Atomic Force Microscopy Imaging of Double Stranded DNA and RNA

Abstract

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

Visiting bags: a labile thermal environment.

OBJECTIVE--To define usual colour and site of storage of visiting bags in general practitioners'' cars and to investigate effect of these variables on temperature inside bag. DESIGN--Questionnaire to general practitioners; serial temperature measurements from paired black visiting bags at different storage sites and from bags of different colour. SETTING--South Devon coastal town during May and June. SUBJECTS--200 general practitioners, of whom 145 returned legible questionnaires. MAIN OUTCOME MEASURES--Bag colour, duration and site of storage, temperature inside black bags at defined storage sites, and effects of bag colour on internal temperature. RESULTS--111 (77%) of the general practitioners carried a black visiting bag, and 76 kept their bag in their car all day. The bag was coolest in the car boot, but irrespective of storage site, maximum internal temperature of the bag was always over 25 degrees C and reached up to 80 degrees C. Spraying a black bag silver significantly reduced the bag''s internal temperature (mean difference 8.37 degrees C (95% confidence interval 6.68 to 9.86 degrees C) df = 59, t = 10.29, P < 0.001). CONCLUSIONS--General practitioners should use a silver coloured visiting bag; when visiting, they should store it in their car boot; at other times they should remove it to a cooler site.     

Molecular characterization of the proteinase-encoding gene, prb1, related to mycoparasitism by Trichoderma harzianum

The soil fungus Trichoderma harzianum is a mycoparasitic fungus known for its use as a biocontrol agent of phytopathogenic fungi. Among other factors, Trichoderma produces a series of antibiotics and fungal cell wall-degrading enzymes. These enzymes are believed to play an important role in mycoparasitism. Among the hydrolytic enzymes, we have identified a basic proteinase (Prb1) which is induced by either autoclaved mycelia, fungal cell wall preparation or chitin; however, the induction does not occur in the presence of glucose. The proteinase was purified and biochemically characterized as a serine proteinase of 31 kDa and pl 9.2. Based on the sequence of three internal peptides, synthetic oligonudeotide probes were designed. These probes allowed subsequent isolation of a cDNA and its corresponding genomic clone. The deduced amino acid sequence indicates that the proteinase is synthesized as a pre-proenzyme and allows its classification as a serine proteinase. Northen analysis shows that the induction of this enzyme is due to an increase in the corresponding mRNA level.     

NUMB does not impair growth and differentiation status of experimental gliomas

The cell fate determinant NUMB orchestrates asymmetric cell division in flies and mammals and has lately been suggested to have a tumor suppressor function in breast and lung cancer. Here, we studied NUMB in the context of malignant gliomas. We used ectopic expression of NUMB in order to inhibit proliferation and induce differentiation in glioma cells by alteration of Notch, Hedgehog and p53 signaling. We found that NUMB is consistently expressed in glioma biopsies with predominance of NUMB2/4 isoforms as determined by isoform-specific real-time PCR and Western blotting. Upon lentiviral overexpression, in vitro proliferation rate and the grade of differentiation as assessed by morphology and expression of neural and glial markers remained unchanged. Orthotopic xenografts of NUMB-transduced human U87 glioma cells could be established in nude rats without impairing engraftment or causing significant changes in morphology based on magnetic resonance imaging (MRI). The previously reported alteration of Hedgehog and p53 signaling by NUMB could not be recapitulated in glioma cells. We thus show that in experimental gliomas, NUMB overexpression most likely does not exert a tumor suppressor function such as seen in epithelial cancers.     

Changes in the amount of lung and airway phosphatidylcholine in 0.5-12.5-day-old rabbits

Newborn rabbits delivered spontaneously at term and cared for by the mothers were studied from 0.5 to 12.5 days of age. Curves are constructed to describe the changes in weight, lung and alveolar wash phosphatidylcholine and saturated phosphatidylcholine, and lung protein. The curves are complex and non-linear. However. expressing the increases in lung and alveolar wash phosphatidylcholine and saturated phosphatidylcholine pool sizes relative to animals weight results in a decreasing linear relationship from 0.5 to 12.5 days of age. By 12.5 days the ratios of lung phosphatidylcholine and saturated phosphatidylcholine to weight approximate the ratios measured for adult rabbits. The ratios of saturated to total phosphatidylcholine in the alveolar washes and lungs remained invarient throughout the study period.     

