The p.T191M mutation of the CBS gene is highly prevalent among homocystinuric patients from Spain, Portugal and South America

Classical homocystinuria is due to cystathionine -synthase (CBS) deficiency. More than 130 mutations, which differ in prevalence and severity, have been described at the CBS gene. Mutation p.I278T is very prevalent, has been found in all European countries where it has been looked for with the exception of the Iberian peninsula, and is known to respond to vitamin B₆. On the other hand, mutation p.T191M is prevalent in Spain and Portugal and does not respond to B₆. We analysed 30 pedigrees from Spain, Portugal, Colombia and Argentina, segregating for homocystinuria. The p.T191M mutation was detected in patients from all four countries and was particularly prevalent in Colombia. The number of p.T191M alleles described in this study, together with those previously published, is 71. The prevalence of p.T191M among CBS mutant alleles in the different countries was: 0.75 in Colombia, 0.52 in Spain, 0.33 in Portugal, 0.25 in Venezuela, 0.20 in Argentina and 0.14 in Brazil. Haplotype analyses suggested a double origin for this mutation. No genotype–phenotype correlation other than the B₆-nonresponsiveness could be established for the p.T191M mutation. Additionally, three new mutations, p.M173V, p.I429del and c.69_70+8del10, were found. The p.M173V was associated with a mild, B₆-responsive, phenotype.     

A cross-over study on the effect of various therapeutic approaches to morning breath odour

OBJECTIVE: The aim of this study was to investigate the effect of tongue scraping and inter-dental flossing on morning bad breath in periodontally healthy subjects. METHODS: A four-step blind, cross-over study was conducted in 19 volunteers, divided into four groups: Group I: tooth brushing; Group II: tooth brushing and inter-dental flossing; Group III: tooth brushing and tongue scraping; and Group IV: tooth brushing, inter-dental flossing and tongue scraping. The volunteers performed these oral hygiene procedures three times a day for 7 days. Seven-day wash-out intervals were observed. Morning mouth breath was assessed organoleptically and by volatile sulphur compound concentrations. RESULTS: The highest mean organoleptic and volatile sulphur compound measurement values were found in the treatment groups in which tongue scraping was not performed and there were statistical differences between the two groups (p < 0.05). In the organoleptic evaluation (p > 0.05), inter-dental flossing did not show any statistical improvement in the effect of tongue hygiene on morning bad breath, but it significantly reduced the concentration of volatile sulphur compounds (p < 0.05). CONCLUSION: The findings suggest that tongue scraping appears to be the most important hygienic procedure to reduce morning bad breath in periodontally healthy subjects.     

Construction of an internal RT-PCR standard control for the detection of human caliciviruses in stool

RT-PCR is the most sensitive assay for the detection of human caliciviruses (HuCV) in stool and environmental samples. However, false negative results are commonly obtained due to the presence of RT-PCR inhibitors. In order to exclude such false negative results, an internal control (IC) was developed for the assay by cloning a 319 nt sequence of the Norwalk virus (NV) polymerase containing a 156 nt cDNA insert. The RT-PCR assay was carried out using RNA derived from the constructed plasmid and a primer set previously described for calicivirus detection, resulting in a 475 nt product. Distinct bands of the internal control and the viral specific RT-PCR products (319 nt) were obtained when the internal control was added to the samples. Similar results were also obtained when both the control RNA and viral RNA were seeded into stool samples from asymptomatic volunteers, or when the internal control was included into positive samples. Since the primer set used in the assays can detect a wide range of strains in both norovirus and sapovirus genera, this internal control should have a broad application for the diagnosis of human caliciviruses diagnosis in both clinical and environmental samples.     

Genetic interactions of a putative Arabidopsis thaliana ubiquitin-ligase with components of the Saccharomyces cerevisiae ubiquitination machinery

The feasibility of using the Saccharomyces cerevisiae genetic tools to get insights into the function of a plant-specific ubiquitin-ligase was examined. ATL2 is a potential ubiquitin-ligase of the RING-H2 type that was originally isolated as a conditionally toxic Arabidopsis cDNA when overexpressed in yeast. ATL2 is a member of an Arabidopsis family that comprises 80 proteins. After testing cDNAs from 25 ATL members for toxicity we found that in addition to ATL2 only ATL63 was toxic, suggesting specific interactions of each one of these two ATLs in yeast. We seek to identify suppressors of the ATL2 toxicity in yeast and we found that toxicity was suppressed by knock-out mutations on different components of the ubiquitination pathway. Suppression was achieved in four deubiquitinating enzyme mutants and in one ubiquitin-conjugating enzyme mutant. A model is proposed in which Ubc4 and ATL2 act together to target for degradation one or more essential yeast proteins, Doa4/Ubp4, Ubp6 and Ubp14 have a role in disassembling ubiquitin chains on the target proteins and Ubp15 protects ATL2 from auto-ubiquitination. We presuppose that our approach can be further utilized to analyze the function of this distinctive class of ubiquitin-ligases in yeast as well as in Arabidopsis.     

