The impact of anthropogenic pressure on the virological quality of water from the Tiber River, Italy.

The objective of the present study was to assess the occurrence of major waterborne enteric viruses (enterovirus, norovirus, adenovirus, rotavirus, hepatitis A and E virus) along the Tiber River in Italy, in areas affected by different kinds of anthropogenic pressure (agricultural, urban, industrial and pristine). Moreover, in light of the recent abundant detection of human bocavirus in urban wastewater samples in Italy, the occurrence of this virus was also assessed. Virus detection was based on nested PCR followed by sequencing, and on real-time PCR. A correlation with anthropogenic pressure was observed. The urban and industrial areas were the most contaminated (100 and 75% of samples were positive for at least one virus respectively). The agricultural area was less contaminated, with 50% of samples positive. None of the samples collected in a pristine area were positive for viruses. The most frequently detected virus was human bocavirus, identified in 37·5% of samples, followed by norovirus and enterovirus (28% each) and adenovirus (21·6%). Rotavirus, and hepatitis A and E viruses were less common (<9%). Although Human Bocavirus is not considered a waterborne pathogen, the widespread contamination of river waters suggests that virus transmission via the water route should not be neglected. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this study constitutes the first attempt to assess the occurrence of enteric viruses in river waters, in areas differentially influenced by anthropogenic pressure. Enteric viruses (enterovirus, norovirus, adenovirus, rotavirus, hepatitis A and E viruses, and bocavirus) were widespread in the industrial and urban areas, and were less frequently detected in the agricultural area. Interestingly, human bocavirus was the most frequently detected virus, outnumbering even adenoviruses, known to be widespread in water environments. The widespread presence of bocavirus in surface waters suggests that a potential role of water in its transmission should not be excluded.

Thymine DNA glycosylase.

More than 50% of colon cancer-associated mutations in the p53 tumor suppressor gene are C-->T transitions. The majority of them locate in CpG dinucleotides and are thought to have arisen through spontaneous hydrolytic deamination of 5-methylcytosine. This deamination process gives rise to G.T mispairs that need to be repaired to G.C in order to avoid C-->T mutation. Similarly, deamination of cytosine generates G.U mispairs that also produce C-->T transitions if not repaired. Restoration of both G.T and G.U mismatches was shown to be mediated by a short-patch excision repair pathway, and one principal player implicated in this process may be thymine DNA glycosylase (TDG). Human TDG was discovered as an enzyme that has the potential to specifically remove thymine and uracil bases mispaired with guanine through hydrolysis of their N-glycosidic bond, thereby generating abasic sites in DNA and initiating a base excision repair reaction. The same protein was later found to interact physically and functionally with the retinoid receptors RAR and RXR, and this implicated an unexpected function of TDG in nuclear receptor-mediated transcriptional activation of gene expression. The objective of this chapter is to put together the results of different lines of experimentation that have explored the thymine DNA glycosylase since its discovery and to critically evaluate their implications for possible physiological roles of this enzyme.

hMSH3 and hMSH6 interact with PCNA and colocalize with it to replication foci.

Proliferating cell nuclear antigen (PCNA) has been implicated in eukaryotic postreplicative mismatch correction, but the nature of its interaction with the repair machinery remained enigmatic. We now show that PCNA binds to the human mismatch binding factors hMutSalpha and hMutSbeta via their hMSH6 and hMSH3 subunits, respectively. The N-terminal domains of both proteins contain the highly conserved PCNA-binding motif Qxx[LI]xx[FF]. A variant of hMutSalpha, lacking this motif because of deletion of 77 N-terminal residues of the hMSH6 subunit, no longer was able to interact with PCNA in vitro and failed to restore mismatch repair in hMSH6-deficient cells. Colocalization of PCNA and hMSH6 or hMSH3 to replication foci implies an intimate link between replication and mismatch correction. We postulate that PCNA plays a role in repair initiation by guiding the mismatch repair proteins to free termini in the newly replicated DNA strands.

Effect of hMSH6 cDNA expression on the phenotype of mismatch repair-deficient colon cancer cell line HCT15.

Mismatch recognition in human cells is mediated primarily by a heterodimer of hMSH2 and hMSH6. Cells mutated in both alleles of the hMSH6 gene are deficient in the correction of base/base mispairs and insertion/deletion loops of one nucleotide and thus exhibit a strong mutator phenotype, evidenced by elevated mutation rates and microsatellite instability, as well as by tolerance to methylating agents. The decrease in replication fidelity associated with a loss of mismatch correction implies that with each division, these cells are likely to acquire new mutations throughout their genomes. Should such secondary mutations occur in genes linked to replication fidelity or involved in the maintenance of genomic stability, they might contribute to the observed mutator phenotype. The human colon tumour line HCT15 represents one such case. Although it carries inactivating mutations in both hMSH6 alleles, it has also been shown to contain a missense mutation in the coding sequence of the proofreading domain of the polymerase-delta gene. In an attempt to find out whether the phenotype of HCT15 cells was indeed brought about solely by the lack of hMSH6, we stably transfected them with a vector carrying the wild-type hMSH6 cDNA. Our results show that although the levels of transgenic hMSH6 were low, expression of the wild-type protein resulted in a substantial restoration of mismatch binding, mismatch repair capacity and the stability of mononucleotide repeats, as well as in the reduction of mutation rates. Although methylation tolerance of the hMSH6-expressing cells was not markedly affected, the G2 cell cycle checkpoint, absent in N-methyl-N'-nitro-N-nitrosoguanidine-treated control cells, was restored.

