Construction of a gorilla fosmid library and its PCR screening system

A gorilla fosmid library of 261,120 independent clones was constructed and characterized. The fosmid vector is similar to the cosmid in average insert size of ca. 40 kb but contains the F factor for replication, and it is more resistant to recombination. This clone library represents about 3.7 times coverage of the gorilla genome. A simple screening system by PCR was established, and we successfully found 9 clones that cover the entire Hox A gene cluster of the gorilla genome. This gorilla fosmid DNA library is a useful resource for comparative genomics of human and apes.     

Analysis of alpha-amylase-derived pyridylamino-dextran sulfate oligomers by the combination of size-exclusion and reversed-phase high-performance liquid chromatography

In a previous study, we reported a novel method for the separation and quantification of a strong negatively charged material, dextran sulfate sodium (DSS), using fluorometric labeling with 2-aminopyridine and size-exclusion high-performance liquid chromatography. In the present study, we developed a method for the separation of pyridylamino-DSS (PA-DSS) using reversed-phase high-performance liquid chromatography (RPLC). In vitro enzymatic degradation of the PA-DSS was carried out using alpha-amylase. In RPLC, depolymerized PA-DSS was eluted on the basis of molecular mass (in the order pentamer, trimer, dimer, and monomer of PA-DSS) and separations were more sharply than in size-exclusion chromatography. The combination of RPLC and size-exclusion chromatography also separated depolymerized PA-DSS as effectively as RPLC alone.     

Differential transduction mechanisms underlying NaCl- and KCl-induced responses in mouse taste cells

The transduction mechanism of salt-induced responses of mouse taste cells was investigated using the patch clamp and the local stimulation techniques under quasi-natural conditions. Apically applied NaCl induced a voltage-independent current, which was partially suppressed by amiloride and Cd2+. In contrast, apically applied 0.5 M KCl induced an inwardly rectifying current (KCl-induced Iir). The KCl-induced Iir was unaffected by amiloride. The Iir was suppressed not only by external Ba2+ and Cs+, but also by a Cl- channel blocker, niflumic acid. The Er of the KCl-induced response was independent of the apical ionic concentration, but rather was close to the equilibrium potential of Cl- (E(Cl)) at the basolateral membrane. The KCl-induced Iir displayed a fast run-down under the conditions of the conventional whole cell clamp method, but not under the perforated patch conditions. Immunohistochemical localization of an inwardly rectifying Cl- channel protein, ClC-2, was observed in taste bud cells of the fungiform papillae. It is concluded that the transduction mechanism of NaCl-induced responses is completely different from that of KCl-induced responses in mouse taste cells.     

Bile acid modulates transepithelial permeability via the generation of reactive oxygen species in the Caco-2 cell line

The barrier functions in epithelial and endothelial cells seem to be very important for maintaining normal biological homeostasis. However, it is unclear whether or how bile acids affect the epithelial barrier. We examined the bile acid-induced disruption of the epithelial barrier. We measured the transepithelial electrical resistance (TEER) of Caco-2 cells as a marker of disruption of the epithelial barrier. Reactive oxygen species (ROS) generation was also measured. Cholic acid (CA) decreased the TEER and increased intracellular ROS generation. PLA2 (phospholipase A2), COX (cyclooxygenase), PKC (protein kinase), ERK 1/2 (extracellular signal-regulated kinase 1/2), PI 3 K (phosphatidylinositol 3-kinase), p38 MAPK (p38 mitogen-activated protein kinase), MLCK (myosin light-chain kinase), NADH dehydrogenase, and XO (xanthine oxidase) inhibitors or ROS scavengers prevented the CA-induced TEER decrease. PLA2, COX, PKC, NADH dehydrogenase, and XO inhibitors prevented the CA-induced ROS generation but not ERK 1/2, PI 3 K, p38 MAPK, and MLCK inhibitors. If the cells were treated with ROS generators such as superoxide dismutase, the TEER decreased. ERK 1/2, PI 3 K, p38 MAPK, and MLCK inhibitors prevent these ROS generators from inducing the TEER decrease. These results suggest that ROS play an important role. In addition, PLA2, COX, PKC, NADH dehydrogenase, and XO are located upstream of the ROS generation, but ERK 1/2, PI 3 K, p38 MAPK, and MLCK are downstream during the signaling of CA-induced TEER alterations.     

