Fluorescence studies on lipoamide dehydrogenases of pig heart. II. Microenvironments of tryptophan residues

The tryptophan residues of two forms of pig heart lipoamide dehydrogenase (LD(I) and LD(II] were investigated fluorometrically. The tryptophan contents of LD(I) and LD(II) determined by the fluorescence method were 3 mol and 2 mol per mol of FAD, respectively. These values were in good agreement with those found by the MCD method. The microenvironments of the tryptophan residues were investigated by fluorescence quenching titration with acrylamide. The tryptophan residues of both enzymes were in heterogeneous microenvironments, and CD spectra showed some differences between these microenvironments in the two enzymes. Energy transfer from tryptophan residues to bound FAD was equally efficient in the two enzymes. It seems probable that the three tryptophan residues in LD(I) are all in different microenvironments, but that two of them are in microenvironments almost identical to those of the corresponding residues in LD(II).     

Re-absorption of chlorophyll fluorescence in leaves revisited. A comparison of correction models.

The application of correction methods to account for re-absorption of chlorophyll fluorescence emission in leaves is subject to a number of controversies in the literature. These uncertainties lead to high discrepancies in the corrected spectral distribution of fluorescence and consequently in the interpretation of related physiological features of plants, according to the chosen method used in the process of correction. In this research, three correction methods, based on transmittance and/or reflectance measurements on leaves, were analysed comparatively. One method gave high values for the corrected fluorescence ratio between 685 nm and 737 nm (F685/F737 approximately 7 to 20 according to the different species of leaves). The two other methods were found to give similar results with corrected fluorescence ratios around a value of two (F685/F737 approximately 2). While the first method was developed in the light of empirical considerations, the latter two models are based upon defined physical approaches depicting interaction between light and matter. The theoretical basis of these methods, the validation methodologies used to support them and the similarity in the spectra corrected by light re-absorption for both models, all showed that they should be treated as confident and suitable approximations to solve the problem of light re-absorption in leaves.     

Characterization of cyclic GMP-binding sites of cyclic GMP-dependent protein kinase by rapid filtration assay.

The kinetics of cyclic [3H]GMP binding to the purified cyclic GMP-dependent protein kinase (cG kinase) were studied by using the rapid filtration assay method with polyethyleneimine-treated glass filters (method A), and the data were compared with those of the (NH4)2SO4 precipitation procedure (method B), which has been used for many previous studies on cyclic GMP binding to cG kinase. Each method gave a similar stoichiometry of approx. 2 mol of cyclic GMP/mol of cG kinase subunit; however, other binding kinetics obtained with these two methods were different. The dissociation of bound cyclic [3H]GMP from the kinase showed a single slow component when method A was used, whereas rapid and slow dissociation components were observed with method B. The Scatchard plot of cyclic [3H]GMP binding with method A was linear with a Kd value of 11 +/- 2 nM, suggesting that the two intrachain binding sites have similar high affinity for cyclic GMP. Results obtained on cyclic nucleotide analogue specificity of the two intrachain cyclic GMP-binding sites were also different between these two methods. These findings suggest that cG kinase has two high-affinity cyclic GMP-binding sites per subunit in the native state, and that when (NH4)2SO4 is added, ostensibly to stop the binding reaction, one low-affinity site is created from one high-affinity site.     

The pH jump study of enzyme proteins. I. Liquefying alpha-amylase from Bacillus subtilis.

Rapid conformational changes due to pH jump were studied kinetically at 25 degrees mainly by the stopped-flow method using liquefying alpha-amylase from Bacillus subtilis [EC 3.2.1-.1, liquefying]. First, the conformational change due to a pH jump produced by mixing with alkali was monitored as a function of time at 245 nm through the ionization of phenolic hydroxyl groups of tyrosine residues which were originally buried and finally become exposed due to the pH jump. Three distinct phases of conformational change were clearly recognized by this method by varying the final pH values. Each phase involved the exposure of an essentially definite number of tyrosine residues, whose rate constant was crucially dependent on pH. Second, these phases of conformational change were subjected to examination in terms of the optical rotation change at 411 nm and the reversibility upon reverse pH jump with respect to conformational reconstitution, as observed through the protonation ofphenolic hydroxyl groups of ionized tyrosine residues and the enzyme activity. The first phase, which occurs above pH 12.5, involves no change in the optical rotation and is reversible as observed by the above two monitoring methods. In contrast, the other two phases, which are observed above pH 12.7, are accompanied by an optical rotation change and no appreciable reversibility was detected by these methods.     