Mutation of the mismatch repair gene hMSH2 and hMSH6 in a human T-cell leukemia line tolerant to methylating agents.

Cell killing by monofunctional methylating agents is due mainly to the formation of adducts at the O6 position of guanine. These methyl adducts are removed from DNA by the O6-alkylguanine DNA alkyltransferase (OGAT). The mechanism by which O6-methylguanine (O6meG) induces cell death in OGAT-deficient cells requires a functional mismatch repair system (MRS). We have previously reported that depletion of OGAT activity in the human T-cell leukemic urkat line does not sensitize these cells to the cytotoxic and apoptotic effects of the methylating triazene temozolomide (Tentori et al., 1995). We therefore decided to establish whether the tolerance of Jurkat cells to O6meG could be associated with a defect in MRS. The results of mismatch repair complementation studies indicated that Jurkat cells are defective in hMutSalpha, a heterodimer of the hMSH2 and hMSH6 proteins. Cytogenetic analysis of two Jurkat clones revealed a deletion in the short arm of chromosome region 2p15-21, indicating an allelic loss of both hMSH2 and hMSH6 genes. DNA sequencing revealed that exon 13 of the second hMSH2 allele contains a base substitution at codon 711, which changes an arginine to a termination codon (CGA-->TGA). In addition, a (C)8-->(C)7 frameshift mutation in codon 1085-1087 of the hMSH6 gene was also found. Although both hMSH2 and hMSH6 transcripts could be detected in Jurkat clones, the respective polypeptides were absent. Taken together, these data indicate that tolerance of Jurkat cells to methylation damage is linked to a loss of functional hMutSalpha.

Mismatch repair deficiency associated with overexpression of the MSH3 gene.

We tested the ability of recombinant hMutSalpha (hMSH2/hMSH6) and hMutSbeta (hMSH2/hMSH3) heterodimers to complement the mismatch repair defect of HEC59, a human cancer cell line whose extracts lack all three MutS homologues. Although repair of both base/base mispairs and insertion-deletion loops was restored by hMutSalpha, only the latter substrates were addressed in extracts supplemented with hMutSbeta. hMutSalpha was also able to complement a defect in the repair of base/base mispairs in CHO R and HL60R cell extracts. In these cells, methotrexate-induced amplification of the dihydrofolate reductase (DHFR) locus, which also contains the MSH3 gene, led to an overexpression of MSH3 and thus to a dramatic change in the relative levels of MutSalpha and MutSbeta. As a rule, MSH2 is primarily complexed with MSH6. MutSalpha is thus relatively abundant in mammalian cell extracts, whereas MutSbeta levels are generally low. In contrast, in cells that overexpress MSH3, the available MSH2 protein is sequestered predominantly into MutSbeta. This leads to degradation of the partnerless MSH6 and depletion of MutSalpha. CHO R and HL60R cells therefore lack correction of base/base mispairs, whereas loop repair is maintained by MutSbeta. Consequently, frameshift mutations in CHO R are rare, whereas transitions and transversions are acquired at a rate two orders of magnitude above background. Our data thus support and extend the findings of Drummond et al. [Drummond, J. T., Genschel, J., Wolf, E. & Modrich, P. (1997) Proc. Natl. Acad. Sci. USA 94, 10144-10149] and demonstrate that mismatch repair deficiency can arise not only through mutation or transcriptional silencing of a mismatch repair gene, but also as a result of imbalance in the relative amounts of the MSH3 and MSH6 proteins.

Chromosomal localizations and molecular analysis of TDG gene-related sequences.

The human mismatch-specific thymine DNA glycosylase gene, TDG, encodes a 60-kDa polypeptide able to correct G/T mispairs arising from the deamination of 5-methylcytosine. We localized by FISH three different TDG-related lambda genomic clones, lambda8, lambda11, and lambda12 on chromosome 12. PCR and sequence analyses revealed that only lambda11, localized at 12q24.1, contained the coding gene. We characterized the intron-exon boundaries of the portion of the gene contained in the lambda clone and identified a CA dinucleotide repeat in one intron. Northern blot analysis showed that TDG is expressed at approximately the same level in all human tissues analyzed. SSCP analysis of 50 tumor and corresponding normal tissue DNAs from lung cancer patients did not reveal the presence of any functional mutation. An abnormal SSCP pattern in one sample proved to be a polymorphism after sequencing and RFLP analysis.

