Crystallization and preliminary X-ray crystallographic studies on a Fab fragment of the mouse anti-human Fas monoclonal antibody HFE7A.

The Fas-Fas ligand system is involved in apoptosis. The mouse anti-human Fas monoclonal antibody HFE7A (m-HFE7A) has a potential use in human therapy against autoimmune diseases such as rheumatoid arthritis. Information on the three-dimensional structure is essential for antibody humanization. Crystals of an antigen-binding fragment (Fab) of m-HFE7A were obtained by the hanging-drop vapour-diffusion method using sodium citrate as a precipitant and 2-methyl-2,4-pentanediol as an additive. Fast optimization to produce single crystals suitable for X-ray analysis was achieved by the streak-seeding technique. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 43.4, b = 74.0, c = 133.8 A. The crystals diffract at least to 2.5 A resolution.

In vitro assembly of phytochrome B apoprotein with synthetic analogs of the phytochrome chromophore.

Phytochrome B (PhyB), one of the major photosensory chromoproteins in plants, mediates a variety of light-responsive developmental processes in a photoreversible manner. To analyze the structural requirements of the chromophore for the spectral properties of PhyB, we have designed and chemically synthesized 20 analogs of the linear tetrapyrrole (bilin) chromophore and reconstituted them with PhyB apoprotein (PHYB). The A-ring acts mainly as the anchor for ligation to PHYB, because the modification of the side chains at the C2 and C3 positions did not significantly influence the formation or difference spectra of adducts. In contrast, the side chains of the B- and C-rings are crucial to position the chromophore properly in the chromophore pocket of PHYB and for photoreversible spectral changes. The side-chain structure of the D-ring is required for the photoreversible spectral change of the adducts. When methyl and ethyl groups at the C17 and C18 positions are replaced with an n-propyl, n-pentyl, or n-octyl group, respectively, the photoreversible spectral change of the adducts depends on the length of the side chains. From these studies, we conclude that each pyrrole ring of the linear tetrapyrrole chromophore plays a different role in chromophore assembly and the photochromic properties of PhyB.

The structure of the C4C4 ring finger of human NOT4 reveals features distinct from those of C3HC4 RING fingers.

The NOT4 protein is a component of the CCR4.NOT complex, a global regulator of RNA polymerase II transcription. Human NOT4 (hNOT4) contains a RING finger motif of the C(4)C(4) type. We expressed and purified the N-terminal region of hNOT4 (residues 1-78) encompassing the RING finger motif and determined the solution structure by heteronuclear NMR. NMR experiments using a (113)Cd-substituted hNOT4 RING finger showed that two metal ions are bound through cysteine residues in a cross-brace manner. The three-dimensional structure of the hNOT4 RING finger was refined with root mean square deviation values of 0.58 +/- 0.13 A for the backbone atoms and 1.08 +/- 0.12 A for heavy atoms. The hNOT4 RING finger consists of an alpha-helix and three long loops that are stabilized by zinc coordination. The overall folding of the hNOT4 RING finger is similar to that of the C(3)HC(4) RING fingers. The relative orientation of the two zinc-chelating loops and the alpha-helix is well conserved. However, for the other regions, the secondary structural elements are distinct.

Light-induced nuclear translocation of endogenous pea phytochrome A visualized by immunocytochemical procedures.

Although the physiological functions of phytochrome A (PhyA) are now known, the distribution of endogenous PhyA has not been examined. We have visualized endogenous PhyA apoprotein (PHYA) by immunolabeling cryosections of pea tissue, using PHYA-deficient mutants as negative controls. By this method, we examined the distribution of PHYA in different tissues and changes in its intracellular distribution in response to light. In apical hook cells of etiolated seedlings, PHYA immunolabeling was distributed diffusely in the cytosol. Exposure to continuous far-red (cFR) light caused a redistribution of the immunolabeling to the nucleus, first detectable after 1.5 hr and greatest at 4.5 hr. During this time, the amounts of spectrally active phytochrome and PHYA did not decline substantially. Exposure to continuous red (cR) light or to a brief pulse of red light also resulted in redistribution of immunolabeling to the nucleus, but this occurred much more rapidly and with a different pattern of intranuclear distribution than it did in response to cFR light. Exposures to cR light resulted in loss of immunolabeling, which was associated with PHYA degradation. These results indicate that the light-induced intracellular location of PHYA is wavelength dependent and imply that this is important for PhyA activity.

