Activity of the antioxidant enzyme paraoxonase in Argentinean children living at high altitude.

BACKGROUND: Children living at high altitude in San Antonio de los Cobres (SAC), Argentina, were shown to have lower high-density lipoprotein cholesterol (HDL-C) levels than Buenos Aires (BA) children. HDL antioxidant capacity is mainly attributed to paraoxonase1 (PON1). OBJECTIVE: To compare PON1 activity in indigenous SAC vs. BA children. METHODS: A cross-sectional study compared 158 SAC vs. 97 BA children (6-16 years). Anthropometric data and lipoprotein profile were measured. PON1 was evaluated employing paraoxon (PON) and phenylacetate (ARE) activity. RESULTS: The prevalence of overweight/obesity was lower in SAC than in BA children (18.3 vs. 30.9%). Triglycerides (1.34 vs. 0.90 mmol/l), apo B (0.84 vs.0.72 g/l), apo A-I (1.33 vs. 1.27 g/l), and ARE activity (100 vs. 90 µmol/ml/min) were higher, while HDL-C (1.16 vs. 1.32 mmol/l) and PON activity (170 vs. 203 nmol/ml/min) were lower in SAC than in BA. Separate multiple linear regression analyses showed that SAC children had significantly higher triglyceride (Beta -0.38), apo B (Beta -0.34), and ARE (Beta -0.36) plus lower HDL-C (Beta 0.33) and PON (Beta 0.25) compared with BA; adjusted for age, gender, and BMI. CONCLUSION: SAC showed an unfavorable lipoprotein profile, lower PON and higher ARE activities compared with BA children, suggesting the presence of altered HDL metabolism and antioxidant capacity.

Observation of Spin-Dependent Charge Symmetry Breaking in ΛN Interaction: Gamma-Ray Spectroscopy of _{Λ}

The energy spacing between the spin-doublet bound state of _{Λ}^{4}He(1^{+},0^{+}) was determined to be 1406±2±2 keV, by measuring γ rays for the 1^{+}→0^{+} transition with a high efficiency germanium detector array in coincidence with the ^{4}He(K^{-},π^{-})_{Λ}^{4}He reaction at J-PARC. In comparison to the corresponding energy spacing in the mirror hypernucleus _{Λ}^{4}H, the present result clearly indicates the existence of charge symmetry breaking (CSB) in ΛN interaction. By combining the energy spacings with the known ground-state binding energies, it is also found that the CSB effect is large in the 0^{+} ground state but is vanishingly small in the 1^{+} excited state, demonstrating that the ΛN CSB interaction has spin dependence.

Short- and long-term outcomes of every graft recovered during a multi-organ procurement procedure including the intestine.

BACKGROUND: The development of intestinal transplant (Tx) programs introduces thymoglobulin donor treatment as well as an almost complete warm dissection of the abdominal organs to allocate them to different recipients. Our aim is to assess the reproducibility and feasibility of the surgical technique of multi-organ procurement with the use of thymoglobulin donor pre-treatment and report the short- and long-term outcomes of every graft harvested as part of multi-organ procurement (MTOp), including the intestine. METHODS: Data were collected of all organs harvested from MTOp, including the intestines allocated to our center from March 2006 to July 2011. Data from 92 recipients and 116 organs procured from 29 MTOp were analyzed. Twelve hearts, 2 lungs, and 1 cardio-pulmonary block were transplanted; primary graft dysfunction developed in 4 of the 12 hearts and in the cardio-pulmonary block. RESULTS: The survival rate was 75% and 100% for hearts and lungs, respectively. Nineteen livers, 9 kidney-pancreas, 19 kidneys, and 29 intestines were transplanted. Delayed graft function (DGF) of the pancreas developed in 3 of 9 kidney-pancreas, and the other 3 exhibited DGF of the kidney; 4 of 19 Tx kidneys had DGF. The survival was 84%, 78%, 95%, and 65.5% for livers, kidney-pancreas, kidneys, and intestines, respectively. CONCLUSIONS: Organs procured during MTOp including the intestine can be safely used, increasing organ availability and transplant applicability without compromising allocation, quality, and long-term results of the non-intestinal-procured organs.

Search for the Θ+ pentaquark via the π(-)p→K(-)X reaction at 1.92 GeV/c.

The Θ(+) pentaquark baryon was searched for via the π(-)p→K(-)X reaction with a missing mass resolution of 1.4 MeV/c(2) (FWHM) at the Japan Proton Accelerator Research Complex (J-PARC). π(-) meson beams were incident on the liquid hydrogen target with a beam momentum of 1.92 GeV/c. No peak structure corresponding to the Θ(+) mass was observed. The upper limit of the production cross section averaged over the scattering angle of 2° to 15° in the laboratory frame is obtained to be 0.26 µb/sr in the mass region of 1.51-1.55 GeV/c(2). The upper limit of the Θ(+) decay width is obtained to be 0.72 and 3.1 MeV for J(Θ)(P)=1/2(+) and J(Θ)(P)=1/2(-), respectively, using the effective Lagrangian approach.