Rapid synthesis of oligodeoxyribonucleotides. II. Machine-aided solid-phase syntheses of two nonanucleotides and an octanucleotide.

Preparation of the two nonanucleotides, d(pG-G-A-G-G-A-G-A-A) and d(pT-T-C-T-G-T-T-G-A), and the octanucleotide, d(pT-G-G-C-C-C-A-T) is described by a rapid solid-phase method on a polyamide support. The syntheses were carried out using a modified solid-phase peptide synthesiser and required one day for each nucleotide addition. A microparticulate anion-exchanger, Partisil 10SAX, was used for the efficient fractionation of synthetic, base-protected oligonucleotides.     

DNA sequence analysis. Terminal sequences of bacteriophage phi80.

Sequences of the cohesive ends and the 3'-terminal regions of phi80 DNA have been determined. Sequences of the cohesive ends were obtained through the use of two standard methods. The first method involved the incorporation of all four labeled deoxyribonucleotides into the phi80 cohesive ends using DNA polymerase I. The DNA was then partially digested with micrococcal nuclease or pancreatic DNase. The products were separated by two-dimensional electrophoresis and characterized by composition, 3'-terminal, and nearest neighbor analyses. The second method involved partial incorporation using one, two, or three labeled deoxyribonucleotides followed by similar analyses. Sequences of the double-stranded regions adjacent to the cohesive ends were determined by three new methods. These methods were: (a) the DNA was specifically labeled at the 3' terminus and then partially degraded. Labeled oligonucleotide products were sequenced by their mobilities on various separation systems. (b) The cohesive ends were enlarged by limited degradation with exonuclease III. After this treatment, the DNA was partially repaired with labeled nucleotides, digested, and the products were analyzed. (c) A synthetic ologonucleotide primer was bound to phi80 DNA which had been repaired with DNA polymerase I, and then partially digested with lambda-exonuclease. The primer was extended into the region of interest by partial repair with labeled nucleotides. The extended primer was isolated and analyzed.     

Comparison of method 1623 and cell culture-PCR for detection of Cryptosporidium spp. in source waters

Analysis of Cryptosporidium occurrence in six watersheds by method 1623 and the integrated cell culture-PCR (CC-PCR) technique provided an opportunity to evaluate these two methods. The average recovery efficiencies were 58.5% for the CC-PCR technique and 72% for method 1623, but the values were not significantly different (P = 0.06). Cryptosporidium oocysts were detected in 60 of 593 samples (10.1%) by method 1623. Infectious oocysts were detected in 22 of 560 samples (3.9%) by the CC-PCR technique. There was 87% agreement between the total numbers of samples positive as determined by method 1623 and CC-PCR for four of the sites. The other two sites had 16.3 and 24% correspondence between the methods. Infectious oocysts were detected in all of the watersheds. Overall, approximately 37% of the Cryptosporidium oocysts detected by the immunofluorescence method were viable and infectious. DNA sequence analysis of the Cryptosporidium parvum isolates detected by CC-PCR showed the presence of both the bovine and human genotypes. More than 90% of the C. parvum isolates were identified as having the bovine or bovine-like genotype. The estimates of the concentrations of infectious Cryptosporidium and the resulting daily and annual risks of infection compared well for the two methods. The results suggest that most surface water systems would require, on average, a 3-log reduction in source water Cryptosporidium levels to meet potable water goals.     

Indices of oxidative stress. 2. Lipid peroxides

Two methods of the determination of lipid peroxidation products have been compared which are based on Fe(II) oxidation by them at acid pH values in the presence of xylenol orange which binds Fe(III) have been compared. The first method uses cumene hydropeoxide as an internal standard. In the second one, lipid peroxides are previously reduced by triphenylphosphine and these substances content is measured as a difference of the production of complexes with xylenol orange and iron ions in the control (with reduction) and experimental sample (without reduction). The optimization of measurement conditions is described. The levels of lipid peroxides in goldfish tissues assayed simultaneously by two methods were similar. The method with cumene hydroperoxide needs less amounts of biological material; moreover, there is no necessity in a calibration curve. Effects of hyperoxia on lipid peroxide levels in goldfish tissues were studied with the cumene method. Within the first hours of hyperoxia this index increased 13-times in the liver and 2-times in the brain and muscle. The further exposure rebounded this parameter to the initial level. Levels of lipid peroxides positively correlated with levels of end products of lipid peroxidation (thiobarbiturate acid reactive substances) in the goldfish tissues. The method of quantification of lipid peroxides with cumene is recommended for wide using in biological investigations.     