Clearance of Cellulosimicrobium cellulans bacteremia in a child without central venous catheter removal

Cellulosimicrobium cellulans (formerly known as Oerskovia xanthineolytica) rarely causes human infection. Infections have been reported in immunocompromised hosts or in patients with foreign bodies, such as catheters, where treatment has generally involved removal of the foreign body. We report on a case in which the organism was isolated in multiple blood cultures from a 13-year-old male. After initial therapy failed, treatment with vancomycin and rifampin resulted in infection clearance without removal of the central venous catheter.     

Validation of string test for diagnosis of Helicobacter pylori infections

The method of recovering Helicobacter pylori DNA or viable cells absorbed on a string that a person has swallowed and that is retrieved an hour later (string test) should be a useful alternative to traditional analysis of cells or DNA obtained by endoscopy, which is invasive, uncomfortable, relatively costly, and ill-suited for community-based and pediatric studies. Here we assayed the sensitivity and validity of the string test versus conventional endoscopic biopsy for detecting and analyzing H. pylori infection. Forty-four people with gastric complaints were studied using both H. pylori culture and urease gene (ureB) PCR. H. pylori organisms cultured from strings and biopsy specimens from the same patients were fingerprinted by the randomly amplified polymorphic DNA (RAPD) method. Biopsy sections were also hematoxylin and eosin and silver stained for H. pylori detection. H. pylori was cultured from 80% of strings and detected by PCR from 91% of strings from participants whose biopsies had been H. pylori positive by culture, PCR, and/or histology. Strains recovered from strings and biopsy specimens yielded identical or closely related RAPD profiles in each of the 24 cases tested. We conclude that the string test is a useful method for H. pylori recovery and analysis when relatively noninvasive procedures are needed.     

Current possibilities for detecting high risk of cardiovascular disease

Current possibilities for better detecting high risk of coronary heart disease (CHD) and stroke and peripheral arterial disease are described in this review. A first step is based on risk factors assessment that allows establishing high-risk diagnostic, either by detecting a condition termed as "CHD risk equivalent" and defined by one or more severe major risk factor, or by calculating multifactorial risk in asymptomatic subjects with a global risk score integrating several moderate risk factors. A second diagnostic step, concerning subjects not considered at high-risk by risk factors assessment, is based on non-invasive detection of sub clinical atherosclerosis via a wide variety of structural and functional arterial markers. A third step focuses on detection of myocardial ischemia that may add diagnostic and prognostic information in subjects with high CHD risk. The implementation of high-risk strategy is not yet standardized but it should allow improving cost-effectiveness of cardiovascular prevention, particularly in asymptomatic subjects.     

Heme oxygenase attenuates oxidative stress and inflammation, and increases VEGF expression in portal hypertensive rats

BACKGROUND/AIMS: The pathophysiological significance of heme oxygenase-1 up-regulation in portal hypertension is not completely understood. In this study, we determined the role of heme oxygenase-1 on oxidative stress, inflammation, angiogenesis, and splanchnic hemodynamics in rats with portal hypertension induced by partial portal vein ligation. METHODS: Rats were treated with the heme oxygenase inhibitor SnMP or vehicle for 7 days. Then, oxidative stress was quantified by superoxide anion production, and inflammatory response was assessed by immunofluorescence. Expression of angiogenesis mediators was determined by western blotting, and the extent of portosystemic collaterals by radioactive microspheres. Hemodynamic studies were performed by flowmetry. RESULTS: Oxidative stress was significantly increased in the mesentery of portal hypertensive rats, as compared with sham-operated controls. In portal hypertensive rats, chronic heme oxygenase inhibition (1) potentiated oxidative stress and inflammation, (2) significantly decreased VEGF expression, without modifying the extent of collaterals or the splanchnic neovascularization, and (3) significantly decreased superior mesenteric artery blood flow and portal pressure. CONCLUSIONS: This study demonstrates that heme oxygenase plays an important (beneficial) role attenuating oxidative stress and inflammation, but it also plays a detrimental role in stimulating VEGF production, and contributing to the development of hyperdynamic splanchnic circulation in rats with portal hypertension.