Early induction of the orphan nuclear receptor NOR-1 during cell death of the human breast cancer cell line MCF-7.

The neuron-derived orphan receptor (NOR-1) is a member of the NGFI-B subfamily within the nuclear receptor superfamily. In T-cell apoptosis, where NGFI-B plays an essential role, a functional redundancy between NGFI-B and NOR-1 has been demonstrated. Here, we examined the regulation and expression of the NOR-1 gene during cell death induced by a calcium ionophore A23187 in the human breast cancer cell line MCF-7. A23187 caused a transient increase in NOR-1 mRNA levels within 6 h after treatment. To delineate the sequences required for the transitional response to A23187, a series of promoter deletion mutants were constructed. From the transient transfection experiments, the element responsive to A23187 was identified between -94 and -42 base pairs upstream from the transcription initiation site. This 53-base pairs region contains three copies of the cAMP response element (CRE). Furthermore, phosphorylation of the CRE-binding protein (CREB), which affects the transcription of the CRE dependent-genes, was detected 30 min after A23187 stimulation. Our findings are consistent with NOR-1 involvement in A23187-induced cell death via the CRE-CREB signaling pathway.

Identification of a novel gene, DAM1, amplified at chromosome 1p13.3-21 region in human breast cancer cell lines.

Screening for differentially expressed genes in cancer cell lines by the RNA differential display (DD) technique identified a novel mRNA that encodes a 26-kDa protein and is up-regulated in MCF-7 and BT-20 breast cancer cells. The predicted amino acid sequence showed 38% homology to Caenorhabditis elegans T12A2.7 protein, indicating that this is a possible human homologue for C. elegans T12A2.7 protein. The mRNA, designated DAM1 (DNA amplified in mammary carcinoma), was mapped at the 1p13.3-21 region, which is frequently altered in human breast cancer. By Southern blot analysis, we confirmed that the up-regulation of this novel gene is associated with gene amplification in MCF-7 (10-fold) and BT-20 (5-fold) cells. Our findings suggest that the DAM1 gene is a novel gene up-regulated by amplification in human breast cancer cell lines.

Identification of a novel gene, LDOC1, down-regulated in cancer cell lines.

By screening for differentially expressed genes in cancer cells, using the RNA differential display (DD) technique, we identified a novel cDNA, LDOC1, that is down-regulated in some cancer cell lines. A Northern blot analysis revealed no expression in pancreatic and gastric cancer cell lines but ubiquitous expression in normal human tissues. This new gene was mapped on chromosome Xq27 and the predicted protein sequence showed no similarity to known sequences in the database except for a leucine zipper-like motif at the N-terminal region and a proline-rich region that shares marked similarity to an SH3-binding domain. In an enhanced green fluorescent protein (EGFP) assay, the EGFP-LDOC1 fusion protein was localized in the nucleus. Although the function of LDOC1 is still unknown, our results suggest that this novel gene codes for a nuclear protein, and down-regulation of LDOC1 may have an important role in the development and/or progression of some cancers.

Staurosporine enhances cAMP-induced expression of neural-specific gene VGF and tyrosine hydroxylase.

Cyclic AMP transiently stimulates transcription of specific genes in the nervous system, including neural-specific gene VGF. The simultaneous presence of staurosporine (SSP) caused a progressive, rather than transient, increase in VGF mRNA levels during the cAMP-induced differentiation of human neuroblastoma cells. This steady increase does not appear to be due to increased stability of VGF mRNA. This synergy between cAMP and SSP was also observed at the protein level for tyrosine hydroxylase. These findings suggest the presence of cellular machinery that counteracts the decline of cAMP-induced gene expression.

