Melioidosis in an Italian tourist injured by the tsunami in Thailand.
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Aged↗
Biomedical subjects
A Ciervo
Publications and source records attributed to A Ciervo.
Sister chromatid exchange and oxidative DNA damage in paving workers exposed to PAHs.
Paving workers are exposed during road paving to several polycyclic aromatic hydrocarbons (PAHs) contained in asphalt fumes. In this study early genotoxic and oxidative effects of exposure to bitumen fumes were evaluated in 19 paving workers and 22 controls. Environmental and biological monitoring of exposure was carried out, measuring, on personal air samples from exposed workers collected during three working days, the concentration of 14 PAHs and urinary OH-pyrene at the end of each of the three working days. Genotoxic effect was evaluated analysing sister chromatid exchange (SCE) frequency and direct-oxidative DNA damage by formamido-pyrimidine-glycosylase (Fpg)-modified comet assay on lymphocytes. Tail moment values from Fpg-enzyme treated cells (TMenz) and from untreated cells (TM) were used as parameters of direct and oxidative DNA damage, respectively. For each subject, the TMenz/TM ratio >2.0 was used to indicate the presence of oxidative damage. DNA damage was also evaluated analysing comet percentage. Personal air samples showed low level of total PAHs (2.843 microg m(-3)) with prevalence of 2-3 ring PAHs (2.693 microg m(-3)). Urinary OH-pyrene after work-shift of the three working days was significantly higher than that found at the beginning of the working week. SCE analysis did not show any difference between two groups while an oxidative DNA damage was found in 37% of exposed with respect to the absence in controls. Comet percentage was significantly higher (P = 0.000 ANOVA) in the exposed than in controls. The results demonstrate the high sensitivity of comet assay to assess early oxidative effects induced by exposure to bitumen fumes at low doses and confirm the suitability of urinary OH-pyrene as a biomarker of PAH exposure. In conclusion the study suggests the use of Fpg-modified comet test as a biomarker of early genotoxic effects and that of urinary OH-pyrene as a biomarker of PAH exposure to furnish indications in terms of characterization, prevention and management of risk in occupational exposure to mixtures of potentially carcinogenic substances.
Adult↗
Chlamydia pneumoniae infection in young stroke patients: a case--control study.
Retrospective and cross-sectional studies have suggested that both bacterial and viral infections may be risk factors for atherosclerosis, ischemic stroke and acute coronary events. The correlation between Chlamydia pneumoniae and atherosclerosis remains a source of controversy. Our case-control study is aimed at evaluating the frequency of C. pneumoniae infection in a cohort of young adults with recent cerebrovascular disease and in particular etiologic stroke subtypes. Chlamydia pneumoniae IgG, IgM and IgA antibodies were evaluated by microimmunofluorescence method and antibody titers to both recombinant antigens chlamydial outer protein 2 and 60-kDa chlamydial heat shock protein (HSP60) by ELISA. The two groups differed with regard to the prevalence of C. pneumoniae IgA (P < 0.001) and IgG (P < 0.0001), as well as the titer of anti-R-HSP60 IgG (P < 0.001). We found an increase in IgA titers, suggestive of persistent, chronic active infection, in 16 patients in whom the etiology of the cerebral ischemic event was large-vessel atherothrombosis. Persistent, active C. pneumoniae infection may be an additional risk factor for ischemic stroke mainly of atherotrombotic origin in young subjects. However, a large-scale prospective confirmation of our findings is required.
Adolescent↗
Absence of the brachial artery: report of a rare human variation and review of upper extremity arterial anomalies.
Variations in the arterial anatomy of the upper extremities, although uncommon, occur in up to one in five patients. Most of these variants occur in either the radial or ulnar artery; brachial artery variations are less common. The case we report is a rare anomaly consisting of brachial artery agenesis or regression. The brachial artery was absent from its origin but reconstituted as a normal-appearing vessel 3 cm above the antecubital fossa. The profunda brachii artery and the superior and inferior ulnar collateral arteries were also absent in this patient. The axillary artery served as the main collateral to the forearm. This constellation of anomalies has not been previously described or explained by developmental models in humans and other primates. We speculate that failure of development or arrest of specific vascular anlage in the upper extremity occurs at particular embryological stages because of unknown factors.