Evidence for heavy hyperhydrogen (Λ)(6)h.

Evidence for the neutron-rich hypernucleus (Λ)(6)H is presented from the FINUDA experiment at DAΦNE, Frascati, studying (π+,π-) pairs in coincidence from the K(stop)(-) + (6)Li →(Λ)(6)H + π+ production reaction followed by (Λ)(6)H → (6)He + π- weak decay. The production rate of (Λ)(6) undergoing this two-body π- decay is determined to be (2.9 ± 2.0) × 10(-6)/K(stop)(-). Its binding energy, evaluated jointly from production and decay, is BΛ((Λ)(6)H) = (4.0 ± 1.1) MeV with respect to (5)H+Λ. A systematic difference of (0.98 ± 0.74) MeV between BΛ values derived separately from decay and from production is tentatively assigned to the (Λ)(6)H 0(g.s.)(+) → 1+ excitation.

Trichothiodystrophy: from basic mechanisms to clinical implications.

Trichothiodystrophy (TTD) is an autosomal recessive disorder with symptoms affecting several tissues and organs. The most relevant features are hair abnormalities, physical and mental retardation, ichthyosis, signs of premature aging and cutaneous photosensitivity. The clinical spectrum of TTD varies widely from patients with only brittle, fragile hair to patients with the most severe neuroectodermal symptoms. To date, four genes have been identified as responsible for TTD: XPD, XPB, p8/TTDA, and TTDN1. Whereas the function of TTDN1 is still unknown, the former three genes encode subunits of TFIIH, the multiprotein complex involved in basal and activated transcription and in nucleotide excision repair (NER). Ongoing investigations on TTD are elucidating not only the pathogenesis of the disease, which appears to be mainly related to transcriptional impairment, but also the modalities of NER and transcription in human cells and how TFIIH operates in these two fundamental cellular processes.

Evidence for a kaon-bound state K(-)pp produced in K(-) absorption reactions at rest.

We have searched for a deeply bound kaonic state by using the FINUDA spectrometer installed at the e(+)e(-) collider DAPhiNE. Almost monochromatic K(-)'s produced through the decay of phi(1020) mesons are used to observe K(-) absorption reactions stopped on very thin nuclear targets. Taking this unique advantage, we have succeeded to detect a kaon-bound state K(-)pp through its two-body decay into a Lambda hyperon and a proton. The binding energy and the decay width are determined from the invariant-mass distribution as 115(+6)(-5)(stat)(+3)(-4)(syst) MeV and 67(+14)(-11)(stat)(+2)(-3)(syst) MeV, respectively.

Different dynamics in nuclear entry of subunits of the repair/transcription factor TFIIH.

We report here the different ways in which four subunits of the basal transcription/repair factor TFIIH (XPB, XPD, p62 and p44) and the damage recognition XPC repair protein can enter the nucleus. We examined their nuclear localization by transiently expressing the gene products tagged with the enhanced green fluorescent protein (EGFP) in transfected 3T3 cells. In agreement with the identification of more than one putative nuclear localization signal (NLS) in their protein sequences, XPB, XPC, p62 and p44 chimeras were rapidly sorted to the nucleus. In contrast, the XPD-EGFP chimeras appeared mainly localized in the cytoplasm, with a minor fraction of transfectants showing the EGFP-based fluorescence also in the nucleus. The ability of the XPD chimeras to enter the nucleus was confirmed by western blotting on fractionated cell extracts and by functional complementation of the repair defect in the UV5 rodent cells, mutated in the XPD homologous gene. By deletion mutagenesis, we were unable to identify any sequence specific for nuclear localization. In particular, deletion of the putative NLS failed to affect subcellular localization and, conversely, the C-terminal part of XPD containing the putative NLS showed no specific nuclear accumulation. These findings suggest that the nuclear entry of XPD depends on its complexation with other proteins in the cytoplasm, possibly other components of the TFIIH complex.

Identical mutations in the CSB gene associated with either Cockayne syndrome or the DeSanctis-cacchione variant of xeroderma pigmentosum.

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are two hereditary disorders in which photosensitivity is associated with distinct clinical and cellular phenotypes and results from genetically different defects. We have identified the primary molecular alteration in two patients in whom clinical manifestations strongly reminiscent of a severe form of XP were unexpectedly associated with the CS cellular phenotype and with a defect in the CSB gene. Sequencing of the CSB -coding region in both cDNA and genomic DNA showed that these patients had identical alterations to those in a patient with the clinical features of the classical form of CS. These data, together with fluorescence in situ hybridization analysis, demonstrated that the two siblings with XP as well as the CS patient were homozygous for the same CSB mutated allele, containing a silent C2830T change and a nonsense mutation C2282T converting Arg735 to a stop codon. The finding that the same inactivating mutation underlies different pathological phenotypes indicates that there is no simple correlation between the molecular defect and the clinical features. Therefore, alterations in the CSB gene give rise to the same repair defect at the cellular level but other genetic and/or environmental factors determine the pathological phenotype.

