Prior Purification Research Articles

Modified FALGPA assay for cell-associated collagenolytic activity

A continuous spectrophotometric assay monitoring the hydrolysis of the synthetic peptide 2-furanacryloyl- l-leucylglycyl- l-prolyl- l-alanine (FALGPA) is used to measure collagenase activity of both bacterial and vertebral collagenases. In the present study, a protocol was developed to adapt this assay to the measurement of cell-associated FALGPA hydrolytic activity in bacteria. The bacteria tested included Bacillus cereus, Streptococcus agalactiae, Streptococcus mutans, Enterococcus faecalis, and Escherichia coli, and various levels of activity were identified. The method presented here allows the detection of FALGPA hydrolysis using a small quantity of cells without the need for prior purification of the collagenolytic enzyme or collection and concentration of a large volume of culture supernatant fluid.

Electrophoretic and FPLC Analysis of Soluble Proteins in Four Portuguese Wines

Two different methodologies were utilized to compare, analyze, and fractionate the soluble proteins of four Portuguese white wines made from Arinto, Bical, Fernão Pires, and Vital grape varieties. First, a new, simple, rapid and sensitive methodology consisting of a desalting step (5 min) of the wine under investigation, to remove salts and other low molecular mass contaminants which may interfere with the proteins during the analytical procedures, and a fractionation step (15 min) involving ion exchange fast protein liquid chromatography (FPLC): anion exchange chromatography on the Mono Q column of the FPLC, previously equilibrated with 50 m<i>M</i> ethanolamine buffer, pH 9.5, or cation exchange chromatography on the Mono S column of the FPLC, previously equilibrated with 50 m<i>M</i> formate buffer, pH 3.5, using suitable, continuous salt gradients to elute the wine proteins. This methodology is rapid (20 min), does not require prior concentration or purification of the proteins (which, besides being time consuming, may cause modification or denaturation of the proteins), and has the additional advantage of allowing the recovery of the isolated proteins in their native form. Second, polyacrylamide gel electrophoresis performed under reducing, denaturing conditions, followed by silver staining. This technique allows the estimation of molecular masses. The wines contained polypeptides with molecular masses ranging from 15.5 to 69 kDa. However, only six polypeptides were common to all four wines, namely the 15.5, 17, 20, 21.5, 24.5, and 69 kDa polypeptides. Both methods are sensitive, but the electrophoresis-based technique gives a higher resolution, is time consuming, destructive and more complex to perform. In contrast, the FPLC technique is non-destructive and very simple and rapid to perform.

In vitro cleavage of serum complement protein C3: a comparison between patients with adult periodontitis and periodontally healthy persons.

This study tested the hypothesis that in vitro cleavage of C3 could be triggered with similar case in serum samples from patients with adult periodontitis (n = 26) as in samples from periodontally healthy subjects (n = 13). A lipoteichoic acid, a lipopolysaccharide and an aggregated IgG served as activators of complement. On the average, the periodontitis group generated significantly (p < 0.01) more C3d activation fragments than did the healthy group, as judged from rocket immunoelectrophoresis measurements. Cleavage of C4 and factor B were then assayed through immunoblotting, without prior purification of the sera. C4c fragments were seen in all activated samples, the healthy group causing significantly (p < 0.05) more C4 conversion than did the periodontitis group. Cleavage of factor B, taken as a measure of soluble amplification convertase formation, was about equal between the groups. We inferred therefore that the 2 groups produced comparable amounts of C3b. The results suggested, however, that periodontitis sera favour breakdown of the opsonin C3b, most likely by activating the regulatory proteins factor H and I. Lipoteichoic acid, causing moderate depletion of C4 and factor B, produced significantly (p < 0.01) more C3d fragments than the other two activators examined. It may be that complement activation is down-regulated in periodontitis sera, perhaps at the expense of adequate local opsonic function.

