Component-specific toxic concerns of the inhalable fraction of urban road dust.

Continuous global urbanisation causes an ever-growing ecological footprint of pollution. Road dust (RD), one of these pollutants, poses a health concern due to carcinogenic and toxic components potentially present in the micron-sized fractions. The literature reports on the concentrations of trace, toxic metals and metalloids present in RD (Hooker and Nathanail in Chem Geol 226:340-351, 2006), but the literature on its molecular composition is limited. Recent reports on the bioaccessibility of platinum group metals are also reported (Colombo et al. in Chem Geol 226:340-351, 2008). In vitro and animal toxicological studies confirmed that the chemical composition of inhaled particles plays a major role in its toxic, genotoxic and carcinogenic mechanisms, but the component-specific toxic effects are still not understood. Particle-bound airborne transition metals can also lead to the production of reactive oxygen species in lung tissue; a special concern amongst particularly susceptible cohorts (children and elderly). The characterisation of the molecular composition of the fine fraction is evidently of importance for public health. During a pilot study, partially characterised size-fractioned RD samples (Barrett et al. in Eviron Sci Technol 44:2940-2946, 2010) were analysed for their elemental concentration using X-ray fluorescence spectrometry and inductively coupled plasma mass spectrometry. In addition, separately dispersed particles (200 particles per size fraction) were analysed individually by means of computer-controlled electron probe X-ray micro-analysis (CC-EPXMA) and their molecular structure probed by studying elemental associations. These were correlated with micro-Raman spectroscopy (MRS) results. It was found that the fine fraction (<38 µm) had the highest Pb (238 ppm) and Cr (171 ppm) concentrations. The CC-EPXMA data showed >50 % association of Cr-rich particles with Pb, and the MRS data showed that the Cr was mostly present as lead chromate and therefore in the Cr(VI) oxidation state. Concentrations of both Pb and Cr decreased substantially (279 (<38 µm)-13 ppm (<1 mm); 171 (<38 µm)-91 ppm (< 1 mm), respectively) in the larger fractions. Apart from rather alarmingly high concentrations of oxidative stressors (Cu, Fe, Mn), the carcinogenic and toxic potential of the inhalable fraction is evident. Preliminary bioaccessibility data indicated that both Cr and Pb are readily mobilised in artificial lysosomal liquid and up to 19 % of Cr and 47 % of Pb were released.

Composition of prehistoric rock-painting pigments from Egypt (Gilf Kébir area).

The composition of rock-painting pigments from Egypt (Gilf Kebia area) has been analyzed by means of molecular spectroscopy such as Fourier transform infrared and micro-Raman spectroscopy and scanning electron microscopy coupled to an energy dispersive X-ray spectrometer and X-ray fluorescence analysis. Red and yellow pigments were recognized as red and yellow ochre with additional rutile.

Argentinean prehistoric pigments' study by combined SEM/EDX and molecular spectroscopy.

Composition of the prehistoric pigments' (from Carriqueo rock shelter, Rio Negro province, Argentina) has been analysed by means of molecular spectroscopy (Fourier transform infrared (FTIR) and micro-Raman) and scanning electron microscopy (SEM) coupled to an energy-dispersive X-ray spectrometer (EDS). Red and yellow pigments were recognized as red and yellow ochre. The matrix of the pigments is composed of one or more substances. According to the matrix composition yellow and red pigments were also divided into two groups-i.e. those containing kaolinite or sulphates. Green pigment was detected as green earth, made up of celadonite as a chromophore.

Acetazolamide prevents vacuolar myopathy in skeletal muscle of K(+) -depleted rats.