Analysis of mutations in the XPD gene in Italian patients with trichothiodystrophy: site of mutation correlates with repair deficiency, but gene dosage appears to determine clinical severity.

Xeroderma pigmentosum (XP) complementation group D is a heterogeneous group, containing patients with XP alone, rare cases with both XP and Cockayne syndrome, and patients with trichothiodystrophy (TTD). TTD is a rare autosomal recessive multisystem disorder associated, in many patients, with a defect in nucleotide-excision repair; but in contrast to XP patients, TTD patients are not cancer prone. In most of the repair-deficient TTD patients, the defect has been assigned to the XPD gene. The XPD gene product is a subunit of transcription factor TFIIH, which is involved in both DNA repair and transcription. We have determined the mutations and the pattern of inheritance of the XPD alleles in the 11 cases identified in Italy so far, in which the hair abnormalities diagnostic for TTD are associated with different disease severity but similar cellular photosensitivity. We have identified eight causative mutations, of which four have not been described before, either in TTD or XP cases, supporting the hypothesis that the mutations responsible for TTD are different from those found in other pathological phenotypes. Arg112his was the most common alteration in the Italian patients, of whom five were homozygotes and two were heterozygotes, for this mutation. The presence of a specifically mutated XPD allele, irrespective of its homozygous, hemizygous, or heterozygous condition, was always associated with the same degree of cellular UV hypersensitivity. Surprisingly, however, the severity of the clinical symptoms did not correlate with the magnitude of the DNA-repair defect. The most severe clinical features were found in patients who appear to be functionally hemizygous for the mutated allele.

Xeroderma pigmentosum and trichothiodystrophy are associated with different mutations in the XPD (ERCC2) repair/transcription gene.

The xeroderma pigmentosum group D (XPD) protein has a dual function, both in nucleotide excision repair of DNA damage and in basal transcription. Mutations in the XPD gene can result in three distinct clinical phenotypes, XP, trichothiodystrophy (TTD), and XP with Cockayne syndrome. To determine if the clinical phenotypes of XP and TTD can be attributed to the sites of the mutations, we have identified the mutations in a large group of TTD and XP-D patients. Most sites of mutations differed between XP and TTD, but there are three sites at which the same mutation is found in XP and TTD patients. Since the corresponding patients were all compound heterozygotes with different mutations in the two alleles, the alleles were tested separately in a yeast complementation assay. The mutations which are found in both XP and TTD patients behaved as null alleles, suggesting that the disease phenotype was determined by the other allele. If we eliminate the null mutations, the remaining mutagenic pattern is consistent with the site of the mutation determining the phenotype.

DNA repair and ultraviolet mutagenesis in cells from a new patient with xeroderma pigmentosum group G and cockayne syndrome resemble xeroderma pigmentosum cells.

Xeroderma pigmentosum (XP)/Cockayne syndrome (CS) complex is a combination of clinical features of two rare genetic disorders in one individual. A sun-sensitive boy (XP20BE) who had severe symptoms of CS, with dwarfism, microcephaly, retinal degeneration, and mental impairment, had XP-type pigmentation and died at 6 y with marked cachexia (weight 14.5 lb) without skin cancers. We evaluated his cultured cells for characteristic CS or XP DNA-repair abnormalities. The level of ultraviolet (UV)-induced unscheduled DNA synthesis was less than 5% of normal, characteristic of the excision-repair defect of XP. Cell fusion studies indicated that his cells were in XP complementation group G. His cells were hypersensitive to killing by UV, and their post-UV recovery of RNA synthesis was abnormally low, features of both CS and XP. Post-UV survival of plasmid pSP189 in his cells was markedly reduced, and post-UV plasmid mutation frequency was higher than with normal cells, as in both CS and XP. Sequence analysis of the mutated plasmid marker gene showed normal frequency of plasmids with multiple base substitutions, as in CS, and an abnormally increased frequency of G:C-->A:T mutations, a feature of XP. Transfection of UV-treated pRSVcat with or without photoreactivation revealed that his cells, like XP cells, could not repair either cyclobutane pyrimidine dimers or non-dimer photoproducts. These results indicate that the DNA-repair features of the XP20BE (XP-G/CS) cells are phenotypically more like XP cells than CS cells, whereas clinically the CS phenotype is more prominent than XP.

A CHO mutant, UV40, that is sensitive to diverse mutagens and represents a new complementation group of mitomycin C sensitivity.

