Pressure cell assisted solution characterization of polysaccharides. 2. Locust bean gum and tara gum

Following the work carried out on guar gum in our first paper of a series, the "pressure cell" solubilization method was applied to two other less highly substituted galactomannans: locust bean gum (LBG) and tara gum. True molecular solution of the polymers was achieved using appropriate temperature, time, and pressure regimes. The technique of capillary viscometry was used to determine the intrinsic viscosity [eta] of the "pressure cell" treated and untreated samples. Molecular weight (M(w)) and radius of gyration (R(g)) were determined by light scattering. The data obtained for LBG and tara gum were compared statistically with reliable data found for guar gum in the literature. The variation in [eta] with M(w) followed the Mark-Houwink-Sakurada relationship, giving the exponent alpha = 0.74 +/- 0.01 for galactomannans consistent with random coil behavior. The characteristic ratio, C(infinity), and the chain persistence length, L(p), were both calculated for LBG and tara gum using the Burchard-Stockmayer-Fixman (BSF) method which is appropriate for flexible to semiflexible chains. A general value of 9 < C(infinity) < 16 and 3 < L(p) < 5 nm can now be estimated with statistical confidence for all galactomannans. According to our statistical analysis, the chain persistence length was found to be insensitive to the degree of galactose substitution.     

Pressure cell assisted solubilization of xyloglucans: tamarind seed polysaccharide and detarium gum

To improve the solubilization of two water-soluble xyloglucans, tamarind seed polysaccharide and detarium gum, by reducing substantially molecular aggregation, a "pressure cell" heating method was used. Conditions allowing solubilization and chain depolymerization were produced by varying appropriately the pressure, time, and temperature applied. The various MW fractions of solubilized xyloglucans were characterized by capillary viscometry and light scattering techniques in order to extract, with reliability, fundamental macromolecular parameters. Mark-Houwink and Flory exponents were found to be 0.67 +/- 0.04 and 0.51 +/- 0.06, respectively for both xyloglucan data combined, consistent with linear random coil behavior. A detailed analysis of the data seems to suggest that tamarind gum solutions are slightly perturbed by the effect of excluded volume, whereas detarium gum samples are close to the theta state. Chain flexibility parameters such characteristic ratio, C( proportional, variant ), and persistence length, L(p), were calculated for tamarind and detarium using the Burchard-Stockmayer-Fixman (BSF) geometric method. L(p) values of 6-8 nm were estimated for xyloglucans. The seemingly linear structure of tamarind and detarium, as suggested by the value of the Mark-Houwink and Flory exponents obtained, follows from analysis of the data by the classical Zimm method but not when employing the square root or Berry method which suggests a more branched chain profile. This was the approach adopted in our previous work on the characterization of detarium samples.     

Detection of hepatitis A virus in sewage sludge by antigen capture polymerase chain reaction.

Antigen capture polymerase chain reaction (PCR) was tested as a sensitive and rapid method for detecting hepatitis A virus (HAV) in raw sewage sludge. The antigen capture PCR was performed both with and without solid-phase virus-catching monoclonal antibodies. Similar results proved that both methods were equally sensitive. Sewage sludge samples from different regions in Germany were examined for evidence of HAV contamination by antigen capture PCR. This method of detection was compared with that used in a previous study of these sewage sludge samples, in which the HAV was detected through indirect immunofluorescence after cell culture inoculation. The results obtained by antigen capture PCR matched those obtained in the earlier cell culture investigations, when HAV was detected in raw as well as digested sewage sludge samples. The advantage of the PCR method, however, lies in the fact that it needs only two days while the cell culture propagation of HAV takes about 8 to 10 weeks.     

Comparison of proteases in DNA extraction via quantitative polymerase chain reaction

We compared four proteases in the QIAamp DNA Investigator Kit (Qiagen) to extract DNA for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate alternate proteases for improved DNA recovery as compared with proteinase K for forensic, biochemical research, genetic paternity and immigration, and molecular diagnostic purposes. The Quantifiler Kit TaqMan quantitative PCR assay was used to measure the recovery of DNA from human blood, semen, buccal cells, breastmilk, and earwax in addition to low-template samples, including diluted samples, computer keyboard swabs, chewing gum, and cigarette butts. All methods yielded amplifiable DNA from all samples.     

A theoretical model for lipid monolayer phase transitions.

We present a theoretical model for the liquid-expanded to liquid-condensed phase transition observed in many phospholipid monolayer films. The total two-dimensional pressure in the model is the sum of the hydrocarbon chain pressure and the surface pressure. The hydrocarbon chain pressure is calculated in an exteded version of a model published earlier. The surface pressure results from a lowering of the surface tension in the monolayer over that of pure water, thus producing a force on a Langmuir float. When these two contributions are added, pi/A isotherms are obtained which have slope discontinuities very similar to those observed experimentally. The results indicate that a successful model for lipid phase behavior must consider the interactions between head groups and water as well as cooperative hydrocarbon chain melting.     