Mode of phytochrome B action in the photoregulation of seed germination in Arabidopsis thaliana.

Arabidopsis thaliana seeds imbibed for a short duration show phytochrome B (PhyB)-specific photo-induction of germination. Using this system, the relationship was determined between the amount of PhyB in seeds and photon energy required for PhyB-specific germination in two transgenic Arabidopsis lines transformed with either the Arabidopsis PhyB cDNA (ABO) or the rice PhyB cDNA (RBO). Immunochemical detection of PhyB apoprotein (PHYB) showed that the expression level of PHYB in ABO seeds was at least two times higher than that in the wild-type seeds, but in RBO seeds the PHYB level was indistinguishable from that in wild-type seeds. The photon fluence required for induction and photoreversible inhibition of germination was examined using the Okazaki large spectrograph. At the wavelengths of 400-710 nm, the ABO seeds required significantly less photon fluence than wild-type seeds for induction of germination, whereas the RBO seeds required similar fluence to wild-type seeds. A critical threshold wavelength for either induction or inhibition of germination of ABO seeds shifted towards the longer wavelengths relative to wild-type seeds. By assuming that PhyA and PhyB are similar in their photochemical parameters, amounts of Pfr at each wavelength were calculated. The photon fluence required for 50% germination was equivalent to the fluence generating a Pfr/Ptot ratio of 0.21-0.43 in wild-type seeds, and of 0.035-0.056 in ABO seeds. These results indicate that PhyB-specific seed germination is not strictly a function of the Pfr/Ptot ratio, but is probably a function of the absolute Pfr concentration.

Solution structure of CINC/Gro investigated by heteronuclear NMR.

Cytokine-induced neutrophil chemoattractant (CINC/Gro) is a chemotactic factor for neutrophils in the rat and a member of the CXC chemokine family. The refined three-dimensional structure of CINC/Gro was derived with the aid of heteronuclear magnetic resonance spectroscopy and a hybrid method of distance geometry and simulated annealing. The backbone atomic r.m.s. differences for 19 structures about the mean coordinates for residues 7 to 70 are 0.69+/-0.15 A for the dimer and 0.56+/-0.13 A for the monomer. The N terminal region containing an ELR sequence, an essential motif for chemotactic activity, is disordered in solution, as in other CXC chemokines. The dimer structure consists of a six-stranded anti-parallel beta sheet with two C-terminal alpha helices on the beta sheet. The overall dimer structure of CINC/Gro is similar to that of human MGSA, though the backbone r.m.s.d.s between CINC/Gro and the two MGSA structures were high (1.81 and 2.34 A for the monomer) in spite of the high sequence homology (67%). The major difference resides in the relative position of the C-terminal alpha helix with respect to the beta sheet. This results in a difference of interhelical distances in the dimer: wider (15 A) in CINC/Gro, as in IL-8, than in MGSA (11.7 and 10 A).

Genetic identification of FIN2, a far red light-specific signaling component of Arabidopsis thaliana.

Phytochrome A (PhyA) mediates most, if not all, various plant responses to far-red (FR) light. Here, we report a novel genetic mutation that impairs a variety of responses in the PhyA-signaling pathway of Arabidopsis thaliana. The mutation was isolated by screening seedlings that show reduced sensitivity to continuous far-red (FRc) light irradiation, but not to continuous red (Rc) light irradiation. The mutation named fin2-1 is not allelic to a PHYA mutation. Furthermore, immunoblot analysis indicated that the amount of the phytochrome A apoprotein in the fin2-1 mutant was comparable to that in wild type. Seedling of the fin2-1 mutant showed defects in hypocotyl growth inhibition and apical hook and cotyledon opening in FRc light but not in Rc light. The results showed that the mutation occurred in a downstream signaling component potentially specific to PhyA. Other PhyA-mediated responses such as FR-preconditioned blocking of greening, anthocyanin accumulation, reduction of gravitropic response, and expression of the CAB and CHS genes were impaired by the fin2-1 mutation: the degree of the mutant effect on the responses was variable. However, FR light-mediated seed germination and photoperiodic flowering responses were not affected significantly in the mutant. These results showed that FIN2 defines an upstream branch point in the PhyA signaling pathway.

