Induction of cytochrome P450 1A1 in rat liver slices by 7-ethoxycoumarin and 4-methyl-7-ethoxycoumarin

7-Ethoxycoumarin (EC) is widely used as a model substrate for monooxygenase function, its O-deethylation representing cytochrome P450 (P450) activity mainly of 1A but also of 2B isoforms. Reports on investigations of its own capacity to induce or suppress P450 activities, however, have not been found in biomedical literature. To avoid the influence of in vivo pharmacokinetics, studies can well be undertaken with liver slice incubation. Therefore in the present investigation precision-cut rat liver slices from male 43-63-day-old male HAN:Wistar outbred rats were incubated at 30 degrees C in carbogen saturated William's Medium E for 24 h. EC was added previously to final concentrations of 10, 25, 50, 75 or 100 microM. After incubation, homogenate was prepared from slices and used for model reactions (7-ethoxyresorufin O-deethylation [EROD] and 7-pentoxyresorufin O-depentylation [PROD]). EROD, indicating activities of 1A isoforms, was enhanced by incubation with EC at 25 and 50 microM to about doublefold but showed control or lower values at 75 and 100 microM. Incubation with beta-naphthoflavone in comparison led to variable increases (3-5-fold of controls). For PROD as an indicator of the phenobarbital inducible P450 isoforms 2B1 and 2B2 no enhancement was found, but a decrease by incubation with 75 and 100 microM EC. To further investigate the correlation between enzyme activity and gene expression after slice incubation, P450 1A1 mRNA content was measured by RT-PCR. Induced gene expression for 1A1 was seen with different EC concentrations to a variable extent, though not as strong as with BNF. Similar incubation with 4-methyl-7-ethoxycoumarin revealed an even stronger induction of EROD activity with maxima at about 10-32 microM, reaching BNF values. In contrast incubation with 7-benzyloxycoumarin had no evident inducing or suppressing effect, neither on EROD nor on PROD activity.     

Probing the structure of complex macromolecular interactions by homolog specificity scanning: the P1 and P7 plasmid partition systems

The P1 plasmid partition locus, P1 par, actively distributes plasmid copies to Escherichia coli daughter cells. It encodes two DNA sites and two proteins, ParA and ParB. Plasmid P7 uses a similar system, but the key macromolecular interactions are species specific. Homolog specificity scanning (HSS) exploits such specificities to map critical contact points between component macromolecules. The ParA protein contacts the par operon operator for operon autoregulation, and the ParB contacts the parS partition site during partition. Here, we refine the mapping of these contacts and extend the use of HSS to map protein-protein contacts. We found that ParB participates in autoregulation at the operator site by making a specific contact with ParA. Similarly, ParA acts in partition by making a specific contact with ParB bound at parS. Both these interactions involve contacts between a C-terminal region of ParA and the extreme N-terminus of ParB. As a single type of ParA-ParB complex appears to be involved in recognizing both DNA sites, the operator and the parS sites may both be occupied by a single protein complex during partition. The general HSS strategy may aid in solving the three-dimensional structures of large complexes of macromolecules.     

Bacillus subtilis DnaG primase stabilises the bacteriophage SPP1 G40P helicase-ssDNA complex

We analyzed our treatment results in 153 patients with histologically verified intracranial germ cell tumors and proposed classifying them into three therapeutic groups with good prognosis, intermediate prognosis, and poor prognosis. In this work, we selected patients treated with chemotherapy (cisplatin or carboplatin combinations) in each subgroup, and we discuss the role of chemotherapy in their treatment. Our combination chemotherapy regimens are: cisplatin-vinblastine-bleomycin, cisplatin-etoposide, and carboplatin-etoposide. We delivered these chemotherapies to the last 33 patients and compared their treatment results with those obtained in the previous 31 patients, who were treated with conventional radiation therapy alone. A combination with chemotherapy and a reduced dose of irradiation with local field was given to 7 patients with germinoma to increase the cure rate and reduce radiation-induced side effects, including anterior pituitary dysfunction. We obtained an excellent initial response to chemotherapy. The chemotherapy we delivered had significantly better effects in the group with intermediate prognosis, but not in the group with poor prognosis. More aggressive chemotherapy and radiation therapy should be given as the initial treatment.     

