Structures of the wild-type MexAB-OprM tripartite pump reveal its complex formation and drug efflux mechanism.

In Pseudomonas aeruginosa, MexAB-OprM plays a central role in multidrug resistance by ejecting various drug compounds, which is one of the causes of serious nosocomial infections. Although the structures of the components of MexAB-OprM have been solved individually by X-ray crystallography, no structural information for fully assembled pumps from P. aeruginosa were previously available.

Induction of the differentiation of human HL-60 promyelocytic leukemia cell line by succinoyl trehalose lipids.

Four analogs of succinoyl trehalose lipid-3 (STL-3)with saturated even-number or odd-number carbonchains, and unsaturated or halogenated fatty acidswere examined for their ability to inhibit the growthand induce the differentiation of HL-60 humanpromyelocytic leukemia cells. The optimalconcentration of STL-3 at which such activities wererecognized was closed to the critical micelleconcentration of STL-3. Analog of STL-3 witheven-number or odd-number carbon chain and unsaturatedfatty acids strongly inhibited growth and induced thedifferentiation of HL-60 cells, as evaluated in termsof nitroblue tetrazilium-reducing activity and theappearance of the CD36 antigen.

Mannosylerythritol lipid increases levels of galactoceramide in and neurite outgrowth from PC12 pheochromocytoma cells.

We report here that a microbial extracellular glycolipid,mannosylerythritol lipid (MEL), induces the outgrowth ofneurites from and enhances the activity of acetylcholinesterase(AChE) in PC12 pheochromocytoma cells. Furthermore, treatment ofPC12 cells with MEL increased levels of galactosylceramide(Galbeta1-1'Cer; GalCer). Exposure of PC12 cells to exogenous GalCer caused the dose-dependent outgrowth ofneurites.

Treatment of mouse melanoma cells with phorbol 12-myristate 13-acetate counteracts mannosylerythritol lipid-induced growth arrest and apoptosis.

Mannosylerythritol lipid (MEL), an extracellularglycolipid from yeast, induces the differentiation ofHL-60 promyelocytic leukemia cells towardsgranulocytes. We show here that MEL is also a potentinhibitor of the proliferation of mouse melanoma B16cells. Flow-cytometric analysis of the cell cycle ofMEL-treated B16 cells revealed the accumulation ofcells in the sub-G(0)/G(1) phase, which is a hallmark ofcells undergoing apoptosis.

Histone acetyltransferase (HAT) activity of ATF-2 is necessary for the CRE-dependent transcription.

ATF-2 is a DNA-binding protein that binds to cAMP-response elements (CREs) and forms a hetrodimer with c-Jun, via binding of the leucine zipper motif and then stimulates the CRE-dependent transcription (1,2). Recently, we have reported that ATF-2 has an intrinsic acetyltransferase activity that is controlled by phosphorylation. Mutant form of either p300 or ATF-2 with mutations in the HAT domain failed to stimulate the CRE-dependent transcription in response to UV irradiation.

Promotion of triplex formation by a fixed N-form sugar puckering: thermodynamic and kinetic studies.

We analyzed the effect of a fixed N-form sugar puckering of TFO (triplex-forming oligonucleotide) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. Both thermodynamic and kinetic analyses revealed that the binding constant of the pyrimidine motif triplex formation at pH 6.8 with modified TFO containing the fixed N-form sugar puckering was about 20-times larger than that observed with unmodified TFO.

Thermodynamic analyses of triplex formation with homopurine oligonucleotide.

We analyzed the thermodynamics of purine motif triplex formation by isothermal titration calorimetry. The signs of calorimetric enthalpy change, delta Hcal, and entropy change, delta S, of the triplex formation were negative in the temperature range between 15 and 35 degrees C. Since an observed negative delta S was unfavorable for the triplex formation, the triplex formation was driven by a large negative delta Hcal.

Role of Flk-1 in mouse hematopoietic stem cells.

It was reported that human hematopoietic stem cells in bone marrow were restricted to the CD34(+)KDR(+) cell fraction. We found that expression levels of Flk-1, a mouse homologue of KDR, were low or undetectable in mouse Lin(-)c-Kit(+)Sca-1(+)CD34(low/-) cells as well as Hoechst33342(-) cells (side population), which have long-term reconstitution capacity. Furthermore, neither Flk-1(+)CD34(low/-) cells nor Flk-1(+)CD34(+) cells had long-term reconstitution capacity in mouse.

