Heat resistance of Campylobacter and Yersinia strains by three methods

Two methods of determining the heat resistance of bacteria, a glass cup and a test tube method, were compared with a method using capillary tubes. Three strains of Yersinia enterocolitica, one of Campylobacter jejuni and two of C. coli were tested in physiological saline. The differences between the results obtained by the glass cup method and the reference method were not statistically significant for five strains and were small also for the other, a Yersinia strain. D values obtained by the glass cup method at 58, 60 and 62 degrees C were 1.4-1.8, 0.40-0.51 and 0.15-0.19 min (zeta values 4.00-4.52 degrees C) for the Yersinia strains, and 0.42, 0.13 and 0.07 min (zeta value 5.07 degrees C) for one C. coli strain. For the other Campylobacter strains, D values of 0.71-0.78, 0.24-0.28 and 0.12-0.14 min (zeta values 4.94 and 5.60 degrees C) were recorded at 56, 58 and 60 degrees C. D values obtained at 60 degrees C by the test tube method were 2.7-5.0 min and were considered to be unrealistic.     

Comparison of methods for estimating percent body fat in distance-runners]

A comparative study was conducted between two independent methods to estimate body fat in 13 college male distance-runners (20.2 +/- 1.1 yrs) and 11 male college students (19.6 +/- 0.7 yrs) as control group. The methods dealt with different body component parameters. Body fat was estimated (1) in terms of total body water based on the analysis of dilution of orally ingested deuterium oxide (D2O) in urine, and (2) in terms of body density based on underwater weighing. The results were as follows: 1) The skinfold thickness at 14 sites in distance-runners were thinner than those in control group. The mean values for subcutaneous fat in distance-runners were 4.3 +/- 0.7 kg (7.2 +/- 1.1%), which were lower than those (8.3 +/- 2.7 kg & 13.3 +/- 3.4%) in control group significantly. Oh the other hand, the mean values for internal fat in distance-runners were 8.7 +/- 1.4 kg (14.4 +/- 1.6%), which were larger than those (6.5 +/- 3.1 kg & 10.2 +/- 4.3%) in control group. 2) The mean values for percent body fat in control group were 16.1 +/- 1.6% by skinfolds method, 20.2 +/- 5.1% by body density method and 23.5 +/- 4.6% by total body water method. The estimated values of percent body fat by the methods of body density and total body water were approximately the same. 3) The mean values for percent body fat in distance-runners were 11.9 +/- 1.4% by skinfolds method, 11.8 +/- 1.7% by body density method and 21.5 +/- 1.9% by total body water method. The estimated values of percent body fat in distance-runners were lower than those in control group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Picric acid methods greatly overestimate serum creatinine in mice: more accurate results with high-performance liquid chromatography

The creatinine levels of blood and urine from humans, rats, and mice were measured by high-performance liquid chromatography. These were compared to the alkaline picrate analysis of creatinine performed by standard colorimetric, kinetic, and AutoAnalyzer techniques. For human serum and urine the values obtained using the HPLC technique gave good agreement with four out of five alkaline picrate techniques. For black or white mice, the serum creatinine concentration was 8.7 +/- 0.4 microM by HPLC but 44.9 +/- 1.9 microM by the lowest alkaline picrate method. Mouse urine creatinine concentrations were 3.24 +/- 0.19 mM by HPLC and 4.59 +/- 0.39 mM by the nearest alkaline picrate method. Rat serum creatinine concentrations analyzed by HPLC were about half the values obtained by AutoAnalyzer. Mouse and rat samples seemed to have substances which gave nonspecific color and thus interfered with the analysis of creatinine by the alkaline picrate methods. While the alkaline picrate analysis of creatinine was adequate for human samples, it was necessary to use HPLC to accurately measure rodent creatinine. The fractional excretion of creatinine was determined by measuring creatinine in mouse urine and plasma by both the kinetic and HPLC methods and comparing these values to urine and plasma inulin. Using the kinetic method, creatinine was cleared at 43 +/- 3% of the rate of inulin. Using the HPLC method, creatinine was cleared at 170 +/- 11% of the rate of inulin.     