Inhibition of bacteriophage lambda, T1, and T7 development by R plasmids of the H incompatibility group

R plasmids from chloramphenicol-resistant salmonella from Ontario are shown to belong to the H(2) incompatibility subgroup and to mediate a broad-spectrum, phage inhibition function.     

Sampling of similar and dissimilar comparison persons and objects as a function of the generality of attribution goal.

Conducted an information-search procedure in which Ss were asked to seek information regarding persons and objects in order to validate a given person or object cause. Four hypotheses were tested: When asked to validate a person cause, Ss are more likely to select distinctiveness information than target-object consensus information. When asked to validate an object cause, Ss are more likely to select target-object consensus information than distinctiveness information. As the generality of person inference increases, progressively dissimilar object comparisons are sought. As the generality of object inference increases, progressively dissimilar person comparisons are sought. In Exp I, 26 undergraduates read attitude statements and answered judgment goals or questions about the statement's generality or object inference. 52 undergraduates in Exp II completed a similar task. The first 3 hypotheses were supported in both Exp I and Exp II, whereas the 4th hypothesis received only mixed support in Exp I and was not supported in Exp II. Unlike Exp I, Exp II did not include cues suggesting the relevant type of information to be sought. (25 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Amphibian FGF-1 is structurally and functionally similar to but antigenically distinguishable from its mammalian counterpart

Recent studies have shown that fibroblast growth factors (FGF) play an important role in the diverse cellular mechanisms involved with vertebrate development. One system which has received a great deal of attention is the developing limb in part because of the extensive epithelial-mesenchymal interactions that take place during this process. Because it closely parallels the developmental process of the limb and is a model for wound repair, the phenomenon of amphibian limb regeneration has been used to investigate the role of FGF in these processes. We have recently reported on the cloning and functional characterization of an FGF receptor (FGFR) isolated from amphibian regenerative tissue. In this report, we describe the isolation and characterization of an FGF-1 molecule from the newt, Notophthalmus viridescens. Amino acid sequence comparisons indicate that the newt FGF-1 exhibits between 79 to 83% identity with FGF-1 from mammalian and avian species. The full length cDNA of the newt FGF-1 was cloned into a prokaryotic expression vector and purified from E. coli. Although the newt FGF-1 shares a high degree of primary amino acid sequence similarity with other FGF-1 molecules, the recombinant protein was not detected in a Western blot analysis using a polyclonal antibody directed against mammalian FGF-1. Despite the antigenic divergence, the newt FGF-1 was capable of binding to NIH/3T3 and Chinese hamster ovary cells overexpressing mammalian and amphibian FGFRs with dissociation constants comparable to those reported for mammalian FGF-1. Newt FGF-1 could also be cross-linked to receptors on the surface of NIH/3T3 cells. In addition, it elicits a mitogenic response in NIH/3T3 cells indistinguishable from human recombinant FGF-1.     

唐钢1号高炉开炉及快速达产实践

对唐钢1号高炉扩容大修改造、开炉及快速达产经验进行总结.经过严密的组织开炉准备和合理的开炉方案,停、开炉历时32 d 7 h 24 min,创造了我国冶金史上同类高炉停产大修时间最短的记录.并在开炉7 d内利用系数突破2.0 t/(m3·d),15 d煤比达70 kg/t·Fe,实现了快速开炉及快速达产.     

A helical coat protein recognition domain of the bacteriophage P22 scaffolding protein

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20 degreesC dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37 degreesC the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR. At the same time, the endosome gives rise to multivesicular endosomes (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surface where they exocytose. Using video microscopy and correlative electron microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contain TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cup-shaped vesicles to which the tubules give rise become enriched in dIgA/pIgR. Taken together with the published information available on pIgR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface of polarized MDCK cells are maintained by a signal-dependent, clathrin-based sorting mechanism that operates along the length of the transcytotic pathway. We propose that the differential sorting of occupied receptors within the MDCK endosome is achieved by this clathrin-based mechanism continuously retrieving receptors like TR from the pathways that deliver pIgR to the apical surface and EGFR to the lysosome.     

