Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells

Patch-clamping and cell imageanalysis techniques were used to study the expression of thevolume-activated Cl current,I_Cl(vol), and regulatory volume decrease (RVD)capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated aCl current with a linear conductance, negligibletime-dependent inactivation, and a reversal potential close to theCl equilibrium potential. The sequence of anionpermeability was I > Br > Cl > gluconate. The Cl channelblockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),and ATP inhibited I_Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by adouble chemical-block (thymidine and hydroxyurea) technique. Theexpression of I_Cl(vol) was cell cycle dependent,being high in G₁ phase, downregulated in S phase, butincreasing again in M phase. Hypotonic solution activated RVD, whichwas cell cycle dependent and inhibited by the Cl channelblockers NPPB, tamoxifen, and ATP. The expression of I_Cl(vol) was closely correlated with the RVDcapacity in the cell cycle, suggesting a functional relationship.Inhibition of I_Cl(vol) by NPPB (100 µM)arrested cells in G₀/G₁. The data also suggest that expression of I_Cl(vol) and RVD capacity areactively modulated during the cell cycle. The volume-activatedCl current associated with RVD may therefore play animportant role during the cell cycle progress.

     

Sequence-dependent flexibility in promoter sequences

The non-neighbor interactions between base-pairs were taken into account to calculate the angular parameters (Omega, rho and tau) describing the orientation of successive base-pair planes and the translation parameters (D(y)) along the long axis of base-pair steps for 36 independent tetramers. A statistical mechanical model was proposed to predict the DNA flexibility that is mainly related to the thermal fluctuations at individual base-pair steps. The DNA flexibility can be described by the root-mean-square deviation of the end-to-end distance of DNA helical structure. The present model was then used to investigate the extreme flexible pattern in prokaryotic and eukaryotic promoter sequences. The results demonstrated several extreme flexible regions related to functionally important elements exist both in prokaryotic promoters and in eukaryotic promoters, DNA flexibility and AT content are highly correlated. The probabilities finding flexibility pattern in promoter sequences were also estimated statistically. The biological implications were discussed briefly.     

Definition of an inhibitory juxtamembrane WW-like domain in the platelet-derived growth factor beta receptor

A variety of tumors contain activating mutations in the cytoplasmic juxtamembrane domain of the type III family of receptor-tyrosine kinases, and some constructed mutations in this domain induce ligand-independent receptor activation. To explore the role of this domain in regulation of receptor activity, we subjected the juxtamembrane domain of the murine platelet-derived growth factor (PDGF) beta receptor to alanine-scanning mutagenesis. The mutant receptors were expressed in Ba/F3 cells and tested for constitutive tyrosine phosphorylation, association with phosphatidylinositol 3'-kinase, and their ability to induce cell survival and proliferation in the absence of interleukin-3. The mutant receptors accumulated to similar levels and appeared to undergo a normal PDGF-induced increase in tyrosine phosphorylation. Alanine substitutions at numerous positions located throughout the juxtamembrane domain caused constitutive receptor activation, as did an alanine insertion in the membrane-proximal segment of the juxtamembrane domain and a six-amino acid deletion in the center of the domain. It is possible to model the PDGF receptor juxtamembrane domain as a short alpha-helix followed by a three-stranded beta-sheet very similar to the known structures of WW domains. Strikingly, the activating mutations clustered in the central portions of the first and second beta strands and along one face of the beta-sheet, whereas the loops connecting the strands were largely devoid of mutationally sensitive positions. These findings provide strong support for the model that the activating mutations in the juxtamembrane region stimulate receptor activity by disrupting an inhibitory WW-like domain.     

ILPIP,a novel anti-apoptotic protein that enhances XIAP-mediated activation of JNK1 and protection against apoptosis

We have previously described a new aspect of the Inhibitor of Apoptosis (IAP) family of proteins anti-apoptotic activity that involves the TAK1/JNK1 signal transduction pathway (1,2). Our findings suggest the existence of a novel mechanism that regulates the anti-apoptotic activity of IAPs that is separate from caspase inhibition but instead involves TAK1-mediated activation of JNK1. In a search for proteins involved in the XIAP/TAK1/JNK1 signaling pathway we isolated by yeast two-hybrid screening a novel X chromosome-linked IAP (XIAP)-interacting protein that we called ILPIP (hILP-Interacting Protein). Whereas ILPIP moderately activates JNK family members when expressed alone, it strongly enhances XIAP-mediated activation of JNK1, JNK2, and JNK3. The expression of a catalytically inactive mutant of TAK1 blocked XIAP/ILPIP synergistic activation of JNK1 thereby implicating TAK1 in this signaling pathway. ILPIP moderately protects against interleukin-1beta converting enzyme- or Fas-induced apoptosis and significantly potentiates the anti-apoptotic activity of XIAP. In vivo co-precipitation experiments show that both ILPIP and XIAP interact with TAK1 and tumor necrosis factor receptor-associated factor 6. Finally, expression of ILPIP did not affect the ability of XIAP to inhibit caspase activation, further supporting the idea that XIAP protection against apoptosis is achieved by two separate mechanisms: one requiring JNK1 activation and a second involving caspase inhibition.     

