Metal-free scanning optical microscopy with a fractal fiber probe.

Scanning Near-field Optical Microscopy (SNOM) is the leading instrument used to image optical fields on the nanometer scale. A metal-coating is typically applied to SNOM probes to define a subwavelength aperture and minimize optical leakage, but the presence of such coatings in the near field of the sample can often cause a substantial change in the sample emission properties. For the first time, the authors demonstrate near-field imaging on a metal substrate with a metal-free probe made from a novel structured optical fiber, designed to maximize optical throughput and potentially remove the need for the metal.

A fractal-based fibre for ultra-high throughput optical probes.

A core component of all scanning near-field optical microscopy (SNOM) systems is the optical probe, which has evolved greatly but still represents the limiting component for the system. Here, we introduce a new type of optical probe, based on a Fractal Fibre which is a special class of photonic crystal fibre (PCF), to directly address the issue of increasing the optical throughput in SNOM probes. Optical measurements through the Fractal Fibre probes have shown superior power levels to that of conventional SNOM probes. The results presented in this paper suggest that a novel fibre design is critical in order to maximize the potential of the SNOM.

Transcriptional repression by nuclear receptors: mechanisms and role in disease.

Co-repressor proteins mediate transcriptional repression by nuclear receptors in the absence of ligand. The identification of a co-repressor-receptor interaction motif, and the finding that co-repressors and co-activators compete for the same site on the receptor, suggests a simple mechanism for the switch from repression to activation upon ligand binding. Defects in this mechanism result in dominant-negative receptors that repress transcription. Such receptors have been implicated in several clinically important diseases, including thyroid hormone resistance and diabetes mellitus.

Mechanism of corepressor binding and release from nuclear hormone receptors.

The association of transcription corepressors SMRT and N-CoR with retinoid and thyroid receptors results in suppression of basal transcriptional activity. A key event in nuclear receptor signaling is the hormone-dependent release of corepressor and the recruitment of coactivator. Biochemical and structural studies have identified a universal motif in coactivator proteins that mediates association with receptor LBDs. We report here the identity of complementary acting signature motifs in SMRT and N-CoR that are sufficient for receptor binding and ligand-induced release. Interestingly, the motif contains a hydrophobic core (PhixxPhiPhi) similar to that found in NR coactivators. Surprisingly, mutations in the amino acids that directly participate in coactivator binding disrupt the corepressor association. These results indicate a direct mechanistic link between activation and repression via competition for a common or at least partially overlapping binding site.

Fabrication and characterization of a planar gradient-index, plasma-enhanced chemical vapor deposition lens.

A thin, one-dimensional, gradient-index slab lens with a parabolic profile was designed and fabricated in fluorine-doped silica by use of plasma-enhanced chemical vapor deposition in a Helicon plasma reactor. The refractive-index profile of the fabricated lens was determined by the application of an inversion technique to the values of modal effective index measured with a prism coupler. The periodic refocusing property of the lens and the independence of the wavelength were measured with the fluorescence of a specially doped, thin polymer layer spin-coated onto the surface of the lens.

Clinical and cost effectiveness of the counter-rotational brush in private practice.

There have previously been no studies on the cost effectiveness of the use of a counter-rotational toothbrush (INTERPLAK Home Plaque-Removal Instrument), which has been demonstrated to be more effective than ordinary toothbrushes in reaching plaque-removal and gingival-health goals. Killoy et al studied the costs of required periodontal treatment for 32 patients with moderate periodontitis at two corporate capitation dental centers. The subjects were divided into two groups, a test group that brushed with a counter-rotational toothbrush and one that brushed with a manual toothbrush. Probing depth, attachment levels, and plaque and bleeding indexes in the test brush group were better than those in the control group. The end result was a mean reduction of $535 in the cost of periodontal treatment that had been planned before using the counter-rotational brush over 18 months, while the group using the manual brush experienced a mean increase of $11 in required treatment over original plans. Furthermore, the test group reached a state of gingival health, but the control group did not. The study concluded that the counter-rotational brush is cost effective.

Clinical assessment of the INTERPLAK toothbrush vs a conventional brush plus floss.

Thirty clinical recall patients from the University of Missouri at Kansas City School of Dentistry participated in a 4-week study to determine the long-term effectiveness of the INTERPLAK Home Plaque-Removal Instrument compared to manual brushing and flossing. Patients were divided into two groups, with each group using the INTERPLAK device for 2 months and the manual brush for 2 months. Patients were assessed according to measured indices for plaque, bleeding, crevice depth, and calculus. The study found that both kinds of brushes significantly reduced plaque, bleeding, and crevice depth; however, only the INTERPLAK brush reduced calculus. Improvements on all indices were greater for the INTERPLAK device than they were for manual brushing and flossing. At the end of the 4 months, most patients indicated that they would continue to use the INTERPLAK device.

