Psoriatic skin lesions contain biologically active amounts of an interleukin 1-like compound

The presence of neutrophil chemoattractant material in aqueous extracts of lesional psoriatic scale has been investigated by use of an agarose microdroplet chemokinesis method in combination with ultrafiltration and high-performance liquid chromatography (HPLC). Fractions were also assayed for murine thymocyte co-stimulating activity. Aqueous extracts of psoriatic scale contained significantly greater neutrophil chemokinetic activity than extracts of scale from normal skin. Successive ultrafiltration of extracts showed that the chemokinetic material was 10 to 30 kd. Heat lability and gel filtration HPLC characteristics suggested that the major chemokinetically active material in aqueous extracts of psoriatic scale is different from C5a des arg. Reversed-phase HPLC of 0.1% trifluoroacetic acid/acetonitrile extracts of psoriatic scale revealed two clearly resolved peaks of chemokinetic activity, the major peak consistently containing thymocyte co-stimulating activity. No significant neutrophil chemokinetic activity was seen in fractions after reversed-phase HPLC of scale from normal skin. These findings suggest that a major portion of the neutrophil chemoattractant activity in aqueous extracts of psoriatic scale is due to interleukin 1-like material, which may play a role in the pathogenesis of this disease.     

Comparison of two methods for the measurement of rat liver methylmalonyl-coenzyme A mutase activity: HPLC and radioisotopic assays

Methylmalonyl-coenzyme A mutase (MCM) is a 5'-deoxyadenosylcobalamin-linked mitochondrial enzyme that catalyzes the isomerization of L-methylmalonyl-coenzyme A to succinyl-coenzyme A. In vitro assays of total and holo-MCM activities are important tools for investigating the cobalamin pathway. Several methods have been described for measuring MCM activity. The most commonly-used method is a radioassay based on the permanganate oxidation of DL[CH(3)-(14)C]methylmalonyl-coenzyme A, but radiometric methods are insensitive, laborious, and time-consuming. Therefore, we have compared this method with a nonradiometric assay, potentially most sensitive, based on the separation of methylmalonyl-coenzyme A and succinyl-coenzyme A by high performance liquid chromatography (HPLC). We determined the optimal assay conditions and the reproducibility and sensitivity of each technique. The results obtained by the two techniques were very different: the specific activities obtained by the permanganate oxidation method (0.039 +/- 0.013 nmol/min/mg protein for the holo-MCM activity and 1.90 +/- 0.69 nmol/min/mg protein for the total-MCM activity) were threefold lower than those obtained with the HPLC method (0.124 +/- 0.011 nmol/min/mg protein for the holo-MCM activity and 6.15 +/- 0.76 nmol/min/mg protein for the total-MCM activity). The coefficients of variation for the radiometric method (18.4-40.6%) were three to five times greater than those for the HPLC assay (3.5-12.2%). This demonstrates the lack of sensibility and reproducibility of the permanganate radioassay. Thus, the radiometric method is not suitable for measuring low mutase activities such as the holo activities in tissues. The intrinsic inconvenience of the radiometric assay indicates that the HPLC method is a method of choice for measuring MCM activity.     

Quantification and comparison of chloramphenicol acetyltransferase activity in transformed plant protoplasts using high-performance liquid chromatography- and radioisotope-based assays.

Rice and petunia leaf and cell suspension protoplasts were transformed by electroporation in the presence of pDW2. This plasmid contains a chloramphenicol acetyltransferase (CAT) coding region under the control of a promoter constructed from sequences of the cauliflower mosaic virus genome. We have compared two different approaches to measuring CAT activity in this system, namely high-performance liquid chromatography (HPLC) and a radioisotope-based method. Our results show that both techniques have a similar detection limit of 10 mU CAT and (with an activity greater than 10 mU CAT) the standard error for measuring known amounts of CAT activity was less than +/- 12% for both assays. The HPLC technique, however, has a greater linear response range of 10-600 mU CAT than the radioisotope method, which has a range of 10-400 mU CAT. The HPLC assay also requires a shorter assay time. As a result of this work we believe that HPLC is a viable alternative to the radioisotope-based assay described.     

Recovery of biologically active enzymes after HPLC separation

The mass and activity recovery of eight different enzymes (two monomeric, six oligomeric) with molecular masses between 25,000 and 240,000 daltons were tested after HPLC separation on three different HPLC instruments (two with stainless steel and one with titanium flow paths). Most of the tested proteins are known to be sensitive to heavy metal ions. Eight wide pore, ion-exchange columns, two size-exclusion columns and two hydrophobic-interaction columns were used. Both stainless steel and glass column hardware were used in all three separation modes. The elution times were between 8 and 12 minutes. In almost all cases, the activity recovery was between 90% and 100% compared with a control sample incubated in the chromatographic elution buffer for the same time at the same temperature. A severe activity loss (about 30%) was observed with only one ion-exchange column and one enzyme. Neither the column hardware nor the material of the HPLC equipment had any negative effect on the activity recovery of the enzymes tested.     