The prevention of seed-borne diseases of flax III. The dusting, short wet and fixation methods of seed disinfection in relation to storage of the seed

Flax seed containing up to 10% of moisture was disinfected with a proprietary dry fungicide containing tetramethylthiuram disulphide (Nomersan) at the rate of 12 oz./cwt. of seed, and stored for periods of up to 18 months, without its germination being impaired.
Experiments were made with samples of seed the moisture content of which, before treatment, varied within the range of 5-8-13-2% and which were kept in a commercial store after disinfection. Treatments were carried out with an 8% solution of a soluble organo-mercurial (Ceresan U. 564) at the rates of 0-67 and 0-9 gal./cwt. applied by the short wet method, and with an organo-mercurial powder (Ceresan UT. 1875 A) at the rate of 12 oz./cwt. applied by the fixation method using 0-9 gal. of separated milk per cwt. The results obtained show that treatment by either method has the effect of lowering the percentage of viable seeds during subsequent storage and that the higher the moisture content of the seed before treatment the earlier this effect becomes apparent. It is suggested that seed to be treated by the short wet method on a commercial scale should be dried to contain about 5% of moisture, that not more than 0-67 gal./cwt. of liquid be applied and that the seed should not be stored for longer than 3 months after treatment. Following upon these suggestions, 10-ton lots of seed were treated commercially by the short wet method using a Kontramix machine and no ill effects on the crop were observed.     

Neoglycoconjugates of mannan with bovine serum albumin and their interaction with lectin concanavalin A.

Neoglycoconjugates were prepared from mannan isolated from yeast Saccharomyces cerevisiae and activated by periodate oxidation to create aldehyde groups. Various degrees of oxidation introduced 11-28 aldehyde groups per mannan molecule and simultaneously resulted in a molar mass decrease from 46 to 44.5-31 kDa. The activated mannans were subsequently conjugated with bovine serum albumin forming neoglycoconjugates. Some parameters of these mannan-bovine serum albumin conjugates were characterized: saccharide content 25-30% w/w, molar mass within the range 169-246 kDa, and polydispersion (M(w)/M(n)) from 2.8 to 3.6. The interaction of these conjugates with lectin concanavalin A was studied using three different methods: (i) quantitative precipitation in solution; (ii) sorption to concanavalin A immobilized on bead cellulose; and (iii) kinetic measurement of the interaction by surface plasmon resonance. Quantitative precipitation assay showed only negligible differences in the precipitation course of original mannan and the corresponding mannan-bovine serum albumin conjugates. Both the sorption method (equilibrium method) and the surface plasmon resonance measurement (kinetic method) demonstrates that the values of dissociation constant K(D) of all synthetic neoglycoconjugates were within the range 10(-7) - 10(-8) mol x L(-1) (close to K(D) = 10(-8) mol x L(-1) determined by the sorption method for the original mannan). In conclusion, characterization of synthetic neoglycoconjugates confirmed that the method used for their preparation retained the ability of mannan moiety to interact with concanavalin A.     

A comparison of three methods of recovery of goat oocytes for in vitro maturation and feritilization

Experiments were conducted to determine an efficient method for recovering a large number of usable oocytes and its effect on subsequent in vitro maturation, fertilization and development. Follicular oocytes were recovered from goat ovaries using 3 different methods: aspiration, puncturing and slicing. The average total number of oocytes recovered per ovary was significantly higher by the aspiration method (2.7+/-0.15) than by puncturing (2.2+/-0.13) or by slicing (2.4+/-0.12). However, significantly more good-quality usable oocytes enclosed with compact cumulus cells were obtained by slicing (0.9+/-0.06) than by aspiration (0.5+/-0.07) or by puncturing (0.5+0.06). Time required for processing each ovary by the slicing method was comparatively less (0.90 min) than that required for puncturing (1.83 min) or for aspiration (1.78 min). Usable oocytes recovered by all three methods were matured, fertilized and developed to the blastocyst stage in vitro. There were no significant differences in the subsequent percentages of oocytes maturing, being fertilized and developing in vitro among the 3 methods of recovering oocytes. In conclusion, the recovery of goat oocytes by the slicing method is simple and efficient compared with the aspiration and puncturing methods.     

Fluorometric assay of 25-hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 in plasma.

The first practical fluorometric assay of plasma 25-hydroxyvitamin D3 (25-OH-D3) and 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3) is described. The method uses a highly fluorescent dienophile, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1, 2,4- triazoline-3,5-dione (DMEQ-TAD), to fluorescence-label vitamin D. Vitamin D metabolites were roughly purified with a short cartridge column followed by HPLC, labeled with DMEQ-TAD, and the product was analyzed on HPLC. In the assay of 25-OH-D3 the new fluorometric method was compared with the HPLC-uv method and was confirmed to be as accurate and reliable (CV, 4-5%) as the HPLC-uv method. Plasma 24,25-(OH)2D3 was accurately assayed by the HPLC-FL method, where the standard addition method was successfully used to calculate the overall recovery.     