Localization,transmission, spontaneous mutations,and variation of function of the Dpp4 (Dipeptidyl-peptidase IV; CD26) gene in rats

Dipeptidyl-peptidase IV (DPPIV) is involved in endocrine and immune functions via cleavage of regulatory peptides with a N-terminal proline or alanine such as incretins, neuropeptide Y, or several chemokines. So far no systematic investigations on the localization and transmission of the Dpp4 gene or the natural variations of DPPIV-like enzymatic function in different rat strains have been conducted. Here we mapped the Dpp4 gene to rat chromosome 3 and describe a semi-dominant mode of inheritance for Dpp4 in a mutant F344/DuCrj(DPPIV-) rat substrain lacking endogenous DPPIV-like activity. This mutant F344/DuCrj(DPPIV-) rat substrain constantly exhibits a nearly complete lack of DPPIV-like enzymatic activity, while segregation of DPPIV-like enzymatic activity was observed in another DPPIV-negative F344/Crl(Ger/DPPIV-) rat substrain. Screening of 12 different inbred laboratory rat strains revealed dramatic differences in DPPIV-like activity ranging from 11 mU/microl (LEW/Ztm rats) to 40 mU/microl (BN/Ztm and DA/Ztm rats). A lack of DPPIV-like activity in F344 rats was associated with an improved glucose tolerance and blunted natural killer cell function, which indicates the pleiotropic functional role of DPPIV in vivo. Overall, the variations in DPPIV-like enzymatic activity probably represent important confounding factors in studies using rat models for research on regulatory peptides.     

Interleukin-12 but not interleukin-18 is required for immunity to Trypanosoma cruzi in mice

Protective immunity to the parasite Trypanosoma cruzi in mice depends on a pro-inflammatory T cell response involving the production of interferon-gamma (IFN-gamma). In conjunction with interleukin-12 (IL-12), IL-18 promotes the synthesis of IFN-gamma and a T helper type 1 immune response. We investigated the requirements of IL-12 and IL-18 in murine T. cruzi infection by use of C57BL/6 mice genetically deficient in either cytokine. IL-12p40(-/-) mice succumbed to infection at doses of 100 parasites, whereas IL-18(-/-) and wild-type mice resisted infectious doses up to 1000 parasites to the same extent. Levels of parasitemia were comparable between the latter groups, as were tissue parasite burdens according to quantitative real-time PCR. In contrast, IL-12p40(-/-) mice displayed vastly increased levels of parasites both in blood and in tissue. IFN-gamma concentrations in the serum of infected mice and in supernatants of splenocytes stimulated in vitro were decreased in IL-18(-/-) mice, whereas in IL-12p40(-/-) mice, IFN-gamma was undetectable in the serum and drastically reduced in cell supernatants. Levels of IL-12 production were generally comparable between wild-type and IL-18(-/-) mice, as were levels of IL-4, IL-2 and nitric oxide. Thus, the requirement for endogenous pro-inflammatory cytokines for a protective murine immune response against T. cruzi is satisfied by the expression of IL-12, while IL-18 is dispensable.     

Drosophila neuroglian: a member of the immunoglobulin superfamily with extensive homology to the vertebrate neural adhesion molecule L1

Drosophila neuroglian is an integral membrane glycoprotein that is expressed on a variety of cell types in the Drosophila embryo, including expression on a large subset of glial and neuronal cell bodies in the central and peripheral nervous systems and on the fasciculating axons that extend along them. Neuroglian cDNA clones were isolated by expression cloning. cDNA sequence analysis reveals that neuroglian is a member of the immunoglobulin superfamily. The extracellular portion of the protein consists of six immunoglobulin C2-type domains followed by five fibronectin type III domains. Neuroglian is closely related to the immunoglobulin-like vertebrate neural adhesion molecules and, among them, shows most extensive homology to mouse L1. Its homology to L1 and its embryonic localization suggest that neuroglian may play a role in neural and glial cell adhesion in the developing Drosophila embryo. We report here on the identification of a lethal mutation in the neuroglian gene.