Evaluation of a Western blot test,Helico blot 2.1, in the diagnosis of Helicobacter pylori infection in a pediatric population

Background. Noninvasive diagnostic tests are useful as screening tools for Helicobacter pylori infection in pediatric populations. The aim of this study was to evaluate performance of the immunoblot assay, Helico Blot 2.1, for the diagnosis of H. pylori infection in symptomatic children. Materials and Methods. Immunoblot assay was used for detection of IgG antibodies to specific H. pylori proteins and to a recombinant H. pylori antigen, CIM marker. The study was performed on sera collected from 134 symptomatic, untreated children (mean age, 9.1 ± 3.2 years; range, 1–14 years). H. pylori infection status was determined by culture, histology and rapid urease test. Results. Immunoblot assay yielded a positive result in 71 of the 72 infected patients (sensitivity 98.6%) and in eight of the 62 noninfected ones (specificity 87.1%). The predictive values for a positive and a negative result were 89.9% and 98.2%, respectively. The performance of the CIM band alone, as a marker for H. pylori infection status, was also evaluated. This band was present on the blot of 71 infected patients and on four of the 62 H. pylori‐negative patients. The sensitivity, specificity, PPV and NPV of the CIM antigen were 98.6%, 93.5%, 94.7% and 98.3%, respectively. Conclusions. The immunoblot assay Helico Blot 2.1 is a suitable noninvasive test for the serodiagnosis of H. pylori infection in children. The good level of performance demonstrated by the novel recombinant antigen CIM suggests it may be a useful contribution to the qualitative and quantitative performance of the Helico Blot 2.1 in pediatric populations.     

The importance of microscopic examination in the management of desquamative diseases of the scalp

After determining the usual malassezic biota of the scalp in adult, normal persons, 259 patients with different desquamative diseases were studied by a simple adhesive tape technique. The main purpose of this study was to investigate the utility of this technique to improve the diagnosis and treatment of patients. Most patients with seborrhoeic dermatitis and sebopsoriasis demonstrated large numbers of {it Malassezia} spp. cells corresponding to the so called ``pityrosporosis'. Only 43.6% of patients with pityriasis capitis (dandruff) presented with such a diagnosis. Symptomatic pityrosporosis of the scalp should be treated with imidazolic derivatives or other antifungal substances. Patients with psoriasis of the scalp showed a typical microscopic picture represented by parakeratosic (nucleated) keratinocytes with absence of lipophilic yeasts which should be attributed to the usual dryness of the scales .Microbial epidermitis (eczema) of the scalp revealed another characteristic picture constituted by abundant leukocytes and bacteria without the presence of yeasts. The different microscopic pictures seen with this simple technique for diseases of the scalp, offer an adjunct to make a proper diagnosis and to establish a convenient treatment in cases which are not clinically well defined. This revised version was published online in June 2006 with corrections to the Cover Date.     

AQR1 gene (ORF YNL065w) encodes a plasma membrane transporter of the major facilitator superfamily that confers resistance to short-chain monocarboxylic acids and quinidine in Saccharomyces cerevisiae

We report results on the functional analysis of Saccharomyces cerevisiae ORF YNL065w, predicted to code for a protein belonging to the poorly characterized major facilitator superfamily (MFS) of transporters that are involved in multidrug resistance (MDR). YNL065w is important for a moderate increase of yeast tolerance to ketoconazole and to the cationic dye crystal violet; it protects the cell against short-chain monocarboxylic acids (C(2)-C(6)), but not against highly liposoluble acids such as octanoic acid or the phenoxyacetic-acid herbicides 2,4-D and MCPA; it is also a determinant of resistance to the antiarrhytmic and antimalarial drug quinidine. The encoding ORF was, thus, denominated the AQR1 gene. Results obtained using an AQR1-lacZ fusion indicate that gene expression is very low and it is not stimulated under weak acid stress. The encoded putative transporter was localized in the plasma membrane by fluorescence microscopy observation of the overproduced Aqr1-GFP fusion protein distribution.     