Angiography↗
Molecular characterization of first human Bartonella strain isolated in Italy.
The aim of this study was to characterize a Bartonella strain (BA-1) isolated from a blood culture of an Italian, human immunodeficiency virus-positive patient with bacillary angiomatosis. We analyzed the isolate using molecular biology methods such as whole-cell fatty acid analysis, PCR-restriction fragment length polymorphism analysis, type-specific 16S rRNA PCRs, sequence analysis of the 16S rRNA, pulsed-field gel electrophoresis, and arbitrarily primed PCR. The BA-1 isolate turned out to be a Bartonella quintana strain, similar but not identical to B. quintana Oklahoma, which was used as a control strain.
Adult↗
Isolation and characterization of Borrelia burgdorferi sensu lato strains in an area of Italy where Lyme borreliosis is endemic.
Between 1993 and 1998, we isolated Borrelia burgdorferi sensu lato from 55 of the 119 patients with clinically diagnosed Lyme borreliosis who were admitted to "San Martino" Hospital in Belluno, Veneto, an Adriatic region in northeastern Italy where Lyme borreliosis is endemic. Upon hospitalization, all patients presented erythema migrans. Isolates were typed using ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) analysis of the rrfA-rrlB intergenic spacer. Of the 41 isolates typed, 37 belonged to Borrelia afzelii, 2 to Borrelia garinii, and 2 to B. burgdorferi sensu stricto. Pulsed-field gel electrophoresis, performed on 21 strains (13 new isolates and 8 controls), revealed different RFLP patterns within the B. garinii and B. afzelii strains; among the five B. garinii strains and the 12 B. afzelii strains, three or two different RFLP patterns were identified, according to the restriction enzyme used. The protein patterns of the new isolates confirmed their genotypic classification and revealed the level of expression of some immunodominant proteins like OspA and other characteristic Osps. These findings constitute the first report of such a high recovery rate of B. burgdorferi from patients in a very restricted area in Italy; they also indicate the predominance of the genospecies B. afzelii in the study area and the heterogeneity of the circulating strains.
Adult↗
Portal mesenteric shunting for reconstruction of the visceral venous system.
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Aged↗
The tourniquet revisited as an adjunct to lower limb revascularization.
PURPOSE: The purpose of this study was to evaluate the role and efficacy of the tourniquet in lower limb revascularization. METHODS: During a 3-year period, 195 patients underwent 205 infrainguinal reconstruction operations in the lower extremity. These patients underwent bypass with a tourniquet and inflow occlusion (group 1) or bypass without a tourniquet (group 2). The type of infrainguinal reconstruction, tourniquet ischemia time, blood loss, and complications related to tourniquet use were recorded. A subset of patients underwent serial muscle biopsies. Specimens from calf muscle were taken just (1) before application of the tourniquet, (2) before tourniquet release, and (3) once wound closure was initiated. These biopsy specimens were studied by histochemical staining and also analyzed for phosphorylase enzyme, a marker for subcellular ischemia. RESULTS: One hundred eleven patients underwent 117 infrainguinal reconstruction procedures in which the tourniquet and inflow occlusion were used. These patients were matched against 84 patients who underwent 88 infrainguinal reconstructions without the use of the tourniquet. Complete hemostatic control in group 1 was obtained in 108 of the procedures (92%). Eight percent of the procedures required minor additional techniques to obtain complete hemostasis; in two instances, the tourniquet was removed because it did not provide hemostasis. Mean tourniquet time was less than 1 hour for all reconstruction groups. There were no instances of neurologic deficit, thrombosis of distal vessels, or vascular injury that was related to the use of a tourniquet. A comparison of the two groups revealed no differences with regard to overall blood loss (P =.63) or duration of operation (P = 0.60), observations that reflect the complexity of the cases rather than the use or nonuse of a tourniquet. When tourniquet control was used, we noted a definite decrease in the time for the distal dissection, because total vascular control with extensive dissection was unnecessary. Histochemical analysis with phosphorylase revealed a conversion of tissue with active enzyme activity to a low level with tourniquet use (P <.05). CONCLUSION: The use of a tourniquet for lower limb revascularization is safe and effective and improves visualization of the operative field. Less dissection of the target vessels is required. With a combination of the nonuse of clamps and other occluding devices, we project a decrease in host hyperplastic response that will, in turn, impact favorably on patency rates. The possibility exists that early failure may be prevented by avoiding the application of traumatic forces to diseased and brittle or calcified arteries. In this study, tourniquet time had no impact on overall operative procedural time, although certain phases of the operation were clearly shortened and facilitated, particularly in complex and difficult reconstructions. Histochemical changes found in muscle biopsy specimens did not adversely impact patients clinically, but further investigation is required to elucidate subcellular events.
