Mechanism of taxol-induced apoptosis in human SKOV3 ovarian carcinoma cells

Taxol is extensively used clinically for chemotherapy of patients with ovarian, breast, and lung cancer. Although taxol induces apoptosis of cancer cells, its exact mechanism of action is not yet known. To determine the mechanism of action of taxol in ovarian cancer, we tested the effects of the drug, on the human ovarian carcinoma cell line, SKOV3. We observed that taxol-induced apoptosis of these cells by phosphatidylserine (PS) externalization and DNA fragmentation. While treatment of cells with taxol resulted in bcl-2 phosphorylation and mitochondrial depolarization, cytochrome c was not released and pro-caspase-3 was not activated. Treatment of SKOV3 cells with taxol, however, resulted in the translocation of AIF from the mitochondria to the nucleus via the cytosol. Taken together, these findings suggest that in SKOV3 cells, taxol induces caspase-independent AIF-dependent apoptosis.     

Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function

Although a great deal is known biochemically about peroxiredoxins (Prxs), little is known about their real physiological function. We show here that two cytosolic yeast Prxs, cPrxI and II, which display diversity in structure and apparent molecular weights (MW), can act alternatively as peroxidases and molecular chaperones. The peroxidase function predominates in the lower MW forms, whereas the chaperone function predominates in the higher MW complexes. Oxidative stress and heat shock exposure of yeasts causes the protein structures of cPrxI and II to shift from low MW species to high MW complexes. This triggers a peroxidase-to-chaperone functional switch. These in vivo changes are primarily guided by the active peroxidase site residue, Cys(47), which serves as an efficient "H(2)O(2)-sensor" in the cells. The chaperone function of these proteins enhances yeast resistance to heat shock.     

Design and synthesis of 3'-ureidoadenosine-5'-uronamides: effects of the 3'-ureido group on binding to the A3 adenosine receptor

On the basis of high binding affinity at the A(3) adenosine receptor of 3'-aminoadenosine derivatives with hydrogen bonding donor ability, novel 3'-ureidoadenosine analogues were synthesized from 1,2:5,6-di-O-isopropylidene-d-glucose in order to lead to stronger hydrogen bonding than the corresponding 3'-aminoadenosine derivatives. However, the synthesized 3'-ureidoadenosine analogues were totally devoid of binding affinity, because 3'-urea moiety caused steric and electrostatic repulsions at the binding site of the A(3) adenosine receptor, leading to conformational distortion.     

Proteomic analysis revealed a strong association of a high level of alpha1-antitrypsin in gastric juice with gastric cancer

To investigate the pathology of gastric disorders, we compared the proteomic patterns of gastric juice from patients with various gastric disorders. In healthy subjects pepsin A, pepsin B and gastric lipase were the major proteins detected by two-dimensional gel electrophoresis. These digestive enzymes were not detected in 60% of gastric cancer cases (18 out of 30 analyzed cases). Interestingly, an extraordinary amount of alpha(1)-antitrypsin was observed in these cases. In contrast to gastric cancer cases, alpha(1)-antitrypsin was detected in only 5% of patients (three out of 56) with chronic atrophic gastritis, and the detection frequency went up as the disease developed (one of four intestinal metaplasia cases, two of seven tubular adenoma cases, a single examined case of hyperplastic polyp and 60% of gastric cancer). Zymography showed that a 60 kDa protease strongly associated with alpha(1)-antitrypsin and mass spectrometric analysis revealed that the gastric alpha(1)-antitrypsin was a protease-cleaved form. Our data suggest that alpha(1)-antitrypsin and 60 kDa protease may serve as good diagnostic and prognostic markers for conditions associated with gastric cancer.     