Mitochondrial,sarcoplasmic membrane integrity and protein degradation in heart and skeletal muscle in exercised rats

Several different exercise regimens varied in the severity of tissue damage induced. Therefore, this study investigated the effects of a single bout of exercise versus endurance training in heart and skeletal muscles with different predominant fiber types on indices of mitochondrial, endoplasmic reticulum (ER) integrity and protein degradation. Male Wistar rats performed different treadmill exercise protocols: exhaustive, maximal exhaustive, eccentric, training and exhaustive exercise after training. The maximal and eccentric exercises resulted in a significant loss of integrity of the sarcoplasmic and ER muscle, while no changes were observed in cardiac muscle. Mitochondrial membrane fluidity measured by the fluorescence polarization method was significantly increased post-acute exercises in heart and oxidative muscles. Regular exercise can stabilize and preserve the viscoelastic nature of mitochondrial membranes in both tissues. The highest increase in carbonyl content was obtained in heart after exhaustive exercise protocol, from 1+/-0.1 to 3.6+/-0.14 nmol mg protein(-1), such increase were not found after regular exercise with values significantly decreased. Nitrate heart levels showed attenuated generation of nitric oxide after training. Muscle protein oxidation was produced in all exhaustive exercises including eccentric exercise.     

Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspects

Although the physiology and metabolism of the growth of yeast strains has been extensively studied, many questions remain unanswered where the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. The strain is able to metabolise the inducer, galactose, which is a much more expensive carbon source than glucose. Both the transport of galactose into the cell-required for the induction of cutinase production-and galactose metabolism are highly repressed by glucose. Different fermentation strategies were tested and the culture behaviour was interpreted in view of the strain metabolism and physiology. A fed-batch fermentation with a mixed feed of glucose and galactose was carried out, during which simultaneous consumption of both hexoses was achieved, as long as the glucose concentration in the medium did not exceed 0.20 g/l. The costs, in terms of hexoses, incurred with this fermentation strategy were reduced to 23% of those resulting from a fermentation carried out using a more conventional strategy, namely a fed-batch fermentation with a feed of galactose.     

Reverse micelles as reaction media for lipases

Reversed micelles are at the present time faced as common organic media to perform biocatalysis. They have been associated to the idea of a microreactor where the enzyme can be sheltered and protected from solvent detrimental effects. This simplistic idea led some investigators to ignore some basic understanding, such as the recognition of the enzyme-specific microenvironment and what the enzyme experiences inside the reversed micelle. To date the number of reactions catalyzed by lipases in reversed micelles is large. This review aims to highlight some of the fundamental aspects of the lipase microencapsulation as well as to resume the outstanding progress of the reversed micellar systems. The properties of the micellar microenvironment are reviewed and related to the lipases' performance both in terms of activity and stability. The heterogeneity of reversed micellar systems is discussed in relation to component distribution models and also to enzymatic kinetics. The new trends and the practical aspects where efforts should be centralized in order to spread out the micellar bioreactor technology over industrial processes are also discussed.     

Role of intrarenal alpha(2)-adrenoceptors in the renal responses to xylazine in rats

This study examined the contribution of intrarenal alpha(2)-adrenoceptor mechanisms to the enhanced urine flow rate (V) and urinary sodium excretion (U(Na)V) responses in ketamine-xylazine-anesthetized rats. Ten minutes after left renal artery (LRA) injection, the alpha(2)-adrenoceptor antagonist yohimbine (5 microg) significantly decreased V from 58 +/- 8 to 35 +/- 7 microl. min(-1). g kidney wt(-1) and U(Na)V from 2.8 +/- 0.4 to 2.1 +/- 0.4 microeq. min(-1). g kidney wt(-1) without altering right kidney function. The renal effects of the LRA injection of yohimbine were completely abolished in chronic bilaterally renal-denervated (RDNX) rats. In RDNX rats, a higher LRA dose of yohimbine (15 microg) significantly reduced left and right kidney V, with no effects on U(Na)V. In separate bladder-catheterized rats, yohimbine (0.5 mg/kg), 20 min after intravenous injection, significantly decreased V from 63 +/- 9 to 13 +/- 2 microl. min(-1). g kidney wt(-1 )and U(Na)V from 4.5 +/- 0.5 to 1.1 +/- 0.1 microeq. min(-1). g kidney wt(-1). In RDNX rats, this dose of yohimbine reduced V and U(Na)V, but the magnitude was blunted compared with intact rats. In contrast, 0.1 mg/kg iv yohimbine significantly reduced V and U(Na)V to similar magnitudes in intact and RDNX groups. Together, these findings indicate that intravenous xylazine acts by renal nerve-dependent and -independent mechanisms to enhance renal excretory function in ketamine-anesthetized rats. Because the effects of the LRA dose of yohimbine were abolished in renal-denervated animals, it appears that xylazine has a direct renal action to augment the renal excretion of water and sodium via a presynaptic alpha(2)-adrenoceptor pathway that inhibits the release of neurotransmitters from renal sympathetic nerve terminals.     