Solution conformation of virginiamycin S. II. The conformation of allohydroxy- and deoxyvirginiamycin S.

The experimental results of 1H- and 13C-NMR studies of allohydroxy-, and of deoxyvirginiamycin S strongly confirm the conformation that was proposed earlier for the parent virginiamycin S (Anteunis, Callens and Tavernier (1975) Eur. J. Biochem. 58, 259--268). The changing nature of dipole-induced dipole interaction is responsible for the specific gradually increasing libration of the N-MePhe side chain along the series virginiamycin S, allohydroxy-, deoxyvirginiamycin S. Previous methods for the estimation of rotameric populations around the alpha, beta bonds are critically evaluated and compared to the present results obtained from interpretation of geminal 2J (beta) coupling constants.     

Analysis of phospholipid molecular species by liquid chromatography--atmospheric pressure chemical ionisation mass spectrometry of diacylglycerol nicotinates.

A method using liquid chromatography - atmospheric pressure chemical ionisation mass spectrometry was evaluated for determining the molecular species composition of phospholipids (phosphatidylcholines from soybean, egg yolk and bovine liver) after conversion to diacylglycerol nicotinate derivatives. The structures could be deduced from pseudo-molecular ions ([MH-123](+)) and three pairs of monoacyl containing fragment ions. All molecular species in mixed peaks were readily identified and many minor components, earlier not encountered in the samples under investigation, were identified. Acyl chain regioisomers were readily distinguished by the ratio of the [MH-RCHCO](+) ions. Molecular species differing only in the position of the double bonds in one polyunsaturated acyl chain were separated on the basis of retention times. A half quantitative estimation of the molecular species composition of complex samples was achieved by a combination of UV detection and, for mixed peaks, the areas of [MH-123](+) ions.     

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 theoretical model for lipid monolayer phase transitions

We present a theoretical model for the liquid-expanded to liquid-condensed phase transition observed in many phospholipid monolayer films. The total two-dimensional pressure in the model is the sum of the hydrocarbon chain pressure and the surface pressure. The hydrocarbon chain pressure is calculated in an extended version of a model published earlier. The surface pressure results from a lowering of the surface tension in the monolayer over that of pure water, thus producing a force on a Langmuir float. When these two contributions are added, $\text{π/A}$ isotherms are obtained which have slope discontinuities very similar to those observed experimentally. These results indicate that a successful model for lipid phase behavior must consider the interactions between head groups and water as well as cooperative hydrocarbon chain melting.     

Comparison of different PCR methods for detection of Brucella spp. in human blood samples

For detection of Brucella species by PCR four DNA extraction methods and four targets were compared using pure culture of Brucella melitensis and the best conditions were applied in clinical samples. It was found that the MagNA Pure LC method was the most efficient and sensitive method showing a positive PCR reaction with DNA extracted from as low as 25 and 100 CFU suspended in one ml blood and one ml water, respectively. Detection of Brucella spp. by conventional PCR was investigated using four different targets. The results indicated that The B4-B5 amplification method was the most sensitive one as it could amplify DNA extracted from as a low as 25 and 100 CFU/ml suspended in one ml water and blood, respectively. Furthermore real-time PCR was able to detect Brucella using DNA extracted from as low as 50 CFU/ml blood and 15 CFU/ml water, respectively. The best and optimum detection conditions were applied to the clinical samples. Evaluation of conventional PCR assays on blood specimens confirmed 72% of the results obtained by conventional blood culture methods with a specificity of 95%, while serum samples had a sensitivity of 54% and specificity of 100%. Real-time PCR was generally found to be more sensitive and specific for detecting Brucella spp. in blood and serum samples compared to conventional PCR. The real-time PCR done on blood specimens confirmed 77.5% of the results obtained by conventional blood culture methods with specificity of 100%, while 60% of serum samples were found to be positive with specificity of 100%. These results suggest that serum and blood analysis by conventional and real time PCR is a convenient and safe method for rapid and accurate diagnosis of brucellosis.     

Solution properties of conventional gum arabic and a matured gum arabic (Acacia (sen) SUPER GUM)

Dilute solution properties of two specially matured gum arabic samples (EM1 and EM2) were compared to the conventional gum (EM0) using static light scattering. The apparent molar mass (M(w,app) and radius of gyration (R(g,app)) for the three samples showed unusual concentration dependence. These data were satisfactorily interpreted by a simple association model that takes into account the repulsive interaction among clusters, which allowed us to obtain the true molar mass (Mw(0)) and radius of gyration (Rg(0)). A common power law relation was observed between Mw(0) and Rg(0) , giving a somewhat higher exponent than expected for linear and branched polymers in a good solvent. Mw(0) and Rg(0) obtained for the three gums do not differ significantly from each other. However, the data showed clearly a constant increase of the association from EM0 to EM2 with increasing concentration. This is in accordance with the previously observed improved functional properties for the matured products.     

