in ABA-mediated stomatal closure and water stress responses. The atpub18-2atpub19-3 double mutant line displayed more sensitivity to ABA and enhanced drought tolerance than each single mutant plant; therefore, AtPUB18 and AtPUB19 are agonistic. Stomatal closure of the atpub18-2atpub19-3 mutant was hypersensitive to hydrogen peroxide (H(2)O(2)) but not to calcium, suggesting that AtPUB18 and AtPUB19 exert negative effects on the ABA signaling pathway downstream of H(2)O(2) and upstream of calcium. AtPUB22 and AtPUB23 are other U-box E3 negative regulators of drought responses. Although atpub22atpub23 was more tolerant to drought stress relative to wild-type plants, its ABA-mediated stomatal movements were highly similar to those of wild-type plants. The atpub18-2atpub19-3atpub22atpub23 quadruple mutant exhibited enhanced tolerance to drought stress as compared with each atpub18-2atpub19-3 and atpub22atpub23 double mutant progeny; however, its stomatal behavior was almost identical to the atpub18-2atpub19-3 double mutant in the presence of ABA, H(2)O(2), and calcium. Overexpression of AtPUB18 and AtPUB19 in atpub22atpub23 effectively hindered ABA-dependent stomatal closure, but overexpression of AtPUB22 and AtPUB23 in atpub18-2atpub19-3 did not inhibit ABA-enhanced stomatal closure, highlighting their ABA-independent roles. Overall, these results suggest that AtPUB18 has a linked function with AtPUB19, but is independent from AtPUB22 and AtPUB23, in negative regulation of ABA-mediated drought stress responses.     

Pollen and phytoliths from fired ancient potsherds as potential indicators for deciphering past vegetation and climate in Turpan, Xinjiang, NW China

It is demonstrated that palynomorphs can occur in fired ancient potsherds when the firing temperature was under 350°C. Pollen and phytoliths recovered from incompletely fired and fully fired potsherds (ca. 2700 yrs BP) from the Yanghai Tombs, Turpan, Xinjiang, NW China can be used as potential indicators for reconstructing past vegetation and corresponding climate in the area. The results show a higher rate of recovery of pollen and phytoliths from incompletely fired potsherds than from fully fired ones. Charred phytoliths recovered from both fully fired and incompletely fired potsherds prove that degree and condition of firing result in a permanent change in phytolith color. The palynological data, together with previous data of macrobotanical remains from the Yanghai Tombs, suggest that temperate vegetation and arid climatic conditions dominated in the area ca. 2700 yrs BP.