Detection of Salmonella spp. in milk by using Felix-O1 bacteriophage and high-pressure liquid chromatography.

A method is described whereby the presence of less than five salmonellae was detected per milliliter of milk within 24 h of sample collection. Salmonellae were removed from milk by means of electropositive large-pore filters. Eluates from the filters were analyzed for the presence of Salmonella spp. by Felix-O1 bacteriophage and high-pressure liquid chromatographic techniques. The method gave only a positive response when salmonellae were present in the milk. Of the serotypes and strains of Salmonella spp. tested, Salmonella dublin (10 strains), Salmonella typhimurium (5 strains), Salmonella anatum, Salmonella krefeld, and Salmonella saint-paul gave positive responses. One strain of Salmonella agona (three strains tested) and three strains of Salmonella enteritidis (seven strains tested) were not detectable by the method described herein.     

Rapid detection of Salmonella spp. by using Felix-O1 bacteriophage and high-performance liquid chromatography

A method is described whereby the presence of Salmonella spp. can be detected within 8 to 24 h of sample collection. The method depends upon the interaction of Salmonella spp. with the Salmonella-specific Felix-O1 bacteriophage. This interaction results in an increase in concentration of the bacteriophage which is detected by high-performance liquid chromatographic techniques.     

Rapid Detection of Salmonella spp. by Using Felix-O1 Bacteriophage and High-Performance Liquid Chromatography

A method is described whereby the presence of Salmonella spp. can be detected within 8 to 24 h of sample collection. The method depends upon the interaction of Salmonella spp. with the Salmonella-specific Felix-O1 bacteriophage. This interaction results in an increase in concentration of the bacteriophage which is detected by high-performance liquid chromatographic techniques.     

Detection of Salmonella in feces by using Felix-01 bacteriophage and high-performance liquid chromatography

Utilizing the Felix-01 bacteriophage and high-performance liquid chromatographic technique, a method was devised making possible the detection of approximately 10⁶Salmonella typhimurium/g of human feces within 8–9 h of sample collection. Using overnight enrichment, as few as 10³S.typhimurium/g of feces were detectable.     

Detection of Salmonella spp. in oysters by PCR.

PCR DNA amplification of a region of the himA gene of Salmonella typhimurium specifically detected Salmonella spp. In oysters, 1 to 10 cells of Salmonella spp. were rapidly detected by the PCR following a pre-enrichment step to increase sensitivity and to ensure that detection was based on the presence of viable Salmonella spp.     

Racemization studies in peptide synthesis using high-pressure liquid chromatography

We have synthesized the dipeptides benzoyl-l-phenylalanyl-l-alanyl benzyl ester and benzoyl-l-phenylalanyl-l-alanyl methyl ester by both the solid-phase and solution coupling methods. A variety of coupling reagents and solvents was employed. Each coupling reaction was analyzed by high-pressure liquid chromatography for extent of racemization. Baseline separations were achieved which allowed the direct, rapid, and reproducible determination of dipeptide diastereomers. Our successes in the separation of dipeptide diastereomers indicate the future value of applying high-pressure liquid chromatography to the separation of larger peptide diastereomers.     

The separation of the mycobactins from Mycobacterium smegmatis by using high-pressure liquid chromatography.

The family of mycobactins from Mycobacterium smegmatis were resolved into seven fractions by high-pressure liquid chromatography. This separation was by virtue of the differences in length and character of the long acyl substituents as shown by g.l.c. of the methyl esters of the isolated fatty acids from the fractions. As t.l.c. could also resolve the individual mycobactin fractions, it too must rely on the same differences to effect separation. As the lengths of the acyl chains were modulated by the growth conditions, a specific range of acyl groups may not be needed for mycobactin to function. This technique provides a simple means of rapidly characterizing crude mycobactins from all mycobacteria.     

Analysis of leukotrienes by high-pressure liquid chromatography

A method is described for the partial synthesis of saturated mixed-chain phosphatidylcholines of a high degree (typically 99 mol%) of purity. This procedure has been designed to eliminate the contamination of the mixed-chain product by symmetric chain phosphatidylcholine and the mixed-chain isomer of the desired product, the two principal impurities introduced by previous techniques. This high degree of purity is obtained by employing a method designed for the complete enzymatic hydrolysis of the C-2 fatty acyl moiety in saturated symmetric phosphatidylcholines and a new technique for the acylation of lysophosphatidylcholines employing the catalyst 4-pyrrolidinopyridine. The versatility of this new procedure is illustrated with the synthesis of several saturated mixed-chain phosphatidylcholines.     

