Pharmacologic methods for identification of receptors

Determinations of apparent equilibrium dissociation constants of drug-receptor interactions are made from both functional and radioligand binding studies. In each type of study, reversible reactions are assumed and the mass action law is applied. Functional studies are frequently used to determine the dissociation constant of a competitive antagonist but are less frequently used to obtain this constant for agonist compounds since the latter determination requires an experimental procedure that irreversibly inactivates a fraction of the receptors. In the present report, values of dissociation constant for prototype agonists and antagonists, determined from binding and from functional studies, are examined in two classical isolated preparations, rabbit aorta and guinea-pig ileum. In each preparation the dissociation constants from binding and functional experiments agree well for the antagonists but differ markedly for the agonists. Further, the dissociation constant values from binding are seen to be greater for the agonists than for the antagonists. When a chronic treatment regimen in the rabbit resulted in a pronounced change in the functional dissociation constant of subsequently administered norepinephrine, there was no significant change in either the binding constant of this agonist or in the pA2 value of the alpha antagonist, phentolamine. These, and the previously described results, are shown to be compatible with a simple two-state receptor model in which agonists bind with high and low affinity to each state while antagonists do not distinguish between the states. In this model, the ratio of low to high affinity states accounts for the failure of the binding procedure to detect changes in the agonists dissociation constant that are highly significant in the functional study. Whereas the model is based on data for these two classical preparations only, and may not be more generally applicable, the findings demonstrate the necessity for employing both functional and radioligand binding experiments when characterizing drug receptors.     

New PCR-based methods for yeast identification

AIMS: To characterize reference yeast strains and identify indigenous strains isolated from wine fermentations by PCR methods. METHODS AND RESULTS: We compared several PCR techniques for yeast identification. We used oligonucleotide primers that are complementary to (i) intron splice sites, (ii) REP and (iii) ERIC elements to produce PCR fingerprints that display specific patterns between the different yeast species. These three techniques were used to characterize 41 reference yeast strains belonging to 15 different species and to identify 40 indigenous strains isolated from grape must and wine fermentations. Species-specific banding patterns were obtained with the three PCR-techniques with different degrees of intraspecific differentiation depending on the method. By comparing the PCR fingerprints of unknown isolates with those produced by reference strains, we identified yeast strains isolated from an industrial wine fermentation. CONCLUSIONS: All three PCR techniques are rapid, reliable and simple methods of yeast identification. As far as we know, this is the first time that the primers designed for amplifying repetitive elements in bacteria have been successfully used in yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Industry needs rapid, reliable and simple methods of yeast identification. The proposed PCR techniques will allow to achieve this objective.     

Current methods for global proteome identification

In a time frame of a few decades, protein identification went from laborious single protein identification to automated identification of entire proteomes. This shift was enabled by the emergence of peptide-centric, gel-free analyses, in particular the so-called shotgun approaches, which not only rely on extensive experiments, but also on cutting-edge data processing methods. The present review therefore provides an overview of a shotgun proteomics identification workflow, listing the state-of-the-art methods involved and software that implement these. The authors focus on freely available tools where possible. Finally, data analysis in the context of emerging across-omics studies will also be discussed briefly, where proteomics goes beyond merely delivering a list of protein accession numbers.     

PCR-based methods for identification of Enterococcus species

Two DNA-based techniques were used for species identification of enterococci.PvuII digestion of the genus-specific PCR product yielded four different restriction profiles among 20 enterococcal species; one of them was species-specific forE. faecium. In the second case, 32 reference strains belonging to 20 enterococcal species were divided to 12 groups by amplification of internal transcribed spacer of rRNA operon. Interspecies and some intraspecies profile variability was determined. Both methods gave similar results.     

Separation and identification methods for metalloproteinase inhibitors

Metalloproteinase inhibitors are being explored for the treatment of a wide variety of human diseases including cancers, arthritis, cardiovascular disorders, human immunodeficiency virus infection, and central nervous system illnesses. This review provides an overview of various analytical sample preparation, separation, detection, and identification techniques employed for the quantitative and qualitative determination of these inhibitor compounds. Special emphasis is placed on biological sample preparation by automated solid-phase extraction, liquid–liquid extraction, and protein precipitation by centrifugation or filtration. Other sample preparation methodologies are also evaluated. Applications of high-performance liquid chromatography, gas chromatography, and capillary electrophoresis to the quantitative determination of metalloproteinase inhibitors are described. Examples of qualitative analysis of metalloproteinase inhibitors by hyphenated liquid chromatography with mass spectrometry and nuclear magnetic resonance are also presented. The advantages and limitations of these separation and identification methodologies as well as other less frequently employed techniques are assessed and discussed.     