Ultraviolet-induced dimerization of non-adjacent pyrimidines in poly[d(A-T)].

The DNA photoproduct responsible for the ultraviolet (UV) light-induced -1 frameshift mutation remains unknown. We recently identified a UV photoproduct consisting of a cyclobutane dimer occurring between non-adjacent thymine residues in the same strand, [sequence: see text] and proposed that replication across this unrepaired photoproduct might result in a -1 frameshift mutation since the intervening base is extrahelical. Until now this novel photoproduct has only been identified in single-stranded DNA polymers and does not occur in UV-irradiated double-stranded polymers due to conformational restraint. This observation suggested that this photoproduct could only occur in vivo in chromosomal sites that were single-stranded. In the current work the cis-syn dithymine cyclobutane dimer has been identified in the self-complementary polymer poly[d(A-T)] when UV irradiated in solution conditions (concentrated manganese chloride or 60% ethanol plus trace salts) wherein this polymer remains double-stranded but the double-helix is partially destabilized. Taken together, the current findings suggest that dipyrimidine photoproducts between non-adjacent residues on the same strand could occur in vivo in double-stranded, but partially destabilized, DNA.

UV-induced interstrand cross-linking of d(GT)n.d(CA)n is facilitated by a structural transition.

Photochemical alterations following ultraviolet irradiation of the alternating copolymer d(GT)n.d(CA)n were studied. We found that in solution conditions which produced circular dichroism spectra compatible with B-form or A-form DNA, no interstrand cross-linking or photoproduct formation could be demonstrated. Zimmer et al. (Zimmer, C., Tymen, S., Marck, C., and Guschlbaumer, W. (1982) Nucleic Acids Res. 10, 1081-1091) and Vorlickova et al. (Vorlickova, M., Kypr, J., Sotkrova, S., Sponar, J. (1982) Nucleic Acids Res. 10, 1071-1080) have reported a number of solution conditions which produce a structural transition of this polymer characterized by a negative deviation of the circular dichroism spectrum in the region of 280 nm. The nature of this transition has not yet been elucidated. Following ultraviolet irradiation of d(GT)n.d(CA)n under two conditions which produce this transition (manganese solution or ethanol plus trace salts solution) we found ultraviolet dose-dependent interstrand cross-linking as well as dose-dependent formation of thymine-containing photoproduct. Interstrand cross-linking is demonstrated by two criteria: increase in polymer size as detected by alkaline agarose gel electrophoresis, and generation of intermediate density material in alkaline cesium sulfate isopycnic gradients. The thymine-containing photo-product was demonstrated by thin layer chromatography of acid hydrolysates of the polymer. The photo-product is at least partially photoreversible. These findings suggest that the geometry of the alternative conformation is such that pyrimidines from different strands are closely approximated, allowing for photodimerization.

Hydrogen peroxide toxicity may be enhanced by heat shock gene induction in Drosophila.

Recent evidence suggests that low dose exposure of cells to hydrogen peroxide and/or induction of heat shock protein (HSP) synthesis will render cells resistant to the lethal effects of a subsequent high dose hydrogen peroxide stress. We explored this possibility in the Drosophila melanogaster Schneider tissue culture line 2. It was found that chronic low dose exposure (1 mM H2O2 for 3 days) resulted in marked potentiation of the toxic effects of a subsequent high dose exposure (50 mM H2O2 for 1 h), as assessed by impairment of uridine incorporation and cell proliferation. Cells preexposed to low dose H2O2 exhibited enhanced heat shock gene transcription upon exposure to high dose H2O2, as compared to cells that did not receive low dose preexposure. Transcriptional induction of the heat shock genes by a mild non-toxic heat shock resulted in marked enhancement of the anti-proliferative effects of a subsequent H2O2 exposure. Thus, low dose hydrogen peroxide exposure or mild heating results in subsequent enhancement of high dose hydrogen peroxide toxicity; this effect correlates with enhanced heat shock gene expression. Possible mechanisms are discussed.

Screening of lambda library for differentially expressed genes using in vitro transcripts.

