Na+/Mg2+ transporter acts as a Mg2+ buffering mechanism in PC12 cells

Mg(2+) buffering mechanisms in PC12 cells were demonstrated with particular focus on the role of the Na(+)/Mg(2+) transporter by using a newly developed Mg(2+) indicator, KMG-20, and also a Na(+) indicator, Sodium Green. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a protonophore, induced a transient increase in the intracellular Mg(2+) concentration ([Mg(2+)](i)). The rate of decrease of [Mg(2+)](i) was slower in a Na(+)-free extracellular medium, suggesting the coupling of Na(+) influx and Mg(2+) efflux. Na(+) influxes were different for normal and imipramine- (a putative inhibitor of the Na(+)/Mg(2+) transporter) containing solutions. FCCP induced a rapid increase in [Na(+)](i) in the normal solution, while the increase was gradual in the imipramine-containing solution. The rate of decrease of [Mg(2+)](i) in the imipramine-containing solution was also slower than that in the normal solution. From these results, we show that the main buffering mechanism for excess Mg(2+) depends on the Na(+)/Mg(2+) transporter in PC12 cells.     

Remarkable stabilization of neutrophil NADPH oxidase using RacQ61L and a p67phox-p47phox fusion protein

Activation of the phagocyte NADPH oxidase occurs via assembly of cytosolic p47(phox), p67(phox), and Rac with the membrane-bound flavocytochrome b(558). Recently, we have found that p67(phox)-(1-210) (p67N) fused with p47(phox)-(1-286) (p47N) or with Rac efficiently stabilizes the oxidase in a cell-free reconstitution system. In an attempt to further stabilize the oxidase, we herein used a constitutively active Rac, RacQ61L, and examined its effect on the oxidase stability. The half-life (t(1/2)) of the activity reconstituted with wild-type Rac was 12 min at 37 degrees C, which was extended 6-fold by RacQ61L. Also, the stability of the oxidase without p47(phox) increased 8-fold using RacQ61L. RacQ61L had a higher affinity for the complex than wild-type Rac and increased the affinity of p67N for the complex. Far-western blotting showed an enhanced binding between RacQ61L and p67N. The oxidase was stabilized by nanomolar FAD, and RacQ61L lowered the FAD concentration required. The combination of RacQ61L and a fusion protein consisting of p67N and p47N produced an extremely stable enzyme (t(1/2) = 184 min at 37 degrees C). The effectiveness of RacQ61L and fusion proteins on stabilization was in the following order: p67N-Rac < p67N + RacQ61L < or = p67N-RacQ61L < p67N-p47N + RacQ61L. These results indicate that a tightly bound ternary complex of p67(phox), Rac, and p47(phox) is very effective in maintaining the oxidase and confirm that the longevity of the activated state requires continuous association of these components. This simple and efficient method of stabilization may provide a useful tool to elucidate the nature of the activated oxidase.     

Reactive oxygen species generation is independent of de novo sphingolipids in apoptotic photosensitized cells

Our recent studies have shown that the de novo sphingolipids play a role in apoptosis of photosensitized cells. To elucidate the involvement of the de novo sphingolipids in reactive oxygen species (ROS) production and mitochondrial depolarization during apoptosis, the stress inducer photodynamic therapy (PDT) with the photosensitizer Pc 4 was used. In Jurkat cells PDT-triggered ROS production or mitochondrial membrane potential (deltapsi(m)) loss was not prevented by the de novo sphingolipid synthesis inhibitor ISP-1. However, PDT + C16-ceramide led to enhanced mitochondrial depolarization and DEVDase activation. The superoxide dismutase mimic manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) protected Jurkat cells from ROS generation and apoptosis, but not from deltapsi(m) reduction. Sphinganine or C16-ceramide counteracted MnTBAP-induced protection from apoptosis in Jurkat, as well as CHO cells. In LY-B cells, CHO-derived mutants deficient in serine palmitoyltransferase (SPT) activity and the de novo sphingolipid synthesis, mitochondrial depolarization, but not ROS generation, was suppressed post-PDT. In LY-B cells transfected with the SPT component LCB1, deltapsi(m) collapse post-PDT was restored. The data support the following hypotheses: MnTBAP protects against apoptosis via steps downstream of deltapsi(m) loss; de novo sphingolipids are not required for ROS generation, but can play a role in deltapsi(m) dissipation in photosensitized apoptotic cells.     