Trehalose delays the reversible but not the irreversible thermal denaturation of cutinase

The effect of trehalose (0.5 M) on the thermal stability of cutinase in the alkaline pH range was studied. The thermal unfolding induced by increasing temperature was analyzed in the absence and in the presence of trehalose according to a two-state model (which assumes that only the folded and unfolded states of cutinase were present). Trehalose delays the reversible unfolding. The midpoint temperature of the unfolding transition (Tm) increases by 4.0 degrees C and 2. 6 degrees C at pH 9.2 and 10.5, respectively, in the presence of trehalose. At pH 9.2 the thermal unfolding occurs at higher temperatures (Tm is 52.6 degrees C compared to 42.0 degrees C at pH 10.5) and a refolding yield of around 80% was obtained upon cooling. This pH value was chosen to study the irreversible inactivation (long-term stability) of cutinase. Temperatures in the transition range from folded to unfolded state were selected and the rate constants of irreversible inactivation determined. Inactivation followed first-order kinetics and trehalose reduced the observed rate constants of inactivation, pointing to a stabilizing effect on the irreversible inactivation step of thermal denaturation. However, if the contribution of reversible unfolding on the irreversible inactivation of cutinase was taken into account, i.e., considering the fraction of cutinase molecules in the reversible unfolded conformation, the intrinsic rate constants can be calculated. Based on the intrinsic rate constants it was concluded that trehalose does not delay the irreversible inactivation. This conclusion was further supported by comparing the activation energy of the irreversible inactivation in the absence and in the presence of trehalose. The apparent activation energy in the absence and in the presence of trehalose were 67 and 99 Kcal/mol, respectively. The activation energy calculated from intrinsic rate constants was higher in the absence (30 Kcal/mol) than in the presence of trehalose (16 Kcal/mol), showing that kinetics of the irreversible inactivation step increased in the presence of trehalose. In fact, trehalose stabilized only the reversible step of thermal denaturation of cutinase.     

Enzymatic esterification of ethanol by an immobilised Rhizomucor miehei lipase in a perforated rotating disc bioreactor

A perforated rotating disc bioreactor was developed to perform the esterification of ethanol with oleic acid, catalyzed by a lipase from Rhizomucor miehei immobilized by adsorption on to a hydrophobic support-Accurel EP700. The bioreactor with total recirculation operated at an optimum agitation rate of 400 rev./min. The experimental results, in this condition, were predict by a kinetic model using the constants obtained in the batch (Erlenmeyer flasks) assays: a catalytic constant, k(cat) = 5.78 mmol/h. mg protein; a Michaelis constant for ethanol, K(m(Et)) = 1.20 M; a Michaelis constant for oleic acid, K(m(Ol)) = 1.16 x 10(-8) M, and a dissociation constant of the ethanol-lipase complex, K((Et)) = 9.46 x 10(7) M. The efficiency of conversion gradually decreased during continuous operation of the reactor. The enzymatic activity decayed according to a first order deactivation model and the integrated equations of a continuous stirred tank reactor (CSTR) and a plug flow reactor (PFR). A half-life time of the lipase of about 10 days and a deactivation constant of 0.003 h(-1) were obtained in the present system.