Synthesis of enantiopure phthalides including 3-butylphthalide, a fragrance component of celery oil, and determination of their absolute configurations

Enantiopure phthalides 2 and 5-8 were synthesized via enantioresolution of the corresponding alcohols with a chiral auxiliary of camphorsultam dichlorophthalic acid, (1S,2R,4R)-(-)-CSDP acid 3, followed by solvolysis with KOH in MeOH and the catalytic oxidation of chiral glycols with iridium complex 28. The absolute configurations of phthalides 2 and 5-8 were determined by applying the (1)H-NMR anisotropy method of MalphaNP acid (4), 2-methoxy-2-(1-naphthyl)propionic acid, to the chiral synthetic precursory alcohols. In the case of 3-phenylphthalide (R)-(-)-7, the absolute configuration determined by the (1)H-NMR anisotropy method using MalphaNP acid 4 agreed with that by the X-ray crystallographic method. By applying these methods, 3-butylphthalide (S)-(-)-2, a fragrance component of essential oil of celery, has been synthesized in an enantiopure form, and its absolute configuration was unambiguously determined.     

Simultaneous measurement of retinol, alpha-tocopherol and six carotenoids in human plasma by using an isocratic reversed-phase HPLC method

A simple and sensitive isocratic reversed-phase high-performance liquid chromatography (HPLC) method for simultaneous determination of retinol, alpha-tocopherol and six carotenoids in human plasma was described. Sample preparation of the earlier published method was further developed by addition of ultrapure water, which enabled aqueous layer to freeze facilitating phase separation without pipetting thus also improving precision of the method. Developed method appeared to be less laborious and time consuming compared to the traditional extraction methods, which require removal of organic layer by pipetting. The recoveries (absolute and relative) were between 80% and 103%. The intra-assay CVs were 1.1-4.0% (normal level) and 3.3-9.0% (low level). Inter-assay CVs were 5.3-8.8%. Reference method for all these analytes was not available, but a comparison with another published method was carried out. The results of the comparison matched satisfactorily. The method is used routinely in our laboratory in a large population-based study.     

Preparation of alkyl (R)-(-)-3-hydroxybutyrate by acidic alcoholysis of poly-(R)-(-)-3-hydroxybutyrate

An efficient method for the preparation of optically active alkyl (R)-(-)-3-hydroxybutyrates by chemical depolymerization of biopolymer, poly-(R)-(-)-(3-hydroxybutyrate), was established. This method consists of simple recovery of poly-(R)-(-)-(3-hydroxybutyrate) from bacterial cells followed by acidic alcoholysis. When poly-(R)-(-)-(3-hydroxybutyrate) was purified by a simple digestion method that used 0.2 N sodium hydroxide, alkyl (R)-(-)-hydroxybutyrates were most efficiently produced by alcoholysis with anhydrous hydrochloric acid.     

Simultaneous determination of paracetamol, caffeine and propyphenazone in ternary mixtures by micellar electrokinetic capillary chromatography

A new micellar electrokinetic capillary chromatographic method has been developed to analyze the pharmaceutical preparations containing ternary combination of paracetamol (PAR), caffeine (CAF) and propyphenazone (PRO). Best results were obtained by using 20mM pH 9.0 borate buffer containing 30mM sodiumdodecylsulphate as the background electrolyte. Diflunisal (DIF) was used as internal standard (IS). The separation was performed through a fused silica capillary (50microm internal diameter, 44cm total length, 35.5cm effective length) at 25 degrees C with the application of 3s of hydrodynamic injection at 50mbar pressure and a potential of 29kV. Detection wavelength was 200nm. Under these conditions, the migration times were found to be 5.174min for PAR, 5.513min for CAF, 7.195min for DIF, and 9.366min for PRO. Linearity ranges for the method were determined as 2-200microgmL(-1) for PAR and CAF and 3-200microgmL(-1) for PRO. Limit of detections were found as 0.6microgmL(-1) for PAR and CAF and 0.8microgmL(-1) for PRO. According to the validation study, the developed method was proved to be accurate, precise, sensitive, specific, rugged and robust. Three pharmaceutical preparations, which are produced by different drug companies in Turkey, were analyzed by the developed method. One of the same preparations was also analyzed by the derivative ratio spectro zero-crossing spectrophotometric method reported in literature. No significant differences were found statistically.     

