Interaction forces and conduction properties between multi wall carbon nanotube tips and Au(111).

We have studied the interaction forces and electrical conduction properties arising between multiwall carbon nanotube tips and the Au(111) surface in air, by means of amplitude modulation scanning force microscopy, also called intermittent contact. We have centered our work on tips with metallic electronic structure and for the specific parameters used we have found a preliminary interaction range where there is no contact between tip and surface. Stable imaging in this non-contact range is possible with multiwall carbon nanotube tips. These tips have also been used to obtain simultaneous topographic and current maps of the surface. They show excellent properties as tips due to their high aspect ratio and durability, as a result of their elastic and non-reactive properties. Correspondingly, multiwall carbon nanotube tips allow high resolution local analysis of electrical conductivity on a nanometer scale.

Patterning DNA on microm scale on mica.

Double-stranded DNA molecules were patterned by selective adsorption to aminosilane patterns on mica surfaces. Line patterns with 10 microm spacing were made by photolithography and transferred to a polymer stamp. The stamp was then used for applying aminosilane molecules by microcontact-printing technique on mica substrates. We applied DNA in Tris-EDTA (TE) buffer solution on the patterned substrate, and incubated it for 5 min at room temperature. The sample was then rinsed with pure water, and dried with nitrogen gas. Tapping mode force microscopy showed that DNA was adsorbed selectively on the aminosilanized parts of the mica substrate. We also tried to bridge two aluminum electrodes with DNA using AC electrophoresis.

Atomic force microscopy of RecA--DNA complexes using a carbon nanotube tip.

We report high resolution images of RecA-double stranded (ds) DNA complexes obtained by atomic force microscopy (AFM). When a carbon nanotube (CNT) tip was used, AFM images visualized the 10-nm pitch of RecA-dsDNA complexes and RecA filaments as three-dimensional surface topography without reconstruction analysis. The depth of the notch between two pitches was less than 1 nm. When adsorbed on a soft surface covered with proteins, naked DNA, RecA monomers, RecA hexamers, and short RecA filaments were all clearly resolved in one image. The high resolution images with a CNT tip provided valuable information on the initiation process of RecA-dsDNA complex formation.

Measurement of the length of the a helical section of a peptide directly using atomic force microscopy.

Using atomic force microscopy (AFM), the length of the alpha-helix structure of poly-L-lysine was investigated by stretching the peptide directly, one molecule at a time. In the absence of urea, many rupturing points that seemed to be due to the breaking of some hydrogen bonds were observed in force-extension curves, while these points were never observed in the presence of 8 M urea. In the presence of 0.4 or 1.6 M urea, both force-extension curve types were observed. Total peptide elongation for each condition was calculated from force-extension curves reflecting the alpha-helix rupturing process. The experimental value of total elongation divided by the theoretical value of total alpha-helix elongation yields the alpha-helix content. This value was compatible with circular dichroism (CD) measurement results. This suggests that peptide conformation and content of the alpha-helix on a single molecule scale can be investigated by direct mechanical measurement using atomic force microscopy.

RecA-double stranded DNA complexes studied by atomic force microscopy.

RecA-double stranded (ds) DNA complexes have been studied by atomic force microscopy (AFM). When the complexes were prepared in the presence of ATP gamma S, fully covered RecA-dsDNA filaments were observed by AFM. When the concentration of RecA proteins was lower, various lengths of filaments were found. The variation of the observed structures may directly reflect the real distribution of the intermediate complexes in the reaction mixture, as the mixture was simply deposited on a mica surface for AFM observation without special fixation or staining. The use of a carbon nanotube (CNT) AFM tip enabled high resolution to reveal the periodicity of RecA-dsDNA filaments. Our observations demonstrated the potential of the AFM method for the structural studies of the RecA-dsDNA complexes, especially their intermediate states.

Growth regulation mechanisms in higher plants under microgravity conditions - changes in cell wall metabolism.

