Molecular Marker Identification for Relapse Prediction in 5-FU-Based Adjuvant Chemotherapy in Gastric and Colorectal Cancers

To confirm the clinical significance of NF-κB and JNK protein expression from experimentally identified candidates for predicting prognosis for patients with 5-FU treatment, we evaluated the protein expression of surgically removed specimens. A total of 79 specimens were obtained from 30 gastric and 49 colorectal cancer patients who underwent R0 resection followed by postoperative 5-FU based adjuvant chemotherapy. Immunohistochemical examinations of NF-κB and JNK on tissue microarrays (TMAs) revealed that significantly shorter time-to-relapse (TTR) in both NF-κB(+) and JNK(−) subgroups in both gastric (NF-κB(+), p = 0.0002, HR11.7. 95%CI3 3.2–43.4; JNK(−), p = 0.0302, HR4.4, 95%CI 1.2–16.6) and colon (NF-κB(+), p = 0.0038, HR36.9, 95%CI 3.2–426.0; JNK(−), p = 0.0098, HR3.2, 95%CI 1.3–7.7) cancers. These protein expression patterns also show strong discriminately power in gastric cancer patients for overall survival rate, suggesting a potential utility as prognostic or chemosensitivity markers. Baseline expression of these proteins using gastric cancer cell lines demonstrated the reciprocal patterns between NF-κB and JNK, while 5-FU exposure of these cell lines only induced NF-κB, suggesting that NF-κB plays a dominant role in the response to 5-FU. Subsequent siRNA experiments confirmed that gene knockdown of NF-κB increased 5-FU-specific sensitivity, whereas that of JNK did not affect the chemosensitivity. These results suggest that the expression of these proteins may aid in the decisions involved with adjuvant chemotherapy for gastrointestinal tract cancers.     

Length and structural effect of signal peptides derived from Bacillus subtilis alpha-amylase on secretion of Escherichia coli beta-lactamase in B. subtilis cells.

The precursor of Bacillus subtilis alpha-amylase contains an NH2-terminal extension of 41 amino acid residues as the signal sequence. The E. coli beta-lactamase structural gene was fused with the DNA for the promoter and signal sequence regions. Activity of beta-lactamase was expressed and more than 95% of the activity was secreted into the culture medium. DNA fragments coding for short signal sequences 28, 31, and 33 amino acids from the initiator Met were prepared and fused with the beta-lactamase structural gene. The sequences of 31 and 33 amino acid residues with Ala COOH-terminal amino acid were able to secrete active beta-lactamase from B. subtilis cells. However beta-lactamase was not secreted into the culture medium by the shorter signal sequence of 28 amino acid residues, which was not cleaved. Molecular weight analysis of the extracellular and cell-bound beta-lactamase suggested that the signal peptide of B. subtilis alpha-amylase was the first 31 amino acids from the initiator Met. The significance of these results was discussed in relation to the predicted secondary structure of the signal sequences.     

Induction of histidine decarboxylase activity in the spleen of mice treated with staphylococcal enterotoxin A and demonstration of its non-mast cell origin

Histidine decarboxylase (HDC) activity increased 13-, 7-, and 2-fold in the spleen, lung and liver, respectively, but not in other tissues of C57BL/6 mice injected i.v. with 50 micrograms/kg of Staphylococcal enterotoxin A (SEA). But even in the spleen, increase in the histamine level was only 1.5 times that of untreated mice. In genetically mast cell-deficient WBB6F1 - W/Wv mice HDC activity in the spleen increased to the same extent as in wild type WBB6F1 - +/+ mice on SEA treatment, but the histamine level in the spleen also increased 20-fold, whereas it increased only 1.4-fold in +/+ mice. These results suggest that the increases in HDC and histamine resulted from interaction of SEA with non-mast cells in tissues.     

