Soybean curd refuse alleviates experimental tumorigenesis in rat colon

Adult Fischer-344 rats which underwent administration of azoxymethane were fed diets containing soybean curd refuse (SCR) or a high-molecular-weight fraction of soy protein digest (HMF), or Hammarsten casein (CAS) as a protein source over a period of 34 weeks. All the living rats of each group at 22, 28 or 34 weeks were endoscopically inspected for tumor incidence in the colon. SCR turned out to be comparable to HMF in anti-tumorigenicity, or rather better than HMF.     

Cell membrane dynamics and the induction of apoptosis by lipid compounds

To investigate the induction of apoptosis by some lipid compounds which are a potent inducer of apoptosis, the plasma membrane fluidity of U937 cells was measured using the fluorescent probe, pyrene. The increase of the membrane fluidity was observed immediately after the treatment of cells with lipid inducers. We also found that the trigger of apoptosis was pulled within 30 min after treatment. Data from the dynamic light scattering experiment indicated that lipid inducers were dissolved to form the emulsion. At the very early stage of apoptosis, possibly, the well-controlled transfer of lipid inducers from the emulsion to the lipid layer of cells can bring about the increase of membrane dynamics which might lead to the induction of apoptosis.     

No braiding of Holliday junctions in positively supercoiled DNA molecules

The Holliday junction is a prominent intermediate in genetic recombination that consists of four double helical arms of DNA flanking a branch point. Under many conditions, the Holliday junction arranges its arms into two stacked domains that can be oriented so that genetic markers are parallel or antiparallel. In this arrangement, two strands retain a helical conformation, and the other two strands effect the crossover between helical domains. The products of recombination are altered by a crossover isomerization event, which switches the strands fulfilling these two roles. It appears that effecting this switch from the parallel conformation by the simplest mechanism results in braiding the crossover strands at the branch point. In previous work we showed by topological means that a short, parallel, DNA double crossover molecule with closed ends did not braid its branch point; however, that molecule was too short to adopt the necessary positively supercoiled topology. Here, we have addressed the same problem using a larger molecule of the same type. We have constructed a parallel DNA double crossover molecule with closed ends, containing 14 double helical turns in each helix between its crossover points. We have prepared this molecule in a relaxed form by simple ligation and in a positively supercoiled form by ligation in the presence of netropsin. The positively supercoiled molecule is of the right topology to accommodate braiding. We have compared the relaxed and supercoiled versions for their responses to probes that include hydroxyl radicals, KMnO4, the junction resolvases endonuclease VII and RuvC, and RuvC activation of KMNO4 sensitivity. In no case did we find evidence for a braid at the crossover point. We conclude that Holliday junctions do not braid at their branch points, and that the topological problem created by crossover isomerization in the parallel conformation is likely to be solved by distributing the stress over the helices that flank the branch point.     

Production of soluble granulocyte colony-stimulating factor receptors from myelomonocytic cells

It has been speculated that a soluble form of G-CSFR might be physiologically present in humans, since G-CSFR mRNA that lacks a transmembrane domain has been identified from a human myelomonocytic cell line. Here, we demonstrate human soluble G-CSFR (sG-CSFR) of two different molecular sizes (80 and 85 kDa) on an immunoblot analysis using Abs generated against the amino-terminal, extracellular domain of the full-length G-CSFR. Both isoforms of sG-CSFR were able to bind recombinant human G-CSF (rhG-CSF). RT-PCR analysis with primers targeted outside of the transmenbrane region revealed that membrane-anchored G-CSFR is expressed at all maturation stages of purified myeloid cells, including CD34+CD13+ cells (blasts), CD11b-CD15+ cells (promyelocytes or myelocytes), CD11b+CD15+ cells (metamyelocytes and mature neutrophils), and CD14+ cells (monocytes). On the other hand, sG-CSFR mRNA was detectable in CD11b-CD15+, CD11b+CD15+, and CD14+ cells, but not in the CD34+CD13+ blast population. The serum concentration of both isoforms of sG-CSFR appeared to be correlated with the numbers of neutrophils/monocytes before and after rhG-CSF treatment in normal individuals. Thus, two isoforms of sG-CSFR are physiologically secreted from relatively mature myeloid cells and might play an important role in myelopoiesis through their binding to serum G-CSF.     

High efficient expression of the functional ligand binding site of the inositol 1,4,5-triphosphate receptor in Escherichia coli

Type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), an inositol 1, 4,5-trisphosphate (IP3)-gated Ca2+ release channel, binds IP3 within the N-terminal ligand-binding region. Here we report an improved Escherichia coli expression system in which large amounts of the IP3 binding sites could be efficiently produced as soluble active proteins. We have found that the structures of IP3 binding constructs expressed in E. coli significantly affect their production as soluble protein. Residues 1-604 (T604), which contain the putative protein folding units, yielded about 4.6% of the total soluble fraction. As a result, soluble active T604 would be 19 mg per liter of culture. The affinity for IP3 of T604 (Kd = 45 nM) is comparable to that of the native IP3R1, whereas that of an R441Q mutant is much higher (8.1 nM). This system should provide an invaluable and powerful means to unveil the molecular recognition of IP3R1 for IP3.     

Ligation errors in DNA computing

DNA computing is a novel method of computing proposed by Adleman (1994), in which the data is encoded in the sequences of oligonucleotides. Massively parallel reactions between oligonucleotides are expected to make it possible to solve huge problems. In this study, reliability of the ligation process employed in the DNA computing is tested by estimating the error rate at which wrong oligonucleotides are ligated. Ligation of wrong oligonucleotides would result in a wrong answer in the DNA computing. The dependence of the error rate on the number of mismatches between oligonucleotides and on the combination of bases is investigated.     

Inhibition of sphingomyelinase activity helps to prevent neuron death caused by ischemic stress

Magnesium-dependent neutral sphingomyelinase (N-SMase) present in plasma membranes is an enzyme that can be activated by stress in the form of inflammatory cytokines, serum deprivation, and hypoxia. The design of small molecule N-SMase inhibitors may offer new therapies for the treatment of inflammation, ischemic injury, and cerebral infarction. Recently, we synthesized a series of difluoromethylene analogues (SMAs) of sphingomyelin. We report here the effects of SMAs on the serum/glucose deprivation-induced death of neuronally differentiated pheochromocytoma (PC-12) cells and on cerebral infarction in mice. SMAs inhibited the enhanced N-SMase activity in the serum/glucose-deprived PC-12 cells, and thereby suppressed the apoptotic sequence: ceramide formation, c-Jun N-terminal kinase phosphorylation, caspase-3 activation, and DNA fragmentation in the nuclei. Administration of SMA-7 (10 mg/kg i.v.) with IC50= 3.3 microM to mice whose middle cerebral arteries were occluded reduced significantly the size of the cerebral infarcts, compared to the control mice. These results suggest that N-SMase is a key component of the signaling pathways in cytokine- and other stress-induced cellular responses, and that inhibiting or stopping N-SMase activity is an important strategy to prevent neuron death from ischemia.