Second-site suppressors of the bacteriophage P1 virs mutant reveal the interdependence of the c4, icd, and ant genes in the P1 immI operon

The immI operon of phage P1 contains the genes c4, icd, and ant, which are transcribed in that order from the same constitutive promoter, P51b. The gene c4 encodes an antisense RNA which inhibits the synthesis of an antirepressor by acting on a target ant mRNA. Interaction depends on the complementarity of two pairs of short sequences encompassing virs+ and the ribosome-binding site involved in ant expression. Accordingly, in a P1 virs mutant phage, antirepressor is synthesized constitutively. We have isolated lysogen-proficient, second-site suppressors of P1 virs in order to evaluate the interdependence of the immI-specific genes. From a total of 17 suppressors analyzed, 15 were found to be located in the icd gene. They were identified as frameshift mutations, containing base insertions or deletions in tandem repeats of a single base pair. One suppressor was identified as a P51b promoter-down mutation; the second site of another suppressor was found to be located in the c4 gene. Furthermore, it was shown that virs cannot be suppressed by ant (icd+) suppressors. The results confirm the model that the immI operon is transcribed as a unit, that the icd and ant genes are translationally coupled, and that the constitutive synthesis of Icd protein alone is lethal to the bacterial cell. The existence of a c4 suppressor of virs, whose effect is not yet known, points to a still more complex regulation of antirepressor synthesis than was anticipated from the model.     

Effect of ultraviolet irradiation of bacteriophage f1 DNA on its conversion to replicative form by extracts of Escherichia coli

When DNA of phage chiX174 or phage f1 is used as a template after exposure to ultraviolet radiation, the conversion of single-stranded DNA to replicative form by cell-free extracts of Escherichia coli is inhibited. The extend of synthesis is proportional to the distance of a pyrimidine dimer from a specific origin of replication as calculated from the random location of dimers at various UV doses. The results therefore indicate that the initiation of DNA synthesis on these phage DNAs occurs normally at a specific site, and that chain elongation is blocked when replication reaches a photo product in the template. Reinitiation of DNA synthesis distal to the lesion does not occur.     

Mechanisms of virus assembly probed by Raman spectroscopy: the icosahedral bacteriophage P22

A microdialysis flow cell has been developed for time-resolved Raman spectroscopy of biological macromolecules and their assemblies. The flow cell permits collection of Raman spectra concurrent with the efflux of small solute molecules into a solution of macromolecules and facilitates real-time spectroscopic detection of structural transitions induced by the effluent. Additionally, the flow cell is well suited to the investigation of hydrogen-isotope exchange phenomena that can be exploited as dynamic probes of viral protein folding and solvent accessibility along the assembly pathway. Here, we describe the application of the Raman dynamic probe to the maturation of the icosahedral capsid of bacteriophage P22, a double-stranded DNA virus. The P22 virion is constructed from a capsid precursor (procapsid) consisting of 420 coat subunits (gp5) in an outer shell and a few hundred scaffolding subunits (gp8) within. Capsid maturation involves expulsion of scaffolding subunits coupled with shell expansion at the time of DNA packaging. Raman static and dynamic probes reveal that the scaffolding subunit is highly alpha-helical and highly thermolabile, and lacks a typical hydrophobic core. When bound within the procapsid, the alpha-helical fold of gp8 is thermostabilized; however, this stabilization confers no apparent protection against peptide NH-->ND exchange. A molten globule model is proposed for the native scaffolding subunit that functions in procapsid assembly. Accompanying capsid expansion, a small conformational change (alpha-helix-->beta-strand) is also observed in the coat subunit. Domain movement mediated by hinge bending is proposed as the mechanism of capsid expansion. On the basis of these results, a molecular model is proposed for assembly of the P22 procapsid.     