Promotion of triplex formation by 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification: thermodynamic and kinetic studies.

We analyzed the effect of 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotide (TFO) on pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The binding constant of the pyrimidine motif triplex formation at pH 6.8 with 2',4'-BNA modified TFO was about 20 times larger than that observed with unmodified TFO.

Factors influencing the stability of alpha-helices and beta-strands in thermophilic ribonuclease H.

Understanding the influence of structural parameters is crucial to enhance the thermal stability of proteins. In this work, the stability (deltaG) of residues in different secondary structures of Ribonuclease H (RNase H) has been analyzed with 48 amino acid properties. The properties reflecting hydrophobicity show a good correlation with stability.

A novel regulator of G-protein signaling bearing GAP activity for Galphai and Galphaq in megakaryocytes.

The regulator of G-protein signaling (RGS) negatively regulates the alpha subunit of G proteins by accelerating their intrinsic guanosine triphosphatase (GTPase) activity. Here are reported the isolation and characterization of a novel mouse RGS, termed RGS18, which is a new member of RGS subfamily B. Northern blot analysis showed that RGS18 messenger RNA was detected predominantly in spleen and hematopoietic cells, and immunohistochemical studies demonstrated that RGS18 was expressed in megakaryocytes, platelets, granulocytes/monocytes, and, weakly, in hematopoietic stem cells, but not in lymphocytes or erythrocytes.

Isolation of a novel interleukin-1-inducible nuclear protein bearing ankyrin-repeat motifs.

We isolated a novel gene termed interleukin (IL)-1-inducible nuclear ankyrin-repeat protein (INAP), of which expression was specifically induced by IL-1 in OP9 stromal cells. The INAP has ankyrin-repeat motifs and shares weak amino acid sequence homology with Bcl-3 and other IkappaB family members. The human genomic INAP gene found in the NCBI data base is located at chromosome 3q3.

Overexpression of EXTL3/EXTR1 enhances NF-kappaB activity induced by TNF-alpha.

EXTL3/EXTR1 is a member of the EXT gene family, which may represent a class of glycosyltransferases involved in heparan sulfate biosynthesis. It is known that heparan sulfate interacts with a variety of proteins and is therefore implicated in various cellular responses. Here, we examined the effect of EXTL3 on nuclear factor-kappaB (NF-kappaB) activity stimulated by tumor necrosis factor-alpha (TNF-alpha), one of heparin-binding cytokine.

Thermodynamic and kinetic effects of N3'-->P5' phosphoramidate modification on pyrimidine motif triplex DNA formation.

I have investigated the thermodynamic and kinetic effects of N3'-->P5' phosphoramidate (PN) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation between a 23-bp target duplex and a 15-mer TFO using electrophoretic mobility shift assay, UV melting, isothermal titration calorimetry, and interaction analysis system. The thermodynamic and kinetic analyses have clearly indicated that the PN modification of TFO not only significantly increased the thermal stability of the pyrimidine motif triplex at neutral pH but also increased the binding constant of the pyrimidine motif triplex formation at room temperature and neutral pH by nearly 2 orders of magnitude. The consideration of the observed thermodynamic parameters has suggested that the more rigidity of the PN TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant of the pyrimidine motif triplex formation at neutral pH.

Genetic reclassification of porcine enteroviruses.

The genetic diversity of porcine teschoviruses (PTVs; previously named porcine enterovirus 1) and most serotypes of porcine enteroviruses (PEVs) was studied. Following the determination of the major portion of the genomic sequence of PTV reference strain Talfan, the nucleotide and derived amino acid sequences of the RNA-dependent RNA polymerase (RdRp) region, the capsid VP2 region and the 3' non-translated region (3'-NTR) were compared among PTVs and PEVs and with other picornaviruses. The sequences were obtained by RT-PCR and 3'-RACE with primers based on the sequences of Talfan and available PEV strains.

Promotion of duplex and triplex DNA formation by polycation comb-type copolymers.