We present an improved approach to the screening of eucaryotic libraries for differential gene expression. Previous techniques have generated probe via the harvesting of cellular poly(A)+ RNA and synthesizing labeled cDNA probe using reverse transcriptase. In our approach we prepare labeled RNA probe via in vitro transcription. Unlike cDNA preparation, in vitro transcription (i) directly reflects the ongoing rate of gene expression, and (ii) allows one to assess expression of genes whose transcripts are not polyadenylated. To make this approach practical for the screening of a large library, we modified and optimized existing in vitro transcription techniques, enhancing manyfold the [alpha-32P]UTP incorporation into mRNA, while almost completely suppressing rRNA incorporation. In addition, we developed a simple procedure for making precise replicate dot blots of very large quantities of lambda-phage library DNA. By combining our techniques of in vitro transcription and replicate blotting, we are able to approach detection of a twofold difference in gene expression over a greater than 1000-fold range in overall expression. Our single-clone amplification and dot-blotting technique resulted in nearly the same number of lambda-phage DNA copies per dot for all members of the library. This feature allows us to assign genes to different expression classes, as well as to detect any alterations in expression. We demonstrate our approach by screening the drosophila genomic DNA library with in vitro transcripts from drosophila tissue culture cells. Screening of the entire drosophila genomic library at the 99% probability level is readily achieved.

Physical mapping and complete nucleotide sequence of the denV gene of bacteriophage T4.

Phage T4 deletion mutants that are folate analog resistant (far) and contain deletions in the region of the T4 genome near denV have been isolated previously. We showed that one of these mutants (T4farP12) expressed normal denV gene activity, whereas another mutant (T4farP13) was defective in the denV gene. The rII-distal (right) physical endpoints of these deletions defined the limits of the interval in which the rII-proximal (left) endpoint of the denV gene should be located. The deletion endpoints were identified by restriction and Southern hybridization analyses of phage derivatives containing deoxycytidine instead of hydroxymethyldeoxycytidine in their DNAs. The results of these analyses localized the rII-proximal (left) end of the denV gene to a region between 62.4 and 64.3 kilobases on the T4 physical map. denV+ phage resulted from marker rescue with two of five denV- alleles tested, using plasmids containing a 1.8-kilobase fragment from this region or a 179-base-pair terminal fragment derived from it. Sequencing of the 179-base-pair fragment from wild-type DNA showed a 130-base-pair open reading frame with its termination codon at the rII-proximal end. Confirmation that this open reading frame is part of the denV coding sequence was obtained by identifying a TAG amber codon in the homologous DNA derived from a denV amber mutant strain. This mutant strain rescued the denV+ allele from plasmids containing the wild-type sequence. An adjacent overlapping restriction fragment was also cloned, permitting determination of the remaining denV gene sequence. Based on these results, the 3' end of the coding region of the denV locus was mapped to kilobase position 64.07 on the T4 physical map, and the 5' end was mapped to position 64.48.

Pyrimidine dimer-DNA glycosylases: studies on bacteriophage T4-infected and on uninfected Escherichia coli.

Pyrimidine dimer (PD)-DNA glycosylase activity has been reported in both the M. luteus and phage T4 UV endonucleases. In the present studies the T4 PD-DNA glycosylase has been purified close to physical homogeneity using an assay that measures the release of free thymine from UV-irradiated poly ([H5] dT):poly (dA), after the photo-reversal of thymine-thymine dimers. The activity has also been demonstrated in vivo following infection of UV-irradiated E. coli uvr- cells with phage T4. Under these conditions the T4 PD-DNA glycosylase accounts quantitatively for all thymine-containing PD excised from [3H] labeled E. coli DNA. In vitro the T4 PD-DNA glycosylase has an associated AP endonuclease activity that incises UV-irradiated DNA 3 to the apyrimidinic sites created by the glycosylase. However, the glycosylase/AP endonuclease reaction mechanism in vitro does not appear to be a concerted one. In addition, a T4 phage with a temperature-sensitive mutation in the denV gene shows wild-type levels of survival at the permissive temperature, despite the fact that in vitro, extracts of E. coli infected with this mutant show no detectable phage-coded AP endonuclease at 28 degrees C. Thus the exact role of the T4 AP endonuclease in the incision of UV-irradiated DNA dimer in vivo is not clear. The ratio of excised non-containing nucleotides to dimer-containing nucleotides following infection of UV-irradiated E. coli with phage T4 denV+ yields a calculated average repair patch size of approximately 7 nucleotides. In contrast, the calculated average patch size in uninfected E. coli is approximately 70 nucleotides. Thus the extent of excision/resynthesis of UV-irradiated DNA may be determined by the specific mode of incision of the DNA at PD. When uninfected E. coli (uvr+) is exposed to UV radiation, a fraction of the excised thymine-containing PD contain photolabile thymine, suggesting the presence of PD-DNA glycosylase in E. coli. The role of this putative activity in the metabolism of UV-irradiated DNA is under investigation.

Use of high-performance liquid chromatography to quantitate thymine-containing pyrimidine dimers in DNA.