Evaluation of an automated fluorescent antibody procedure for detection of Salmonella in foods and feeds.

A prototype automated system using fluorescent antibody (FA) was evaluated for rapid detection of salmonellae in foods. Samples were enriched in selenite cystine and tetrathionate broths. After incubation, both were transferred into fresh selenite cystine for a 4-h "post-enrichment" to dilute possible background fluorescence from product. These cultures were then analyzed automatically, and results were compared with those obtained by the methods of the Association of Official Analytical Chemists (AOAC). Initially, 167 samples of milk powder, dried yeast, and imported frog legs were examined. The AOAC and automated FA methods correlated well with all samples but frog legs. Difficulty with the latter was caused by procedural and mechanical problems coupled with high numbers of competing microorganisms in post-enrichment cultures. Modification of procedure and partial redesign of equipment corrected these difficulties, and excellent correlation was obtained with another 116 frog leg samples. All 89 AOAC-confirmed positives were also detected by the automated FA method, and there were only 4% false FA positives. The system shows potential for screening products for salmonellae; however, all positives should be confirmed by manual biochemical and serological methods.     

Molecular characterisation of Sargassum polycystum and S. siliquosum (Phaeophyta) by polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD) primers

Genomic DNA was extracted from 13 samples of Sargassum polycystum and S. siliquosum collected from various localities around Peninsular Malaysia and Singapore by using four different extraction methods. The yields and the suitability of the DNA to be used as template for the polymerase chain reaction (PCR) was compared. DNA samples were subjected to PCR analysis by using random primers. Only DNA samples that were extracted using the CTAB method were successfully amplified by random amplified polymorphic DNA (RAPD)-PCR. Five of 31 random primers (OPA02, OPA03, OPA04, OPA13 and OPM10) tested amplified sequences of DNA from the DNA samples. Reproducible, amplified products were obtained using these primers and showed some potential to be useful in discriminating individual samples within the genus, in determining relationships between species within a genus and in developing individual fingerprints for individual samples.     

Robust Discriminant Functions in Assisting Medical Diagnosis. Application to the Chronic Obturative Lung Disease Data

The results of assisting medical diagnosis in bronchial asthma and chronic bronchitis via robust discriminant functions are presented in the paper. The robust discriminant functions are obtained by replacing classical estimates of mean vectors and covariance matrices by their robust equivalents. The new methods were compared with classical ones. These methods resulted in the improvement of automatic diagnosis especially when the “new” data set was classified on the basis of individuals collected earlier.     

Diagnosis of urogenital Chlamydia trachomatis infection in women based on mailed samples obtained at home: multipractice comparative study.

OBJECTIVE: To compare urine and vaginal flush samples collected by women at home with endocervical and urethral swabs obtained by general practitioners for their efficacy in the diagnosis of urogenital Chlamydia trachomatis infection. DESIGN: Multipractice comparative study. SETTING: 33 general practices and a central department of clinical microbiology in Aarhus County, Denmark. SUBJECTS: 222 women aged 18-25 years who for any reason had a gynaecological examination. INTERVENTIONS: Endocervical and urethral swabs were obtained by the women''s general practitioners. The same women when at home then collected a first void urine sample, a midstream urine sample, and a vaginal flush sample (using a vaginal pipette) and mailed them to the laboratory. MAIN OUTCOME MEASURES: C trachomatis defected by the polymerase chain reaction and the ligase chain reaction. Eight tests for C trachomatis were performed for every woman. When two of the eight yielded positive results the patient was considered infected. RESULTS: The overall prevalence of C trachomatis infection was 11.2% (23/205 women). Test sensitivities in samples obtained by general practitioners, samples obtained at home subjected to polymerase chain reaction, and samples obtained at home subjected to ligase chain reaction were 91%, 96%, and 100% respectively. The corresponding specificities were 100%, 92.9%, and 99.5%. CONCLUSIONS: The diagnostic efficacy of samples obtained by women at home and mailed to the laboratory was as good as for samples obtained by a general practitioner when using the ligase chain reaction. This may have important implications for the practicability of screening for this common, often asymptomatic, and treatable infection.     