Oligomeric interaction of hepatitis C virus NS5B is critical for catalytic activity of RNA-dependent RNA polymerase.

HCV NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme for HCV replication, which has the "palm and fingers" substructure. We recently identified five novel residues critical for RdRP activity (Qin, W., Yamashita, T., Shirota, Y., Lin, Y., Wei, W., and Murakami, S. (2001) Hepatology 33, 728-737). Among them, GLU-18 and His-502, far from the catalytic center, may be involved in conformational change(s) for RdRP activity as addressed in some palm and fingers enzymes. We examined the possibility that NS5B is oligomerized, and we could detect the interaction between two different tagged NS5B proteins in vitro and transiently expressed in mammalian cells. By scanning 27 clustered and then point alanine substitutions in vivo and in vitro, Glu-18 and His-502 were found to be critical for the homomeric interaction in vivo and in vitro, strongly suggesting a close relationship between the oligomerization and RdRP activity of NS5B. All mutants with substitutions at these two residues failed to bind wild type NS5B, however E18H interacted with H502E in vitro and in vivo. Interestingly, the NS5B protein with E18H or H502E did not exhibit RdRP activity, but a mixture of the two mutant proteins did. These results clearly indicate that two residues of HCV NS5B are critical for the oligomerization that is prerequisite to RdRP activity.     

Representation of the glomerular olfactory map in the Drosophila brain

We explored how the odor map in the Drosophila antennal lobe is represented in higher olfactory centers, the mushroom body and lateral horn. Systematic single-cell tracing of projection neurons (PNs) that send dendrites to specific glomeruli in the antennal lobe revealed their stereotypical axon branching patterns and terminal fields in the lateral horn. PNs with similar axon terminal fields tend to receive input from neighboring glomeruli. The glomerular classes of individual PNs could be accurately predicted based solely on their axon projection patterns. The sum of these patterns defines an "axon map" in higher olfactory centers reflecting which olfactory receptors provide input. This map is characterized by spatial convergence and divergence of PN axons, allowing integration of olfactory information.     

Differential expression of methanogenesis genes of Methanothermobacter thermoautotrophicus (formerly Methanobacterium thermoautotrophicum) in pure culture and in cocultures with fatty acid-oxidizing syntrophs

The expression of genes involved in methanogenesis in a thermophilic hydrogen-utilizing methanogen, Methanothermobacter thermoautotrophicus strain TM, was investigated both in a pure culture sufficiently supplied with H(2) plus CO(2) and in a coculture with an acetate-oxidizing hydrogen-producing bacterium, Thermacetogenium phaeum strain PB, in which hydrogen partial pressure was constantly kept very low (20 to 80 Pa). Northern blot analysis indicated that only the mcr gene, which encodes methyl coenzyme M reductase I (MRI), catalyzing the final step of methanogenesis, was expressed in the coculture, whereas mcr and mrt, which encodes methyl coenzyme M reductase II (MRII), the isofunctional enzyme of MRI, were expressed at the early to late stage of growth in the pure culture. In contrast to these two genes, two isofunctional genes (mtd and mth) for N(5),N(10)-methylene-tetrahydromethanopterin dehydrogenase, which catalyzes the fourth step of methanogenesis, and two hydrogenase genes (frh and mvh) were expressed both in a pure culture and in a coculture at the early and late stages of growth. The same expression pattern was observed for Methanothermobacter thermoautotrophicus strain DeltaH cocultured with a thermophilic butyrate-oxidizing syntroph, Syntrophothermus lipocalidus strain TGB-C1. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole proteins of M. thermoautotrophicus strain TM obtained from a pure culture and a coculture with the acetate-oxidizing syntroph and subsequent N-terminal amino acid sequence analysis confirmed that MRI and MRII were produced in the pure culture, while only MRI was produced in the coculture. These results indicate that under syntrophic growth conditions, the methanogen preferentially utilizes MRI but not MRII. Considering that hydrogenotrophic methanogens are strictly dependent for growth on hydrogen-producing fermentative microbes in the natural environment and that the hydrogen supply occurs constantly at very low concentrations compared with the supply in pure cultures in the laboratory, the results suggest that MRI is an enzyme primarily functioning in natural methanogenic ecosystems.     

Rapid detection of Shigella species in environmental sewage by an immunocapture PCR with universal primers

In the present study, we developed a quick, highly specific method for detection of Shigella species by combining immunocapturing of the bacteria and a universal primer PCR. The method drastically enhances test sensitivity, and it can be used not only for identification of Shigella species in the environment but also for rapid detection of other pathogens.