A new mitomycin C (MMC)-sensitive rodent line, UV40, has been identified in the collection of ultraviolet light- (UV-) sensitive mutants of Chinese hamster ovary (CHO) cells isolated at the previous Facility for Automated Experiments in Cell Biology (FAECB). It was isolated from an UV mutant hunt using mutagenesis of AA8 cells with the DNA intercalating frameshift mutagen ICR170. It is complemented by CHO-UV-1, irsl, irs3, irslSF, MC5, V-C8 and V-H4 with respect to its MMC sensitivity based on cell survival. Despite having approx. 4 X normal UV sensitivity and increased sensitivity to UV inhibition of DNA replication, it has near-normal incision kinetics of UV irradiated DNA, and normal (6-4) photoproducts removal. It also is not hypermutable by UV, and shows near normal levels of UV inhibition of RNA synthesis. UV40 also has approx. 11 x .10 x .5 x and 2 x AA8 sensitivity to MMC, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and X-rays, respectively. Thus, its defect apparently does not involve nucleotide excision repair but rather another process, possibly in replicating past lesions. The spontaneous chromosomal aberration frequency is elevated to 20% in UV40, and the baseline frequency of sister chromatid exchange is also approximately 4-fold increased. The phenotype of UV40 appears to differ from all other rodent mutants that have so far been described.

Genetic analysis of twenty-two patients with Cockayne syndrome.

Cockayne syndrome (CS) is an autosomal recessive disorder with dwarfism, mental retardation, sun sensitivity and a variety of other features. Cultured CS cells are hypersensitive to ultraviolet (UV) light, and following UV irradiation, CS cells are unable to restore RNA synthesis rates to normal levels. This has been attributed to a specific deficiency in CS cells in the ability to repair damage in actively transcribed regions of DNA at the rapid rate seen in normal cells. We have used the failure of recovery of RNA synthesis, following UV irradiation of CS cells, in a complementation test. Cells of different CS donors are fused. Restoration of normal RNA synthesis rates in UV-irradiated heterodikaryons indicates that the donors are in different complementation groups, whereas a failure to effect this recovery implies that they are in the same group. In an analysis of cell strains from 22 CS donors from several countries and different racial groups, we have assigned five cell strains to the CS-A group and the remaining 17 to CS-B. No obvious racial, clinical or cellular distinctions could be made between individuals in the two groups. Our analysis will assist the identification of mutations in the recently cloned CSA and CSB genes and the study of structure-function relationships.

Molecular and cellular analysis of the DNA repair defect in a patient in xeroderma pigmentosum complementation group D who has the clinical features of xeroderma pigmentosum and Cockayne syndrome.

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are quite distinct genetic disorders that are associated with defects in excision repair of UV-induced DNA damage. A few patients have been described previously with the clinical features of both disorders. In this paper we describe an individual in this category who has unusual cellular responses to UV light. We show that his cultured fibroblasts and lymphocytes are extremely sensitive to irradiation with UV-C, despite a level of nucleotide excision repair that is 30%-40% that of normal cells. The deficiency is assigned to the XP-D complementation group, and we have identified two causative mutations in the XPD gene: a gly-->arg change at amino acid 675 in the allele inherited from the patient's mother and a -1 frameshift at amino acid 669 in the allele inherited from his father. These mutations are in the C-terminal 20% of the 760-amino-acid XPD protein, in a region where we have recently identified several mutations in patients with trichothiodystrophy.

Genetic heterogeneity of the excision repair defect associated with trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare autosomal recessive disease characterized by brittle hair with reduced sulfur content, mental and physical retardation, a peculiar face and ichthyosis. Photosensitivity has been reported in approximately 20% of the cases in the literature. DNA repair investigations demonstrated that clinical photosensitivity is usually associated with an enhancement of the cellular UV-sensitivity and that the repair defect is in the same gene as in patients from group D of xeroderma pigmentosum (XP). In this paper we describe the characterization of 13 further TTD patients; a defect in the nucleotide-excision repair was observed in fibroblast strains from 10 patients, confirming that TTD is frequently associated with DNA repair defects. Genetic analysis based on complementation studies demonstrated the presence of the XP-D defect in seven repair-defective TTD cases, indicating definitively that the concurrence of TTD with XP-D is not a sporadic or casual event. However, three further cell strains (TTD4VI and TTD6VI from two French siblings and TTD1BR from an English patient) showed restoration of normal UV-induced DNA repair synthesis after fusion with XP or TTD cells belonging to XP group D. These observations, which give the first indication that TTD is associated with repair defects behaving differently in the functional test of complementation, suggest some kind of causal connection between defective excision-repair factors and clinical features diagnostic for TTD. A peculiar aspect of TTD in which repair deficiencies are not related to an increased susceptibility to cancer is confirmed also in all the repair-defective TTD patients investigated in this paper.