Determination of adenosine deaminase activity using high-pressure liquid chromatography

A method of assaying adenosine deaminase, using high-pressure liquid chromatography to isolate products, has been developed, which has several advantages over available procedures. The method has inherently low background values, affording high sensitivity. Ten picomoles of product can be reliably detected, so that as little as 0.008 unit of enzyme can be determined. Up to six samples per hour can be assayed. The procedure has been applied to erythrocytes and lymphocytes.     

Determination of E. gracilis mRNA base composition using high-pressure liquid chromatography.

The purine and pyrimidine bases of E. gracilis have been separated using a high-pressure liquid chromatograph (HPLC). Each base was unambiguously identified by its characteristic elution profile and UV absorption spectrum. This method allows for the study of the base composition of mRNAs altered by pathological processes, exemplified here by the analysis and comparison of mRNAs from zinc sufficient and deficient organisms.     

Analysis of sugars in glycoproteins by high-pressure liquid chromatography

A method for analyzing the carbohydrate composition of glycoproteins and similar glycoconjugates by methanolysis followed by reverse-phase high-pressure liquid chromatography of the perbenzoylated methyl glycosides has been developed. As described, the method is capable of quantifying sugars in the 1- to 10-nmol range while further optimization of procedures may increase the usable sensitivity by a factor of 10 or greater. Improved yields of the sugar derivatives have been achieved by incorporating several modifications of the original methanolysis procedure. This, together with the use of high-pressure liquid chromatography rather than gas chromatography for separating the sugar derivatives, eliminates the need for empirically determined molar response ratios.     

Selective analysis for adenosine using reversed-phase high-pressure liquid chromatography

A high-pressure liquid chromatographic procedure for the selective determination of adenosine in the presence of other nucleic acid components is reported. Reversed-phase microparticle columns and an isocratic elution mode of dilute potassium dihydrophosphate and anhydrous methanol were used. The analysis is specific for adenosine and is achieved in less than 10 min. An example of the use of this analysis in a biomedical study is reported.     

Determination of cyclooxygenase products and prostaglandin metabolites using high-pressure liquid chromatography and radioimmunoassay.

A method is described for measurement of the cyclooxygenase products, thromboxane,prostacyclin, and prostaglandins (PG), and several prostaglandin metabolites. The procedure involves separation of the compounds by high-pressure liquid chromatography combined with identification and estimation by serologic analysis. These combined procedures have been used to identify and estimate five such products, PGE₂, PGE₁ PGF2α, PGF_1α, and 6-keto-PGF_1α, in the culture fluids of dog kidney cells stimulated by a tumor-promoting phorbol diester. The prostaglandin metabolites, 13,14-dihydro-15-keto-PGE₂, 13,14-dihydro-15-keto-PF2_2α, 13,14-dihydro-PGE₂, and 13,14-dihydro-PGF_2α, were not found in these culture fluids.     

Purification of tyrosine hydroxylase by high-pressure liquid chromatography

Our study of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) from rabbit adrenals has identified two major requirements which are likely to be of general application for the optimal purification and recovery of enzyme activity consequent to high-pressure liquid chromatography: (i) recovery of activity is maximized by pretreatment of the high-pressure liquid chromatography column before each use with protein to saturate high affinity, nonspecific sites exposed by the methanol used for washing, and storage of the column. (ii) Both purification and recovery are critically dependent upon the molarity of the mobile phase buffer. Examination of high-pressure liquid chromatography purified rabbit adrenal tyrosine hydroxylase by nondenaturing gel electrophoresis indicated that tyrosine hydroxylase activity was associated with one of the two protein bands in the gel. Thus, the convenient purification procedure described in this report leads to preparative amounts of tyrosine hydroxylase which is approximately 50% homogeneous.     

Rapid separation of acetylated oligosaccharides by reverse-phase high-pressure liquid chromatography.

Peracetylated saccharides were separated by chromatography on a reverse-phase support, eluting with mixtures of acetonitrile-water. Gradient elution for 2.5 h gave significant separations of all linear glucose oligomers containing up to 35 sugar residues. With isocratic elution retention was exponentially related to molecular mass and only slightly affected by linkage or anomeric configuration. The presence of glucosamine in various saccharides markedly reduced their retention.     