Antigenicity of cystatin-binding proteins from parasitic protozoan Detection by a proteinase inhibitor based capture immunoassay (PINC-ELISA)

A novel immunoassay (PINC-ELISA) was designed using proteinase inhibitors of the cystatin superfamily (PINC) in the solid phase, to promote the selective capture of cysteine proteinases. The method was applied in the identification of papain-like antigens from parasitic protozoa. PINC of human origin, namely recombinant cystatin C (r-cystatin C) or low molecular weight kininogen were used in the assays to adsorb proteases contained in cell lysates from various trypanosomatids. The PINC-ELISA was at first optimized with the major cysteine proteinase from Trypanosoma cruzi (known as GP57 51 or cruzipain), an antigen whose serodiagnostic properties were previously established. Cruzipain is selectively adsorbed from crude extracts of T. cruzi onto PINC-coated wells; the finding that antibodies bind to epitopes located away from the sites of interaction with r-cystatin or low molecular weight kininogen has allowed for the screening of antibodies in chagasic sera, the methodology being advantageous in that it dispensed prior purification of the proteinase antigen. The PINC-ELISA was then carried out with lysates originating from Leishmania m. amazonensis (amastigotes) or Leishmania donovani (promastigotes). Complexes between solid-phase r-cystatin C and antigenic ligands in the lysates were again detected. The Leishmania molecules which bound to r-cystatin C, were respectively recognized by serum antibodies from mice chronically infected with L. amazonensis or from patients with visceral leishmaniasis. Direct evidence for the presence of cysteine proteinases in lysates from L. donovani was then obtained, using synthetic fluorogenic substrates. Due to the broad inhibition profile of r-cystatin C and the marked antigenicity of parasitic cysteine proteinases, such enzymes can be readily detected by PINC-ELISA, without requirement for prior knowledge of their substrate specificities.

Characterization of leukocytes by filtration of diluted blood.

From the known numbers and properties of normal blood cells, it is apparent that the analysis of the flow of diluted blood, through micropore filters, should allow calculation of the properties of leukocytes without the need for their prior purification. The number and transit time of a "slow" leukocyte population can be deduced by fitting the flow, of diluted blood through 5 microns membranes over about 150 s, to the appropriate mathematical model, which is chosen by the use of a suitable statistical test--the runs test. This population of leukocytes equates numerically with the monocyte population of normal blood; cells have a transit time, through 5 microns pores, of 27.7 +/- 10.9 s. The remaining "fast" leukocytes represent the sum of granulocytes and lymphocytes; their flow properties can be deduced from the measured initial flow rate of diluted blood and the estimated properties of the red blood cells. The properties of the red cells can be assessed from filtration of purified suspensions with any concentration of cells from 0.52 x 10(9)/ml to 3.0 x 10(9)/ml. The transit times for red cells and granulocytes/lymphocytes, in blood diluted with about an equal volume of buffer, are 1.36 +/- 0.17 ms and 1.48 +/- 0.33 s, respectively. The transit times of blood cells, through 5 microns pores, are therefore inversely related to their numbers in blood.

A PCR-based sex-determination assay in cattle based on the bovine amelogenin locus.

A method for determining the sex of bovine embryos has been established. Primers for a portion of the bovine amelogenin locus (AMX/Y) were used to amplify DNA present in either 0.1 microliter of blood or biopsies taken from 6-7-day-old embryos. The primers amplify a 280 bp band in females and a 280 and 217 bp bands in males. The method is rapid, does not require prior purification of DNA and contains an internal control which detects PCR failure.

Cloning by function: expression cloning in mammalian cells

malian protein, the most important decision is the selection of a suitable strategy. Until recently, cloning re- quired prior purification of the pro- tein, and the corresponding cDNA was isolated by one of two approaches: (1) Micro-sequencing of all or parts of the purified protein was used to generate nucleic acid probes for screening

Flow Injection-Immunometric Assay of 17α-Hydroxyprogesterone Using Fluorescein-Labeled Fab Fragment

The flow injection-immunoassay has been used for the determination of trace bioactive materials with high specificity and simplicity.1'2 The principle of this method is almost the same as that of solid phase immunoassay, which is based on a competitive reaction between antigen (the target compound) and labeled antigen with immobilized antibody. The antigen bound on immobilized antibody thus must be purged out prior to the subsequent analysis. The continuous flow analysis using immobilized antigen to remove unbound labeled antibody after the competitive reaction with antigen has also been developed, but a highly purified monospecific antibody such as monoclonal antibody is required to minimize the background level.3,4 If we employ a reactor consisting of labeled antibody bound on immobilized antigen, the analyte bound with labeled antibody in an eluent from the reactor can be determined successively (Fig. 1). Moreover, in this method, polyclonal antibody is applicable without any prior purification. The present paper deals with the development of a novel rapid and convenient method named flow injection-immunometric assay (FIIMA).

