By its functional interaction with a RecA polymer, the mutagenic UmuD'C complex possesses an antirecombination activity. We show here that MucA'B, a functional homolog of the UmuD'C complex, inhibits homologous recombination as well. In F- recipients expressing MucA'B from a Ptac promoter, Hfr x F- recombination decreased with increasing MucA'B concentrations down to 50-fold. In damage-induced pKM101-containing cells expressing MucA'B from the native promoter, recombination between a UV-damaged F lac plasmid and homologous chromosomal DNA decreased 10-fold. Overexpression of MucA'B together with UmuD'C resulted in a synergistic inhibition of recombination. RecA[UmuR] proteins, which are resistant to UmuD'C inhibition of recombination, are inhibited by MucA'B while promoting MucA'B-promoted mutagenesis efficiently. The data suggest that MucA'B and UmuD'C contact a RecA polymer at distinct sites. The MucA'B complex was more active than UmuD'C in promoting UV mutagenesis, yet it did not inhibit recombination more than UmuD'C does. The enhanced mutagenic potential of MucA'B may result from its inherent superior capacity to assist DNA polymerase in trans-lesion synthesis. In the course of this work, we found that the natural plasmid pKM101 expresses around 45,000 MucA and 13,000 MucB molecules per lexA(Def) cell devoid of LexA. These molecular Muc concentrations are far above those of the chromosomally encoded Umu counterparts. Plasmid pKM101 belongs to a family of broad-host-range conjugative plasmids. The elevated levels of the Muc proteins might be required for successful installation of pKM101-like plasmids into a variety of host cells.

Bacterial Proteins↗

PubMed1999

Severity of diabetic retinopathy is linked to lipoprotein (a) in type 1 diabetic patients.

To determine the relationship between plasma Lp(a) concentration and the risk of developing diabetic retinopathy, 341 Type 1 diabetic patients underwent an annual retinal fluorescein angiography and were assigned to one of 3 groups according to the stage of their diabetic retinopathy: no retinopathy (NR), non-proliferative diabetic retinopathy (N-PDR), or proliferative diabetic retinopathy (PDR). One hundred and twenty-three Type 1 diabetic patients had no retinopathy, 188 had N-PDR and 30 had PDR. The ages of the three groups and the duration of diabetes were significantly different. Hypertension, microalbuminuria and diabetic nephropathy were more frequent in PDR than in NR or N-PDR (p < 0.0001). Plasma HbA1c was higher in PDR than in NR or N-PDR (p < 0.01). Type 1 patients who had been diabetic for at least 20 years included 30 NR, 108 N-PDR and 24 PDR. Type 1 diabetic patients with PDR had microalbuminuria and macroproteinuria more frequently than other patients (p < 0.0001 and 0.01, respectively). Type 1 diabetic patients with PDR had the highest median plasma Lp(a) and the highest frequency of Lp(a) above 30 mg/dl (p < 0.05). Multivariate analysis carried out in Type 1 diabetic patients with a duration of diabetes of at least 20 years showed that microproteinuria, HbA1c and Lp(a) accounted significantly for 21% of variance in retinal status. Lp(a) above 30 mg/dl was related to the risk of developing PDR (OR = 8.40, p < 0.05). Lipoprotein(a) appears to be associated with the severity of diabetic retinopathy in Type 1 diabetic patients, and particular attention should be paid to those with Lp(a) above 30 mg/dl and pre-proliferative retinopathy.

Adult↗

PubMed1998

Methylation of CpG dinucleotides in the lacI gene of the Big Blue transgenic mouse.

Cytosine residues at CpG dinucleotides can be methylated by endogenous methyltransferases in mammalian cells. The resulting 5-methylcytosine base may undergo spontaneous deamination to form thymine causing G/C to A/T transition mutations. Methylated CpGs also can form preferential targets for environmental mutagens and carcinogens. The Big Blue(R) transgenic mouse has been used to investigate tissue and organ specificity of mutations and to deduce mutational mechanisms in a mammal in vivo. The transgenic mouse contains approximately 40 concatenated lambda-like shuttle vectors, each of which contains one copy of an Escherichia coli lacI gene as a mutational target. lacI mutations in lambda transgenic mice are characterized by a high frequency of spontaneous mutations targeted to CpG dinucleotides suggesting an important contribution from methylation-mediated events. To study the methylation status of CpGs in the lacI gene, we have mapped the distribution of 5-methylcytosines along the DNA-binding domain and flanking sequences of the lacI gene of transgenic mice. We analyzed genomic DNA from various tissues including thymus, liver, testis, and DNA derived from two thymic lymphomas. The mouse genomic DNAs and methylated and unmethylated control DNAs were chemically cleaved, then the positions of 5-methylcytosines were mapped by ligation-mediated PCR which can be used to distinguish methylated from unmethylated cytosines. Our data show that most CpG dinucleotides in the DNA binding domain of the lacI gene are methylated to a high extent (>98%) in all tissues tested; only a few sites are partially (70-90%) methylated. We conclude that tissue-specific methylation is unlikely to contribute significantly to tissue-specific mutational patterns, and that the occurrence of common mutation sites at specific CpGs in the lacI gene is not related to selective methylation of only these sequences. The data confirm previous suggestions that the high frequency of CpG mutations in lacI transgenes is related to the presence of 5-methylcytosine bases.