The two-wavelength method of microspectrophotometry. I. A microspectrophotometer and tests on model systems

In spite of a number of examples of the apparently successful application of the two-wavelength method of microspectrophotometry to biological materials, a demonstration of the effective transition from the theoretical to the practical aspects of the method has never been given. The following tests were carried out on model systems in order to confirm the practical validity of the method: (1) Spherical droplets of Sudan blue or Sudan IV (varying in volume by as much as 400 times) were measured with the surrounding blank field included and were found to have amounts of chromophore which conformed to the estimated droplet volume to within 3 to 6 per cent. (2) Repeated determinations on the same isolated nucleus gave results which did not vary in spite of wide variations in the area of the photometric field. (3) The results of repeated determinations on the same droplet of dye were unaffected by gross changes in the focus of the objective. The microspectrophotometer on which these measurements were made has been briefly described; it was designed specifically for the two-wavelength method. Some of the details of instrumentation have been discussed, in particular, the effects of non-uniform illumination and the contrasting demands of the two integral parts of this type of photometry. Although it has certain limitations, the two-wavelength method conforms to its theoretical promise and is a practical method of measuring light absorption in complex objects.     

Robust regression of enzyme kinetic data.

A method described previously [Cornish-Bowden & Endrenyi (1981) Biochem. J. 193, 1005-1008] for fitting theoretical equations to enzyme kinetic data without prior knowledge of weights or error distribution has been tested by computer simulation. With the equations for various kinds of linear inhibition as an example, the method performed well under all of the conditions examined, giving results that were often much better than those given by widely used least-squares alternatives, and were never appreciably worse. Although equations for two-substrate kinetics were not explicitly tested, the results for inhibition equations can be generalized to include two-substrate equations because the two are formally equivalent for simulation purposes. As a check on the results with inhibition equations the method was also tested for fitting bell-shaped pH-activity profiles and gave correspondingly good results.     

Theory of excitable membranes. I. A simple model for a three-state artificial membrane.

The effect of the thermal fluctuation on the orientation distribution pattern of globular protein molecules in a two-dimensional lattice was investigated by the method of computer simulation. A set of interaction parameters was assigned to interaction sites on each molecule and the interaction energy between two molecules was given by the product of the parameters of facing sites. Orientation fluctuation was assumed to take place with the probability proportional to the Boltzmann factor. Patterns having different degrees of order appeared with the change of temperature. The entropy and other thermodynamic quantities of these patterns were calculated, and gradual and transitional changes of the pattern were discussed in comparison with the case of simple atoms or molecules.     

Membrane solubility of chlorpromazine. Hygroscopic desorption and centrifugation methods yield comparable results.

Binding of the positively charged drug chlorpromazine to artificial and erythrocyte bilayer membranes was investigated by the filtration method called hygroscopic desorption [Conrad & Singer (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5202-5206] and by the conventional centrifugation method. Only minor differences in the partition coefficients were observed using the two methods. Our finding is not consistent with the observation of Conrad & Singer that amphipaths are completely excluded from biological membranes. However, the partition coefficient is dependent on membrane composition, which means dependent on the physical properties of a membrane.     

Extraction of Clostridium perfringens spores from bottom sediment samples.

Two extraction-separation procedures were developed and evaluated for use in conjunction with the mCP membrane filter method for the enumeration of Clostridium perfringens spores in bottom sediments. In the more facile of the two procedures, a distilled-water suspension of the sediment sample is pulse sonicated for 10 s and allowed to settle. Portions of the supernatant are then removed for membrane filtration. This procedure is recommended for general use. The more complicated procedure is recommended for situations in which the presence of high levels of toxic materials is suspected or in which relatively low spore densities are present in fine silts. In this procedure, sonication is followed by a distilled water wash. The centrifuged sediment is resuspended in distilled water and mixed with the components of a two-phase separation system (50% polyethylene glycol in distilled water and 25% sucrose in 3 M phosphate buffer [pH 7.1]). After equilibration of the system and low-speed centrifugation, the top phase and interphase are removed, mixed, and membrane filtered. The recoveries of C. perfringens spores by the two procedures, when used in conjunction with the mCP method, were comparable to each other and significantly greater than those by the British most-probable-number method. It was estimated that more than 85% of the spores were recovered by the procedures. The precision of the sonicate-and-settle-mCP procedure was markedly better than that obtained theoretically by the most-probable-number method and approached that theoretically attributable to counting an average of 85 colonies on each of two plates.     