Aged↗
Mapping of protein domains of hepatitis A virus 3AB essential for interaction with 3CD and viral RNA.
The small hydrophobic protein 3AB of the picornaviruses, encompassing the replication primer 3B, has been suggested to anchor the viral replication complex to membranes. For hepatitis A virus (HAV) 3AB, we have previously demonstrated its ability to form stable homodimers, to bind to membranes, and to interact specifically with RNA, implicating its multiple involvement in viral replication. In the present report, we show that HAV 3AB additionally interacts with HAV protein 3CD, a feature also described for the corresponding polypeptide of poliovirus. By assessing the interactions of three deletion mutants, distinct domains of HAV 3AB were mapped. The hydrophobic domain and the 3B moiety were found to be essential for the 3AB interaction with 3CD. Both electrostatic and hydrophobic forces are involved in this interaction. The cluster of charged amino acid residues at the C terminus of 3A seems to determine the specificity of 3AB interaction with RNA structures formed at either terminus of the HAV genome. Furthermore, our data implicate that 3A can interact with HAV RNA. Compared with poliovirus 3AB, which by itself is a nonspecific RNA-binding protein, HAV 3AB specifically recognizes HAV RNA structures that might be of relevance for initiation of viral RNA replication.
3C Viral Proteases↗
Polypeptide 3AB of hepatitis A virus is a transmembrane protein.
Hepatitis A virus (HAV) protein 3AB is a membrane-interacting protein containing a stretch of 21 hydrophobic amino acid residues. The nature of its membrane association was studied in detail by analysing various deletion mutants. In vivo and in vitro expression of the wild-type protein and its mutants allowed to demonstrate that the hydrophobic domain interacts with membranes and to define the portions essential for this feature. Furthermore, the results suggest that 3AB behaves as an integral membrane protein. Expression in Escherichia coli showed that 3AB can be isolated, in association with membranes, both in monomeric and in dimeric form. This finding was confirmed in vitro after post-translational incubation of the protein with microsomal membranes. Analysis of deletion mutants demonstrated that the dimerization region colocalises with the hydrophobic transmembrane domain, implicating that HAV 3AB could form oligomers mediated by the interaction of transmembrane alpha-helices.
Amino Acid Sequence↗
Site-directed mutagenesis of hepatitis A virus protein 3A: effects on membrane interaction.
Due to a stretch of hydrophobic amino acids, protein 3A of hepatitis A virus (HAV) has been suggested to act as a membrane anchor or a carrier of the genome-linked protein 3B (VPg) during viral RNA synthesis. Mutagenesis analysis was performed in order to elucidate the role of the N- and C-terminal tracts of protein 3A in cell membrane interaction. Expression of the mutated proteins in E. coli cells demonstrated that the presence of positively charged residues at the C-terminus is not required for membrane anchoring. Changes in the primary sequence involving charged amino acids at the N- and C-termini critically influenced the ability of the protein 3A of a cytopathic strain of HAV to change bacterial membrane permeability. This result demonstrates the strict correlation between the structure and pore-forming potential of HAV protein 3A.
Amino Acid Sequence↗
Cochlear dysfunction in IDDM patients with subclinical peripheral neuropathy.
