Chromatographic methods for amylases

This review surveys recent developments in chromatographic methods for the separation of amylases from complex extracts, including the separation of isozymes. It contains two tables with the properties and molecular characteristics of α- and β-amylases from different sources as well as an updated review of methods for the determination of amylase activity. The main subject of this review is a detailed evaluation of the application of newly developed chromatographic methods for the purification of amylases.     

Identification and Characterization of a New Serotonergic Recognition Site with High Affinity for 5-Carboxamidotryptamine in Mammalian Brain

Abstract: We analyzed the existence of an additional serotonin (5-HT) receptor subtype, sensitive to 5-carboxamidotryptamine, in the mammalian brain. Radioligand binding studies with [³H]5-HT were carried out in rat, guinea pig, and human brain membranes, in the presence of unlabeled drugs to mask the binding to all known 5-HT receptors, with the exception of 5-HT_1E sites. Under these conditions, unlabeled 5-carboxamidotryptamine still showed a biphasic competition curve with a nanomolar affinity component. Saturation studies with 5-[³H]carboxamidotryptamine were carried out in the presence of (±)-8-hydroxy-2-(di- n -propylamino)tetralin, mesulergine, and ergotamine, to mask the binding to all receptors known to be labeled by 5-carboxamidotryptamine. These studies showed the existence in cortex and hippocampus from guinea pig and human brain of a remaining binding site with high affinity ( pK _D = 7.8–8.1) and a unique pharmacological profile. 5-HT and 5-carboxamidotryptamine showed nanomolar affinity, whereas 5-methoxytryptamine recognized this binding site with intermediate affinity. Other drugs exhibited low or very low potency in inhibiting this binding. The addition of 5'-guanylylimidodiphosphate significantly reduced the number of binding sites labeled by 5-[³H]carboxamidotryptamine, in the presence of the masking drugs described above, indicating the interaction with a GTP-binding protein. Preliminary autoradiographic studies in human brain appear to indicate that this 5-HT binding site is present in areas such as the globus pallidus, neocortex, and hippocampus, among others.     

In silico two-hybrid system for the selection of physically interacting protein pairs

Deciphering the interaction links between proteins has become one of the main tasks of experimental and bioinformatic methodologies. Reconstruction of complex networks of interactions in simple cellular systems by integrating predicted interaction networks with available experimental data is becoming one of the most demanding needs in the postgenomic era. On the basis of the study of correlated mutations in multiple sequence alignments, we propose a new method (in silico two-hybrid, i2h) that directly addresses the detection of physically interacting protein pairs and identifies the most likely sequence regions involved in the interactions. We have applied the system to several test sets, showing that it can discriminate between true and false interactions in a significant number of cases. We have also analyzed a large collection of E. coli protein pairs as a first step toward the virtual reconstruction of its complete interaction network.     

Early diagnosis of invasive aspergillosis in neutropenic patients with bi-weekly serial screening of circulating galactomannan by Platelia Aspergillus

The diagnosis of invasive aspergillosis in neutropenic individuals is difficult and lengthy since non-invasive diagnostic tests lack sensitivity and specificity. The diagnosis of invasive aspergillosis in 154 prolonged neutropenic patients was prospectively bi-weekly validated by screening circulating galactomannan. The global sensitivity was 73% and specificity was 96%. The positive and negative predictive values were 73% and 98% respectively. False positive reactions occurred at a rate of 2%. Antigenemia was detected before clinical suspicion of invasive aspergillosis (median, 6 days before) in 30% of patients and anticipated the onset of radiologic signs 9 days in 60% of patients. CONCLUSION: the prospective screening of galactomannan is a sensitive and non-invasive tool for early diagnosis of invasive aspergillosis in high-risk adult hematology patients.     

