Pancreatic exocrine enzyme-producing cell differentiation via embryoid bodies from human embryonic stem cells

Mouse embryonic stem cells (ESCs) can be induced to form pancreatic exocrine enzyme-producing cells in vitro in a stepwise fashion that recapitulates the development in vivo. However, there is no protocol for the differentiation of pancreatic-like cells from human ESCs (hESCs). Based upon the mouse ESC model, we have induced the in vitro formation of pancreatic exocrine enzyme-producing cells from hESCs. The protocol took place in four stages. In Stage 1, embryoid bodies (EBs) were formed from dissociated hESCs and then treated with the growth factor activin A, which promoted the expression of Foxa2 and Sox17 mRNAs, markers of definitive endoderm. In Stage 2, the cells were treated with all-trans retinoic acid which promoted the transition to cells that expressed gut tube endoderm mRNA marker HNF1b. In Stage 3, the cells were treated with fibroblast growth factor 7 (FGF7), which induced expression of Pdx1 typical of pancreatic progenitor cells. In Stage 4, treatment with FGF7, glucagon-like peptide 1, and nicotinamide induced the expression amylase (AMY) mRNA, a marker for mature pancreatic exocrine cells. Immunohistochemical staining showed the expression of AMY protein at the edges of cell clusters. These cells also expressed other exocrine secretory proteins including elastase, carboxypeptidase A, chymotrypsin, and pancreatic lipase in culture. Production of these hESC-derived pancreatic enzyme-producing cells represents a critical step in the study of pancreatic organogenesis and in the development of a renewable source of human pancreatic-like exocrine cells.     

Discovery of novel arylpyrazole series as potent and selective opioid receptor-like 1 (ORL1) antagonists

The synthesis and biological evaluation of new potent opioid receptor-like 1 antagonists are presented. A structure–activity relationship (SAR) study of arylpyrazole lead compound 1 obtained from library screening identified compound 31, (1S,3R)-N-{[1-(3-chloropyridin-2-yl)-5-(5-fluoro-6-methylpyridin-3-yl)-4-methyl-1H-pyrazol-3-yl]methyl}-3-fluorocyclopentanamine, which exhibits high intrinsic potency and selectivity against other opioid receptors and hERG potassium channel.     

Growth hormone-releasing factor reduces voltage-gated Ca2+ channel current in Rat GH3 cells

Summary The action of GRF on GH₃ cell membrane was examined by patch electrode techniques. Under current clamp with patch elecrtrode, spontaneous action potentials were partially to totally eliminated by application of GRF. In the case of partial elimination, the duration of remaining spontaneous action potentials was prolonged and the amplitude of afterhyperpolarization was decreased. The evoked actiion potential in the cells which did not show spontaneous action potentials was also eliminated by GRF. In order to examine what channels were affected by GRF, voltage-clamp analysis was performed. It was revealed that voltage-gated Ca²⁺ channel current and Ca²⁺-induced K⁺ channels current were decreased by GRF, while voltage-gated Na⁺ channel and delayed K⁺ channel current was considered to be a consequence of he decrease of voltage-gated Ca²⁺ channels current. Therefore it is likely that the effect of GRF on GH₃ cells was due to the block of voltage-gated Ca²⁺ channels. The elimination of action potential under current clamp corresponded to the block of voltage-gated Ca²⁺ channels and the prolongation of action potential could be explained by the decrease of Ca²⁺-induced K⁺ channel current. The amplitude decrease of afterhyperpolarization could also be explained by the reduction of Ca²⁺-induced K⁺ channel current. Thus the results under current clamp well coincide with the results under voltage clamp. Hormone secretion from GH₃ cells was not stimulated by GRF. However, the finding that GRF solely blocked voltage-gated Ca²⁺ channel suggested the specific action of GRF on GH₃ cell membranes.     

The IkappaB kinase (IKK) inhibitor, NEMO-binding domain peptide, blocks osteoclastogenesis and bone erosion in inflammatory arthritis

Activation of NF-kappaB leads to expression of ample genes that regulate inflammatory and osteoclastogenic responses. The process is facilitated by induction of IkappaB kinase (IKK) complex that phosphorylates IkappaB and leads to its dissociation from the NF-kappaB complex, thus permitting activation of NF-kappaB. The IKK complex contains primarily IKKalpha, IKKbeta, and the regulatory kinase IKKgamma, also known as NEMO. NEMO regulates the IKK complex activity through its binding to carboxyl-terminal region of IKKalpha and IKKbeta, termed NEMO-binding domain (NBD). In this regard, a cell-permeable NBD peptide has been shown to block association of NEMO with the IKK complex and inhibit activation of NF-kappaB. Given the pivotal role of cytokine-induced NF-kappaB in osteoclastogenesis and inflammatory bone loss, we deduced that cell-permeable TAT-NBD peptide may hinder osteoclastogenesis and bone erosion in inflammatory arthritis. Using NBD peptides, we show that wild type, but not mutant, NBD blocks IKK activation and reduces cytokine-induced promoter and DNA binding activities of NF-kappaB and inhibits cytokine-induced osteoclast formation by osteoclast precursors. Consistent with the key role of NF-kappaB in osteoinflammatory responses in vivo, wild type TAT-NBD administered into mice prior to induction of inflammatory arthritis efficiently block in vivo osteoclastogenesis, inhibits focal bone erosion, and ameliorates inflammatory responses in the joints of arthritic mice. The mutant NBD peptide fails to exert these functions. These results provide strong evidence that IKKs are potent regulators of cytokine-induced osteoclastogenesis and inflammatory arthritis. More importantly, blockade of NEMO assembly with the IKK complex is a viable strategy to avert inflammatory osteolysis.     