Enhancing effect of low culture temperature on specific antibody productivity of recombinant Chinese hamster ovary cells: clonal variation

To understand the different responses of recombinant Chinese hamster ovary (rCHO) cells to low culture temperature regarding specific productivity (q), 12 parental clones and their corresponding amplified clones producing a humanized antibody were cultivated at 32 and 37 degrees C. The specific growth rate of all clones, including both parental and amplified clones, decreased by 30-63% at 32 degrees C, compared to rates at 37 degrees C. In contrast, their specific antibody productivity (qAb) was significantly enhanced at 32 degrees C. Furthermore, the degree of qAb enhancement at 32 degrees C varied a lot from 4- to 25-fold among the parental clones. At 32 degrees C, most of the amplified clones, regardless of methotrexate (MTX) levels, also showed enhanced qAb but to a lesser extent than their parental clones. However, clone 14 amplified at 0.32 microM MTX (clone 14-0.32) and clone 20 amplified at 1 microM MTX (clone 20-1.00), unlike their parental clones, did not show enhanced qAb at 32 degrees C. Thus, it was found that the enhancing effect of low culture temperature on q of rCHO cells depends on clones. Taken together, the results obtained here emphasize the importance of clonal selection for the successful application of low culture temperature to the enhanced foreign protein production in rCHO cells.     

Description of characteristics of humic substances from different waste materials

Humic acids (HAs) extracted from different organic wastes have been characterised by chemical methods. The chemical properties of HAs showed differences depending on the source from which they were obtained. The C content in HAs from organic wastes (41.1-63.2%) fluctuated around the C value in soil HA with the exception of composted bark and tobacco dust. Compared with soil HA, the N contents of HAs from sewage sludge and brewery sludge were found much higher than the others. E4:E6 ratios for HAs in organic wastes were generally greater than that for soil HA, which indicated a low degree of condensation and humification. The carboxyl and phenolic-OH group contents ranged 0.51-2.23 and 11.1-20.7 meq g(-1), respectively. High values of carboxyl and phenolic-OH contents indicated that these materials were still within early stages of humification.     

The water-soluble fraction of bee venom produces antinociceptive and anti-inflammatory effects on rheumatoid arthritis in rats

We recently demonstrated that bee venom (BV) injection into the Zusanli acupoint produced a significantly more potent anti-inflammatory and antinociceptive effect than injection into a non-acupoint in a Freund's adjuvant induced rheumatoid arthritis (RA) model. However, the precise BV constituents responsible for these antinociceptive and/or anti-inflammatory effects are not fully understood. In order to investigate the possible role of the soluble fraction of BV in producing the anti-arthritic actions of BV acupuncture, whole BV was extracted into two fractions according to solubility (a water soluble fraction, BVA and an ethylacetate soluble fraction, BVE) and the BVA fraction was further tested.Subcutaneous BVA injection (0.9 mg/kg/day) into the Zusanli acupoint was found to dramatically inhibit paw edema and radiological change (i.e. new bone proliferation and soft tissue swelling) caused by Freund's adjuvant injection. BVA treatment also reduced the increase in serum interleukin-6 caused by RA induction to levels observed in non-arthritic animals. In addition, BVA therapy significantly reduced arthritis-induced nociceptive behaviors (i.e. nociceptive scores for mechanical hyperalgesia and thermal hyperalgesia). Finally, BVA treatment significantly suppressed adjuvant-induced Fos expression in the lumbar spinal cord at 3 weeks post-adjuvant injection. In contrast, BVE treatment (0.05 mg/kg/day) failed to show any anti-inflammatory or antinociceptive effects on RA.The results of the present study demonstrate that BVA is the effective fraction of whole BV responsible for the antinociception and anti-inflammatory effects of BV acupuncture treatment. Thus it is recommended that this fraction of BV be used for long-term treatment of RA-induced pain and inflammation. However, further study is necessary to clarify which constituents of the BVA fraction are directly responsible for these anti-arthritis effects.     

Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library

Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.     

ProNGF induces p75-mediated death of oligodendrocytes following spinal cord injury

The neurotrophin receptor p75 is induced by various injuries to the nervous system, but its role after injury has remained unclear. Here, we report that p75 is required for the death of oligodendrocytes following spinal cord injury, and its action is mediated mainly by proNGF. Oligodendrocytes undergoing apoptosis expressed p75, and the absence of p75 resulted in a decrease in the number of apoptotic oligodendrocytes and increased survival of oligodendrocytes. ProNGF is likely responsible for activating p75 in vivo, since the proNGF from the injured spinal cord induced apoptosis among p75(+/+), but not among p75(-/-), oligodendrocytes in culture, and its action was blocked by proNGF-specific antibody. Together, these data suggest that the role of proNGF is to eliminate damaged cells by activating the apoptotic machinery of p75 after injury.