Various methods to assay carbenicillin activity against Pseudomonas aeruginosa]

It was shown that the cultures of Pseudomonas aeruginosa were sensitive to 75-100 micrograms/ml and resistant to 25-50 micrograms/ml of carbenicillin when tested by the method of serial dilutions in the synthetic medium developed by the authors and in Jorgensen's medium. The cultures were incubated for 4 and 16 hours, respectively. The data did not confirm the resistance of the cultures to 25-75 and 100 micrograms/ml carbenicillin when it was determined with the serial dilution method in Pal's medium and the disk diffusion method, respectively. The advantages of the medium developed by the authors for testing P. aeruginosa sensitivity to carbenicillin are discussed.     

Models for the duo-trio and triangular methods

Duo-trio and triangular method models by Ura (1960, Japanese Union of Scientists and Engineers 7, 107-119) and David and Trivedi (unpublished Technical Report #55, Department of Statistics, Virginia Polytechnic Institute, 1962) for examining perceptual processes have proven to be very useful, but are limited to univariate phenomena. Recent Monte Carlo studies by Ennis and Mullen (1985, Chemical Senses 10, 605-608; 1986, Journal of Mathematical Psychology 30, 206-219), on the probabilities of correct decisions for multivariate responses, showed how they depend on discriminal distance, variance-covariance structure, and orientation in n-space. Mathematical models for the triangular and duo-trio method in the bivariate case were developed by Mullen and Ennis (1987, Psychometrika 52, 235-249). Formulation of the n-dimensional triangular method model was accomplished and problems in numerical integration were resolved by Kapenga et al. (1987, in Numerical Integration, P. Keast and G. Fairweather (eds), 321-328; Dordrecht: Reidel). The n-dimensional duo-trio method model is given in this paper and previous work on the triangular method is reviewed briefly.     

Isolation of circulating human monocytes with high purity for proteomic analysis

We describe a simple method for isolation of human blood monocytes with the high purity (95-98%) required for proteomic analysis, which avoids contamination by other blood cells (platelets and lymphocytes) and the most abundant plasma proteins (albumin and immunoglobulins). Blood monocytes were purified by gradient centrifugation followed by positive selection with specific monoclonal antibodies coupled to paramagnetic beads. The elution conditions of the positive selection step were modified to avoid contamination with albumin. This method is compatible with flow cytometry which was used to assess the purity of the cell population. From 28 mL of blood, 10(7) monocytes with > 96% purity are routinely obtained. From the isolated monocytes 200-250 microg of protein could be recovered. The whole method can be performed in three hours. Similar results were obtained using a negative selection step but with lower purity (92%), increased cost and longer time. After solubilization of monocytes, the proteins were analyzed by two-dimensional gel electrophoresis (2-DE) in the 3-10, 4-7, 6-9 and 6-11 pH range. DNA was the main contaminant that interfered with the 2-DE and it was removed by treatment with DNAse. Image analysis of gels allowed the reproducible detection and quantification of 1500 spots in the 4-7 pH range and more than 2000 spots in total by combining (overlapping) 2-D gels in the 4-7, 6-9 and 6-11 pH range. This method is useful for clinical studies of monocytes from a large number of patients due to its rapidity and reproducibility, which permits comparative analysis of normal versus pathological samples and which allows follow up of the expressed proteins of monocytes from each patient.     

Determination of bevantolol enantiomers in human plasma by coupled achiral-chiral high performance-liquid chromatography

A coupled achiral-chiral high performance liquid chromatographic method was developed and fully validated for the determination of bevantolol enantiomers, (-)-(S)-bevantolol and (+)-(R)-bevantolol, in human plasma. Plasma samples were prepared by solid phase extraction with Sep-Pak Plus C18 cartridges followed by HPLC. Bevantolol enantiomers and (+)-(R)-Propranolol as internal standard (IS) were preseparated from interfering components in plasma on a Phenomenex silica column and bevantolol enantiomers and IS were resolved and determined on a Chiralcel OJ-H chiral stationary phase. The two columns were connected by a switching valve equipped with silica precolumn. The Precolumn was used to concentrate bevantolol in the eluent from the achiral column before back flushing onto chiral phase. A detailed validation of the method was performed accordingly to FDA guidelines. For each enantiomer the assay was linear between 20 and 1600 ng/ml. The quantification limits of both bevantolol enantiomers were 20 ng/ml. The intraday variation was between 1.07 and 12.64% in relation to the measured concentration and the interday variation was 0.91 and 11.79%. The method has been applied to the determination of (-)-(S)- and (+)-(R)-bevantolol in plasma from healthy volunteers dosed with racemic bevantolol hydrochloride.     

