Thermal denaturation of distamycin a - DNA Complexes as followed by hyperchromic spectra

Interaction of distamycin A with calf spleen DNA is investigated by the method of hyperchromic spectra. Hyperchromic spectra of complexes are partitioned into the components corresponding to the denaturation A-T and G+C base pairs and dissociation of the ligand, fractions of respective components are found as a function of temperature. A scheme of melting of successive regions of DNA -with different G+C content together with the scheme of distamycin A redistribution in the course of thermal denaturation is presented.     

Selection of quantum dot wavelengths for biomedical assays and imaging

Fluorescent semiconductor nanocrystals (quantum dots [QDs]) are hypothesized to be excellent contrast agents for biomedical assays and imaging. A unique property of QDs is that their absorbance increases with increasing separation between excitation and emission wavelengths. Much of the enthusiasm for using QDs in vivo stems from this property, since photon yield should be proportional to the integral of the broadband absorption. In this study, we demonstrate that tissue scatter and absorbance can sometimes offset increasing QD absorption at bluer wavelengths, and counteract this potential advantage. By using a previously validated mathematical model, we explored the effects of tissue absorbance, tissue scatter, wavelength dependence of the scatter, water-to-hemoglobin ratio, and tissue thickness on QD performance. We conclude that when embedded in biological fluids and tissues, QD excitation wavelengths will often be quite constrained, and that excitation and emission wavelengths should be selected carefully based on the particular application. Based on our results, we produced near-infrared QDs optimized for imaging surface vasculature with white light excitation and a silicon CCD camera, and used them to image the coronary vasculature in vivo. Taken together, our data should prove useful in designing fluorescent QD contrast agents optimized for specific biomedical applications.     

Selection of optimal model for the DNA histogram by analysis of error of estimated parameters.

The ability of four different mathematical models of the DNA histogram to give accurate estimates for the fractions of cells in G1, S, and G2 + M has been investigated. The models studied differ in the form and number of parameters of the function used to represent cells in S-phase. Results obtained from simulated DNA histograms suggest that the standard deviations of the model parameters increase exponentially with the width of the G1 and G2 + M peaks of the histogram. Error analysis is presented as a method to select a model of optimal complexity in relation to the resolution provided by the data in a given set of DNA histograms. Introduction of additional parameters improves the agreement between model and data but may result in a less well-posed model. A model with an optimal number of parameters can therefore be found that will yield parameter estimates with the smallest possible standard deviations.     

Interpretation of hypochromic and hyperchromic intensity changes in the Raman spectra of polypeptides and polynucleotides undergoing transition.

The hyperchromic and hypochromic changes in the intensity of the amide-I and amide-III lines of polypeptides and certain ring vibrations of the bases of polynucleotides are shown to be related to similar changes in the lower energy uv absorption bands. The selection rules strictly limit the pairs of excited electronic states that can contribute to the elements of the polarizability matrix. An energy-dependent term in this equation weights the contribution of the pairs of electronic transitions in favor of those involving the lower energy transitions. For both polypeptides and polynucleotides, there is a large hypochromic inensity change in the first π → π* exciton band upon the coil-to-helix transition. Through the selection rules, certain conformationally sensitive Raman lines are shown to derive their intensity predominantly from this band and hence also display hypochromism. Again, through an application of the selection rules, certain Raman lines can be demonstrated to depend predominantly for their intensity upon the n → π* transition, and consequently have the opposite hyperchromic intensity change upon the same conformational transition.     

Selection of optimal DNA oligos for gene expression arrays.

MOTIVATION: High density DNA oligo microarrays are widely used in biomedical research. Selection of optimal DNA oligos that are deposited on the microarrays is critical. Based on sequence information and hybridization free energy, we developed a new algorithm to select optimal short (20-25 bases) or long (50 or 70 bases) oligos from genes or open reading frames (ORFs) and predict their hybridization behavior. Having optimized probes for each gene is valuable for two reasons. By minimizing background hybridization they provide more accurate determinations of true expression levels. Having optimum probes minimizes the number of probes needed per gene, thereby decreasing the cost of each microarray, raising the number of genes on each chip and increasing its usage. RESULTS: In this paper we describe algorithms to optimize the selection of specific probes for each gene in an entire genome. The criteria for truly optimum probes are easily stated but they are not computable at all levels currently. We have developed an heuristic approach that is efficiently computable at all levels and should provide a good approximation to the true optimum set. We have run the program on the complete genomes for several model organisms and deposited the results in a database that is available on-line (http://ural.wustl.edu/~lif/probe.pl). AVAILABILITY: The program is available upon request.     

