Rapid hydrolysis of proteins and peptides by means of microwave technology and its application to amino acid analysis

A rapid heating method of hydrolysis by the use of microwave oven has been applied to amino acid analysis of proteins and peptides. This convenient method has been compared with the conventional 6 N HCl hydrolysis at 110 degrees for 24 h. The advantages of this new method are its expedition and the accurate and comparable results as compared to the tedious conventional technique. The method provides a rapid processing of multiple samples within minutes instead of days and inexpensive access to the important data of amino acid compositions of proteins by the commonly used microwave oven. The necessary change in the design of hydrolysis vials and the safety precautions accompanying this novel use of microwave acid-digestion method are also described.     

Reverse line blotting: a new technique for the sensitive detection of tick borne pathogens

Recently, a new molecular diagnostic technique for tick borne pathogens has been developed and is called Reverse Line Blotting. This paper outlines the basis of this method and then illustrates its use for the analysis of blood samples obtained from Ugandan cattle and buffalo. The sensitivity and the specificity are discussed in relation to the use of this technique in the Maghreb     

Two-dimensional gel electrophoretic method for mapping DNA replicons.

We describe in detail a method which allows determination of the directions of replication fork movement through segments of DNA for which cloned probes are available. The method uses two-dimensional neutral-alkaline agarose gel electrophoresis followed by hybridization with short probe sequences. The nascent strands of replicating molecules form an arc separated from parental and nonreplicating strands. The closer a probe is to its replication origin or to the origin-proximal end of its restriction fragment, the shorter the nascent strands that are detected by the probe. The use of multiple probes allows determination of directions of replication fork movement, as well as locations of origins and termini. In this study, we used simian virus 40 as a model to demonstrate the feasibility of the method, and we discuss its applicability to other systems.     

Retrieval of rat aortic smooth muscle cells as intact cell sheet for regenerative medicine: a cost effective approach using photo polymerization

Cell-based therapeutics are promising routes for the regeneration of damaged cells and organs. The recovery of cells cultured in vitro for such applications requires the use of proteolytic enzymes which deteriorate its property by disruption of cell–cell and cell–matrix interactions. Intact cell sheets can be retrieved with the use of thermo responsive polymer grafted on to the culture plates. Our study presents the use of photo-polymerization as a simple and inexpensive way to create thermo-responsive culture surfaces for the detachment of intact cell sheet. Poly (N-isopropyl acrylamide) (PNIPAAm) was synthesized by photo-polymerization and characterized by NMR spectroscopy, differential scanning calorimetry and gel permeation chromatography. Thermo-responsive culture dishes were prepared by the coating method and characterized for its thermo-responsive efficacy using FTIR spectroscopy and water contact angle measurements. Atomic force microscopy depicted the thin coating achieved with this method is similar to the conventional grafting method. Suitability for cell culture and cell sheet retrieval was assessed by culturing rat aortic smooth muscle cells in the PNIPAAm coated tissue culture plates. The cells remained viable as evident from the live dead assay and the cell sheet was detached by low temperature treatment. The results demonstrate a versatile method for creating thermo responsive culture surfaces while eliminating the use of expensive radiation sources for the conventional grafting method.     

Determination of protein secondary structure using factor analysis of infrared spectra

A method is presented for determining the secondary structural composition of a protein in aqueous solution from its infrared spectrum. A factor analysis approach is used to analyze the infrared spectra of 18 proteins whose crystal structures are known from X-ray studies. Factor analysis followed by multiple linear regression identifies those eigenspectra that correlate with the variation in properties described by the calibration set. The properties of interest in this study are % alpha-helix, % beta-sheet, and % turns. In the analysis of an unknown, the factor loadings required to reproduce its spectrum are substituted in the regression equation for each property to predict its secondary structural composition. The accuracy of the method was determined by removing each standard, in turn, from the calibration set and using a calibration set generated from the remainder to predict its composition. By this method we obtain standard errors of prediction of 3.9% for alpha-helix, 8.3% for beta-sheet, and 6.6% for turns. The method may also be applied to the spectra of proteins in 2H2O. The method has important advantages over those currently in use for the quantitative analysis of the infrared spectra of proteins. Manipulation of the spectrum is kept to a minimum, no curve-fitting is necessary, and the several amide I band components need not be assigned.     

Cellular associations and the differential spermiogram: making sense of stallion spermatozoal morphology

Morphologic assessment of spermatozoa is one of the most objective measures in a Breeding Soundness Examination of a stallion. There are different systems for morphologic assessment of spermatozoa. The objectives of this article are to review spermatogenesis, describe clinical sample preparation, discuss previous methods of morphologic classification and explain the use of a differential spermiogram. The advantages of the differential spermiogram method of analysis are discussed, along with its use in delineating intrinsic and extrinsic disturbances in spermatogenesis. Case examples of specific cellular associations and their diagnostic relevance are discussed.     