Interaction of the receptor binding domains of Pseudomonas aeruginosa pili strains PAK, PAO, KB7 and P1 to a cross-reactive antibody and receptor analog: implications for synthetic vaccine design

The four synthetic peptide antigens, PAK 128-144, PAO 128-144, KB7 128-144 and P1 126-148, correspond in amino acid sequence to the C-terminal receptor binding regions of four strains (PAK, PAO, KB7, P1) of Pseudomonas aeruginosa pilin. The NMR solution structures of the trans forms of the peptides show conserved beta-turns which have been implicated in antibody and receptor recognition. The interactions between these peptides and a cross-reactive monoclonal antibody, PAK-13, have been studied using two-dimensional (1)H NMR spectroscopy in order to map the antigenic determinants recognized by the antibody. Residues for which spectral changes were observed upon antibody binding differed from peptide to peptide but were mostly confined to one or both of the turn regions and to the hydrophobic pockets. Conformational changes in the beta-turns and hydrophobic pockets of these peptides upon antibody binding were also monitored by examination of the pattern of nuclear Overhauser effects (NOEs) versus transferred nuclear Overhauser effects (TRNOEs) for the free versus the bound peptides. Although TRNOEs developed strongly between side chain resonances in the hydrophobic pockets of the peptides, no additional backbone TRNOEs were observed in the presence of antibody, suggesting no major conformational changes in the secondary structures of the peptides upon binding. This implies a flexible antibody combining site, a feature which is discussed with respect to cross-reactivity, strain specificity, and the design of a synthetic peptide vaccine effective against a broad spectrum of P. aeruginosa strains. The binding of the PAK peptide to a disaccharide receptor analog, (beta GalNAc(1-4)beta Gal), was also studied using (1)H NMR in order to map the "adhesintope" recognized by the receptor. Spectral changes observed in the peptide spectrum with the binding of receptor were similar to those seen for the binding of antibody, suggesting that the epitope recognized by the antibody is structurally coincident with the adhesintope recognized by the receptor. The relevancy of this result is discussed with respect to immunogenicity versus pathogenicity, and the proper design of a vaccine which could prevent the mutational escape of the pathogen away from the host's defence systems.     

DNA cleavage by 7-methylbenzopentathiepin: a simple analog of the antitumor antibiotic varacin

The compound 7-methylbenzopentathiepin, a simple analog of the benzopentathiepin antitumor antibiotic varacin, was shown to be a potent thiol-dependent DNA-cleaving agent. Biological experiments previously suggested that DNA cleavage might play a role in the cytotoxicity of varacin; however, this is the first direct evidence that benzopentathiepins can cause DNA strand breaks under physiologically relevant conditions.     

Effects of estradiol and progesterone on cytochrome P4501A1 expression in Hepa 1c1c7 cells

The effects of estradiol and progesterone on the expression of cytochrome P4501A1 were investigated in Hepa 1c1c7 cells. Both steroids, at 10 microM concentration, increased P4501A1-mediated 7-ethoxyresorufin O-deethyalase activity and amounts of its immunoreactive protein and CYP1A1 mRNA. Gel shift assay revealed that the steroids could induce both AhR transformation and binding of the ligand-AhR complex to its specific DNA recognition site. Transient transfection demonstrated that 5'flanking region of CYP1A1 could respond to the steroid action. The competitive binding assay showed that the steroids bound to AhR with moderate affinity. These results suggested that steroidal structure can be AhR ligands and induce CYP1A1 expression in AhR-dependent manner.     

A novel poxvirus gene and its human homolog are similar to an E. coli lysophospholipase