Library-based methods for identification of soluble expression constructs

When expression or crystallisation of a protein target in its wild-type full-length form proves problematic, a common strategy is to divide it into subconstructs comprising one or more domains. Rational construct design is not always successful, especially with targets for which there are few similar sequences to generate multiple sequence alignments. Even when this is possible, expression constructs may still fail to yield soluble protein, commonly expressing insolubly or at unusable yields. To address this, several new methods have been described that borrow concepts from the field of directed evolution whereby a random library is generated encompassing construct diversity; this is then screened to identify soluble constructs empirically. Here, we review progress in this area.     

General methods of sequence comparison

Mathematical methods for comparison of nucleic acid sequences are reviewed. There are two major methods of sequence comparison: dynamic programming and a method referred to here as the regions method. The problem types discussed are comparison of two sequences, location of long matching segments, efficient database searches and comparison of several sequences. This work was supported by a grant from the System Development Foundation.     

General methods for enriching aldoses with oxygen isotopes

Synthetic methods are described for enriching 4-, 5-, and 6-carbon aldoses with oxygen isotopes. The general approach includes exchange between H2(1)8O and the aldehyde group of an aldose, exchange of O-1 onto C-2 of both of the 2-epimeric aldoses formed by molybdate-resin epimerization, and chain extension using cyanide addition. These methods make possible the production of all 16 aldohexoses enriched at 5 of the 6 oxygen atoms, all 8 aldopentoses enriched at 4 of the 5 oxygen atoms, and the four aldotetroses enriched at 2 of the 4 oxygen atoms. The general applicability of these methods is illustrated by the synthesis of a group of 22 different, 18O-enriched, biologically important D-aldoses having 4, 5, and 6 carbon atoms. The group includes D-[1-, 2-, 3-, 4-, O]glucose, D-[1-, 2-, 3-, 4-, and 6-18O]mannose, D-[1-, 2-, 3-, and 5-18O]arabinose, D-[1- abd 2- 18O] erythorose, and D-[1- and 2-18O]threose. The g.l.c.-m.s. characterization of these sugars with respect to the position and degree of 18O-enrichment is reported. The potential of the methods for producing aldoses having oxygen labels at multiple positions, or aldoses labeled simultaneously with oxygen, hydrogen, and carbon isotopes is discussed.     

General methods for the synthesis of glycopyranosyluronic acid azides

Per-O-acetylated D-glycopyranoses derived from both mono- and disaccharides were first converted to glycosyl iodides and subsequently reacted with an azide source to achieve the stereoselective synthesis of beta-D-glycosyl azides after deacetylation. Low-temperature (4 degrees C) TEMPO oxidation of the monosaccharides provided the corresponding uronic acids, which were purified as the free acids. Oxidation of the lactosyl- and cellobiosyl azides resulted in diacid formation. However, 4',6'-O-benzylidene protection enabled selective oxidation of the C-6 hydroxyl. 2-Acetamido-2-deoxy-D-glycopyranosyl azides were also prepared and converted to uronic acids completing the library synthesis.     

Comparison of methods for the identification of coagulase-negative staphylococci

Coagulase-negative staphylococci (CNS) species identification is still difficult for most clinical laboratories. The scheme proposed by Kloos and Schleifer and modified by Bannerman is the reference method used for the identification of staphylococcal species and subspecies; however, this method is relatively laborious for routine use since it requires the utilization of a large number of biochemical tests. The objective of the present study was to compare four methods, i.e., the reference method, the API Staph system (bioMérieux) and two methods modified from the reference method in our laboratory (simplified method and disk method), in the identification of 100 CNS strains. Compared to the reference method, the simplified method and disk method correctly identified 100 and 99% of the CNS species, respectively, while this rate was 84% for the API Staph system. Inaccurate identification by the API Staph method was observed for Staphylococcus epidermidis (2.2%), S. hominis (25%), S. haemolyticus (37.5%), and S. warneri (47.1%). The simplified method using the simple identification scheme proposed in the present study was found to be efficient for all strains tested, with 100% sensitivity and specificity and proved to be available alternative for the identification of staphylococci, offering, higher reliability and lower cost than the currently available commercial systems. This method would be very useful in clinical microbiology laboratory, especially in places with limited resources.     

