The progesterone receptor (PR) in the chicken oviduct is a phosphoprotein that regulates gene transcription in the presence of progesterone. Treatment with progesterone in vivo stimulates phosphorylation of the progesterone receptor. With transient transfection assays, the present work has tested whether phosphorylation participates in the regulation of PR-mediated transcription. Treatment with 8-bromo-cyclic adenosine monophosphate (8-Br cAMP), a stimulator of cAMP-dependent protein kinase [protein kinase A (PKA)], mimicked progesterone-dependent, receptor-mediated transcription in the absence of progesterone. Inhibition of PKA blocked hormone action. Treatment with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, stimulated transcription in a manner similar to that of progesterone. These observations suggest that phosphorylation of the PR or other proteins in the transcription complex can modulate PR-mediated transcription in vivo.

Advanced glycation end product (AGE) activation of the signal-transducing receptor for AGE (RAGE) has been linked to a proinflammatory phenotypic change within cells. However, the precise intracellular signaling pathways involved have not been elucidated. We demonstrate here that human serum albumin modified with N -(carboxymethyl)lysine (CML), a major AGE adduct that progressively accumulates with aging, diabetes, and renal failure, induced nuclear factor (

When will embryonic stem cells reach the clinic? The answer is simple -- not soon! To produce large quantities of homogeneous tissue for transplantation, without feeder layers, and with the appropriate recipient's immunological phenotype, is a significant scientific hindrance, although adult stem (ADS) cells provide an alternative, more ethically acceptable, source. The annual global 100 million human birth rate proposes umbilical cord blood (UCB) as the largest untouched stem cell source, with advantages of naive immune status and relatively unshortened telomere length. Here, we report the world's first reproducible production of cells expressing embryonic stem cell markers, - cord-blood-derived embryonic-like stem cells (CBEs). UCB, after elective birth by Caesarean section, has been separated by sequential immunomagnetic removal of nucleate granulocytes, erythrocytes and haemopoietic myeloid/lymphoid progenitors. After 7 days of high density culture in microflasks, (10(5) cells/ml, IMDM, FCS 10%, thrombopoietin 10 ng/ml, flt3-ligand 50 ng/ml, c-kit ligand 20 ng/ml). CBE colonies formed adherent to the substrata; these were maintained for 6 weeks, then were subcultured and continued for a minimum 13 weeks. CBEs were positive for TRA-1-60, TRA-1-81, SSEA-4, SSEA-3 and Oct-4, but not SSEA-1, indicative of restriction in the human stem cell compartment. The CBEs were also microgravity--bioreactor cultured with hepatocyte growth medium (IMDM, FCS 10%, HGF 20 ng/ml, bFGF 10 ng/ml, EGF 10 ng/ml, c-kit ligand 10 ng/ml). After 4 weeks the cells were found to express characteristic hepatic markers, cytokeratin-18, alpha-foetoprotein and albumin. Thus, such CBEs are a viable human alternative from embryonic stem cells for stem cell research, without ethical constraint and with potential for clinical applications.