Methods of measuring swimming speed of spermatozoa.

Three basic approaches for determining the mean swimming speed of a suspension of microorganisms were compared, using bull and ram spermatozoa. Number fluctuation counting was performed automatically on a Quantimet 720 image analysing computer, the mean speed being obtained using 'probability after' statistics. The other two approaches were photomicrographic: number flux counting was performed on single photomicrographs; on the same photomicrographs, the mean speed was estimated from measurement of 'whole' and 'half' track lengths. These results were compared with each other and with the Quantimet results. The 'probability after' method was also compared, on additional samples, with cine-photomicrographic tracking. The mean speeds predicted by the 'probability after' method compared favourably with the other methods (range 68 mum/sec to 162 mum/sec). The results also suggested that, on single photomicrographs, measurement of 'half' track lengths or number flux counting were generally preferable to measurement of whole track lengths.     

A comparison of two procedures used for complexing Fe(III) with human apotransferrin. II. Uptake of Fe(III) by K-562 cells from 55Fe . transferrins and Fe . [3H]transferrins

Samples of human apotransferrin (apo . HTr) were saturated with Fe(III) by two different techniques, a method employing excess trisodium citrate to chelate Fe(III) and a nonchelating approach which involves the ferroxidase activity of ceruloplasmin to convert Fe(II)----Fe(III). The samples were radiolabelled with either 55Fe or 3H. Using an initial molar Fe/apo . HTr ratio of 2.0-2.1, preparations of human transferrin with bound Fe (Fe . HTr) using the citrate method invariably contained 2.2-2.4 atoms Fe/molecule, whereas Fe . HTr (ceruloplasmin method) contained 2.0 atoms/molecule as shown by spectrophotometric and radioactivity measurements. Uptake of Fe from these Fe . HTr preparations by K-562 cells grown in a serum-free medium was marginally, but consistently, more rapid from 55Fe . HTr (citrate) than from 55Fe . HTr (ceruloplasmin). Taking account of the different Fe contents of the Fe . HTr preparations, the rate measured over a 2-h period amounted to approximately 12,700 and 16,100 Fe atoms/(cell . min) for Fe . HTr (ceruloplasmin) and Fe . HTr (citrate), respectively. However, cell binding by the two Fe . [3H]HTr preparations did not differ significantly over the 8-h incubation period. Furthermore, from the 3H distribution, the quantities of Fe . HTr bound reversibly at the cell surface and contained within the cell were similar for the two Fe . HTr preparations. The results indicate that apo . HTr may bind Fe in different ways depending on the method of Fe presentation and that the Fe . HTr product can donate Fe to K-562 cells at a rate which may reflect the method used for Fe-complex formation.     

Novel family shuffling methods for the in vitro evolution of enzymes.

It has recently been shown that shuffling of the amino acid sequences of family enzymes allows the generation of improved enzymes. Family shuffling is generally achieved by a DNase I treatment and then by PCR. Shuffling of the xylE and nahH genes, both encoding catechol 2,3-dioxygenases, was carried out by the published method. However, nahH-xylE hybrids were only formed at a very low frequency (less than 1%). Therefore, we developed improved methods for family shuffling by which DNA was cleaved by restriction enzymes instead of by DNase I. With the first improved method, five nahH fragments and five xylE fragments that had been generated by restriction enzyme digestion were subjected to the PCR reactions in two steps, the first being without a primer and the second with a set of primers. This method enabled nahH-xylE hybrid genes to be formed at a high frequency (almost 100%). With the second improved method, nahH and xylE were cleaved by several sets of restriction enzymes, and these digests were then reassembled in two steps. The nahH and xylE DNAs were each cleaved by two (or three) sets of restriction enzymes, and one type of nahH digest and one type of xylE digest were mixed, thus making four (or nine) different mixtures of the nahH and xylE digests. These mixtures were used as templates to carry out PCR without a primer. After the first PCR reaction, all the mixtures were combined, and a second PCR reaction was carried out without a primer. Following these two PCR assembly steps, a third PCR reaction was carried out with two primers to amplify the full-length nahH-xylE hybrid genes. This second method also yielded nahH-xylE hybrids at a frequency of 100%. The degree of recombination of the products with the second method was higher than that with the first method. These methods were used to isolate catechol 2,3-dioxygenases exhibiting relatively high stability at high temperature, one of them being respectively 13- and 26-fold more thermostable than XylE and NahH at 50 degrees C.     