Osteogenic differentiation of the mesenchymal progenitor cells, Kusa is suppressed by Notch signaling

Notch receptor plays a crucial role in proliferation and differentiation of many cell types. To elucidate the function of Notch signaling in osteogenesis, we transfected the constitutively active Notch1 (Notch intracellular domain, NICD) into two different osteoblastic mesenchymal cell lines, KusaA and KusaO, and examined the changes of their osteogenic potentials. In NICD stable transformants (KusaA(NICD) and KusaO(NICD)), osteogenic properties including alkaline phosphatase activity, expression of osteocalcin and type I collagen, and in vitro calcification were suppressed. Transient transfection of NICD attenuated the promoter activities of Cbfa1 and Ose2 element. KusaA was capable of forming trabecular bone-like tissues when injected into mouse abdomen, but this in vivo bone forming activity was significantly suppressed in KusaA(NICD). Osteoclasts were induced in the KusaA-derived bone-like tissues, but lacked in the KusaA(NICD)-derived tissues. These results suggest that Notch signaling suppresses the osteoblastic differentiation of mesenchymal progenitor cells.     

Targeted disruption of LIG-1 gene results in psoriasiform epidermal hyperplasia

We have designed a chimeric promoter that can be stimulated by various pro-inflammatory mediators and so drive the expression of therapeutic genes under inflammatory conditions. The promoter has two parts, the [-247/+20] fragment of the human type IIA secreted phospholipase A2 gene promoter, which is stimulated by the pro-inflammatory cytokine interleukin-1beta (IL-1beta), and a double peroxisome proliferator-activated receptor response element that is activated by some eicosanoids and by non-steroidal anti-inflammatory drugs (NSAIDs). Transfection experiments using rabbit articular chondrocytes in primary culture showed that this chimeric promoter produced a low basal activity and was induced by NSAIDs, WY-14643, IL-1beta, and 15-deoxy Delta12,14 prostaglandin J2. The latter two compounds stimulated the promoter synergistically.     

The meiotic recombination checkpoint is regulated by checkpoint rad+ genes in fission yeast

During the course of meiotic prophase, intrinsic double-strand breaks (DSBs) must be repaired before the cell can engage in meiotic nuclear division. Here we investigate the mechanism that controls the meiotic progression in Schizosaccharomyces pombe that have accumulated excess meiotic DSBs. A meiotic recombination-defective mutant, meu13Delta, shows a delay in meiotic progression. This delay is dependent on rec12+, namely on DSB formation. Pulsed-field gel electrophoresis analysis revealed that meiotic DSB repair in meu13Delta was retarded. We also found that the delay in entering nuclear division was dependent on the checkpoint rad+, cds1+ and mek1+ (the meiotic paralog of Cds1/Chk2). This implies that these genes are involved in a checkpoint that provides time to repair DSBs. Consistently, the induction of an excess of extrinsic DSBs by ionizing radiation delayed meiotic progression in a rad17(+)-dependent manner. dmc1Delta also shows meiotic delay, however, this delay is independent of rec12+ and checkpoint rad+. We propose that checkpoint monitoring of the status of meiotic DSB repair exists in fission yeast and that defects other than DSB accumulation can cause delays in meiotic progression.     

Casein digestion by Debaryomyces hansenii isolated from cheese

To understand the possible proteolytic contribution of yeast during cheese ripening, Debaryomyces hansenii 212 was isolated from commercial blue-veined cheese and incubated in a medium containing casein. Growth and casein degradation were recognized at the cheese-ripening temperature. Proteolytic activity was found in the intracellular fraction, and the enzyme, which was attached to the cell wall, primarily acted on beta-casein. The cytosol contained more than 90% of the total proteolytic activity which was responsible for the degradation of both alpha(s)- and beta-casein. These results suggest that the contribution of yeast to cheese ripening would depend on the susceptibility to cell lysis in addition to its proteolytic activity.     

Molecular cloning of the gene encoding thermostable endo-1,5-alpha-L-arabinase of Bacillus thermodenitrificans TS-3 and its expression in Bacillus subtilis

The gene that encodes a thermostable endo-arabinase (called ABN-TS) from Bacillus thermodenitrificans TS-3 was cloned, sequenced, and expressed in the mesophilic B. subtilis. The gene contained an open reading frame consists of 939 bp, which encodes 313 amino acids. The deduced amino acid sequence of the enzyme showed 50, 46, and 36% similarity with endo-arabinase from B. subtilis IFO 3134 (PPase-C), Pseudomonas fluorescens (ArbA), and Aspergillus niger (ABNA), respectively. The hydrophobic and acidic amino acids making up ABN-TS outnumbered those in PPase-C. The gene product expressed in B. subtilis, as the host, had substantially the same characteristics, and was stable up to 70 degrees C, and the reaction was optimal around 70 degrees C, as well as native ABN-TS.