Seasonal changes in lipid classes and fatty acid composition in the digestive gland of Pecten maximus

Seasonal variations in lipid classes and fatty acid composition of triacylglycerols and phospholipids in the digestive gland of Pecten maximus were studied over a period of 16 months. Acylglycerols predominated (19-77% of total lipids), in accordance with the role of the digestive gland as an organ for lipid storage in scallops. Seasonal variations were mainly seen in the acylglycerol content, while phospholipids (2.5-10.0% of total lipids) and sterols (1.9-7.4% of total lipids) showed only minor changes. The most abundant fatty acids were 14:0, 16:0, 18:0, 16:1(n-7), 18:1(n-9), 18:1(n-7), 18:4(n-3), 20:5(n-3) and 22:6(n-3) and these showed similar seasonal profiles in both, triacylglycerol and phospholipid fractions. In contrast to the phospholipid fraction, the triacylglycerol fraction contained more 20:5(n-3) than 22:6(n-3). In three phospholipid samples we noted a high percentage of a 22-2-non-methylene-interrupted fatty acid, previously described to have a structural role in several bivalve species. The main polyunsaturated fatty acids displayed important seasonal variations parallel to those of the acylglycerols, suggesting good nutritional conditions. A positive correlation existed between the level of saturated fatty acids and temperature, whereas the levels of polyunsaturated fatty acids correlated negatively with temperature.     

Selective Increase of α2A-Adrenoceptor Agonist Binding Sites in Brains of Depressed Suicide Victims

Abstract: The α_2A- and α_2C-adrenoceptor subtypes were evaluated in postmortem brains from suicides with depression (n = 22), suicides with other diagnoses (n = 12), and controls (n = 26). Membrane assays with the antagonist [³H]RX821002 (2-[³H]methoxyidazoxan) suggested the presence of α_2A-adrenoceptors in the frontal cortex and both α_2C-adrenoceptors and α_2A-adrenoceptors in the caudate. The proportions in caudate were similar in controls (α_2A, 86%; α_2C, 14%), depressed suicides (α_2A, 91%; α_2C, 9%), and suicides with other diagnoses (α_2A, 88%; α_2C, 12%). Autoradiography of [³H]RX821002 binding under α_2B/C-adrenoceptor-masking conditions confirmed the similar densities of α_2A-adrenoceptors in the cortex, hippocampus, and striatum from controls and suicides. In the frontal cortex of depressed suicides, competition of [³H]RX821002 binding by (?)-adrenaline revealed a greater proportion (61 ± 9%) of α_2A-adrenoceptors in the high-affinity conformation for agonists than in controls (39 ± 5%). Simultaneous analysis with the agonists [³H]clonidine and [³H]UK14304 and the antagonist [³H]RX821002 in the same depressed suicides confirmed the enhanced α_2A-adrenoceptor density when evaluated by agonist, but not by antagonist, radioligands. The results indicate that depression is associated with a selective increase in the high-affinity conformation of the brain α_2A-adrenoceptors.     

Protein functional features are reflected in the patterns of mRNA translation speed

Background

The degeneracy of the genetic code makes it possible for the same amino acid string to be coded by different messenger RNA (mRNA) sequences. These “synonymous mRNAs” may differ largely in a number of aspects related to their overall translational efficiency, such as secondary structure content and availability of the encoded transfer RNAs (tRNAs). Consequently, they may render different yields of the translated polypeptides. These mRNA features related to translation efficiency are also playing a role locally, resulting in a non-uniform translation speed along the mRNA, which has been previously related to some protein structural features and also used to explain some dramatic effects of “silent” single-nucleotide-polymorphisms (SNPs). In this work we perform the first large scale analysis of the relationship between three experimental proxies of mRNA local translation efficiency and the local features of the corresponding encoded proteins.

Results

We found that a number of protein functional and structural features are reflected in the patterns of ribosome occupancy, secondary structure and tRNA availability along the mRNA. One or more of these proxies of translation speed have distinctive patterns around the mRNA regions coding for certain protein local features. In some cases the three patterns follow a similar trend. We also show specific examples where these patterns of translation speed point to the protein’s important structural and functional features.

Conclusions

This support the idea that the genome not only codes the protein functional features as sequences of amino acids, but also as subtle patterns of mRNA properties which, probably through local effects on the translation speed, have some consequence on the final polypeptide. These results open the possibility of predicting a protein’s functional regions based on a single genomic sequence, and have implications for heterologous protein expression and fine-tuning protein function.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1734-7) contains supplementary material, which is available to authorized users.     