Immunochemical study of the antigenic structure of bacteria of genus Bordetella. IV. Fractionation of B. pertussis extracts and study of the immunochemical and biological properties of the isolated fractions]

Fractionation of an extract of pertussis microbes was carried out with the aid of gel-filtration on Sephadex G-100, ion-exchange chromatography on DEAE-cellulose, and preparative electrophoresis. Fractions differing in serological, immunogenic activity and the content of antigenic components were isolated. In using the method of gel-filtration of sefadex G-100 the greatest serological, immunogenic and histamine-sensitizing activity was possessed by the high-molecular fraction containing 8 of 11 antigenic components detected in the initial extract. The antigenic components were distributed into 5 fractions by the ion exchange chromatography on DEAE-cellulose. The greatest serological activity was possessed by fractions exiting from the column at the 0.01--0.04M interval of the phosphate buffer concentration. A method of preparative electrophoresis from the pertussis microbes extract was applied and two fractions were isolated from the anode and the cathode zones, each containing 4 antigenic components only, but possessing serological and immunogenic activity and having no histamine-sensitizing properties.     

Lipid peroxidation and inguinal hernia repair. Tension-free vs. Andrews technique

AIM OF THE STUDY: To evaluate the early effect of inguinal hernia repair by the tension-free method compared to the conventional Andrew's technique on lipid peroxidation. PATIENTS-METHODS: Thirty-four patients subjected to elective hernia repair were enrolled in the study divided in two groups. Group A (n=18) underwent hernia repair by the tension-free method using a polypropylene mesh. Group B (n=16) underwent hernia repair by the Andrew's technique (i.e. a modification of the Bassini's technique). Venous blood samples were drawn preoperatively and at 12, 24 and 48 h postoperatively. Malondialdehyde (MDA) was estimated by the thiobarbiturate assay. RESULTS: Neutrophil counts were significantly higher in patients of group B compared to group A at 12 and 48 h postoperatively. Concentrations of fibrinogen were similar between the two groups. MDA was significantly higher in patients of group B hours compared to group A at 12, 24 and 48 h postoperatively. Positive correlation was found between neutrophil counts and MDA at 12 h (r: +0.43, P: 0.015) and 48 h (r: +0.496, P: 0.005) but not at 24 h. No correlation was found between serum fibrinogen and MDA. CONCLUSION: Hernia repair by the Andrews's technique elicits a sustained triggering of lipid peroxidation, compared to the tension-free method.     

Matrix representation of the Haseman-Elston method.

The Hasemann-Elston method of linkage detection is based on the probabilities of a sib pair having 0, 1, or 2 alleles identical by descent (IBD) at a marker and a trait locus. These probabilities form a 3x3 matrix. Here, the characteristic values and characteristic vectors of this matrix were used to clarify the structure of the equations and to simplify calculations. As examples, the regression coefficients were derived for three genetic systems: a trait and a marker, two epistatic traits and two markers, and one trait locus and two markers. The last model was studied under the assumption of no crossover interference, the expression for allele IBD sharing at a trait locus was derived as a function of allele IBD sharing at two marker loci, and the regression is shown to be non-linear.     

Isolation procedures for blood lymphocytes produce artifacts. Detection by changes of electrophoretic mobilities of lymphocytes.

The changes induced in the distribution of the electrophoretic mobility (EPM) of human peripheral blood lymphocytes (HPBL), by various methods used to prepare the lymphocyte suspensions and eliminate platelets from them, were investigated on blood samples collected from healthy individuals and thrombopenic patients. Data showed that the distribution of the lymphocyte EPMs, i.e., the "lymphocyte electrophoregram," was dependent on the method chosen to enrich the suspension in the cell type of interest. The relative percentages of the low and high mobility cells, the two main subpopulations defined by lymphocyte electrophoresis, were different. The most striking artifactual differences in the lymphocyte electrophoregram were induced by the method of elimination of platelets; the distribution was unimodal and asymmetric when thrombin was used and bimodal when the blood sample, or the lymphocyte suspension, was placed on ice for 30 min (as is the practice in some laboratories). The "split" of the lymphocyte electrophoregram was found to be reversible within 90 min. Similar changes were observed on lymphocyte suspensions and blood samples of thrombopenic patients when the step for the elimination of platelets was not involved.