Animals↗

PubMed1998

Ab Initio Calculations and High-Resolution Spectroscopy of the Bending Pentad of SiH2D2 in the 10-16 µm Region.

The SiH2D2 asymmetric top has nine vibrational modes, five of them forming a pentad strongly perturbed by Coriolis interactions. High-level ab initio calculations of SiH2D2 have been performed which yield numerous spectroscopic parameters related to the harmonic and anharmonic force fields. The bending pentad comprising nu4(A1), nu7(B1), nu5(A2), nu9(B2), and nu3(A1) has been studied by high-resolution Fourier transform spectroscopy; the region 600-1050 cm-1 has been investigated with a resolution of ca. 4 x 10(-3) cm-1. Raman BOXCARS spectroscopy has been used for the infrared inactive nu5 band. The Raman apparatus function was 0.0054 cm-1. Assignments of about 4000 transitions including all bands have been made, mostly employing ground state combination differences techniques, and a global fit has been performed. The fundamentals nu4 (681.624 cm-1), nu7 (742.640 cm-1), nu5 (842.381 cm-1), nu9 (859.750 cm-1), and nu3 (942.741 cm-1) are strongly coupled by A-, B-, and C-type Coriolis interactions, and ab initio predictions of these interaction parameters were used to set up a network of interactions that was refined by the experimental data. The global standard deviation for the entire body of data is 7.1 x 10(-4) cm-1. Satisfactory synthetic spectra which are very sensitive to relative signs of dipole moment derivatives and Coriolis interaction constants were obtained with the guidance of ab initio calculations. Finally, fair to good agreement of experimental and ab initio calculated molecular parameters was obtained. For the first time, a complete analysis of the pentad of SiH2D2 in the 10-16 µm region has been carried out. A full set of rovibrational parameters is given for these five interacting levels, including first and second order Coriolis interaction constants. Copyright 1998 Academic Press.

Journal Article↗

PubMed1998

The sensitivity of denaturing gradient gel electrophoresis: a blinded analysis.

Denaturing gradient gel electrophoresis (DGGE) is considered one of the most sensitive and specific of the mutational scanning techniques, yet blinded analyses have not been reported. We report the results of a blinded study of the efficiency of DGGE to detect mutations in the Human Coagulation Factor IX. Two overlapping genomic DNA sequences from exon 8 of Factor IX (290 bp and 539 bp length) with an unknown number of mutations were amplified with a 40 bp GC-clamp and tested blindly by DGGE. DGGE detected all mutations in the 290 bp genomic DNA segment. DGGE detected all but one mutation in the 539 bp genomic segment after experimental conditions were fully optimized but missed multiple mutations in an initial blinded experiment. These results demonstrate the utility of blinded analyses and confirm the exquisite power of DGGE for detecting mutations.

DNA↗

PubMed1998

The Koebner phenomenon in vitiligo following treatment of a port-wine stain naevus by pulsed dye laser.

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Adolescent↗

PubMed1998

Ruby laser treatment for hirsutism: clinical response and patient tolerance.

We report the results of a study of the ruby laser in normal mode for treatment of hirsutism in 43 patients with skin types I-IV. A pulse width of 950 microseconds, a 4 mm spot and a fluence sufficient to produce minimal whitening of the epidermis were used (mean 48 J/cm2). In each patient, one site was treated once only, and a second site received four treatments at monthly intervals. After a single treatment, hair counts had reduced to a mean of 46% (median 67, reduced to 26) of the pretreatment values at 1 month, but increased to 80% (median 42) at 3 months and were 66% (median 37) at 6 months. One month after three treatments, hair counts had reduced to a mean of 29% (median 60, reduced to 15) of pretreatment values, and 3 months after four treatments at the same site, counts had increased to 44% (median 21). Patient tolerance of the treatment was good and higher fluences tended to be tolerated at consecutive visits. The first treatment was followed by mild, short-lived erythema and swelling in 60% (25 of 42) patients. Fourteen per cent (six of 42) experienced mild blistering and 33% (14 of 42) crusting. More severe reactions were seen infrequently. These results indicate that normal mode ruby laser treatment is well tolerated. Sustained reduction in hair counts can be achieved for at least 6 months, and multiple treatments produce greater clinical effects.