Non-radioactive DNA probe and polymerase chain reaction procedures for the specific detection of Acanthmoeba

Acanthamoebae are potential pathogens which can cause serious infections of humans. A non-radioactive rDNA probe and polymerase chain reaction (PCR) amplification procedures which are specific, rapid, sensitive and safe for the detection of Acanthamoeba have been developed. A restriction fragment (126 bp; ArDNA-a) from a variable region of the cloned 26S rDNA unit of Acanthamoeba castellanii (from plasmid pAR2) was labelled by biotinylation. Cells and DNAs were incubated with the labelled rDNA probe to define conditions providing the highest hybridization specificity for Acanthamoeba by both colorimetric and chemiluminescent assays. Four recent isolates of Acanthamoeba, Acanthamoeba polyphaga, various bacteria, Herpes simplex virus, other eukaryotic amoebae and human cell lines, were sources of DNA for testing. The rDNA probe was found to be highly specific for Acanthamoeba and is capable of directly detecting about 250 cells without prior DNA purification. PCR primers for this unique ArDNA-a fragment have also been designed. Amplification of the targeted sequence by PCR using those primers yielded a single product which was specifically generated for Acanthamoeba template DNA and not DNA from the other control cells. This PCR procedure provided increased sensitivity with the direct detection of as few as 10 Acanthamoeba cells.

4 Receptors for gut regulatory peptides

Receptors for regulatory peptides (hormones or neurotransmitters) play a pivotal role in the ability of cells to taste the rich neuroendocrine environment of the gut. Recognition of low concentration of peptides with a high specificity and translation of the peptide-receptor interaction into a biological response through different signalling pathways (adenylyl cyclase-cAMP or phospholipase C-phosphatidylinositol) are crucial properties of receptors. While many new receptors have been identified and thereafter characterized functionally during the 1980s, molecular biology now emerges as the privileged way for the structural characterization and discovery of receptors. Different strategies of receptor cloning have been developed which may or may not require prior receptor purification. Among cloning strategies that do not require receptor purification, homology screening of cDNA libraries, expression of receptor cDNA or mRNA in Xenopus laevis oocytes or in COS cells, and the polymerase chain reaction method achieved great success, e.g. cloning of receptors for cholecystokinin, gastrin, glucagon-like peptide 1, gastrin-releasing peptide/bombesin, neuromedin K, neuropeptide Y, neurotensin, opioids, secretin, somatostatin, substance K, substance P and vasoactive intestinal peptide. All these receptors belong to the superfamily of G-protein-coupled receptors which consist of a single polypeptide chain (350-450 amino acids) with seven transmembrane segments, an N-terminal extracellular domain and a C-terminal cytoplasmic domain. In this chapter, we have detailed the properties of three receptors which play an important role in digestive tract physiology and illustrate various signal transduction pathways: pancreatic beta-cell galanin receptors which mediate inhibition of insulin release and intestinal epithelial receptors for vasoactive intestinal peptide and peptide YY, which mediate the stimulation and inhibition of water and electrolyte secretion, respectively.

A Direct HPLC Separation of Wine Phenolics

A direct HPLC technique that does not require prior purification was developed for the analysis of the phenolic components in red wine and musts. This novel procedure utilizes a ternary HPLC solvent system for effective separation of cinnamic acid derivatives, flavonoids, and anthocyanins, monitoring at three different wavelengths.

Functional activity of plasma fibronectin in patients with diabetes mellitus.

Decreased wound healing and increased infection are major problems in patients with diabetes mellitus. Fibronectin plays a fundamental role in wound healing and acts as an opsonin for the phagocytosis of foreign antigens. The aim of this study was to ascertain the functional activity of plasma fibronectin from patients with diabetes mellitus. Initially, a modified Boyden chamber technique was used to measure cell migration on fibronectin purified from patient's plasma and an enzyme-linked immunosorbent assay was used to measure the binding of gelatin. A sandwich assay was developed that enabled the capture of fibronectin directly from patient's plasma without prior purification. With the use of a 96-well format, the binding of two different monoclonal antibodies could be compared simultaneously with the binding of gelatin and cell adhesion. In this way, differences in the function of particular domains of fibronectin from diabetic patients and control subjects could be measured. Results showed no difference between fibronectin from diabetic patients and control subjects with respect to the monoclonal antibodies binding in 1) the cell adhesion domain and 2) the heparin-binding domain. Furthermore, no detectable differences were noted with respect to cell adhesion, cell migration, or gelatin binding. These results suggest that diabetic patients receiving insulin treatment show no modulation of plasma fibronectin function, despite raised levels of circulating glucose.

Renin mRNA quantification using polymerase chain reaction in cultured juxtaglomerular cells. Short-term effects of cAMP on renin mRNA and secretion.