In previous work it was shown that the immune cytokine interferon-gamma (IFN-gamma) inhibits hormone secretion in anterior pituitary (AP) cell cultures, an action most likely mediated by folliculostellate (FS) cells. In the present study, we wanted to investigate whether nitric oxide (NO) is involved in this inhibitory action of IFN-gamma. NO synthase (NOS) inhibitors with affinity for the inducible (iNOS) and the constitutive (cNOS) isoform such as N(G)-monomethyl-L-arginine (L-NMMA) and S-methyl-L-thiocitrulline (SMLT) dose-dependently blocked the inhibitory action of IFN-gamma on GHRH-stimulated GH secretion, and partially reversed the inhibitory effect on basal prolactin (PRL) release. In the absence of IFN-gamma these inhibitors significantly augmented basal PRL release and slightly enhanced GHRH-stimulated GH release. L-N6-(1-iminoethyl)lysine (L-NIL), a NOS inhibitor with preferential affinity for iNOS, abrogated the IFN-gamma effect on GHRH-stimulated GH secretion and partially reversed IFN-gamma inhibition of PRL release. However, L-NIL did not exert a stimulatory effect on basal PRL and GHRH-stimulated GH release by its own. 2,4-diamino-6-hydroxypyrimidine (DAHP), a NOS inhibitor by interfering with tetrahydrobiopterin (BH4) cofactor availability, showed the same activity profile as L-NIL. NOS inhibitors blocked or reduced the production of NO as detected by measuring nitrite (NO2-) levels in AP cell cultures and cGMP levels in the NO-reporter cell line RFL-6. The NOS inhibiting action of L-NMMA was confirmed by competition experiments with the natural NOS substrate L-arginine. Thus, in culture medium with lower amounts of L-arginine, L-NMMA blocked the IFN-gamma-induced inhibition of GHRH-stimulated GH release at a lower dose. The inhibition of PRL and GH release by IFN-gamma was markedly reduced in L-arginine-depleted medium. The NO donor sodium nitroprusside (SNP) mimicked the inhibitory action of IFN-gamma on GHRH-stimulated GH and basal PRL release. Similarly to IFN-gamma, SNP did not affect basal GH release. As previously reported, inhibition by IFN-gamma occurred only in AP cell populations containing a minimal proportion of FS cells. As studied in different cell populations obtained by unit gravity sedimentation in a serum albumin gradient, L-NMMA reversed the IFN-gamma effect in the same populations enriched in FS cells. Interestingly, in the absence of IFN-gamma L-NMMA strongly stimulated basal PRL release in the population most enriched in FS cells. It is concluded that IFN-gamma through activation of the iNOS pathway probably in FS cells enhances the production of NO and that this effect is responsible for the inhibitory action of IFN-gamma on GHRH-stimulated GH release and partially for the IFN-gamma-induced decrease in basal PRL release. On the other hand, NO, likely produced by cNOS, appears to exert a tonic inhibitory effect on GHRH-stimulated GH and basal PRL release. It seems therefore that low amounts of NO produced constitutively may take charge of subtle physiological adaptations, and higher levels of NO produced by iNOS under the influence of IFN-gamma may attenuate PRL and GH release during emergency conditions of immune and inflammatory reactions.     

Long-lasting mnemotropic effect of substance P and its N-terminal fragment (SP1-7) on avoidance learning

Obstruction of a prosthetic valve by an infective vegetation is a rare and life-threatening complication of endocarditis that demands emergent surgical intervention. In our patient's case, transthoracic echocardiography showed the large vegetation, transthoracic Doppler imaging showed severe obstruction of diastolic flow through the bioprosthetic valve, and transesophageal echocardiography showed that no perivalvular abscess was present. Rapid diagnosis of prosthetic valve infection and obstruction demanded application of all three major echocardiographic modalities and proved critical to the patient's recovery.     

Binding of interleukin-18 to the interleukin-1 receptor homologous receptor IL-1Rrp1 leads to activation of signaling pathways similar to those used by interleukin-1

Interleukin-18 (IL-18) is an inflammatory cytokine that has been shown to enhance a variety of Th1 type T cell responses. Because IL-18 is homologous to IL-1, we tested binding of IL-18 to the known IL-1R family members. We could show binding of IL-18 to the orphan receptor IL-1Rrp1 but not to other IL-1R homologous proteins. IL-1Rrp1 and IL-1RI share highly conserved domains within their cytoplasmic regions. Comparison of the IL-1 and IL-18 signaling mechanisms showed that they activate identical cytoplasmic messengers. IL-18, like IL-1, induced association of its receptor with IRAK and subsequent recruitment of TRAF6. IL-18 activated p38 MAP kinase, jun kinase, and beta casein kinase (TIP kinase), an apparently novel kinase previously thought to be specifically activated by IL-1 and tumor necrosis factor (TNF). IL-18 activated NF-kappaB in EL4/6.1 thymoma cells but not in COS-7 cells, even though the latter presumably contain all components required for the IL-1 signaling pathway. From our binding and signaling studies, we conclude that the IL-18 receptor complex consists of IL-18, the IL-1Rrp1, and another thus far unidentified receptor molecule.