We have developed two high-performance liquid chromatographic systems for the measurement of pyrimidine dimers in hydrolysates of DNA. Normal-phase chromatography on an NH2 column in methanol-ethyl acetate (3.97) at an elution rate of 2.0 ml/min allowed quantitation of thymine-containing (thymine-thymine plus thymine-uracil) pyrimidine dimers at levels as low as 0.1% of the total radioactivity as thymine in DNA. This system was unaffected by the presence of up to 1 mg of contaminating protein (bovine serum albumin) or 40 micrograms of DNA in hydrolysates prepared for chromatography. Reversed-phase chromatography on a muBondapak C18 column allowed measurement of thymine-thymine dimers at concentrations as low as 0.02% of the total radioactivity. With 0.1% tetrahydrofuran in water as the solvent at a flow-rate of up to 0.6 ml/min, thymine-thymine, thymine-uracil, and uracil-uracil dimers were completely resolved. We were not able to quantitate the latter two dimeric forms, however, owing to the presence of other radioactive components of undefined origin that eluted concomitantly with the uracil-containing dimers.

Nonspecific cleavage of phi X174 RFI deoxyribonucleic acid by bleomycin.

The covalently closed circular duplex deoxyribonucleic acid (DNA) of phi X174 underwent progressive conversion to nicked and linear DNA with increasing bleomycin/phi X174 RFI DNA molecule ratios. The formation of linear DNA (a double-strand break) occurred under limited reaction conditions as low as an average of 0.2 single-strand break/phi X174 RFI DNA molecule. As bleomycin-produced linear DNA was further fragmented by bleomycin, a broad distribution of DNA fragments without notable concentrations of unique size was formed. Restriction enzymes PstI and SstII did not generate discrete fragments from bleomycin-produced full-length linear phi X174 DNA, nor did bleomycin cleavage generate discrete fragments from HpaII or PstI digests of phi X174 RFI. These findings suggest that bleomycin does not act at a few specific sites on phi X174 RFI DNA. The single-strand nick appeared to be the preferred site for bleomycin action for a second cleavage in a phi X174 molecule.

Molecular mechanisms of pyrimidine dimer excision in Saccharomyces cerevisiae: excision of dimers in cell extracts.

Cell-free extracts prepared from rad1-19, rd2-2, rad3-1, rad4-3, rad7-1, rad10-1, rd14-1, rad16-1, and cyc1-1 (rad7) mutants of Saccharomyces cerevisiae all catalyze the preferential excision of thymine-containing pyrimidine dimers from ultraviolet-irradiated DNA specifically incised with M. luteus ultraviolet deoxyribonucleic acid incising activity.

The repair of DNA damage: recent developments and new insights.

This brief review presents the salient features of new developments in the enzymatic repair of base damage to DNA. DNA glycosylases and apurinic/apyrimidinic (AP) endonucleases are reviewed and evidence is presented that in at least two prokaryote systems incision of UV-irradiated DNA occurs by the sequential action of these two classes of enzymes. In contradistinction, the uvrA, uvrB, and uvrC gene products of E coli appear to function as a multi-protein complex that catalyzes hydrolysis of phosphodiester bonds in damaged DNA directly. The inducible rapid repair of O6-methylguanine in E coli is also reviewed.

The relationship between human serum and human pancreatic DNase I.

Deoxyribonuclease (DNase) activities have been partially purified from human serum and pancreas. Several of their physical and enzymatic characteristics were determined and compared in order to evaluate their relatedness. Human serum deoxyribonuclease has an isoelectric point in the range of 3.9 to 4.3 and a molecular weight of 33,000 to 38,000. Optimal enzymatic activity at pH 7.0 was dependent on both Mg2+ and Ca2+, whereas a pH optimum of from 5.5 to 5.8 was observed in the presence of Mg2+ and ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). The proportion of single strand or double strand breakage products at early stages of DNA digestion were variable functions of the composition of the buffers employed for the reactions. Single strand break age was predominant under all reaction conditions. Double strand breakage occurred with greatest frequency under neutral conditions in the presence of Mg2+ and Ca2+, was inhibited by the inclusion of 0.15 M NaCl, and did not occur at pH 5.8 in the presence of Mg2+, EGTA, and 0.15 M NaCl. Human pancreas deoxyribonuclease exhibited essentially the same physical properties and enzymatic characteristics as those of the human serum enzyme. Thus, human serum deoxyribonuclease may originate in this pancreas.

Refracting leaky rays in graded-index fibers.

A local plane wave analysis is employed to derive the ray power transmission and attenuation coefficients for refracting leaky rays on graded-index fibers. These coefficients also reduce to established forms for tunneling rays and are compared with modal attenuation coefficients in the limit of strongly refracting rays.