Background and Purpose-Nuclear factor-B (NF-B) is a ubiquitous transcription factor that, when activated, translocates to the nucleus, binds to DNA, and promotes transcription of many target genes. Its activation has been demonstrated in chronic inflammatory conditions, cerebral ischemia, and apoptotic cell death. The present study evaluated the presence and activation of NF-B in relation to cell death surrounding intracerebral hemorrhage (ICH). Methods-Striatal ICH was induced in rats by the double blood injection method. Animals were killed 2, 8, and 24 hours and 4 days after ICH. To examine changes in NF-B protein, Western blot was performed on brain extract. We determined NF-B activity using electrophoretic mobility shift assay (EMSA) and immunohistochemistry, using an antibody that only recognizes active NF-B. DNA fragmentation was detected with terminal deoxynucleotidyl transferase-mediated uridine 5Ј-triphosphate-biotin nick end-labeling (TUNEL) staining. Results-Western blot analysis of the NF-B p65 subunit showed that there was no difference in p65 protein levels in the control, 2-hour, 8-hour, or 24-hour groups. However, ipsilateral perilesional samples from the 4-day group revealed a 1.8-to 2.5-fold increase compared with the contralateral hemisphere. Western blotting showed no differences in the inhibitor of NF-B, IB␣, in any group. EMSA showed 1.3-, 2.1-, and 3.6-fold increased NF-B activation in the ipsilateral striatum from the 8-hour, 24-hour, and 4-day groups, respectively, compared with the contralateral hemisphere. Immunohistochemistry, in which an activation-dependent anti-NF-B antibody was used, demonstrated perivascular NF-B activation as early as 2 hours after ICH with more generalized activation at 8 hours, in agreement with the EMSA results. NF-B activation colocalized to cells containing fragmented DNA measured by TUNEL. Conclusions-The present study suggests a relationship between NF-B and the pathobiology of perilesional cell death after ICH. (Stroke. 1999;30:2472-2478.)

Advanced glycation end products (AGEs) have been implicated in the pathogenesis of diabetic kidney disease. The actions of AGEs are mediated both through a non-receptor-mediated pathway and through specific receptors for AGE (RAGEs). To explore a specific role for RAGE in renal changes in type 2 diabetes, we examined the renal effects of a neutralizing murine RAGE antibody in db/db mice, a model of obese type 2 diabetes. One group of db/db mice was treated for 2 months with the RAGE antibody, and another db/db group was treated for the same period with an irrelevant IgG. Two groups of nondiabetic db/؉ mice were treated with either RAGE antibody or isotype-matched IgG for 2 months. Placebo-treated db/db mice showed a pronounced increase in kidney weight, glomerular volume, basement membrane thickness (BMT), total mesangial volume, urinary albumin excretion (UAE), and creatinine clearance compared with nondiabetic controls. In RAGE antibody-treated db/db mice, the increase in kidney weight, glomerular volume, mesangial volume, and UAE was reduced, whereas the increase in creatinine clearance and BMT was fully normalized. Notably, these effects in db/db mice were seen without impact on body weight, blood glucose, insulin levels, or food consumption. In conclusion, RAGE is an important pathogenetic factor in the renal changes in an animal model of type 2 diabetes. Diabetes 53: 166 -172, 2004 T he prevalence of type 2 diabetes is increasing worldwide. The development of diabetic nephropathy is seen in up to 30 -40% of all type 2 diabetic subjects followed by an increased morbidity and mortality. Furthermore, diabetic nephropathy is the most common cause of end-stage renal failure in the western world. Among the many potential pathogenic mechanisms responsible for the development of diabetic kidney disease (1,2), advanced glycation end products (AGEs) have been suggested to have measurable effects on the development of diabetic kidney changes as they appear in animal models of type 1 and type 2 diabetes (3-12). Recently, the receptor for AGEs (RAGE) has been proposed to play a key role in the development of diabetic renal changes in experimental type 1 diabetes (11). Accordingly, streptozotocin (STZ)-induced diabetic mice overexpressing RAGE showed a pronounced increase in renal damage compared with changes seen in nontransgenic diabetic animals (11). Furthermore, in a recent study, administration of soluble RAGE (sRAGE), a truncated form of RAGE, was shown to blunt the renal changes seen in a mouse model of type 2 diabetes (12).The aim of the present study was to explore the role of RAGE in the development of renal changes in type 2 diabetes. Accordingly, a specific neutralizing murine RAGE antibody (RAGE antibody) was administered for 2 months in db/db mice, a genetic model of type 2 diabetes characterized by obesity, sustained hyperglycemia, hyperinsulinemia, lack of ketonuria, and progressive renal kidney disease (11-17). RESEARCH DESIGN AND METHODSAdult female db/db mice (C57BLKS/J-lepr db /lepr db ) and their...