During Space Shuttle STS-95 mission, we cultivated seedlings of rice (Oryza sativa L. cv. Koshihikari and cv. Tan-ginbozu) and Arabidopsis (Arabidopsis thaliana L. cv. Columbia and cv. etr1-1) for 68.5, 91.5, and 136 hr on board, and then analyzed changes in the nature of their cell walls, growth, and morphogenesis under microgravity conditions. In space, elongation growth of both rice coleoptiles and Arabidopsis hypocotyls was stimulated. Also, the increase in the cell wall extensibility, especially that in the irreversible extensibility, was observed for such materials. The analyses of the amounts, the structure, and the physicochemical properties of the cell wall constituents indicated that the decreases in levels and molecular masses of cell wall polysaccharides were induced under microgravity conditions, which appeared to contribute to the increase in the wall extensibility. The activity of certain wall enzymes responsible for the metabolic turnover of the wall polysaccharides was increased in space. By the space flight, we also confirmed the occurrence of automorphogenesis of both seedlings under microgravity conditions; rice coleoptiles showed an adaxial bending, whereas Arabidopsis hypocotyls elongated in random directions. Furthermore, it was shown that spontaneous curvatures of rice coleoptiles in space were brought about uneven modifications of cell wall properties between the convex and the concave sides.

Analysis of HLA-DRB1-related alleles in Japanese patients with lung cancer--relationship to genetic susceptibility and resistance to lung cancer.

PURPOSE: To investigate differences in HLA status among lung cancer patients, patients with hematological malignancies, and healthy controls in order to determine the genetic susceptibility and resistance features of HLA-DRB1-related alleles in Japanese patients with lung cancer. METHODS: HLA class I (HLA-A, -B, and -C) antigens and HLA class II (HLA-DRB1) alleles were determined in 36 patients with lung cancer, 35 patients with hematological malignancies, and 90 healthy controls. HLA class I status was investigated by serological techniques, and HLA class II by polymerase chain reaction/restriction-fragment-length polymorphism analysis. RESULTS: Lung cancer patients showed an increased frequency of HLA-DRB1*0901, and a decreased frequency of HLA-DRB1*1302 and DRB1*14-related alleles when compared to the other subjects. CONCLUSION: These results suggest that genetic factors influence the susceptibility and resistance to lung cancer. However, this study should be considered preliminary because of the relatively small number of patients examined and the possibility of racial differences in HLA status of lung cancer patients between Japan and other countries.

Spinocerebellar ataxia type 6: MRI of three Japanese patients.

We describe the MRI findings in three Japanese patients with spinocerebellar ataxia type 6 (SCA6) in which a polymorphic CAG repeat was identified in the gene encoding the alpha 1A voltage-dependent P/Q-type Ca2+ channel subunit (CACNL1A4). All showed slowly progressive cerebellar ataxia and mild pyramidal signs. Neuroradiologically, they had moderate cerebellar atrophy, most prominently in the superior vermis, whereas the brain stem appeared to be spared. No abnormal signal intensity was identified.

Design and racemic synthesis of conformationally restricted carbocyclic pyrimidine nucleoside analogs based on the structure of the L-nucleoside residue in heterochiral DNA.

Carbocyclic pyrimidine nucleoside analogs which have restricted glycosidic conformation at chi approximately 180 degrees were designed, based on the conformational features of the L-nucleotide residue in heterochiral DNA, and synthesized. The synthesis of (+/-)-carbocyclic 6,6'-O-cyclo-2'-deoxyuridine was achieved via bromination and subsequent intramolecular cyclization of carbocyclic 6'beta-hydroxy-2'-deoxyuridine. (+/-)-Carbocyclic 6,6'-O-cyclo-2'-deoxycytidine was synthesized from protected carbocyclic 6,6'-O-cyclo-2'-deoxyuridine via the 4-triazole intermediate.

Adult-onset Krabbe disease with homozygous T1853C mutation in the galactocerebrosidase gene. Unusual MRI findings of corticospinal tract demyelination.

A 51-year-old woman developed a slowly progressive spastic paraparesis and diminished vibration sense beginning at age 38. Intellectual capacity was normal. Krabbe disease was confirmed by markedly reduced leukocyte galactocerebrosidase (GALC) activity, typical inclusions in Schwann cell cytoplasm, and an identification of the homozygous point mutation T1835C (Leu618Ser) in the GALC gene. T2-weighted MRI of the brain showed symmetric high-signal-intensity lesions in the bilateral frontoparietal white matter, the centrum semiovale, and the posterior limb of the internal capsule with sparing of the periventricular white matter. This case is unusual because of the late onset, protracted clinical course, and MRI findings of demyelination confined to the corticospinal tracts.

[Primary pulmonary hemangiopericytoma detected on routine chest X-ray examination].