Interaction specificity of violamycin BI and BII with DNA using the hyperchromic spectra method

Interaction specificity of the anthracycline antibiotics violamycin BI and violamycin BII in respect to A.T and G.C pairs was investigated. For comparison denaturation of complexes with A.T and G.C specific ligands distamycin A and actinomycin D are presented. Making use of the least squares hyperchromic spectra measured in the course of thermal denaturation were partitioned into the components corresponding to the melting of A.T and G.C base pairs and dissociation of ligand. The mutual dependence of AT and GC denaturation allows one to draw conclusions about specificity of interaction. In case of both violamycins only slight preference of interaction with AT-rich regions was detected. The dissociation of violamycin BII in the latest stage of thermal denaturation was found to be cooperative.     

Quantum counter for correcting fluorescence excitation spectra at 320- to 800-nm wavelengths

A procedure for recording corrected fluorescence excitation spectra to wavelengths as long as 800 nm is described. The procedure involves the use of a commercial spectrofluorometer, which is modified by substituting 1,1',3,3,3',3'-hexamethylindotricarbocyanine perchlorate in place of rhodamine B as the quantum counter dye. This modification is applicable to spectrofluorometers supplied by several different manufacturers and can be accomplished by a user having only modest technical skills. A study of the fluorescence excitation spectrum of bacteriochlorophyll a is presented as an illustration of the use of the procedure. The procedure will be valuable in biological and biochemical studies that involve the use of long-wavelength fluorescent probes of either natural or synthetic origin.     

大鼠睾丸组织固定法的优选研究

目的选择一种最优势、合理的固定方式以提高对睾丸组织制片效果,以配合不同种类的科学研究。方法选用10％甲醛溶液、NBF—Bouin’s、Bouin’s和改良Davidson’s四种不同的固定液对大鼠睾丸进行充分固定后,制作石蜡切片,进行HE染色,比较不同固定液中的睾丸组织学形态的差异;利用糖原特殊染色（PAS）,探讨不同固定液对睾丸糖原观察的影响;采用免疫组织化染色,测评睾丸组织内雄激素受体的固定效果。结果改良Davidson’8固定液较NBF—Bouin’s引起的曲细精管萎缩轻,形态更为清晰,用免疫组织化学方法检测雄激素更为敏感,并且改良Davidson＇s固定液在需要对精子发生进行分期时,其PAS染色的效果与Bouin＇s液固定后等同。结论与苴守圈常浦相№曲冉David0Rnn浦对女宙奥由的圈索特罩掂杯     

Selection of an optimal system for measurement of blood pressure

The "pop-test" method was used to design an optimal system for precise measurement of fast arterial pressure oscillations by means of selecting optimal parameters of cardiovascular catheters and pressure transducers.     

Selection of optimal reference genes for normalization in quantitative RT-PCR

Background  

Normalization in real-time qRT-PCR is necessary to compensate for experimental variation. A popular normalization strategy employs reference gene(s), which may introduce additional variability into normalized expression levels due to innate variation (between tissues, individuals, etc). To minimize this innate variability, multiple reference genes are used. Current methods of selecting reference genes make an assumption of independence in their innate variation. This assumption is not always justified, which may lead to selecting a suboptimal set of reference genes.     

Model-based gene set analysis for Bioconductor

Gene Ontology and other forms of gene-category analysis play a major role in the evaluation of high-throughput experiments in molecular biology. Single-category enrichment analysis procedures such as Fisher's exact test tend to flag large numbers of redundant categories as significant, which can complicate interpretation. We have recently developed an approach called model-based gene set analysis (MGSA), that substantially reduces the number of redundant categories returned by the gene-category analysis. In this work, we present the Bioconductor package mgsa, which makes the MGSA algorithm available to users of the R language. Our package provides a simple and flexible application programming interface for applying the approach. AVAILABILITY: The mgsa package has been made available as part of Bioconductor 2.8. It is released under the conditions of the Artistic license 2.0. CONTACT: peter.robinson@charite.de; julien.gagneur@embl.de.     

Regression trees for analysis of mutational spectra in nucleotide sequences.

MOTIVATION: The study and comparison of mutational spectra is an important problem in molecular biology, because these spectra often reveal important features of the action of various mutagens and the functioning of repair/replication enzymes. As is known, mutability varies significantly along nucleotide sequences: mutations often concentrate at certain positions in a sequence, otherwise termed 'hotspots'. RESULTS: Herein, we propose a regression analysis method based on the use of regression trees in order to analyse the influence of nucleotide context on the occurrence of such hotspots. The REGRT program developed has been tested on simulated and real mutational spectra. For the G:C-->T:A mutational spectra induced by Sn1 alkylating agents (nine spectra), the prediction accuracy was 0. 99. AVAILABILITY: The REGRT program is available upon request from V.Berikov.     

A new method to find a set of energetically optimal RNA secondary structures.