OBJECTIVE: To investigate the function of the auditory pathway from the cochlea to the auditory cortex in subjects with IDDM. RESEARCH DESIGN AND METHODS: Brain stem, middle-, and long-latency auditory-evoked responses and evoked otoacoustic emissions were measured in 48 normally hearing IDDM patients and in age- and sex-matched nondiabetic subjects. Peripheral neuropathy was diagnosed by nerve conduction velocity (NCV) at the peroneal and sural nerves. Auditory brain stem responses (ABRs) reflect auditory pathway function within the brain stem; middle-latency responses (MLRs) and long-latency responses (LLRs) originate from the auditory cortex; and evoked otoacoustic emissions (EOAEs) give objective information about preneural, mechanical elements of the cochlear function. RESULTS: A subclinical peripheral neuropathy was found in 12 diabetic patients. We found higher latencies of waves I (t = 4.4, P < 0.0001), III (t = 3.7, P = 0.0004), and V (t = 2.7, P = 0.008) of ABRs in diabetic patients (I: 1.7 +/- 0.13 ms; III: 3.9 +/- 0.17 ms; V: 5.7 +/- 0.24 ms), compared with those of the control group (I: 1.6 +/- 0.13 ms; III: 3.7 +/- 0.18 ms; V: 5.6 +/- 0.17 ms). However, neither central transmission time (i.e., the wave interpeak I-V) nor MLRs and LLRs were found to be significantly different in diabetic and control subjects. Mean EOAE amplitude was found to be significantly reduced (F = 4.2, P = 0.02) in diabetic patients with a reduced NCV (7.6 +/- 3.9 dB; Scheffé test: P = 0.03), but not in those without neuropathy (9.1 +/- 4.2 dB), compared with the control group (10.8 +/- 3.1 dB). No correlations were found between duration of diabetes and EOAEs or between sural NCV and peroneal NCV and metabolic control. EOAEs were not correlated with peroneal and sural NCVs. CONCLUSIONS: Our results indicate that the early preneural dysfunction of cochlear receptors causes a prolonged activation of the peripheral portion of the auditory pathway, while signal conduction along the central auditory pathway was shown to be normal in diabetes.
Adult↗
Iron-regulated transcription of the pvdA gene in Pseudomonas aeruginosa: effect of Fur and PvdS on promoter activity.
The pvdA gene, encoding the enzyme L-ornithine N5-oxygenase, catalyzes a key step of the pyoverdin biosynthetic pathway in Pseudomonas aeruginosa. Expression studies with a promoter probe vector made it possible to identify three tightly iron-regulated promoter regions in the 5.9-kb DNA fragment upstream of pvdA. The promoter governing pvdA expression was located within the 154-bp sequence upstream of the pvdA translation start site. RNA analysis showed that expression of PvdA is iron regulated at the transcriptional level. Primer extension and S1 mapping experiments revealed two 5'termini of the pvdA transcript, 68 bp (T1) and 43 bp (T2) 5' of the PvdA initiation. The pvdA transcripts were monocystronic, with T1 accounting for 90% of the pvdA mRNA. Fur box-like sequences were apparently absent in the regions 5' of pvdA transcription start sites. A sequence motif resembling the -10 hexamer of AlgU-dependent promoters and the iron starvation box of pyoverdin genes controlled by the sigmaE -like factor PvdS were identified 5' of the T1 start site. The minimum DNA region required for iron-regulated promoter activity was mapped from bp -41 to -154 relative to the ATG translation start site of pvdA. We used pvdA'::lacZ transcriptional fusions and Northern (RNA) analyses to study the involvement of Fur and PvdS in the iron-regulated expression of pvdA. Two fur mutants of P. aeruginosa were much less responsive than wild-type PAO1 to the iron-dependent regulation of pvdA expression. Transcription from the pvdA promoter did not occur in a heterologous host unless in the presence of the pvdS gene in trans and was abrogated in a pvdS mutant of P. aeruginosa. Interaction of the Fur repressor with a 150-bp fragment encompassing the pvdS promoter was demonstrated in vivo by the Fur titration assay and confirmed in vitro by gel retardation experiments with a partially purified Fur preparation. Conversely, the promoter region of pvdA did not interact with Fur. Our results support the hypothesis that the P. aeruginosa Fur repressor indirectly controls pvdA transcription through the intermediary sigma factor PvdS; in the presence of sufficient iron, Fur blocks the pvdS promoter, thus preventing PvdS expression and consequently transcription of pvdA and other pyoverdin biosynthesis genes.