A new approach for the determination of oligosaccharide structures

A new method for the determination of oligosaccharide chains, known as the D60 one-dimensional TOCSY method is introduced in this paper. The results show that the use of this method enables a more effective coherent long-range magnetic relay transfer compared with that of existing DIPSI-2 and MMDY methods. Further, the method is easy to use and is not sensitive to the error of the pulse width. Without complex z-filtering steps, the high-quality sub-spectrum of pure absorption can be quickly obtained, which facilitates sub-spectroscopy detection for the existence of weak spin-coupling sugar components in the saccharide ring. Glycosides are taken as examples to discuss the characteristics of this method and its application in the determination of oligosaccharides in spectrum peak height.     

A reliable method for preparation and electron microscopic visualization of nucleosomes in higher plants

An improved and reliable method is given for the preparation and electron microscopic visualization of nucleosomes in higher plants. The critical steps of the technique are indicated and enough details are given to allow for its use without any prior experience.     

An efficient system for markerless gene replacement applicable in a wide variety of enterobacterial species

We describe an improved allelic-exchange method for generating unmarked mutations and chromosomal DNA alterations in enterobacterial species. Initially developed for use in Salmonella enterica, we have refined the method in terms of time, simplicity, and efficiency. We have extended its use into related bacterial species that are more recalcitrant to genetic manipulations, including enterohemorrhagic and enteropathogenic Escherichia coli and Vibrio parahaemolyticus. Data from over 50 experiments are presented including gene inactivations, site-directed mutagenesis, and promoter exchanges. In each case, desired mutations were identified by polymerase chain reaction screening typically from as few as 10-20 colonies up to a maximum of 300 colonies. The method does not require antibiotic nor nutritional markers in target genes and works efficiently in wild-type strains, obviating the need for specialized hosts or genetic systems. The use is simple, requiring basic laboratory materials, and represents an alternative to existing methods for gene manipulation in the Enterobacteriaceae.     

Significance of refractory period in the method of counter flows

The results of model tests accomplished for precision and evaluation of present restrictions in the collision method are demonstrated in this work. The reduction of induced antidromic potential due to its "collision" takes place in the interelectrode area. This fact is of a great importance for the use of the collision method as it increases probable encounters of anti- and orthodromic impulses and possible intervals between stimulation and exploring electrodes.     

Simultaneous determination of 1-chloro-2,4-dinitrobenzene, 2,4-dinitrophenyl-S-glutathione and its metabolites for human placental disposition studies by high-performance liquid chromatography

We developed and validated an HPLC method for determination of 1-chloro-2,4-dinitrobenzene (CDNB) and its glutathione conjugate 2,4-dinitrophenyl-S-glutathione (DNP-SG) to study the kinetics and mechanisms involved in DNP-SG formation and efflux, as a probe for human placental metabolism and transport. This method combines use of 3 microm solid phase, rapid mobile phase gradient with dual wavelength ultraviolet detection to permit determination of a lipophilic parent compound and its hydrophilic metabolites in a single short run. The selectivity, linearity, accuracy, precision, relative recovery and stability of the assay are sufficient for determining CDNB, DNP-SG and its metabolites from buffer and tissue samples to support placental drug metabolism and transport studies.     

An Improved Colorimetric Method to Quantify Sugar Content of Plant Tissue

A modification of the phenol-sulphuric acid method to determinethe bulk of soluble or insoluble sugars in plant tissue is proposed.The method is based on optimizing the phenol concentration toobtain equal colour yields for glucose, fructose and sucrose.The optimal phenol concentration is found to depend on the ethanolconcentration of the measuring solution. The method is quick,easy and its costs are low, permitting its use in routine analysis. Key words: Free sugars, colorimetry, routine analysis, plant tissue     

In vitro studies of the transmission barrier

Protein misfolding cyclic amplification (PMCA) has proved to be an efficient method mimicking in vitro some of the fundamental steps involved in prion replication in vivo. Thus, it can be used to efficiently replicate a variety of prion strains/species. The in vitro generated prions possess key prion features, i.e., they are infectious in vivo and maintain their strain specificity. One of the big challenges is its use for studying prion transmission barriers. PMCA has been efficiently used for adapting different prion species through a range of species barriers; however its capacity for overcoming purportedly unbreakable species barriers compels us to adapt it in order to use it as a reliable technique. In addition, this in vitro method might be a crucial tool in evaluating the potential risks of different prion strains (natural or experimentally generated in vitro) to humans and animals.     

Immunologic studies on nucleic acids and their components. II. Reversible inhibition of anti-nucleoside antibodies in aqueous pyridine and its application in antibody purification

The effect of aqueous pyridine on a hapten-antihapten system was investigated by the quantitative precipitin reaction and by the membrane filtration method. It was found that dilute solutions of pyridine inhibited the reaction between isopentenyladenosine and its antiserum. Other solvents examined were less effective. The effect of pyridine was reversible at concentrations where complete inhibition occurred, thus indicating its use for the dissociation of antigen-antibody complexes. The inhibitory effect of pyridine was exploited in a single-step purification method for anti-isopentenyladenosine and anti-deoxy-adenylate antibodies. In addition, generally applicable methods for linking nucleosides and nucleotides to aminoethyl-Sepharose are described.     