Hemangiopericytoma is a rare tumor originating from pericytes. In May 1988, a 33-year-old man was found to have a well-defined nodular shadow in the right upper lobe during a routine chest X-ray examination. Although the mass had been thought to be benign, in December 1992 it was found to have grown. In May 1993, the patient was referred to our hospital for further examination. A chest X-ray film and a high-resolution CT scan revealed a well-defined nodule in the right upper lung field without vascular gathering or pleural puckering. The tumor was slightly less dense than was soft tissues. There was no evidence suggesting another primary tumor or metastasis. In July 1993, because the mass was suspected to be a low-grade malignant tumor, a segementectomy (rt-S2) was done. On the basis of histologic, immunohistochemical and electronmicroscopic findings, pulmonary hemangiopericytoma was diagnosed. The postoperative course was uneventful. The patient has been well for 2 years and five months after the operation, with no sign of recurrence or metastasis.

Lipocortin-1 and the control of arachidonic acid release in cell signalling. Glucocorticoids (changed from glucorticoids) inhibit G protein-dependent activation of cPLA2 activity.

In pre-labelled A549 cells epidermal growth factor (EGF) (10 nM) stimulates the release of [5,6,8,9,11,12,14,15-3H(N)]-arachidonic acid (3H-AA) by approximately 70%. Increasing Ca2+i with thapsigargin (50 nM) stimulates 3H-AA release by approximately 120%. However, the combined use of these two agents results in a synergistic stimulation of 3H-AA release by over 700%. The EGF stimulated release is sensitive to pertussis toxin (10 ng/mL) and guanosine 5'-O-(2-thiodiphosphate) suggesting a G protein-mediated event. This is supported by the fact that the G protein activators AlF-4 and guanosine 5'-O-(2-thiotriphosphate) both stimulate 3H-AA release. The stimulation of 3H-AA release by both EGF or direct G protein activation is completely blocked following pre-treatment for 3 hr with 1 nM dexamethasone. This effect is reversed with a neutralizing antibody to lipocortin-1 (1 microgram/mL) suggesting that this protein mediates the inhibitory effects of glucocorticoids on agonist activated 3H-AA release. Thapsigargin stimulation of 3H-AA release is insensitive to dexamethasone treatment. A peptide fragment from the N-terminus of lipocortin-1-Lc13-25 (20-200 micrograms/mL) mimics the effect of glucocorticoid in suppressing both EGF and G protein activated 3H-AA release. A peptide with Me-Tyr substituting Tyr21 is much reduced in activity suggesting that the presence of this residue is essential. As peptide Lc13-25 is not derived from the Ca2+/phospholipid binding domain of the native protein then sequestration of phospholipid substrate for PLA2 remains an unlikely mechanism of action for this peptide.

Differential role of extra- and intracellular calcium in bradykinin and interleukin 1 alpha stimulation of arachidonic acid release from A549 cells.

The release of arachidonic acid in A549 cells was stimulated in a time- and dose-dependent manner by the Ca2+ ionophore ionomycin (t1/2 = 4 min), thapsigargin (t1/2 = 8 min), bradykinin (t1/2 = 12 min, EC50 = 3 nM), and interleukin 1 alpha (t1/2 = 28 min, EC50 = 0.3 ng/ml). Bradykinin (10 nM) and interleukin 1 alpha (1 ng/ml) stimulation was blocked by the bradykinin B2 receptor antagonist, D-Arg,[Hyp3,Thi5,8, D-Phe7]bradykinin and interleukin 1 receptor antagonist (IC50 = 30 mM and 20 ng/ml, respectively), suggesting receptor mediation. Diacylglycerol release was < 10% of total arachidonic acid release in all cases, suggesting activation of phospholipase A2 activity was greater than phospholipase C activation by these agents. The effects of ionomycin (3 microM) and thapsigargin (0.3 microM) were abolished in Ca(2+)-free buffer with and without 0.5 mM EGTA. Bradykinin (10 nM) stimulation was reduced by 50% in Ca(2+)-free buffer whereas interleukin 1 alpha (1 ng/ml) stimulation remained unaffected. However, the presence of EGTA completely abolished bradykinin stimulation and partially blocked the effect of interleukin 1 alpha (43% inhibition). In the presence of extracellular Ca2+, ionomycin (3 mM), thapsigargin (0.3 mM), bradykinin (10 nM), and interleukin 1 alpha (1 ng/ml) stimulation of arachidonic acid release was blocked by the Ca2+ influx blocker LaCl3 (29, 44, 35, and 41% inhibition, respectively). Nifedipine also blocked ionomycin and thapsigargin stimulation but only partially blocked bradykinin and interleukin 1 alpha stimulation. These results suggest that following B2 receptor activation, cytosolic phospholipase A2 is stimulated by a rise in intracellular Ca2+ levels which are sensitive to the action of EGTA, whereas interleukin 1 alpha stimulation of cytosolic phospholipase A2 is mediated by a rise in intracellular Ca2+ from both EGTA-sensitive and resistant pools. Furthermore the results of ionomycin and thapsigargin indicate that extracellular Ca2+ is important for activation of cytosolic phospholipase A2 in A549 cells.