The mutagenic modulating effect of p-phenylenediamine on the oxidation of o- or m-phenylenediamine with hydrogen peroxide in the Salmonella test

The mutagenicity of o- and m-phenylenediamine (PD) was remarkedly enhanced by oxidation; their major mutagenic oxidation products were 2,3- and 2,7-diaminophenazine, respectively. In order to evaluate the modulation effect of p-PD on the oxidation of m- or o-PD, p-PD and mixtures of m- and p-PD (m-PD/p-PD) and o- and p-PD (o-PD/p-PD) were oxidized with hydrogen peroxide and their mutagenicity was tested in Salmonella typhimurium TA98 in the presence or absence of a mammalian metabolic activation system (S9 mix). The H2O2-oxidized m-PD/p-PD and o-PD/p-PD were potent mutagens with S9 mix, whereas H2O2-oxidized p-PD was slightly mutagenic. The major mutagenic oxidation products of m-PD/p-PD and o-PD/p-PD were identified as 2,7- and 2,3-diaminophenazine, respectively, by TLC and HPLC. 2,8-Diaminophenazine was also found as a reaction product in oxidized m-PD/p-PD, and it was weakly mutagenic. The mutagenic potency of oxidized m-PD/p-PD or o-PD/p-PD was lower than that of singly oxidized m-PD or o-PD. The yield of 2,7- and 2,3-diaminophenazine was obviously decreased with increases in p-PD, and it was concluded that the declined mutagenic potency of oxidized m-PD/p-PD or o-PD/p-PD was due to the decrease in diaminophenazines. But the formation of diaminophenazines was not completely inhibited by the addition of a 9-fold molar ratio of p-PD to m-PD or o-PD, 8.6 nmole of 2,7-diaminophenazine and 1882.4 nmole of 2,3-diaminophenazine were formed from 1 mmole of m-PD and o-PD, respectively.     

Studies on cellulases of a phytopathogenic fungus, Pyricularia oryzae cavara. II. Purification and properties of a beta-glucosidase.

Three components (GA, GB-1, and GB-2) of beta-glucosidase were detected in the culture filtrate of Pyricularia oryzae grown in a cellulose or cellulose derivative medium. Among them, GB-1 was induced most strongly. Purified GB-1 was homogeneous on polyacrylamide gel electrophoresis and showed an approximately 1,400-fold increase of specific activity over the starting material. The molecular weight was determined to be 240,000 by sodium dodecyl sulfate-gel electrophoresis. A similar value was also obtained by sucrose density gradient centrifugation. The enzyme contained a high proportion of acidic amino acids and mannose, and the isoelectric point of the enzyme was pH 4.15. The enzyme had a pH optimum of 5.5 and a temperature optimum at 55 degrees C. beta-Glucosidase activity was inhibited by Mn2+, Cu2+, Hg2+, p-chloromercuribenzoate, and glucono-delta-lactone. The enzyme split off glucose units one by one from the nonreducing ends of not only beta-glucooligosaccharides but also some beta-glucans, such as carboxymethylcellulose, laminaran, pustulan, and zeagallan. The affinity for cello- and laminari-oligosaccharides tended to increase in parallel with the chain length.     

Electrocardiographic findings in Mexican chagasic subjects living in high and low endemic regions of Trypanosoma cruzi infection

In México the first human chronic chagasic case was recognized in 1940. In spite of an increasing number of cases detected since that time, Chagas disease in México has been poorly documented. In the present work we studied 617 volunteers subjects living in high and low endemic regions of Trypanosoma cruzi infection with seroprevalence of 22% and 4% respectively. Hemoculture performed in those seropositive subjects failed to demonstrate circulating parasites, however polymerase chain reaction identified up to 60% of them as positives. A higher level of anti-T. cruzi antibodies was observed in seropositive residents in high endemic region, in spite of similar parasite persistence (p < 0.05). On standard 12 leads electrocardiogram (ECG) 20% to 22% seropositive individuals from either region showed right bundle branch block or ventricular extrasystoles which were more prevalent in seropositive than in seronegative individuals (p < 0.05). In conclusion, the frequency or type of ECG abnormality was influenced by serologic status but not by endemicity or parasite persistence. Furthermore, Mexican indeterminate patients have a similar ECG pattern to those reported in South America.     

Evolutionary dynamics of intratumor heterogeneity

Intraneoplastic diversity in human tumors is a widespread phenomenon of critical importance for tumor progression and the response to therapeutic intervention. Insights into the evolutionary events that control tumor heterogeneity would be a major breakthrough in our comprehension of cancer development and could lead to more effective prevention methods and therapies. In this paper, we design an evolutionary mathematical framework to study the dynamics of heterogeneity over time. We consider specific situations arising during tumorigenesis, such as the emergence of positively selected mutations ("drivers") and the accumulation of neutral variation ("passengers"). We perform exact computer simulations of the emergence of diverse tumor cell clones over time, and derive analytical estimates for the extent of heterogeneity within a population of cancer cells. Our methods contribute to a quantitative understanding of tumor heterogeneity and the impact of heritable alterations on this tumor trait.