遗传算法支持下土地利用空间分形特征尺度域的识别

针对土地利用空间分形特征只存在于有限尺度域的现象,采用无标度区内离散点拟合的离差平方和平均值最小作为目标函数,提出了一种基于遗传算法的土地利用空间分形特征尺度域的识别方法,用于准确计算分形维数的有效区间范围。以武汉市武昌区水域空间分形特征为例,利用Quickbird多光谱遥感影像提取土地利用空间信息,重点讨论了基于遗传算法识别土地利用空间分形特征尺度域范围的总体思路、适应度函数和遗传算子等环节;然后分别从测定系数、标准差和无标度区间3个角度,将其同人工判断法、相关系数法以及强化系数法进行对比讨论;并结合研究区域实际的水域空间分布格局,分析不同方法计算所得半径维数的合理性。结果表明,土地利用分形特征尺度域的范围对分形维数计算结果有较大影响;相对于传统计算方法来说,遗传算法在尺度无标度区间识别上具有更高的精度,可以为土地利用空间格局分形特征的研究提供客观指导意见。     

Elimination of affinity reagent interference for the mass spectrometric detection of low-abundance proteins following immunoprecipitation

The presence of affinity reagents such as immunoglobulin in preparations for sensitive mass spectrometry analyses can preclude the identification of low-abundance proteins of interest. We report a method whereby antisera are purified and biotinylated prior to use in immunoprecipitation that allows for its efficient removal from proteomic samples via streptavidin capture. This method can similarly be extended to other affinity reagents such as recombinant fusion proteins for enhanced identification of interacting proteins.     

Molecular mechanisms of insertional mutagenesis in yeasts and mycelium fungi

Random insertional mutagenesis is an efficient tool for studying molecular mechanisms of many genetically determined processes. An improved variant of this method is REMI (Restriction Enzyme Mediated Integration) mutagenesis. In this method, the insertion cassette is introduced into the recipient cell together with restriction endonuclease. As a result, the REMI cassette insertion occurs in sites recognized by the restriction enzyme. The use of restriction endonucleases enhances transformation rate and provides cassette insertion in virtually any locus. A mutation is tagged by the insertion cassette, which can be identified by isolating the REMI cassette together with the flanking genomic DNA regions. The review describes general requirements to REMI. The mechanisms of REMI mutagenesis are surveyed with special reference to yeast Saccharomyces cerevisiae. Special attention is given to the development and use of REMI for other lower eukaryotes (yeasts and mould fungi). Drawbacks of the method and perspectives of its use are discussed.     

PRESENT STATUS OF DERMABRASION

The use of dermabrasion for cosmetic purposes is becoming less popular due to limitations inherent in the method despite the fact that it is still the best method available for the minimizing of acne scarring. Planing for precancerous skin is increasing in demand because of definite benefits to be gained from its use. While the method has not attained universal acceptance for the latter purpose, 80 per cent of dermatologists who have tried this approach, and who answered a questionnaire, rate the benefits obtained as excellent or good. Only 3.5 per cent considered the results as poor. In a five-year period between two questionnaires, there was in general a trend away from enthusiasm for this modality, but esteem for it as a way of dealing with precancerous skin held up better than opinion of its use for cosmetic purposes.     

Molecular mechanisms of insertional mutagenesis in yeasts and mycelium fungi

Random insertional mutagenesis is an efficient tool for studying molecular mechanisms of many genetically determined processes. An improved variant of this method is REMI (Restriction Enzyme Mediated Integration) mutagenesis. In this method, the insertion cassette is introduced into the recipient cell together with restriction endonuclease. As a result, the REMI cassette insertion occurs in sites recognized by the restriction enzyme. The use of restriction endonucleases enhances transformation rate and provides cassette insertion in virtually any locus. A mutation is tagged by the insertion cassette, which can be identified by isolating the REMI cassette together with the flanking genomic DNA regions. The review describes general requirements to REMI. The mechanisms of REMI mutagenesis are surveyed with special reference to yeast Saccharomyces cerevisiae. Special attention is given to the development and use of REMI for other lower eukaryotes (yeasts and mould fungi). Drawbacks of the method and perspectives of its use are discussed.     

Mesure par μTDM et dilution de nanocolloïdes-99mTc de la surface péritonéale chez le rat insuffisant rénal au cours d’une expérience de dialyse péritonéale

Peritoneal dialysis takes an important place for the management of patients with end-stage renal failure, especially for young children. The optimization of its efficiency needs to use biocompatible dialysis solutions but also to improve peritoneal surface area (PSA) recruitment. Small animals experimental models are an essential step for PSA measurements. We propose a fast method using in vivo microcomputerized tomography (μCT) and isotopic dilution method to evaluate the PSA.