Analysis of metallonucleoside hydrolysis by high-pressure liquid chromatography

A number of ammine complexes of transition metals in the platinum group exhibit antitumor and mutagenic activities, which probably result from in vivo metal ion coordination to nucleic acids. Coordination at N-7 purine sites on nucleic acids may induce depurination through a general acid-catalyzed cleavage of the sugar-purine bond. This possibility was tested by observing the hydrolysis of [dGuo)(NH₃)₅Ru]³⁺ under physiological conditions. Since multiple products result from this reaction, a high-pressure liquid chromatography technique was developed for separating various ammineruthenium(III) complexes with purine, pyrimidine, and nucleoside ligands. Using this technique it was possible to identify and follow the relative concentrations of several of the hydrolysis products. These data were used to develop a preliminary reaction scheme for the decomposition of (dGuo)(NH₃)₅Ru(III). The net rate constant (1.8 × 10^?6 s^?1) for the disappearance of this complex yields an upper limit for the rate of metal-induced sugar hydrolysis. While the half-life ($\text{5}\text{days}\text{< t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{< 28}\text{days}$) for the sugar hydrolysis reaction is substantially shorter than that for the free nucleoside under the same conditions, it cannot be concluded that this represents a major contribution to the mutagenicity of Ru(III) complexes.     

Detection of Salmonella spp. in food by a rapid PCR-hybridization procedure.

A rapid and sensitive PCR-hybridization procedure for detection of Salmonella serovars in food samples was developed. This method is based on three subsequent steps: (1) extraction of nucleic acids from a 2 ml aliquot of the pre-enrichment medium used for the conventional culture method after 6 h of incubation at 37 degrees C; (2) amplification with primers selected from the sequences of invE and invA genes; (3) Southern blot and hybridization with a biotin labeled oligonucleotide probe. The entire procedure requires 30 h. The PCR-hybridization assay was able to detect as little as 50 fg of purified chromosomal DNA of S. typhimurium and 0.2 cfu g-1 of an artificially contaminated food sample. Of 245 food samples analyzed by culture and PCR-hybridization, 20 were positive by both methods and 16 were positive by PCR-hybridization only. None of the 209 PCR-negative samples tested positive by culture. The sensitivity, specificity, alpha and beta error values of the results of the PCR-hybridization procedure, compared with those of culture, were 100, 92.9, 0 and 7.1%, respectively. These results indicate that a short pre-enrichment and PCR-hybridization could be used as a screening test for the detection of Salmonella in food samples.     

Isolation and purification of angiotensin II using affinity and high-pressure liquid chromatography

Peptides have been found in a variety of tissues including brain. To purify the peptide angiotensin II, a three-step method for the isolation and purification has been developed using extraction, affinity chromatography, and high-pressure liquid chromatography. Angiotensin II antiserum purified by affinity chromatography was covalently coupled to Affi-gel 10 (Affi-gel 10-AB). The efficiency and usefulness of this column for the purification of angiotensin II from biological sources were tested with 125I- and 3H-labeled (Ile5)-angiotensin II added to rat brains prior to extraction. After extraction, the recoveries for both peptides were 74 and 75%, respectively. Recovery after the purification on Affi-gel 10-AB was 84 and 82%. Thirty-two percent of the radioactivity was not retained and 50% of the radioactivity could be eluted with 0.1 M Na citrate buffer containing 1 M NaCl using a stepwise pH gradient. Characterization by HPLC of the unretained radioactivity from the Affi-gel 10-AB column showed one peak for [125I]angiotensin II, coeluting with the [125I]angiotensin II standard and two minor peaks. Only 30% of unretained [3H]angiotensin II could be identified as intact [3H]angiotensin II on HPLC. Both [125I]angiotensin II and [3H]angiotensin II elutable at pH 5.0 and 4.0 on Affi-gel 10-AB could be demonstrated as highly purified [125I]angiotensin II and [3H]angiotensin II on HPLC with a purity of more than 90%. On HPLC, the recovery was 81% for [125I]angiotensin II and 99% for [3H]angiotensin II. The recovery for the entire three-step procedure was about 60%. The loading capacity of the Affi-gel 10-AB column for (Ile5)-angiotensin II was 550 ng.(ABSTRACT TRUNCATED AT 250 WORDS)