MAPK signaling is involved in camptothecin-induced cell death

Camptothecin, a topoisomerase I inhibitor, is a well-known anticancer drug. However, its mechanism has not been well studied in human gastric cancer cell lines. Camptothecin induced apoptotic cell death in human gastric cancer cell line AGS. Z-VAD-fmk, pan-caspase inhibitor, blocked apoptotic phenotypes induced by camptothecin suggesting that caspases are involved in camptothecin-induced cell death. An inhibitor of caspase-6 or -8 or -9 did not prevent cell death by camptothecin. Various protease inhibitors failed to prevent camptothecin-induced cell death. These results suggest that only few caspases are involved in camptothecin-induced cell death. Camptothecin induced phosphorylation of ERK1/2, JNK, and p38 MAPK, in a dose and time-dependent manner in AGS. Z-VAD-fmk did not affect MAPK signaling induced by camptothecin suggesting that caspase signaling occurs downstream of MAPK signaling. Blocking of p38 MAPK, but not ERK1/2, resulted in partial inhibition of cell death and PARP cleavage by camptothecin in AGS. Taken together, MAPK signaling is associated with apoptotic cell death by camptothecin.     

An improved helper phage system for efficient isolation of specific antibody molecules in phage display

Phage display technology has been applied in many fields of biological and medical sciences to study molecular interactions and especially in the generation of monoclonal antibodies of human origin. However, extremely low display level of antibody molecules on the surface of phage is an intrinsic problem of a phagemid-based display system resulting in low success rate of isolating specific binding molecules. We show here that display of single-chain antibody fragment (scFv) generated with pIGT3 phagemid can be increased dramatically by using a genetically modified Ex-phage. Ex-phage has a mutant pIII gene that produces a functional wild-type pIII in suppressing Escherichia coli strains but does not make any pIII in non-suppressing E.coli strains. Packaging phagemids encoding antibody-pIII fusion in F⁺ non-suppressing E.coli strains with Ex-phage enhanced the display level of antibody fragments on the surfaces of recombinant phage particles resulting in an increase of antigen-binding reactivity >100-fold compared to packaging with M13KO7 helper phage. Thus, the Ex-phage and pIGT3 phagemid vector provides a system for the efficient enrichment of specific binding antibodies from a phage display library and, thereby, increases the chance of obtaining more diverse antibodies specific for target antigens.     

Differential identification of mycoplasma pulmonis and M. arthritidis using PCR-based RFLP.

Mycoplasma pulmonis and Mycoplasma arthritidis were differentially identified using PCR-restriction fragment length polymorphism (RFLP). A genus-specific sequence of mycoplasma was amplified by PCR and the PCR products were digested with the restriction enzyme SmaI. Each PCR product from the four isolates of M. pulmonis was digested with SmaI into two fragments; however, there was no digestion in the PCR product from M. arthritidis. This method might be useful to differentiate infection of M. pulmonis from that of M. arthritidis.     

Deubiquitinating enzymes: their diversity and emerging roles

A growing number of important regulatory proteins within cells are modified by conjugation of ubiquitin, a well-conserved 76-amino-acid polypeptide. The ubiquitinated proteins are targeted to proteasome for degradation or alternative metabolic fates, such as triggering of plasma membrane endocytosis and trafficking to vacuoles or lysosomes. Deubiquitination, reversal of this modification, is being recognized as an important regulatory step. Deubiquitinating enzymes are cysteine proteases that specifically cleave off ubiquitin from ubiquitin-conjugated protein substrates as well as from its precursor proteins. Genome sequencing projects have identified more than 90 deubiquitinating enzymes, making them the largest family of enzymes in the ubiquitin system. This review will concentrate on recent important findings as well as new insights into the diversity and emerging roles of deubiquitinating enzymes in the ubiquitin-dependent pathway.     

Purification and characterization of a serine protease with fibrinolytic activity from Tenodera sinensis (praying mantis)

Mantis egg fibrolase (MEF) was purified from the egg cases of Tenodera sinensis using ammonium sulfate fractionation, gel filtration on Bio-Gel P-60 and affinity chromatography on DEAE Affi-Gel blue gel. The protease was assessed homogeneous by SDS-polyacrylamide gel electrophoresis and has a molecular mass of 31500 Da. An isoelectric point of 6.1 was determined by isoelectric focusing. Amino acid sequencing of the N-terminal region established a primary structure composed of Ala-Asp-Val-Val-Gln-Gly-Asp-Ala-Pro-Ser. MEF readily digested the Aalpha- and Bbeta-chains of fibrinogen and more slowly the gamma-chain. The nonspecific action of the enzyme results in extensive hydrolysis of fibrinogen and fibrin releasing a variety of fibrinopeptide. The enzyme is inactivated by Cu2+ and Zn2+ and inhibited by PMSF and chymostatin, yet elastinal, aprotinin, TLCK, TPCK, EDTA, EGTA, cysteine, beta-mercaptoethanol, iodoacetate, E64, benzamidine and soybean trypsin inhibitor do not affect activity. Antiplasmin was not sensitive to MEF but antithrombin III inhibited the enzymatic activity of MEF. Among chromogenic protease substrates, the most sensitive to MEF hydrolysis was benzoyl-Phe-Val-Arg-p-nitroanilide with maximal activity at pH 7.0 and 30 degrees C. MEF preferentially cleaved the oxidized B-chain of insulin between Leu15 and Tyr16. D-Dimer concentrations increased on incubation of cross-linked fibrin with MEF, indicating the enzyme has a strong fibrinolytic activity.     