The Expression of Fn14 via Mechanical Stress-activated JNK Contributes to Apoptosis Induction in Osteoblasts

Bone mass is maintained by the balance between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. It is well known that adequate mechanical stress is essential for the maintenance of bone mass, whereas excess mechanical stress induces bone resorption. However, it has not been clarified how osteoblasts respond to different magnitudes of mechanical stress. Here we report that large-magnitude (12%) cyclic stretch induced Ca²⁺ influx, which activated reactive oxygen species generation in MC3T3-E1 osteoblasts. Reactive oxygen species then activated the ASK1-JNK/p38 pathways. The activated JNK led to transiently enhanced expression of FGF-inducible 14 (Fn14, a member of the TNF receptor superfamily) gene. Cells with enhanced expression of Fn14 subsequently acquired sensitivity to the ligand of Fn14, TNF-related weak inducer of apoptosis, and underwent apoptosis. On the other hand, the ASK1-p38 pathway induced expression of the monocyte chemoattractant protein 3 (MCP-3) gene, which promoted chemotaxis of preosteoclasts. In contrast, the ERK pathway was activated by small-magnitude stretching (1%) and induced expression of two osteogenic genes, collagen Ia (Col1a) and osteopontin (OPN). Moreover, activated JNK suppressed Col1a and OPN induction in large-magnitude mechanical stretch-loaded cells. The enhanced expression of Fn14 and MCP-3 by 12% stretch and the enhanced expression of Col1a and OPN by 1% stretch were also observed in mouse primary osteoblasts. These results suggest that differences in the response of osteoblasts to varying magnitudes of mechanical stress play a key role in switching the mode of bone metabolism between formation and resorption.     

Novel benzo-fused lactam scaffolds as factor Xa inhibitors.

Rigid benzolactam P3-P2 dipeptide mimics were designed and prepared as potential inhibitors of blood coagulation factor Xa. Methoxy substitution of the tetrahydrobenzazepinone scaffold led to potent and selective inhibitors. The synthesis and biological activities of these derivatives are reported herein.     

The ventilatory threshold gives maximal lactate steady state

The purpose of this study was to examine whether the ventilatory threshold (Thv) would give the maximal lactate steady state ([la]ss, max), which was defined as the highest work rate (W) attained by a subject without a progressive increase in blood lactate concentration [la]b at constant intensity exercise. Firstly, 8 healthy men repeated ramp-work tests (20 W.min-1) on an electrically braked cycle ergometer on different days. During the tests, alveolar gas exchange was measured breath-by-breath, and the W at Thv (WThv) was determined. The results of two-way ANOVA showed that the coefficient of variation of a single WThv determination was 2.6%. Secondly, 13 men performed 30-min exercise at WThv (Thv trial) and at 4.9% above WThv (Thv + trial), which corresponded to the 95% confidence interval of the single determination. The [la]b was measured at 15 and 30 min from the onset of exercise. The [la]b at 15 min (3.15 mmol.l-1, SEM 0.14) and at 30 min (2.95 mmol.l-1, SEM 0.18) were not significantly different in Thv trial. However, the [la]b of Thv + trial significantly increased (P less than 0.05) from 15 min (3.62 mmol.l-1, SEM 0.36) to 30 min (3.91 mmol.l-1, SEM 0.40). These results indicate that Thv gives the [la]ss, max, at which one can perform sustained exercise without continuous [la]b accumulation.     

MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences

The Molecular Evolutionary Genetics Analysis (MEGA) software is a desktop application designed for comparative analysis of homologous gene sequences either from multigene families or from different species with a special emphasis on inferring evolutionary relationships and patterns of DNA and protein evolution. In addition to the tools for statistical analysis of data, MEGA provides many convenient facilities for the assembly of sequence data sets from files or web-based repositories, and it includes tools for visual presentation of the results obtained in the form of interactive phylogenetic trees and evolutionary distance matrices. Here we discuss the motivation, design principles and priorities that have shaped the development of MEGA. We also discuss how MEGA might evolve in the future to assist researchers in their growing need to analyze large data set using new computational methods.