BACKGROUND AND PURPOSE: Acetazolamide and dichlorphenamide are carbonic anhydrase (CA) inhibitors effective in the clinical condition of hypokalemic periodic paralysis (hypoPP). Whether these drugs prevent vacuolar myopathy, which is a pathogenic factor in hypoPP, is unknown. The effects of these drugs on the efflux of lactate from skeletal muscle were also investigated. EXPERIMENTAL APPROACH: For 10 days, K(+)-depleted rats, a model of hypoPP, were administered 5.6 mg kg(-1) day(-1) of acetazolamide, dichlorphenamide or bendroflumethiazide (the last is not an inhibitor of CA). Histological analysis of vacuolar myopathy and in vitro lactate efflux measurements were performed in skeletal muscles from treated and untreated K(+)-depleted rats, and also from normokalemic rats. KEY RESULTS: About three times as many vacuoles were found in the type II fibres of tibialis anterioris muscle sections from K(+)-depleted rats as were found in the same muscle from normokalemic rats. In ex vivo experiments, a higher efflux of lactate on in vitro incubation was found in muscles of K(+)-depleted rats compared with that found in muscles from normokalemic rats. After treatment of K(+)-depleted rats with acetazolamide, the numbers of vacuoles in tibialis anterioris muscle decreased to near normal values. Incubation with acetazolamide in vitro inhibited efflux of lactate from muscles of K(+)-depleted rats. In contrast, bendroflumethiazide and dichlorphenamide failed to prevent vacuolar myopathy after treatment in vivo and failed to inhibit lactate efflux in vitro. CONCLUSIONS AND IMPLICATIONS: Acetazolamide prevents vacuolar myopathy in K(+)-depleted rats. This effect was associated with inhibition of lactate transport, rather than inhibition of CA.

COP1D, an alternatively spliced constitutive photomorphogenic-1 (COP1) product, stabilizes UV stress-induced c-Jun through inhibition of full-length COP1.

COP1 is an evolutionarily conserved RING-finger ubiquitin ligase acting within a Cullin-RING ligase (CRL) complex that promotes polyubiquitination of c-Jun and p53. Stability of the above substrates is affected by post-translational changes priming the proteins for polyubiquitination and proteasome-dependent degradation. However, degradation of both substrates is controlled indirectly by signaling pathways affecting the E3 ligases involved in their polyubiquitination. Here, we report the identification of COP1D, a ubiquitously expressed splice variant of COP1 lacking a portion of a coiled-coil region involved in intermolecular associations. While being unable to associate with other components of the CRL complex, COP1D exerts a dominant-negative function over the full-length protein, due to its ability to heterodimerize with COP1 and sequester it from the enzymatically active complex. Ectopic expression of COP1D antagonizes the function of COP1, while its selective downregulation by RNA interference promotes more efficient degradation of c-Jun and p53 by the full-length protein. The COP1/COP1D mRNA ratio is modulated by UV stress and a decreased COP1/COP1D ratio correlates with elevated c-Jun, but not p53 protein levels in invasive ductal breast cancer. Thus, dynamic changes of the COP1/COP1D ratio provide an additional level of regulation of the half-life of the substrates of this E3 ligase under homeostatic or pathological conditions.

Muscleblind-like protein 1 nuclear sequestration is a molecular pathology marker of DM1 and DM2.

Myotonic dystrophies (DM) are repeat expansion diseases in which expanded CTG (DM1) and CCTG (DM2) repeats cause the disease. Mutant transcripts containing CUG/CCUG repeats are retained in muscle nuclei producing ribonuclear inclusions, which can bind specific RNA-binding proteins, leading to a reduction in their activity. The sequestration of muscleblind-like proteins (MBNLs), a family of alternative splicing factors, appears to be involved in splicing defects characteristic of DM pathologies. To determine whether MBNL1 nuclear sequestration is a feature of DM pathologies, we have examined the in vivo distribution of MBNL1 in muscle sections from genetically confirmed DM1 (n=7) and DM2 (n=9) patients, patients with other myotonic disorders (n=11) and from patients with disorders caused by repeat expansions, but not DM1/DM2 (n=3). The results of our immunofluorescence study indicate that, among patients examined, MBNL1 nuclear sequestration in protein foci is a molecular pathology marker of DM1 and DM2 patients where ribonuclear inclusions of transcripts with expanded CUG/CCUG repeats are also present. These findings indicate that MBNLs might be important targets for therapeutic interventions to correct some of the specific features of DM pathology.