Subunit association and monomer structure of CINC/Gro revealed by 1H-NMR.

Rat CINC/Gro is a 72 residue chemotactic factor of neutrophils, and a member of the CXC chemokine family, that includes IL-8 and MGSA/GRO. Although the three-dimensional structure of CINC/Gro had previously been determined to be that of a dimer with 200 mM NaCl, it was shown on both ultracentrifugation analysis and 1H-NMR spectral analysis that CINC/Gro exists mainly as a monomer at a physiological concentration, similar to other proteins belonging to this family. By reducing the NaCl concentration, the equilibrium could be shifted to the monomer, making it possible to observe the monomer and dimer resonances in 1H-NMR spectra. There were no significant chemical shift changes of alpha protons in the beta sheet between the monomer and dimer, suggesting that the beta sheet structure was retained in the monomer. Instead, the chemical shift changes of a protons were significant at I18 and K21, which are located in the long loop region interacting with the alpha helix, and V59 at the beginning of the a helix, indicating structural changes in the relative positions of the alpha helix and beta sheet.

Fluence and wavelength requirements for Arabidopsis CAB gene induction by different phytochromes.

The roles of different phytochromes have been investigated in the photoinduction of several chlorophyll a/b-binding protein genes (CAB) of Arabidopsis thaliana. Etiolated seedlings of the wild type, a phytochrome A (PhyA) null mutant (phyA), a phytochrome B (PhyB) null mutant (phyB), and phyA/phyB double mutant were exposed to monochromatic light to address the questions of the fluence and wavelength requirements for CAB induction by different phytochromes. In the wild type and the phyB mutant, PhyA photoirreversibly induced CAB expression upon irradiation with very-low-fluence light of 350 to 750 nm. In contrast, using the phyA mutant, PhyB photoreversibly induced CAB expression with low-fluence red light. The threshold fluences of red light for PhyA- and PhyB-specific induction were about 10 nmol m-2 and 10 mumol m-2, respectively. In addition, CAB expression was photoreversibly induced with low-fluence red light in the phyA/phyB double mutant, revealing that another phytochrome(s) (PhyX) regulated CAB expression in a manner similar to PhyB. These data suggest that plants utilize different phytochromes to perceive light of varying wave-lengths and fluence, and begin to explain how plants respond so exquisitely to changing light in their environment.

Hydrophilicity of quinolones is not an exclusive factor for decreased activity in efflux-mediated resistant mutants of Staphylococcus aureus.

The elevated expression of the norA gene is responsible for efflux-mediated resistance to quinolones in Staphylococcus aureus (E.Y.W. Ng, M. Trucksis, and D.C. Hooper, Antimicrob. Agents Chemother. 38:1345-1355, 1994). For S. aureus transformed with a plasmid containing the cloned norA gene, SA113(pTUS20) (H. Yoshida, M. Bogaki, S. Nakamura, K. Ubukata, and M. Konno, J. Bacteriol. 172:6942-6949, 1990), and an overexpressed mutant, SA-1199B (G.W. Kaatz, S.M. Seo, and C.A. Ruble, J. Infect. Dis. 163:1080-1086, 1991), the MICs of norfloxacin increased 16 and 64 times compared with its MICs for the recipient and wild-type strains, SA113 and SA-1199, respectively. MICs of CS-940, however, increased only two and eight times, even though these two fluoroquinolones are similarly hydrophilic (apparent logPs of approximately -1). No good correlation was found, among 15 developed and developing quinolones, between the increment ratio in MICs and hydrophobicity (r = 0.61). Analysis of the quantitative structure-activity relationship among 40 fluoroquinolones revealed that the MIC increment ratio was significantly correlated with the bulkiness of the C-7 substituent and bulkiness and hydrophobicity of the C-8 substituent of fluoroquinolones (r = 0.87) and not with its molecular hydrophobicity (r = 0.47). Cellular accumulation of norfloxacin in SA-1199B was significantly lower than that in SA-1199, and it was increased by addition of carbonyl cyanide m-chlorophenyl hydrazone. On the other hand, accumulations of CS-940 in these strains were nearly identical, and they were not affected by addition of the protonophore.