Bacterial Proteins↗
Cloning and nucleotide sequence of the pvdA gene encoding the pyoverdin biosynthetic enzyme L-ornithine N5-oxygenase in Pseudomonas aeruginosa.
The enzyme L-ornithine N5-oxygenase catalyzes the hydroxylation of L-ornithine (L-Orn), which represents an early step in the biosynthesis of the peptidic moiety of the fluorescent siderophore pyoverdin in Pseudomonas aeruginosa. A gene bank of DNA from P. aeruginosa PAO1 (ATCC 15692) was constructed in the broad-host-range cosmid pLAFR3 and mobilized into the L-Orn N5-oxygenase-defective (pvdA) P. aeruginosa mutant PALS124. Screening for fluorescent transconjugants made it possible to identify the trans-complementing cosmid pPV4, which was able to restore pyoverdin synthesis and L-Orn N5-oxygenase activity in the pvdA mutant PALS124. The 17-kb PAO1 DNA insert of pPV4 contained at least two genetic determinants involved in pyoverdin synthesis, i.e., pvdA and pvdC4, as shown by complementation analysis of a set of mutants blocked in different steps of the pyoverdin biosynthetic pathway. Deletion analysis, subcloning, and transposon mutagenesis enabled us to locate the pvdA gene in a minimum DNA fragment of 1.7 kb flanked by two SphI restriction sites. Complementation of the pvdA mutation was under stringent iron control; both pyoverdin synthesis and L-Orn N5-oxygenase activity were undetectable in cells of the trans-complemented mutant which had been grown in the presence of 100 microM FeCl3. The entire nucleotide sequence of the pvdA gene, from which the primary structure of the encoded polypeptide was deduced, was determined. The pvdA structural gene is 1,278 bp; the cloned DNA fragment contains at the 5' end of the gene a putative ribosome-binding site but apparently lacks known promoterlike sequences. The P. aeruginosa L-Orn N5-oxygenase gene codes for a 426-amino-acid peptide with a predicted molecular mass of 47.7 kDa and an isoelectric point of 8.1. The enzyme shows approximately 50% homology with functional analogs, i.e., L-lysine N6-hydroxylase of aerobactin-producing Escherichia coli and L-Orn N5-oxygenase of ferrichrome-producing Ustilago maydis. The pvdA gene was expressed in P. aeruginosa under the control of the T7 promoter. Induction of the T7 RNA polymerase system resulted in parallel increases of the L-Orn N5-oxygenase activity and of the amount of a 47.7-kDa polypeptide. We also constructed a site-specific pvdA mutant by insertion of a tetracycline-resistance cassette in the chromosomal pvdA gene of P. aeruginosa PAO1. Similarly to strain PALS124, the pvdA mutant obtained by gene disruption also disclosed no pyoverdin synthesis, lacked L-Orn N5-oxygenase activity, was complemented by the cloned pvdA gene, and produced pyoverdin at wild-type levels when fed with the biosynthetic precursor L-N5-OH-Orn. Southern blot analysis indicated that genes homologous to pvdA could be located within a 1.7-kb DNA fragment from SphI-digested genomic DNA of different hydroxamate-producing Pseudomonas spp. Our results suggest that omega-amino acid oxygenases have been conserved over a wide evolutionary range and probably evolved from a common ancestor.
Amino Acid Sequence↗
Iron-regulated salicylate synthesis by Pseudomonas spp.