We present a computer method to determine nucleic acid secondary structures. It is based on three steps: 1) the search for all possible helical regions relied on a mathematical approach derived from the convolution theorem; it uses a tetradimensional complex vector representation of the bases along the sequence; 2) a 'tree' search for a set of minimum free energy structures, by the aid of an approximate energy evaluation to reduce the computer time requirements; 3) the exact calculation and refinement of the energies. A method to introduce the experimental data and reach an arrangement between them and the free energy minimization criterion is shown. In order to demonstrate the confidence of the program a test on four RNA sequences is performed. The method has computer time requirement proportional to N2, where N is the length of the sequence and retrieves a set of optimal free energy structures.     

Selection of a representative set of structures from Brookhaven Protein Data Bank.

Reliable structural and statistical analyses of three dimensional protein structures should be based on unbiased data. The Protein Data Bank is highly redundant, containing several entries for identical or very similar sequences. A technique was developed for clustering the known structures based on their sequences and contents of alpha- and beta-structures. First, sequences were aligned pairwise. A representative sample of sequences was then obtained by grouping similar sequences together, and selecting a typical representative from each group. The similarity significance threshold needed in the clustering method was found by analyzing similarities of random sequences. Because three dimensional structures for proteins of same structural class are generally more conserved than their sequences, the proteins were clustered also according to their contents of secondary structural elements. The results of these clusterings indicate conservation of alpha- and beta-structures even when sequence similarity is relatively low. An unbiased sample of 103 high resolution structures, representing a wide variety of proteins, was chosen based on the suggestions made by the clustering algorithm. The proteins were divided into structural classes according to their contents and ratios of secondary structural elements. Previous classifications have suffered from subjective view of secondary structures, whereas here the classification was based on backbone geometry. The concise view lead to reclassification of some structures. The representative set of structures facilitates unbiased analyses of relationships between protein sequence, function, and structure as well as of structural characteristics.     

Continuous spectrophotometric assay for restriction endonucleases using synthetic oligodeoxynucleotides and based on the hyperchromic effect.

A continuous spectrophotometric assay for the EcoRV restriction endonuclease has been developed. The synthetic self-complementary oligonucleotide d(GACGATATCGTC) (which is double stranded under the assay conditions) is used as the substrate. The EcoRV endonuclease recognizes d(GATATC) sequences cutting between the central T and dA bases. Thus d(GACGATATCGTC) is converted to d(GACGAT) and d(pATCGTC) during catalysis. Both of the hexameric products are single stranded under the assay conditions. The conversion of the dodecameric substrate to the two hexameric products and the concomitant change from double- to single-stranded DNA is associated with an increase in absorbance at 254 nm due to the hyperchromic effect. This change can be used to monitor column effluents for endonuclease activity and also for Km and kcat determination under steady-state kinetic conditions.     

Autoradiographic analysis of the incorporation of uridine-H3 into hyperchromic motoneurons of mouse spinal cord]

Under conditions of local administration of uridine-H3 solution a study was made of the dynamics of the isotope incorporation into the RNA of the nuclei and the cytoplasm of the perikarion of the hyperchromic motor neurons of the ventro-lateral nucleus of mouse spinal cord. An increase in the content of hyperchromic neurocytes in the population under study was reached in mice as a result of swimming for four hours. A different character of the label regression curves for the hyperchromic and normochromic motor neurons was shown.     

Selection of optimal cardiac phases for ECG-triggered coronary CT angiography in pediatrics

PurposeThe use of coronary computed tomography angiography (CCTA) in children remains limited by patient’s irradiation, and motion artefacts impairing image quality. Triggering the acquisition at the appropriate moment, and acquiring only necessary components of the cardiac cycle could overcome these limitations. Yet, optimal cardiac intervals to perform CCTA as a function of heart rate (HR) have not yet been addressed in pediatrics.MethodsFifty children with coronary artery anomalies underwent a CCTA on a wide-coverage single-beat CT scanner. Multiple phases from 25% to 85% of the R-R interval were acquired and reconstructed with 10% increments. Two radiologists independently assessed motion artifacts on each cardiac phase using a 4-point semi-quantitative scale.ResultsAt patient level, the best phase for acquisition was found in diastole for patients with HR ≤ 75 bpm and in systole for patients with HR > 85 bpm. At coronary segments and structures level, median optimal phases were reported at 70%, 80%, 47%, 50%, and 54% of the R-R interval for patients with HR ≤ 60, 61–75, 86–100, 101–130, and >130 bpm respectively. For patients with HR between 76 and 85 bpm, no clear trend could be observed. Optimal acquisition durations represented 10% (2 phases), 20% (3 phases), 50% (multiphase), 20% (3 phases), and 10% (2 phases) of the R-R interval for patients with HR ≤ 60, 61–75, 76–100, 101–130, and >130 bpm, respectively.ConclusionsOptimal positioning and duration of CCTA acquisition intervals were investigated as a function of children’s HR, to reduce motion artifacts and patient’s irradiation.