Biomolecular identification of (CCTG)n mutation in myotonic dystrophy type 2 (DM2) by FISH on muscle biopsy.

Myotonic dystrophy type 2 (DM2) is a dominantly inherited disorder with multisystemic clinical features, caused by a CCTG repeat expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. The mutant transcripts are retained in the nucleus forming multiple discrete foci also called ribonuclear inclusions. The size and the somatic instability of DM2 expansion complicate the molecular diagnosis of DM2. In our study fluorescence-labeled CAGG-repeat oligonucleotides were hybridized to muscle biopsies to investigate if fluorescence in situ hybridization (FISH), a relatively quick and simple procedure, could be used as a method to diagnose DM2. When FISH was performed with (CAGG)5 probe, nuclear foci of mutant RNA were present in all genetically confirmed DM2 patients (n=17) and absent in all patients with myotonic dystrophy type 1 (DM1; n=5) or with other muscular disease (n=17) used as controls. In contrast, foci were observed both in DM1 and DM2 myonuclei when muscle tissue were hybridized with (CAG)6CA probe indicating that this probe is not specific for DM2 identification. The consistent detection of ribonuclear inclusions in DM2 muscles and their absence in DM1, in agreement with the clinical diagnosis and with leukocyte (CCTG)n expansion, suggests that fluorescence in situ hybridization using (CAGG)5 probes, may be a specific method to distinguish between DM1 and DM2. Moreover, the procedure is simple, and readily applicable in any pathology laboratory.

Muscle biopsy and cell cultures: potential diagnostic tools in hereditary skeletal muscle channelopathies.

Hereditary muscle channelopathies are caused by dominant mutations in the genes encoding for subunits of muscle voltage-gated ion channels. Point mutations on the human skeletal muscle Na+ channel (Nav1.4) give rise to hyperkalemic periodic paralysis, potassium aggravated myotonia, paramyotonia congenita and hypokalemic periodic paralysis type 2. Point mutations on the human skeletal muscle Ca2+ channel give rise to hypokalemic periodic paralysis and malignant hyperthermia. Point mutations in the human skeletal chloride channel CIC-1 give rise to myotonia congenita. Point mutations in the inwardly rectifying K+ channel Kir2.1 give rise to a syndrome characterized by periodic paralysis, severe cardiac arrhythmias and skeletal alterations (Andersen's syndrome). Involvement of the same ion channel can thus give rise to different phenotypes. In addition, the same mutation can lead to different phenotypes or similar phenotypes can be caused by different mutations on the same or on different channel subtypes. Bearing in mind, the complexity of this field, the growing number of potential channelopathies (such as the myotonic dystrophies), and the time and cost of the genetic procedures, before a biomolecular approach is addressed, it is mandatory to apply strict diagnostic protocols to screen the patients. In this study we propose a protocol to be applied in the diagnosis of the hereditary muscle channelopathies and we demonstrate that muscle biopsy studies and muscle cell cultures may significantly contribute towards the correct diagnosis of the channel involved. DNA-based diagnosis is now a reality for many of the channelopathies. This has obvious genetic counselling, prognostic and therapeutic implications.

A genomic approach of the hepatitis C virus generates a protein interaction map.

The hepatitis C virus (HCV) causes severe liver disease, including liver cancer. A vaccine preventing HCV infection has not yet been developed, and, given the increasing number of infected people, this virus is now considered a major public-health problem. The HCV genome is a plus-stranded RNA that encodes a single polyprotein processed into at least 10 mature polypeptides. So far, only the interaction between the protease NS3 and its cofactor, NS4A, which is involved in the processing of the non-structural region, has been extensively studied. Our work was aimed at constructing a protein interaction map of HCV. A classical two-hybrid system failed to detect any interactions between mature HCV polypeptides, suggesting incorrect folding, expression or targetting of these proteins. We therefore developed a two-hybrid strategy, based on exhaustive screens of a random genomic HCV library. Using this method, we found known interactions, such as the capsid homodimer and the protease dimer, NS3-NS4A, as well as several novel interactions such as NS4A-NS2. Thus, our results are consistent with the idea that the use of a random genomic HCV library allows the selection of correctly folded viral protein fragments. Interacting domains of the viral polyprotein are identified, opening the possibility of developing specific anti-viral agents, based on their ability to modulate these interactions.