Two iron-regulated compounds have been found in acidified ethyl acetate extracts from culture supernatants of Pseudomonas aeruginosa and Pseudomonas cepacia type-strains. Synthesis of both compounds paralleled iron-deficient growth, and was repressed in the presence of 100 microM-FeCl3. Yields of these substances varied among different strains and attained maximum levels during stationary phase. Thin layer chromatographic analysis in five different solvent systems revealed that the slower-moving compound chromatographed as two distinct bands, and showed RF values and spectral properties similar to pyochelin. The faster-moving compound co-migrated as a single band with a standard of commercial salicylic acid in each of the chromatographic systems tested. Moreover, a molecule with an identical RF was also produced by Pseudomonas fluorescens CHA401, which is known to synthesize salicylic acid as the only siderophore during iron-limited growth. Spectrophotometric and spectrofluorometric titrations led to the identification of this iron-regulated compound as salicylic acid, in agreement with the structure deduced from 1H-NMR and mass spectroscopy. The identity of the P. cepacia siderophore azurechelin as salicylic acid was also conclusively demonstrated. Salicylic acid, like pyochelin and pyoverdin, promoted P. aeruginosa growth in an iron-depleted medium. These results are consistent with a putative siderophore activity for salicylic acid, i.e. azurechelin, as has been demonstrated for P. aeruginosa, P. fluorescens and P. cepacia. Thus, salicylic acid is likely to act as a siderophore in more than one species belonging to the genus Pseudomonas.
Burkholderia cepacia↗
Multiple sclerosis. Magnetic resonance imaging, evoked potentials and cerebrospinal fluid analysis.
We have studied 27 patients with multiple sclerosis (22 definite, 5 probable) by magnetic resonance imaging (MRI), visual evoked potentials (VEPs), brainstem auditory evoked potentials (BAEPs), and oligoclonal bands (OBs) in cerebrospinal fluid (CSF), MRI was altered in 92.5% of the cases, the presence of OBs in CSF was revealed in 73.1% of the examined patients, the frequency of evoked potentials (EPs) alteration was VEPs = 59.3% and BAEPs = 29.6%, at least one of the two EPs occurred positive in 66.6% of the cases. Our data confirm the more sensitive value of MRI compared with OBs and EPs studies in assessing MS, and stress the utility of the combined use of these tests.
Adolescent↗
[Molecular diagnosis of Chlamydia pneumoniae diseases].
Chlamydia pneumoniae (C. p.) is an intracellular parasite directly involved in respiratory disease and more recently in chronic degenerative pathologies as atherosclerosis and asthma. Its peculiar life cycle makes cultural isolation difficult, thus, troublesome the diagnosis of the disease. Serology is so far the most common method of diagnosis of the, although the indirect based evidence of the serology may give clinically misleading results. Nucleic acid amplification methods offer indeed rapid, reliable and low cost assessment pathogen bacteria isolation, with relevant benefits for the patient's management. These molecular methods are nowadays essential in presence of bacteria of difficult cultivation or method inconsistent with temporal clinical needs, for they allow to rapidly detect even nucleic acid traces of the infectious agent, providing direct evidence of its presence in the biological samples and hence the relevant therapy. Nucleic acid methods are extensively applied in laboratory diagnosis of Chlamydia trachomatis bringing about the development of sensitive and reliable commercial kits. This review analyses the literature of the genic amplification methods in the search of C. p. in clinical samples highlighting methodological and diagnostic aspects. Although genic amplification methods have been implemented presently by the clinical research labs only, it is anticipated that through their standardisation they could be used by most clinical microbiology laboratories.
Chlamydophila Infections↗
[Study of genotoxic and oxidative effects induced by PAH exposure in paving workers].
Paving workers are exposed during road paving to several PAHs contained in asphalt fumes. We aimed to evaluate early genotoxic and oxidative effects in 19 paving workers and 22 controls. We analysed sister chromatide exchange (SCE) frequency as marker of genotoxicity. Moreover we assessed oxidative DNA damage by Fpg-modified comet assay on lymphocytes calculating tail moment values from fpg-enzyme treated cells (TMenz) and from untreated cells (TM). For each subject the TMenz/TM ratio higher than 2.0 was used to indicate the presence of oxidative damage. We also evaluated DNA damage analysing comet percentage. SCE analysis didn't show any difference between exposed and control groups. We found oxidative DNA damage in 37% of exposed in respect to the absence in controls. Comet percentage was significantly higher in the exposed than in controls. The results demonstrate the high sensitivity of comet assay to assess early oxidative effects induced by exposure to PAH mixtures at low doses and suggest the use of this biomarker in the characterization, prevention and management of risk induced by occupational exposure to mixtures of potentially carcinogenic substances.
Adult↗