Cloning and expression of human G/T mismatch-specific thymine-DNA glycosylase.

Hydrolytic deamination of 5-methylcytosine leads to the formation of G/T mismatches. We have shown previously that these G/T mispairs are corrected to G/C pairs by a mismatch-specific thymine-DNA glycosylase, TDG, which we subsequently purified from human cells. Here we describe the cloning of the human cDNA encoding TDG. We have identified two distinct cDNA species that differ by 100 nucleotides at the 3'-untranslated region. These cDNAs contain a 410-amino acid open reading frame that encodes a 46-kDa polypeptide. The G/T glycosylase, expressed both in vitro and in Escherichia coli, migrated in denaturing polyacrylamide gels with an apparent size of 60 kDa. The substrate specificity of the recombinant protein corresponded to that of the cellular enzyme, and polyclonal antisera raised against the recombinant protein neutralized both activities. We therefore conclude that the cDNA described below encodes human TDG. Data base searches identified a serendipitously cloned mouse cDNA sequence that could be shown to encode the murine TDG homologue. No common amino acid sequence motifs between the G/T-specific enzyme and other DNA glycosylases could be found, suggesting that TDG belongs to a new class of base-excision repair enzymes.

GTBP, a 160-kilodalton protein essential for mismatch-binding activity in human cells.

DNA mismatch recognition and binding in human cells has been thought to be mediated by the hMSH2 protein. Here it is shown that the mismatch-binding factor consists of two distinct proteins, the 100-kilodalton hMSH2 and a 160-kilodalton polypeptide, GTBP (for G/T binding protein). Sequence analysis identified GTBP as a new member of the MutS homolog family. Both proteins are required for mismatch-specific binding, a result consistent with the finding that tumor-derived cell lines devoid of either protein are also devoid of mismatch-binding activity.

Light scattering by sinusoidal surfaces: illumination windows and harmonics in standards.

Sinusoidal surfaces can be used as material standards to help calibrate instruments that measure the angular distribution of the intensity of light scattered by arbitrary surfaces, because the power in the diffraction peaks varies over several orders of magnitude. The calculated power in the higher-order diffraction peaks from sinusoidal surfaces expressed in terms of Bessel functions is much smaller than the values determined from angular distributions that are measured or computed from measured profiles, both of which are determined mainly by the harmonic contents of the profile. The finite size of the illuminated area, represented by an illumination window, gives rise to a background that is much larger than the calculated power in the higher-order peaks. For a rectangular window of a size equal to an even number of periods of the sinusoid, a computation of the power distribution produces minima at or near the location of the diffraction angles for higher-order diffraction angles.

Enhanced activity of human G6PD promoter transfected in HeLa cells producing high levels of HIV-1 Tat.

Glucose 6-phosphate dehydrogenase (G6PD) enzyme activity has an important role in the cell defence against oxidative damage produced by hydrogen peroxide, a compound known to activate HIV-1 expression through the intermediate of NF-kappa B. By means of CAT assays, in this paper we show that the Tat protein increases the rate of transcription from the human G6PD promoter in HeLa cells; furthermore, we report a similar effect of Tat on transcription driven from the viral RSV promoter. We did not observe Tat stimulation with the human CMV, SV40 or the basic RSV promoters. Dose-response curves indicate that Tat activates G6PD and RSV through a mechanism different from the major one operating on the HIV-1 LTR promoter. TAR-like structures are not involved, instead a short sequence close to the G6PD transcription start site and the RSV LTR enhancer is a good candidate for mediating the phenomena described in this paper. This sequence has some features in common with the NF-kappa B motif.

Autocorrelation functions from optical scattering for one-dimensionally rough surfaces.

The relationship between the height autocorrelation function of a one-dimensionally rough surface and the Fourier transform of the intensity distribution of the light scattered by that surface is tested experimentally. The theory is derived by using the Fraunhofer approximation, without recourse to the inconsistent Kirchhoff boundary conditions. In spite of the limitations imposed by the approximations used, the results obtained from optical data agree well with those obtained from stylus data, even for an autocorrelation length as small as the optical wavelength. However, this method should be limited to surfaces with rms roughness smaller than approximately 0.14 times the wavelength of light.

Regimes of surface roughness measurable with light scattering.