Process development and economic evaluation of recombinant human lactoferrin expressed in rice grain

In this paper, we show that recombinant human lactoferrin (rhLF) has been stably expressed at 0.5% brown rice flour weight for nine generations. Process development indicates that rhLF can be efficiently extracted from rice flour in 20 mM phosphate buffer (pH 7.0) containing up to 0.5 M NaCl and at a ratio of 1 kg flour to 10 L buffer. After solid/liquid separation, the extract can then be loaded directly onto an ion-exchange column and rhLF can be eluted using 0.8 M NaCl. The resulting rhLF is about 95 pure. A range of biochemical and biophysical analyses were carried out and results indicated that the purified rhLF was identical to its native human counterpart other than its glycosylation. Economic analysis shows that at 600 kg/year scale, the cash cost to produce 1 g of rhLF of pharmaceutical grade is US$ 5.90. Analysis also indicates that the expression level has profound impact on costs related to planting, milling, extraction and purification, thus high level expression of recombinant protein in plants is one of the key parameters for the success of plant made pharmaceuticals.     

Isolation and proteomic characterization of human Parvulin-associating preribosomal ribonucleoprotein complexes

Human parvulin (hParvulin; Par14/EPVH) belongs to the third family of peptidylprolyl cis-trans isomerases that exhibit an enzymatic activity of interconverting the cis-trans conformation of the prolyl peptide bond, and shows sequence similarity to the regulator enzyme for cell cycle transitions, human Pin1. However, the cellular function of hParvulin is entirely unknown. Here, we demonstrate that hParvulin associates with the preribosomal ribonucleoprotein (pre-rRNP) complexes, which contain preribosomal RNAs, at least 26 ribosomal proteins, and 26 trans-acting factors involved in rRNA processing and assembly at an early stage of ribosome biogenesis. Since an amino-terminal domain of hParvulin, which is proposed to be a putative DNA-binding domain, was alone sufficient to associate in principle with the pre-rRNP complexes, the association is probably through protein-RNA interaction. In addition, hParvulin co-precipitated at least 10 proteins not previously known to be involved in ribosome biogenesis. Coincidentally, most of these proteins are implicated in regulation of microtubule assembly or nucleolar reformation during the mitotic phase of the cell. Thus, these results, coupled with the preferential nuclear localization of hParvulin, suggest that hParvulin may be involved in ribosome biogenesis and/or nucleolar re-assembly of mammalian cells.     

Extracellular signal-regulated kinases 1 and 2 participate in interleukin-17 plus tumor necrosis factor-alpha-induced stabilization of interleukin-6 mRNA in human pancreatic myofibroblasts

In human pancreatic myofibroblasts, interleukin (IL)-17 markedly enhances tumor necrosis factor (TNF)-alpha-induced IL-6 secretion through the induction of IL-6 mRNA stabilization. Induced stability of IL-6 mRNA was markedly decreased by the inhibitors of extracellular signal-regulated kinase (ERKs), PD98059 and U0216. This indicates that activation of the ERK pathway is involved in the induction of IL-6 mRNA stabilization by IL-17 plus TNF-alpha.     

Differential activation of yeast adenylate cyclase by wild-type and mutant RAS proteins

In these experiments we demonstrate that purified RAS proteins, whether derived from the yeast RAS1 or RAS2 or the human H-ras genes, activate yeast adenylate cyclase in the presence of guanine nucleotides. These results confirm the prediction of earlier genetic and biochemical data and for the first time provide a complete biochemical assay for RAS protein function. Furthermore, we observe a biochemical difference between the RAS2 and RAS2val19 proteins in their ability to activate adenylate cyclase after preincubation with GTP.