MIND-BEST: Web server for drugs and target discovery; design, synthesis, and assay of MAO-B inhibitors and theoretical-experimental study of G3PDH protein from Trichomonas gallinae

Many drugs with very different affinity to a large number of receptors are described. Thus, in this work, we selected drug-target pairs (DTPs/nDTPs) of drugs with high affinity/nonaffinity for different targets. Quantitative structure-activity relationship (QSAR) models become a very useful tool in this context because they substantially reduce time and resource-consuming experiments. Unfortunately, most QSAR models predict activity against only one protein target and/or they have not been implemented on a public Web server yet, freely available online to the scientific community. To solve this problem, we developed a multitarget QSAR (mt-QSAR) classifier combining the MARCH-INSIDE software for the calculation of the structural parameters of drug and target with the linear discriminant analysis (LDA) method in order to seek the best model. The accuracy of the best LDA model was 94.4% (3,859/4,086 cases) for training and 94.9% (1,909/2,012 cases) for the external validation series. In addition, we implemented the model into the Web portal Bio-AIMS as an online server entitled MARCH-INSIDE Nested Drug-Bank Exploration & Screening Tool (MIND-BEST), located at http://miaja.tic.udc.es/Bio-AIMS/MIND-BEST.php . This online tool is based on PHP/HTML/Python and MARCH-INSIDE routines. Finally, we illustrated two practical uses of this server with two different experiments. In experiment 1, we report for the first time a MIND-BEST prediction, synthesis, characterization, and MAO-A and MAO-B pharmacological assay of eight rasagiline derivatives, promising for anti-Parkinson drug design. In experiment 2, we report sampling, parasite culture, sample preparation, 2-DE, MALDI-TOF and -TOF/TOF MS, MASCOT search, 3D structure modeling with LOMETS, and MIND-BEST prediction for different peptides as new protein of the found in the proteome of the bird parasite Trichomonas gallinae, which is promising for antiparasite drug targets discovery.     

Resonant mirror biosensor detection method based on yessotoxin-phosphodiesterase interactions

Yessotoxin (YTX) is a generic name for a group of lipophilic compounds recently discovered and chemically characterized. Association measurements were done in a resonant mirror biosensor. The instrument detects changes in the refractive index and/or thickness occurring within a few hundred nanometers form the sensor surface where a molecule is attached. We used aminosilane surfaces where phosphodiesterase 3',5'-cyclic-nucleotide-specific from bovine brain (PDEs) was immobilized. Over this immobilized ligand different amounts of YTX were added and typical association curve profiles were observed. These association curves fit a pseudo-first-order kinetic equation where the apparent association rate constant (k(on)) can be calculated. The value of this constant increases with YTX concentration. From the representation of k(on) versus YTX concentration we obtained the association rate constant (k(ass)) 248+/-40 M(-1)s(-1) and the dissociation rate constant (k(diss)) 9.36 x 10(-4)+/-1.72 x 10(-4)s(-1). From these values the kinetic equilibrium dissociation constant (K(D)) for YTX-PDEs association can be calculated. The value of this last constant is 3.74 x 10(-6)+/-8.25 x 10(-8)M YTX. The PDE-YTX association was used as a method suitable for determination of the toxin concentration in a shellfish sample. The assay had sufficient sensitivity and can be used on simple shellfish extracts.     

Diagnostic potential of (1,3)-beta-D-glucan and anti-Candida albicans germ tube antibodies for the diagnosis and therapeutic monitoring of invasive candidiasis in neutropenic adult patients

The usefulness to diagnose and monitor invasive candidiasis (IC) using beta-glucan (BG) and antibodies against Candida albicans germ tubes (CAGT) was evaluated in a twice-weekly screening of 35 episodes in neutropenic adults at high risk. Three proven IC and three probable IC were assessed. Diagnostic levels of both markers were detected in 100% of proven IC and in 66% of probable IC. Sensitivity, specificity, positive and negative predictive values of BG and anti-CAGT antibodies detection were 83.3%, 89.6%, 62.5% and 96.3%, and 83.3%, 86.2%, 55.5%, 96.1%, respectively. False positive reactions occurred at a rate of 10.3% and 13.8% for the detection of BG and anti-CAGT antibodies, respectively. However, the patients with false positive results were different by each test. Both tests anticipated the clinical and radiological diagnosis, and the initiation of antifungal therapy in most patients. Combination of both tests improved specificity and positive predictive value to 100%.