Occurrence of induced vitamin-dependent mutants of Yersinia pestis

Vitamin-dependent mutants were isolated from Yersinia pestis after the treatment with supermutagens. From 13 mutants obtained 10 appeared to be unstable. The stable vitamin-dependent mutants were revealed with the frequency of 2.5.10(-11) divided by 4.9.10(-11) by the indirect method of selection and 5.10(-7) by the use of Davis's method. The frequency of amino acid and nitrogen base dependent auxotroph isolation was significantly higher than that of the vitamin-dependent mutants.     

High performance liquid chromatographic determination of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE), a novel organoselenium compound, in dog plasma using pre-column derivatization and its application in pharmacokinetic study

A novel HPLC-UV method with pre-column derivatization by using 2-mercaptoethanol was established for determination of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE) in dog plasma. The derivatives were identified by mass spectrometry. The method had a good linear range of 0.05-2 microg/ml (r(2)=0.9995). The lower limit of quantification (LOQ) was 0.05 microg/ml. The precision and accuracy were less than 7%. After dosing of BBSKE (30 mg/kg, p.o. and 0.79 mg/kg, i.v.) in dogs, AUC(0-t) were 5.72+/-2.42 and 1.35+/-0.41 microg h/ml; t(1/2) were 4.6+/-2.1 and 1.7+/-0.6h, respectively. The method was successfully applied to the pharmacokinetic study in dogs.     

Analytical chiral separation of a new quinolone compound in biological fluids by high-performance liquid chromatography

Two methods for the separation of a new racemic quinolone compound, temafloxacin (TMFX), in biological fluids by high-performance liquid chromatography (HPLC) were studied. The first method was coupling of TMFX to S-(−)-N-1-(2-naphthyl sulfonyl)-2-pyrrolidine carbonylchloride (L-NSPC). The diastereomeric derivatives were separated on a silica gel column. The second method was separation on a chiral stationary phase with an ovomucoid conjugated to aminopropyl silica gel. Two enantiospecific methods gave a satisfactory result concerning both accuracy and precision, and the second method was superior to the first one for chromatographic separation. Furthermore, the pharmacokinetics of the enantiomers after oral administration of racemic TMFX to healthy volunteers was investigated by the second method.     

A method for the identification of human peripheral blood T lymphocytes by sequential immunogold and esterase double staining

A double staining method involving the sequential use of monoclonal OKT hybridoma antibodies applied in the colloidal immunogold method and followed by a simultaneously capturing azo dye method for the detection of acid alpha-naphthyl acetate esterase (ANAE) is described. Mononuclear leukocytes isolated from human peripheral blood using a Ficoll-Hypaque density gradient were stained. M-pattern ANAE-positive monocytes (diffuse staining) were excluded from the lymphocyte counts. 80 +/- 5% of all lymphocytes were T-pattern ANAE positive (dot-like staining) and 77 +/- 3% were OKT3 positive. 86 +/- 6% of all ANAE-positive T-pattern lymphocytes were also OKT3 positive, and 89 +/- 6% of all OKT3-positive lymphocytes were also ANAE positive. This indicates that ANAE is a good marker for total human T lymphocytes. 53 +/- 10% of human peripheral blood lymphocytes were OKT4 positive and 87 +/- 8% of all OKT4-positive lymphocytes were also ANAE positive. 30 +/- 6% of all lymphocytes were OKT8 positive, and only 26 +/- 18% of all OKT8-positive lymphocytes were ANAE negative. This indicates that ANAE cannot be used to distinguish T-helper and T-suppressor lymphocytes as identified by monoclonal antibodies.     

Determination of cortisol in human saliva using liquid chromatography-electrospray tandem mass spectrometry

The aim of this work was to develop a method for determination of cortisol in saliva by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Saliva was sampled on Salivette tubes. These were centrifuged, deuterium-labeled cortisol was added as internal standard and the proteins precipitated by acetonitrile. The supernatant was evaporated, dissolved in methanol acidified with acetic acid and analyzed by LC-MS-MS. The with-in run precision, tested by pooling saliva samples from volunteers and then analyzing these in a single run, was found to be 7% at 0.7 microgram l(-1). The between-run precision was tested by analysis of the same samples at different days and found to be 11% at 2.5 microgram l(-1). The limit of quantification was 0.5 microgram l(-1). The method was applied for analysis of saliva samples from three volunteers during their last week before vacation and the first and second week on vacation. In addition, the method was compared to analysis by an immunological method. The values from the immunological method were 2.7 times higher than the LC-MS-MS results.