Establishment and characterization of <Emphasis Type="Italic">Prosopis laevigata</Emphasis> (Humb. &; Bonpl. ex Willd) M.C. Johnst. cell suspension culture: a biotechnology approach for mesquite gum production

This study presents a protocol for the establishment of Prosopis laevigata cell suspension culture as a strategy to obtain an in vitro mesquite gum productive cell line. The callus used for this purpose was obtained with hypocotyls from 15-day-old plantlets, placed on Murashige–Skoog medium with two different plant growth regulators (PGRs), 2,4,5-trichlorophenoxy acetic acid (2,4,5-T; 5.0 μM) and kinetin (KIN; 5.0 μM). With this PGRs treatment, after four subcultures (30 days each) an exuded gum-like substance was observed on the callus surface. The growth kinetics of the cell suspension culture showed a specific cell growth rate (μ) of 0.14 d⁻¹ and doubling time (t _d) of 6.6 days, respectively. The gum-like substance from callus culture and the broth from cell suspension culture were subjected to chemical analysis and compared with the mesquite gum exuded from wild trees. Both, gum-like substance from callus culture and the broth from cell suspension culture showed the presence of Arabinogalactan-proteins, and their polysaccharide fraction presented the same monosaccharides as those isolated from mesquite gum. In addition, the emulsifying properties of gum-like substance from callus culture and the broth from cell suspension culture were compared to those of mesquite gum and all three samples exhibited similar emulsifying capacity and emulsification stability.     

Comparison between modified DNA extraction protocols and commercial isolation kits in grapevine (Vitis vinifera L.)

Various protocols have been developed and used for DNA extraction in grapevine. However, owing to the long duration of the isolation steps in previously developed protocols, researchers have preferred to use isolation kits for studies in recent years. In our study, the DNA yield and purity obtained using six methods - namely three DNA isolation protocols and three commercial DNA isolation kits - were compared. Modifications were made and the isolation steps were shortened in the previously developed DNA isolation protocols to achieve more rapid and practical protocols. The samples were taken from plants grown under vineyard and greenhouse conditions in two periods during spring and autumn. The best results among the six DNA isolation methods were discussed. The results were also supported with polymerase chain reaction analyses conducted with isolated DNAs.     

Orientation of silk III at the air-water interface.

A threefold helical crystal structure of Bombyx mori silk fibroin has been observed in films prepared from aqueous silk fibroin solutions using the Langmuir Blodgett (LB) technique. The films were studied using a combination of transmission electron microscopy and electron diffraction techniques. Films prepared at a surface pressure of 16.7 mN/m have a uniaxially oriented crystalline texture, with the helical axis oriented perpendicular to the plane of the LB film. Films obtained from the air-water interface without compression have a different orientation, with the helical axes lying roughly in the plane of the film. In both cases the d-spacings observed in electron diffraction are the same and match a threefold helical model crystal structure, silk III, described in previous publications. Differences in the relative intensities of the observed reflections in both types of oriented samples, as compared to unoriented samples, allows estimations of orientation distributions and the calculations of orientation parameters. The orientation of the fibroin chain axis in the plane of the interfacial film for uncompressed samples is consistent with the amphiphilic behavior previously postulated to drive the formation of the threefold helical silk III conformation.     

Direct comparison of four bacterial source tracking methods and use of composite data sets

AIMS: Four bacterial source tracking (BST) methods, enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR), automated ribotyping using HindIII, Kirby-Bauer antibiotic resistance analysis (KB-ARA) and pulsed-field gel electrophoresis (PFGE) were directly compared using the same collection of Escherichia coli isolates. The data sets from each BST method and from composite methods were compared for library accuracy and their ability to identify water isolates. METHODS AND RESULTS: Potential sources of faecal pollution were identified by watershed sanitary surveys. Domestic sewage and faecal samples from pets, cattle, avian livestock, other nonavian livestock, avian wildlife and nonavian wildlife sources were collected for isolation of E. coli. A total of 2275 E. coli isolates from 813 source samples were screened using ERIC-PCR to exclude clones and to maximize library diversity, resulting in 883 isolates from 745 samples selected for the library. The selected isolates were further analysed using automated ribotyping with HindIII, KB-ARA and PFGE. A total of 555 E. coli isolates obtained from 412 water samples were analysed by the four BST methods. A composite data set of the four BST methods gave the highest rates of correct classification (RCCs) with the fewest unidentified isolates than any single method alone. RCCs for the four-method composite data set and a seven-way split of source classes ranged from 22% for avian livestock to 83% for domestic sewage. Two-method composite data sets were also found to be better than individual methods, having RCCs similar to the four-method composite and identification of the same major sources of faecal pollution. CONCLUSIONS: The use of BST composite data sets may be more beneficial than the use of single methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first comprehensive comparisons using composite data from several BST methods. While the four-method approach provided the most desirable BST results, the use of two-method composite data sets may yield comparable BST results while providing for cost, labour and time savings.