In this paper we summarize a number of previous experiments on the measurement of the roughness of metallic surfaces by light scattering. We identify several regimes that permit measurement of different surface parameters and functions, and we establish approximate limits for each regime. Using a straightforward criterion, we calculate that the smooth-surface regime, in which the angular distribution of scattered light is closely related to the power spectral density of the roughness, ranges over 0 < σ/λ ≲ 0.05, where σ is the rms roughness and λ is the opitcal wavelength. Above that the surfaceautocorrelation function may be calculated from a Fourier transform of the angular distribution over 0 < σ/λ ≲ 0.14. Then comes the specular regime where the specular beam can still be identified andmeasured over 0 < σ/λ ≲ 0.3. For all these regimes and for rougher surfaces too, the rms width of thescatter distribution is proportional to the rms slope of the surface.

The CpG island in the 5' region of the G6PD gene of man and mouse.

The nucleotide (nt) sequence of the entire CpG island in the 5' region of the human glucose-6-phosphate dehydrogenase-encoding gene (G6PD) and of the corresponding region in mouse was determined. In comparison to the human gene, the 5' region of the mouse G6PD gene has highly reduced G + C and CpG dinucleotide content, but maintains the functional features of a CpG island, as it is differentially methylated on the active vs. the inactive X chromosome. In addition to the expected conservation of exons, nt sequence comparison showed that several boxes are highly conserved between the two species in the 5' flanking DNA and in the first intron. Moreover, the conservation of the position of most CpG dinucleotides in the promoter region and in one of the upstream boxes, at about -900, gives support to the hypothesis that, in each island, specific CpGs play a major role in the regulation of gene expression.

Light-scattering measurement of the rms slopes of rough surfaces.

Angle-resolved light scattering (ARLS) is used to estimate the root-mean-square (rms) slopes of rough surfaces having a well-defined lay, and the effect on slope measurements caused by changing the angles of incidence and scattering is investigated. The ARLS patterns are taken with the Detector Array for Laser Light Angular Scattering (Dallas) research instrument, and the rms slopes are obtained from the angular widths of these patterns. In general, it was found that the angular width, and thus the estimated rms slope, is surprisingly insensitive to relatively large changes in both the incident and scattering angles of light. These results are independent of surface material and are valid for both sinusoidal and random rough surfaces with lay. The principles, experiments, analyses, and conclusions involved in using ARLS to estimate rms surface slopes are described.

Light scattering from glossy coatings on paper.

The application of angle-resolved light scattering (ARLS) to the measurement of the surface roughness of glossy coatings on paper was investigated. To this end, ARLS patterns were measured for laser light scattered from several glossy paper samples, and these patterns were compared with those calculated using a theoretical model based on plane-wave scattering from an isotropic rough surface. Mechanical stylus profilometry data for the rms roughnesses and the autocorrelation functions of the coatings were used as input to calculate the patterns. For all the paper samples measured, as well as for all the incidence angles used, there was good agreement between the experimental and the calculated patterns when all the rms roughnesses measured by profilometry were reduced by 30%. The indication from these experiments is that ARLS may be used to determine the roughness parameters of the coatings. As a check on these results, measurements were also performed with a commercial optical surface probe; these data agreed well with both the ARLS and the stylus profilometry results.

Development of a One-Micrometer-Diameter Particle Size Standard Reference Material.

The average diameter of the first micrometer particle size standard (Standard Reference Material 1690), an aqueous suspension of monosized polystyrene spheres with a nominal 1 µm diameter, was accurately determined by three independent techniques. In one technique the intensity of light scattered by a diluted suspension of polystyrene spheres was measured as a function of scattering angle, using a He-Ne laser polarized in the vertical direction. The second technique consisted of measuring as a function of angle the intensity of light scattered from individual polystyrene spheres suspended in air, using a He-Cd laser with light polarized parallel and perpendicular to the scattering plane. The measurement of row length by optical microscopy for polystyrene spheres arranged in close-packed, two-dimensional hexagonal arrays was the basis of the third technique. The measurement errors for each technique were quantitatively assessed. For the light scattering experiments, this required simulation with numerical experiments. The average diameter determined by each technique agreed within 0.5% with the most accurate value being 0.895±0.007 µm based on light scattering by an aqueous suspension. Transmission electron microscopy, flow through electrical sensing zone counter measurements, and optical microscopy were also used to obtain more detailed information on the size distribution including the standard deviation (0.0095 µm), fraction of off-size particles, and the fraction of agglomerated doublets (1.5%).

Sizing of individual optically levitated evaporating droplets by measurement of resonances in the polarization ratio.

Resonances observed in the polarization ratio of light scattered at 90 degrees from single optically levitated evaporating droplets are shown to provide a means for continuous high-resolution monitoring of droplet size. Due to the distinctive character of the individual features in the polarization ratio, each experimentally measured feature could be clearly identified with a specific calculated one. For evaporating droplets of glycerol from 6.6 to 11.5 microm in diameter, the sharp features which appeared in the calculations at ~0.03-microm intervals allowed measurement of droplet diameter to a resolution of 0.003 microm.