Processing of a dicistronic small nucleolar RNA precursor by the RNA endonuclease Rnt1.

Small nucleolar RNAs (snoRNAs) are intron encoded or expressed from monocistronic independent transcription units, or, in the case of plants, from polycistronic clusters. We show that the snR190 and U14 snoRNAs from the yeast Saccharomyces cerevisiae are co-transcribed as a dicistronic precursor which is processed by the RNA endonuclease Rnt1, the yeast ortholog of bacterial RNase III. RNT1 disruption results in a dramatic decrease in the levels of mature U14 and snR190 and in accumulation of dicistronic snR190-U14 RNAs. Addition of recombinant Rnt1 to yeast extracts made from RNT1 disruptants induces the chase of dicistronic RNAs into mature snoRNAs, showing that dicistronic RNAs correspond to functional precursors stalled in the processing pathway. Rnt1 cleaves a dicistronic transcript in vitro in the absence of other factors, separating snR190 from U14. Thus, one of the functions of eukaryotic RNase III is, as for the bacterial enzyme, to liberate monocistronic RNAs from polycistronic transcripts.

Oculopharyngeal muscular dystrophy in Italy.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant myopathy particularly frequent in Québec. The few Italian cases thus far described with bilateral ptosis, dysphagia and variable muscle weakness, show non-specific dystrophic findings on muscle biopsies by light microscopy. We describe a 70-year-old Italian woman with an adult-onset ptosis, mild dysphagia and proximal muscle weakness belonging to a family segregating OPMD according to an autosomal dominant mode of inheritance. Clinical features of four of her relatives are reviewed. Muscle biopsy studied by electron microscopy showed the typical 8.5 nm in diameter intranuclear filamentous inclusions (INI). To our knowledge, this is the first Italian report of OPMD with INI. The identification of nuclear inclusions is mandatory in order to confirm the diagnosis prior to linkage analysis.

Substrate structure requirements of the Pac1 ribonuclease from Schizosaccharmyces pombe.

The Pac1 ribonuclease of Schizosaccharomyces pombe is a member of the RNase III family of double-strand-specific ribonucleases. To examine RNA structural features required for efficient cleavage by the Pac1 RNase, we tested a variety of double-stranded and hairpin RNAs as substrates for the enzyme. The Pac1 RNase required substrates that have a minimal helix length of about 20 base pairs. The enzyme cut both strands of the helix at sites separated by two base pairs. However, Pac1 was also able to make a single-stranded cleavage within an internal bulge of an authentic Escherichia coli substrate at the same site chosen by RNase III. Pac1 efficiently degraded the structurally complex adenovirus VA RNA(I), but was inactive against the short HIV-1 TAR RNA hairpin. These results indicate that the Pac1 RNase prefers straight, perfect helices, but it can tolerate internal bulges that do not distort the helix severely. Like its homologue from Saccharomyces cerevisiae, the Pac1 RNase cleaved at two in vivo RNA processing sites in a hairpin structure in the 3' external transcribed spacer of the S. pombe pre-rRNA, suggesting a role for the enzyme in rRNA maturation.

Computerized tomography and magnetic resonance muscle imaging in Miyoshi's myopathy.

We describe clinical, pathological, and muscle imaging findings in a patient with an early adult-onset progressive muscular weakness in association with atrophy beginning in the legs and involving both gastrocnemi in particular. Muscle biopsy findings showed a severe dystrophic process with no vacuoles, consistent with Miyoshi's myopathy. Computerized tomography and magnetic resonance imaging scans were used to provide an ongoing permanent record of the various stages of the disease.