The aim of the present study was to set up a method to quantify renin mRNA levels in mouse renal juxtaglomerular cells, the main physiological site of renin synthesis. Because of the scarcity of the cells, a quantitative polymerase chain reaction had to be developed to measure renin mRNA. Juxtaglomerular cells were isolated and cultured for 2 days under various conditions, and renin mRNA was measured directly from the cytoplasm of the cultured cells without prior RNA purification. An internal standard consisting of a mutated renin mRNA with an insertion of 60 bp was designed to quantify the reaction, ensuring an identical detection and amplification efficiency to the target RNA. Renin mRNA could be precisely quantified between 0.6 and 20 pg, thus allowing its detection in approximately 5000 juxtaglomerular cells. Forskolin, an activator of adenylate cyclase, led to a concentration-dependent maximal threefold increase in renin mRNA in the cultures after 20 hours of incubation. The half-maximal effective dose was 3 x 10(-7) mol/L. The effect of forskolin was mimicked by 10(-5) mol/L isoproterenol, a beta-receptor agonist, and by 10(-5) mol/L isobutylmethylxanthine. A time-course study showed a rapid increase in renin mRNA within 3 hours after forskolin and isoproterenol addition. Renin secretion in the culture medium was measured in parallel and found to be stimulated by both agents. These results show that quantitative polymerase chain reaction is a suitable tool for studying renin gene expression in cultured juxtaglomerular cells. Our findings indicate that cAMP is a potent and fast activator of renin gene transcription and renin secretion in renal juxtaglomerular cells.

Measurement of Plasma Angiotensin II: Purification by Cation-Exchange Chromatography

Measurement of plasma angiotensin II (AII) by radioimmunoassay (RIA) usually requires prior purification of the plasma to remove substances that cross-react in the RIA, most notably angiotensin III (AIII). Purification of AII is generally accomplished by solid-phase extraction (SPE) followed by reverse-phase HPLC, with tedious evaporation and resuspension steps in between, and requires collection of many HPLC fractions per sample for RIA. In this report, we describe a rapid two-step SPE procedure for the purification of plasma AII, including an improved protease inhibitor cocktail for preventing the formation or degradation of AII in vitro. Plasma is first extracted on an S-Sepharose cation-exchange column, in which AII is separated from AIII by virtue of their difference in net charge, and then extracted on a C8 SPE column, without need for intermediate sample handling. The two-step SPE method is fast, results in only a single fraction for RIA per sample, and yields consistently high recoveries (77-86%) of AII, reducing the volume of plasma needed from 2 to 0.5 ml. Rat plasma was used in the present study, but the complete conservation of angiotensin peptide sequences (except angiotensinogen) in mammals suggests that this method will be applicable for other species including humans. In summary, the two-step SPE method offers the speed and simplicity of solid phase extraction while achieving a purity in AII (i.e., free of AIII) previously only obtained by laborious procedures involving HPLC.

Analysis of microsomal cholesteryl ester hydrolases by radiation inactivation.

Radiation inactivation by high energy electrons, a method for determining the size of a protein without prior purification, was used to study the acid and neutral cholesteryl ester hydrolase (CEH) activities of rat liver microsomes. The same preparations were also assayed for the microsomal, "nonspecific" carboxylesterases using o-nitrophenyl acetate as substrate. Non-specific esterase activity surviving radiation could be fit to a single exponential function, the slope of which yielded a target size of 47 +/- 5 kDa (mean +/- S.D., n = 7). Surviving CEH activity assayed at pH 5 could also be fit to a single exponential that yielded a target size of 71 +/- 14 kDa (n = 5). In contrast, the surviving CEH activity assayed at pH 7 was more complex. The data from six experiments were described as the sum of two exponentials, indicating that most of the activity is due to an entity that is three to four times larger and a minor amount to one that is half the size of the pH 5 enzyme. The results are consistent with the suggestion that the acid and neutral microsomal CEH activities are due to distinct enzymes, which are not the "nonspecific" carboxylesterases. Their sizes also differ from those previously determined for lysosomal acid lipase and other lipases in the liver.

Liquid chromatographic evaluation of purine production in the donor human heart during transplantation.