Adolescent↗

PubMed1998

Specific RecA amino acid changes affect RecA-UmuD'C interaction.

The UmuD'C mutagenesis complex accumulates slowly and parsimoniously after a 12 Jm(-2) UV flash to attain after 45 min a low cell concentration between 15 and 60 complexes. Meanwhile, RecA monomers go up to 72,000 monomers. By contrast, when the UmuD'C complex is constitutively produced at a high concentration, it inhibits recombinational repair and then markedly reduces bacterial survival from DNA damage. We have isolated novel recA mutations that enable RecA to resist UmuD'C recombination inhibition. The mutations, named recA [UmuR], are located on the RecA three-dimensional structure at three sites: (i) the RecA monomer tail domain (four amino acid changes); (ii) the RecA monomer head domain (one amino acid change, which appears to interface with the amino acids in the tail domain); and (iii) in the core of a RecA monomer (one amino acid change). RecA [UmuR] proteins make recombination more efficient in the presence of UmuD'C while SOS mutagenesis is inhibited. The UmuR amino acid changes are located at a head-tail joint between RecA monomers and some are free to possibly interact with UmuD'C at the tip of a RecA polymer. These two RecA structures may constitute possible sites to which the UmuD'C complex might bind, hampering homologous recombination and favouring SOS mutagenesis.

Amino Acid Sequence↗

PubMed1998

Expression of bacterial chorismate pyruvate-lyase in tobacco: evidence for the presence of chorismate in the plant cytosol.

The bacterial gene ubiC encodes chorismate pyruvate-lyase, an enzyme which converts chorismate to 4-hydroxybenzoate and which is not normally present in plants. The UbiC protein was expressed in tobacco, with targeting of the gene product either to the plastids or to the cytosol. In both cases, chorismate pyruvate-lyase activity and a resulting formation of 4-hydroxybenzoate was detected. This suggests that chorismate, a metabolite of the shikimate pathway, is present not only in the plastids but also in the cytosol of plant cells.

Agrobacterium tumefaciens↗

PubMed1998

Contact dermatitis caused by phenoxybenzamine hydrochloride.

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Adult↗

PubMed1998

Airborne contact dermatitis caused by microscopy immersion fluid containing epoxy resin.

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Adult↗

PubMed1998

[Bilateral ischemic optic neuropathy secondary to acute ergotism].

We report a case of a 31 year-old man who presented a bilateral ischemic optic neuropathy associated with headaches and severe systemic hypertension. This episode appeared after administration of ergotamine tartrate and macrolides. This medication probably led to a vasospasm which occurs in patients with hypertension. The cardiovascular and serum lipid evaluations were normal. A migraine optic neuropathy can be evoked.

Adult↗

PubMed1998

[Uveitis in Whipple disease: Identification of Trophyrema whippelii by PCR].

Whipple's disease is a multisystem bacterial disease usually characterized by malabsorption, diarrhea and polyarthritis. Ocular manifestations include uveitis, vitreitis, retinis, myositis, papilledema and optic atrophy. We report a case of a chronic bilateral vitreitis in a 63 year-old man who had been treated for a Whipple's disease with gastrointestinal involvement 30 years before. The jejunal biopsy was negative but the polymerase-chain-reaction (PCR) revealed the presence of Trophyrema Whippelii in the vitreous prelevement of both eye. This new, original strategy of PCR is specific and more sensitive than histological diagnosis.

Actinomyces↗

PubMed1997

A population-based case-control study of the tumor necrosis factor alpha-308 polymorphism in multiple sclerosis.

Tumor necrosis factor alpha (TNF alpha) expression is enhanced in patients with active MS and other inflammatory diseases. A guanine-to-adenine polymorphism at position -308 of the TNF alpha promoter region (the TNF2 allele) is associated with increased TNF alpha expression. We evaluated 110 MS patients derived from an Olmsted County, MN, prevalence study. Three other patient cohorts (autoimmune, serious infectious illness, and other neurologic diseases) and matched controls were derived from a Mayo Clinic DNA bank. We used polymerase chain reaction amplification of specific alleles to screen for the TNF2 allele and to determine zygosity. We found one homozygote and 29 heterozygotes in the MS patients. There was no association between the presence of the TNF2 allele and MS or the other disease categories by matched-pair and group analyses.

Alleles↗