Purification and characterization of the Pac1 ribonuclease of Schizosaccharomyces pombe.

The pac1+ gene of the fission yeast Schizosaccharomyces pombe is essential for viability and its overexpression induces sterility and suppresses mutations in the pat1+ and snm1+ genes. The pac1+ gene encodes a protein that is structurally similar to RNase III from Escherichia coli, but its normal function is unknown. We report here the purification and characterization of the Pac1 protein after overexpression in E. coli. The purified protein is a highly active, double-strand-specific endoribonuclease that converts long double-stranded RNAs into short oligonucleotides and also cleaves a small hairpin RNA substrate. The Pac1 RNase is inhibited by a variety of double- and single-stranded polynucleotides, but polycytidylic acid greatly enhances activity and also promotes cleavage specificity. The Pac1 RNase produces 5'-phosphate termini and requires Mg2+; Mn2+ supports activity but causes a loss of cleavage specificity. Optimal activity was obtained at pH 8.5, at low ionic strength, in the presence of a reducing agent. The enzyme is relatively insensitive to N-ethylmaleimide but is strongly inhibited by ethidium bromide and vanadyl ribonucleoside complexes. The properties of the Pac1 RNase support the hypothesis that it is a eukaryotic homolog of RNase III.

Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease.

The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P. We report here the isolation of the pac1+ gene as a multi-copy suppressor of snm1. The pac1+ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed. The pac1+ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli. To investigate the essential structural features of the Pac1 RNase, we altered the pac1+ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility. These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347. These amino acids are conserved in all RNase III-like proteins. The glycine and glutamic acid residues were previously identified as essential for E. coli RNase III activity. The valines are conserved in an element found in a family of double-stranded RNA binding proteins. Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.

Mutation in the S4 segment of the adult skeletal sodium channel gene in an Italian paramyotonia congenita (PC) family.

The periodic paralyses are a group of autosomal dominant muscle diseases sharing the common feature of episodic stiffness and weakness, usually occurring with muscle cooling (as in the case of paramyotonia congenita, PC phenotype) or changes in extracellular K+ levels resulting from various precipitating factors (hyperkalemic periodic paralysis, HYPP and hypokalemic periodic paralysis, HypoPP). It is now known that HYPP maps to chromosome 17q, and that PC and a form of myotonia congenita without periodic paralysis also map to the 17q locus, thus indicating that they derive from allelic variants. So far, these disorders have been described in various ethnic groups but, to our knowledge, have never been reported in Italy. We describe a mutation in an S4 segment of the adult skeletal muscle sodium channel in a clinically-defined Italian family that leads to the paramyotonia congenita (PC) phenotype with dominant autosomal inheritance and temperature-related symptoms (regional weakness following cooling and exercise), present since childhood in all of the affected family members.

Neural regulation of acid maltase in an unusual adult onset deficiency.

In a 48-year-old female, the first symptoms apparently manifested themselves 18 years before, with occasional tripping and weakness in both legs. During the next 18 years, weakness progressed and the patient developed a waddling gait; she became unable to rise from a lying or seated position unassisted and the shoulder girdle also became affected. Neurological examination revealed limb and shoulder girdle predominantly involving the lower extremities. We established cell cultures from muscle biopsy specimens obtained from our patient and carried out morphological analysis which, although aspecific, demonstrated clear signs of neurogenic suffering. This was confirmed in EMG studies performed. Biochemical analysis revealed very low acid maltase residual activity. We describe an unusual case of adult-onset acid maltase deficiency (AMD) with neurogenic atrophy and low residual activity. Innervated myofibres prepared by co-culturing the patient's myoblasts, with spinal cord foetal mouse explants were not associated with an abnormal in vitro maturation of the innervated myofibres as expected by the very low residual enzymatic activity found both in the muscle biopsy specimens and in the muscle cultures. There is strong suggestion that factors other than the amount of residual activity must be involved to determine the clinical manifestation of this disease.