A reversed phase high performance liquid chromatographic technique is presented allowing purine catabolite determination in a whole blood extract without a prior purification step. The method was applied to determine the timing and the profile of myocardial nucleotide catabolite release during reperfusion of the transplanted human heart. Samples of arterial and coronary sinus blood collected at various times within 1 h after aortic declamping during heart or heart-lung transplantations were used for nucleotide catabolite determination. Massive release of inosine and hypoxanthine from the heart was demonstrated. Production of adenosine was also shown but there was no liberation of xanthine or uric acid. Nucleotide catabolite release was greatest in the first 5 min (coronary sinus-arterial difference = 15-20 microM), but was still significant after 30 min of reperfusion. The determination of inosine and hypoxanthine--major catabolites released--was found to be reproducible in coronary sinus blood (coefficient of variation < 10%). However, immediate protein precipitation afer blood sample collection was necessary, as rapid metabolism of both exogenous and endogenous adenosine and inosine was demonstrated.

Determination of Size of Antigenic Fragments after Treatment of Enveloped Viruses with Non-ionic Detergents

Abstract. The two-dimensional double immunodiffusion, the so-called 'two-cross' method, is introduced for determination of the size of antigenic subunits released after solubilization of enveloped viruses with non-ionic detergents. The method enables the determination of diffusion coefficients and calculation of relative molecular masses of immunoreacting components without using any standard and without prior isolation, purification or labelling of material to be analysed. The molecular masses of surface antigenic fragments of the Newcastle disease virus (NDV) disrupted by non-ionic detergent Triton X-100 were determined. Two antigenic fragments, the larger having a molecular mass between 350 and 187 kDa and the smaller from 140 to 86 kDa, were released by the action of 0·1-1·0% detergent at 20°C. One fragment, whose molecular mass varied from 210 to 187 kDa, was obtained at 37°C after treatment of NDV with 0·2-1·0% detergent, respectively The detergent disruption of purified NDV is studied for comparison. No difference was found whether purified or NDV in allantoic fluid was subjected to the same detergent extraction.

Detection of Chlamydia trachomatis in semen of artificial insemination donors by the polymerase chain reaction

To assess the feasibility of detecting Chlamydia trachomatis in cryopreserved donor semen by a specific, direct polymerase chain reaction (PCR). Cryopreserved donor semen was tested for the presence of C. trachomatis by a specific PCR, directly applied to semen without prior DNA purification. Tertiary care fertility center in a teaching hospital and university-based laboratory for molecular pathology. Cryopreserved semen from 30 donors was investigated. These semen samples had previously given negative results in cell culture for C. trachomatis. Two different ejaculates of each donor, cryopreserved with an interval of 2 years, were retrospectively analyzed. None. The presence of C. trachomatis as demonstrated by PCR. In 3 of 30 donors C. trachomatis was detected in both ejaculates, whereas in 2 additional donors only one of the two samples tested positive. Additional samples from 2 positive donors, together with samples from 3 negative donors, were studied more extensively, to test the reproducibility and reliability of PCR results. All ejaculates of the donors, previously positive for C. trachomatis by PCR, indeed appeared to be positive, whereas the samples of the negative donors remained negative. The direct PCR is a reliable, sensitive, and valuable method for detection of C. trachomatis in semen. The incidence of contamination of donor semen with C. trachomatis in the donor population in this study stresses the need for rigorous screening of donor semen before artificial insemination, preferably using a sensitive method such as the PCR.

Fructo-oligosaccharides from Urginea maritima

Fructo-oligosaccharides from red squill ( Urginea maritima) were isolated by precipitation with methanol, GPC on Biogel P2/P4, and reversed-phase HPLC. Structures of the tri- and tetra-saccharides were verified by the reductive cleavage method. A tetrasaccharide that contained both (2 → 1)- and (2 → 6)-linked β- d-Fru f residues was isolated. The higher fractions from GPC were analysed by the reductive cleavage method without prior purification by reversed-phase HPLC. The mode of biosynthesis of sinistrin is discussed.

Maternal RNA content in oocytes of several mammalian and avian species

The amount of maternal RNA in the oocytes of several mammalian (mouse, rabbit, cow, pig, sheep) and avian (hen, Japanese quail, guinea hen, turkey) species was determined. For mammals these were in the range of 0.47 ng/oocyte (mouse) to 0.98 ng/oocyte (cow), with the exception of the rabbit, where it was up to 15 ng/oocyte. Birds' oocytes contain much higher levels of RNA, from 1.0 microgram (Japanese quail) up to 2.1 micrograms/oocyte (hen). The RNA levels were determined by direct measurement of EtBr-induced fluorescence of the oocyte homogenates without prior extraction and purification, and correlate well with standard spectrophotometric OD260 measurements. This method is useful when there are limited amounts of a sample, and it permits nucleic acid determination in the presence of proteins.