Currently used methods for identification and characterization of hemichannels

Connexins and pannexins are vertebrate transmembrane proteins that form hexameric conduits termed hemichannels. Functional hemichannels allow the diffusional transport of ions and small molecules across the plasma membrane and serve as paracrine and autocrine communication pathways. During the last decade, interest in the hemichannel field increased substantially. Today, there is evidence for the existence of connexin hemichannels in vertebrate cells and bulk of information supports their function in diverse physiological and pathological responses. Controversy regarding the molecular identity of the hemichannel type mediating many responses arose recently with the identification of pannexin-based hemichannels. Here, the authors describe the most frequently used methods for studying hemichannels in living mammalian cells and focus on those with which they have more experience. Although the available in vitro evidence is substantial, further studies and possibly new experimental approaches are required to understand the role and properties of connexin and pannexin hemichannels in vivo.     

Comparison of three rapid methods for identification of Salmonella spp

A study was carried out to compare three rapid methods for detection of Salmonella spp. The fluorogenic MUCAP test (Biolife, Italy), the SM-ID agar test (bioMérieux, France) and the Rambach agar test (Merck, Germany) were used in this study to examine 103 strains (69 Salmonella strains and 34 non- Salmonella strains). Two conventional culture media, Hektoen and Leifson agars, were also included. The sensitivities of the MUCAP, SM-ID, Rambach and Hektoen agar tests for pure strains were 100, 93, 88 and 99%, respectively, and their specificities were 74, 97, 76 and 59%, respectively. A total of 100 stool samples from patients with acute diarrhoea was also tested and showed great discrepancy between the different methods. In agreement with other investigators, it was found that the discriminating capacity of Rambach and SM-ID as primary plating media was very restricted. The MUCAP test was very sensitive, rapid and easy to perform but not very specific. In view of these results, it is essential to combine different methods for the accurate and reliable detection of Salmonella strains.     

Computational methods for protein identification from mass spectrometry data

Protein identification using mass spectrometry is an indispensable computational tool in the life sciences. A dramatic increase in the use of proteomic strategies to understand the biology of living systems generates an ongoing need for more effective, efficient, and accurate computational methods for protein identification. A wide range of computational methods, each with various implementations, are available to complement different proteomic approaches. A solid knowledge of the range of algorithms available and, more critically, the accuracy and effectiveness of these techniques is essential to ensure as many of the proteins as possible, within any particular experiment, are correctly identified. Here, we undertake a systematic review of the currently available methods and algorithms for interpreting, managing, and analyzing biological data associated with protein identification. We summarize the advances in computational solutions as they have responded to corresponding advances in mass spectrometry hardware. The evolution of scoring algorithms and metrics for automated protein identification are also discussed with a focus on the relative performance of different techniques. We also consider the relative advantages and limitations of different techniques in particular biological contexts. Finally, we present our perspective on future developments in the area of computational protein identification by considering the most recent literature on new and promising approaches to the problem as well as identifying areas yet to be explored and the potential application of methods from other areas of computational biology.     

Principal methods for isolation and identification of soil microbial communities

Soil microbial populations play crucial role in soil properties and influence below-ground ecosystem processes. Microbial composition and functioning changes the soil quality through decomposition of organic matter, recycling of nutrients, and biological control of parasites of plants. Moreover, the discovery that soil microbes may translate into benefits for biotechnology, management of agricultural, forest, and natural ecosystems, biodegradation of pollutants, and waste treatment systems maximized the need of scientists for the isolation and their characterization. Operations such as the production of antibiotics and enzymic activities from microorganisms of soil constitute objectives of industry in her effort to cope with the increase of population of earth and disturbance of environment and may ameliorate the effects of global climate change. In the past decades, new biochemical and molecular techniques have been developed in our effort to identify and classify soil bacteria. The goal of measuring the soil microbial diversity is difficult because of the limited knowledge about bacteria species and classification through families and orders. Molecular techniques extend our knowledge about microbial diversity and help the taxonomy of species. Measuring and monitoring soil microbial communities can lead us to better understanding of their composition and function in many ecosystem processes.     

Comparison of different methods for the identification of groups of streptococci

The Streptococci, isolated from 500 mucus-pharyngeal tampons, have been tested, for a group identification, by means of four different techniques in order to value the specificity and reliability in comparison with more traditional and, sometimes, more complex tests; such as Maxted and Lancefield. The most suitable method for routine researchers of microbiology laboratories is the one based on the extraction, by means of enzyme obtained from Streptomyces Griseus, of streptococci antigens before starting their serum identification, possible for A-B-C-D-F-G groups (Streptex). On the contrary, the method based on the links of group-specific antibodies with the A protein of the surface of Staphylococci Cowan I, has resulted more defective because Streptococci D and F cannot be grouped, and less specific because of frequent co-agglutinations.