Triplex DNA has attracted considerable interest recently because of its possible biological functions in vivo and its wide variety of potential applications, such as regulation of gene expression, site-specific cleavage of duplex DNA, mapping of genomic DNA, and gene-targeted mutagenesis (1-3). A triplex is usually formed through the sequence-specific interaction of a single-stranded homopyrimidine or homopurine triplex-forming oligonucleotide (TFO) with the major groove of the homopurine-homopyrimidine stretch in duplex DNA (1-5). In the pyrimidine motif triplex, a homopyrimidine TFO binds parallel to the homopurine strand of the target duplex by Hoogsteen hydrogen bonding to form T[Symbol: see text]A:T and C(+[Symbol: see text])G:C triplets ([Symbol: see text] and : represent Hoogsteen hydrogen bonding and Watson Crick base pairing, respectively).

Gibberellin biosynthesis: metabolic evidence for three steps in the early 13-hydroxylation pathway of rice.

[14C4]GA53, [14C4]GA44, and [2H2/14C4]GA19 were injected separately into seedlings of rice (Oryza sativa) using a dwarf mutant (d35) that has low levels of endogenous gibberellins (GAs). After 8 h incubation, the shoots were extracted and the labeled metabolites were identified by full-scan gas chromatography mass spectrometry (GC-MS) and Kovats retention indices (KRIs). Our results document the metabolic sequence, GA53-->GA44-->GA19-->GA20 and the presence of endogenous GA53, GA44, GA19, GA20 and GA1.

Identification of IkappaBalpha as a substrate of Fas-associated phosphatase-1.

Fas (APO-1/CD95), a member of the tumor necrosis factor receptor (TNFR)/nerve growth factor receptor (NGFR) superfamily, is a cell-surface molecule that induces apoptosis upon activation. Fas-associated phosphatase-1 (FAP-1) is a 250-kDa protein tyrosine phosphatase (PTP) that is associated with the negative regulatory domain of Fas (C-terminal 15 amino acids). Human tumor cell lines become resistant to Fas-mediated apoptosis when transfected with FAP-1, indicating that FAP-1 functions as a negative regulator in Fas-mediated death signaling.

Morphological changes in the nucleus and actin cytoskeleton in the process of Fas-induced apoptosis in Jurkat T cells.

To investigate the early event of apoptosis, we monitored the morphological changes in the early stage of Fas-induced apoptosis in the human T-cell lymphoma cell line Jurkat, using confocal microscopy. Morphological changes in the nuclei were observed from 30 min after stimulation, and preceded the changes in the cytoskeleton. This kind of change was enhanced in the presence of EGTA but decreased in the presence of dihydrocytochalasin B, without any changes in caspase-3 activation.

Human CENP-H multimers colocalize with CENP-A and CENP-C at active centromere--kinetochore complexes.

Centromere and kinetochore proteins have a pivotal role in centromere structure, kinetochore formation and sister chromatid separation. However, the molecular architecture and the precise dynamic function of the centromere-kinetochore complex during mitosis remain poorly understood. Here we report the isolation and characterization of human CENP-H.

2'-O,4'-C-methylene bridged nucleic acid modification promotes pyrimidine motif triplex DNA formation at physiological pH: thermodynamic and kinetic studies.

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in an artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is, therefore, crucial in improving its therapeutic potential. To this end, we have investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotide (TFO), on pyrimidine motif triplex formation at physiological pH.

An Arabidopsis gene encoding a Ca2+-binding protein is induced by abscisic acid during dehydration.

An Arabidopsis thaliana RD20 cDNA, which was isolated as one of drought-inducible genes, encodes a putative protein with a conserved EF-hand Ca2+-binding domain. The recombinant RD20 protein was shown to bind Ca2+. The transcription of RD20 gene was induced not only by drought but also by ABA and high salinity.

A novel Arabidopsis thaliana dynamin-like protein containing the pleckstrin homology domain.

A full-length cDNA encoding a novel type of plant dynamin-like protein, ADL3, was isolated from Arabidopsis thaliana. ADL3 is a high molecular weight GTPase whose GTP-binding domain shows a low homology to those of other plant dynamin-like proteins. ADL3 contains the pleckstrin homology domain as is in mammalian dynamins, although other plant dynamin-like proteins reported lack this domain.