Phospholipase A2-induced stimulation of A549 lung adenocarcinoma cell line proliferation.

Phospholipase A2 (PLA2) activity was found in the cytosolic fraction of the A549 human lung adenocarcinoma line. This PLA2 had a molecular mass of approximately 70 kDa as assessed by gel filtration chromatography and required submicromolar concentrations of calcium concentrations for optimal activity. These characteristics are consistent with the cytosolic PLA2 recently reported in other cell types, such as U937 cells. We have now demonstrated that A549 cell PLA2 (PLA2 activity: 1 unit/ml) partially purified by gel filtration stimulated proliferation of A549 cells by 50% after 3 days of culture. Similarly, porcine pancreatic PLA2 (0.1 unit/ml) also promoted proliferation of A549 cell cultures by 42%. Furthermore, A549 cell PLA2 stimulated prostaglandin E2 release (approx. 7-fold increase). Both PLA2s lost activity when treated with p-bromophenacyl bromide. Neither porcine pancreatic PLA2 nor A549 cell PLA2 reversed the inhibitory activities of dexamethasone and indomethacin on cell growth. These results suggest that both of these PLA2s stimulate A549 cell growth, and that this is likely to be mediated by increased eicosanoid production.

Detection of IgA binding site by flow cytometry with fluorescent microspheres.

To establish a sensitive method to detect IgA Fc receptors (Fc alpha R) on human and murine lymphoid cells, fluorescent microspheres (FMS) were used in a flow cytometric assay. The following three assays with FMS were tested and compared with a conventional indirect Fc alpha R assay using FITC-labeled anti-IgA: (1) direct cell binding assay with murine myeloma IgA(MOPC315)-coated FMS(IgA-FMS assay), (2) indirect assay with TNP-BSA-coated FMS which bind to cells preincubated with MOPC315 IgA bearing anti-TNP activity (TNP-BSA-FMS assay), and (3) indirect assay with anti-IgA coated FMS after preincubation of the cells with IgA(anti-IgA-FMS assay). In these three assays for Fc alpha R using FMS, the binding of IgA to the cells was not affected by purified IgM or IgG preparations. In both indirect assays using TNP-BSA-FMS and anti-IgA-FMS, sharp and dose dependent IgA binding was obtained at lower IgA concentrations ranging from 4 to 125 micrograms/ml as compared with the conventional indirect assay. The background MFI levels in all these FMS assays remained as low as those in the conventional assay. These findings suggest that FMS coupled with TNP-BSA or anti-IgA antibodies is suitable for the detection of Fc alpha R on both murine and human T cells.

Interleukin 4 (IgG1 induction factor): a multifunctional lymphokine acting also on T cells.

A cDNA encoding the murine interleukin 4 (IL4) (IgG1 induction factor/B cell-stimulating factor no. 1) was recently cloned (Noma et al., Nature 1986.319: 640; Lee et al., Proc. Natl. Acad. Sci. USA 1986. 83: 2061). In this report we tested recombinant IL 4 in various T cell assays. It was found that IL 4 activated the murine T cell line CTLL to increased DNA synthesis but not to growth. It also activated normal concanavalin A (Con A)-stimulated T cells both to increased DNA synthesis and to growth. These T cell growth factor-like activities were not inhibitable by anti-IL 2 receptor antibodies. Evidence is given that both Lyt-2+ and L3T4+ T cells responded to IL 4. Finally, IL 4 acted synergistically with phytohemagglutinin or Con A on normal T lymphocytes as well as on thymocytes. These data, as well as those of others, imply that lymphokines have a broader range of activity than previously anticipated.