Synthesis of new oligosaccharides from raffinose by Aspergillus niger fructosyltransferase

Purified fructosyltransferase from Aspergillus niger exhibited transfructosylation activities, producing fructose, DP2, DP4, and DP5 from raffinose. The structures of two products synthesized from raffinose were identified as O--d-galactopyranosyl (16)--d-glucopyranose and O--d-galactopyranosyl (16)--d-glucopyranosyl-[O--d-fructofuranosyl (21)]--D-fructofuranoside, which means that C-2 hydroxyl group of fructose released from one raffinose molecule were linked to the C-1 hydroxyl group of fructose of another raffinose.     

Ca2+-dependent membrane currents in vascular smooth muscle cells of the rabbit.

The membrane potential in vascular smooth muscle cells contributes to the regulation of cytosolic [Ca2+], which in turn regulates membrane potential by means of Ca2+i-dependent ionic currents. We investigated the characteristics of Ca2+i-dependent currents in rabbit coronary and pulmonary arterial smooth muscle cells. Ca2+i-dependent currents were recorded using the whole-cell patch-clamp technique while cytosolic [Ca2+] was increased by caffeine. The reversal potentials of caffeine-induced currents were between -80 and -10 mV under normal ionic conditions, whereas they were about 0 mV when K+-free NaCl solutions were used both in pipette and bath. The total substitution of extracellular Na+ with membrane-impermeable cation N-Methyl-D-glucamine did not affect caffeine-induced currents, implying no significant contribution of Na+ as a permeant ion to the currents. The substitution of extracellular NaCl with sucrose reduced outward component of the currents and shifted the reversal potentials according to the change in Cl- equilibrium potential. Upon application of the niflumic acid under K+-free conditions, most of the current induced by caffeine was inhibited. Taken together, the results of the present study indicate that K+ and Cl- currents are major components of Ca2+i-dependent currents in vascular smooth muscles isolated from coronary and pulmonary arteries of the rabbit, and the relative contribution of each type of current to total currents are not different between the two arteries.     

Effect of irradiation on cytokine secretion and nitric oxide production by inflammatory macrophages

This study explored the effects of low-dose and high-dose irradiation on inflammatory macrophage cells, specifically inflammatory cytokine secretion and nitric oxide (NO) production after irradiation. To elucidate the effect of irradiation on active and inactive macrophages, we exposed LPS-treated or untreated murine monocyte/macrophage RAW 264.7 cell lines to low-dose to high-dose radiation (0.01–10 Gy). We analyzed the effects of irradiation on RAW 264.7 cell proliferation by MTT assays and analyzed cytokine secretion and NO production related to inflammation by ELISA assays. Low-to-high doses of radiation did not significantly affect the proliferation of LPS-treated or untreated RAW 264.7 cells. Pro-inflammatory cytokine IL-1ß was generally increased in RAW 264.7 cells at 3 days after radiation. Especially, IL-1ß was significantly increased in only high dose-irradiation (2 and 10 Gy irradiation) groups in LPS-untreated RAW 264.7 cells but increased in both low and high dose-irradiation groups (0.01–10 Gy) in LPS-treated RAW 264.7 cells at 3 days after irradiation. Whereas, the expression of IL-1ß was prolonged in high-dose irradiation group at 5 days after irradiation. The production of anti-inflammatory cytokine IL-10 did not change significantly at 3 days after radiation but was significantly reduced at 5 days after 10 Gy radiation. The effect of irradiation on the secretion of IL-1ß and IL-10 was not significantly different between RAW 264.7 cells treated or not treated with LPS. The effect of irradiation on NO secretion by RAW 264.7 cells showed a specific pattern. NO was produced after low-dose irradiation but reduced in a high-dose irradiation group at 3 days after irradiation. However, NO production was not changed after low-dose irradiation and reduced at 5 days after high-dose irradiation. These results showed that irradiation affected the inflammatory system and regulated NO production in both activated and inactivated macrophages through different regulation mechanisms, depending on irradiation dose.