Action spectra for phytochrome A- and B-specific photoinduction of seed germination in Arabidopsis thaliana.

We have examined the seed germination in Arabidopsis thaliana of wild type (wt), and phytochrome A (PhyA)- and B (PhyB)-mutants in terms of incubation time and environmental light effects. Seed germination of the wt and PhyA-null mutant (phyA) was photoreversibly regulated by red and far-red lights of 10-1,000 micromol m-2 when incubated in darkness for 1-14 hr, but no germination occurred in PhyB-null mutant (phyB). When wt seeds and the phyB mutant seeds were incubated in darkness for 48 hr, they synthesized PhyA during dark incubation and germinated upon exposure to red light of 1-100 nmol m-2 and far-red light of 0.5-10 micromol m-2, whereas the phyA mutant showed no such response. The results indicate that the seed germination is regulated by PhyA and PhyB but not by other phytochromes, and the effects of PhyA and PhyB are separable in this assay. We determined action spectra separately for PhyA- and PhyB-specific induction of seed germination at Okazaki large spectrograph. Action spectra for the PhyA response show that monochromatic 300-780 nm lights of very low fluence induced the germination, and this induction was not photoreversible in the range examined. Action spectra for the PhyB response show that germination was photoreversibly regulated by alternate irradiations with light of 0.01-1 mmol m-2 at wavelengths of 540-690 nm and 695-780 nm. The present work clearly demonstrated that PhyA photoirreversibly triggers the germination upon irradiations with ultraviolet, visible and far-red light of very low fluence, while PhyB controls the photoreversible effects of low fluence.

Carbohydrate structures of the glycoprotein allergen Cry j I from Japanese cedar (Cryptomeria japonica) pollen.

The glycoprotein allergen Cry j I from Japanese cedar (Cryptomeria japonica) pollen was treated with pepsin and glycopeptidase A to release asparagine-linked oligosaccharides. The reducing ends of the oligosaccharides were aminated with the fluorescent reagent 2-aminopyridine. The oligosaccharide derivatives were purified by gel permeation chromatography and reversed-phase HPLC. Their structures were determined by sequential exoglycosidase digestion and 500 MHz 1H-NMR spectroscopy. Four oligosaccharide structures, A, B, C, and D, were identified as the xylose-containing complex-type. They were present at a molar ratio of 8:1:6:1. By amino acid sequence analyses of the tryptic peptides, Asn-170 and Asn-333 of Cry j I were found to carry asparagine-linked oligosaccharides. [formula: see text]

The three dimensional structure of rat cytokine CINC/Gro in solution by homonuclear 3D NMR.

The solution conformation of rat cytokine-induced neutrophil chemoattractant (CINC/Gro), a small protein consisting of 72 amino acid residues with proinflammatory activities, and a member of the interleukin 8 family corresponding to a counterpart of human Gro, was investigated with homonuclear 2D and 3D NMR spectroscopy. At each phase of the structural analysis, the homonuclear 3D NOESY-HOHAHA and HOHAHA-NOESY spectra afforded valuable data, removing ambiguities intractable by conventional 2D NMR techniques. CINC/Gro exists as a dimer in solution and contains a triple stranded anti-parallel beta-sheet and C-terminal alpha-helix in the monomer structure, as observed in human IL-8, but non-trivial differences are also observed.