Determination of aldosterone secretion rate utilizing mixed mode and high performance liquid chromatography

A simpler method for determining aldosterone secretion rate (ASR) has several applications. High performance liquid chromatography (HPLC) has several advantages over traditional chromatographic methods for purification to constant specific activity of aldosterone liberated from its 18-glucuronide by acid hydrolysis. We found it necessary to introduce several modifications to remove urochromes before HPLC. Two methods for determining ASR were developed. With Method A a more traditional initial procedure was followed, and Sephadex LH-20 chromatography allowed removal of considerable urochromes before HPLC. However, aldosterone recovery was improved with Method B, which employed several bonded phase silica derivatives (Sepralytes) and a PBE 94 column to remove urochromes before HPLC. With this procedure the Sephadex LH-20 chromatography was not required. Aldosterone purification to constant specific activity was achieved by HPLC on a diol column with a normal phase system, and quantification was performed by RIA. ASR determinations were equivalent with both methods. This methodology should be applicable to other steroid secretory rate determinations and to applications involving purification of steroid conjugates.     

Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay

Quantitation of plasma lipopolysaccharides (LPSs) might be used to document Gram-negative bacterial infection. In the present work, LPS-derived 3-hydroxymyristate was extracted from plasma samples with an organic solvent, separated by reversed phase HPLC, and quantitated by MS/MS. This mass assay was combined with the limulus amebocyte lysate (LAL) bioassay to monitor neutralization of LPS activity in biological samples. The described HPLC/MS/MS method is a reliable, practical, accurate, and sensitive tool to quantitate LPS. The combination of the LAL and HPLC/MS/MS analyses provided new evidence for the intrinsic capacity of plasma lipoproteins and phospholipid transfer protein to neutralize the activity of LPS. In a subset of patients with systemic inflammatory response syndrome, with documented infection but with a negative plasma LAL test, significant amounts of LPS were measured by the HPLC/MS/MS method. Patients with the highest plasma LPS concentration were more severely ill. HPLC/MS/MS is a relevant method to quantitate endotoxin in a sample, to assess the efficacy of LPS neutralization, and to evaluate the proinflammatory potential of LPS in vivo.     

Purification and stability characterization of a cell regulatory sialoglgycopeptide inhibitor

Previous attempts to physically separate the cell cycle inhibitory and protease in activities in preparations of a purified cell regulatory sialoglycopeptide (CeReS) inhibitor were largely unsuccessful. Gradient elution of the inhibitor preparation from a DEAE HPLC column separated the cell growth inhibitor from the protease, and the two activities have been shown to be distinct and non-overlapping. The additional purification increased the specific biological activity of the CeReS preparation by approximately two-fold. The major inhibitory fraction that eluted from the DEAE column was further analyzed by tricine-SDS-PAGE and microbore reverse phase HPLC and shown to be homogeneous in nature. Two other fractions separated by DEAE HPLC, also devoid of protease activity, were shown to be inhibitory to cell proliferation and most likely represented modified relatives of the CeReS inhibitor. The highly purified CeReS was chemically characterized for amino acid and carbohydrate composition and the role of the carbohydrate in cell proliferation inhibition, stability, and protease resistance was assessed. © 1995 Wiley-Liss, Inc.     

Fast determination of low-level cytochrome P-450 1A1 activity by high-performance liquid chromatography with fluorescence or visible absorbance detection

A method to determine the activity of the cytochrome P-450 1A1 enzyme, by measuring 7-ethoxyresorufin-O-deethylase (EROD) activity using high-performance liquid chromatography (HPLC) with fluorescence or with visible absorbance detection of resorufin, is described. The lowest quantifiable activity (0.2 pmol/mg min) is obtained by incubation of 0.3 mg of human duodenal microsomal proteins using HPLC fluorescence detection. Using HPLC with visible absorbance detection, sensitivity was ten times lower. However, the equipment for this last method is available in most laboratories. The use of both HPLC assays allows determination of the low EROD activity level in samples of small size, such as two or three human duodenal biopsies obtained by routine endoscopy. These methods will be a useful tool to study the role of drug intestinal metabolism by cytochrome P-450 1A1.     

Development of high performance liquid chromatography with immobilized enzyme onto magnetic nanospheres for screening enzyme inhibitor

A novel-immobilized enzyme strategy created by magnetic nanospheres for monitoring enzyme activity and screening inhibitors followed by high performance liquid chromatography (HPLC) has been demonstrated. Through the reaction of the aldehyde groups with amine groups, alpha-glycosidase was simply and stably immobilized onto magnetic nanospheres by the cross-linking agent glutaraldehyde. In order to profiling the activity of the immobilized alpha-glucosidase, the natural substrate was hydrolyzed by it and the yield of product was determined by HPLC. Compared with traditional bioassay approach, the prepared immobilized alpha-glucosidase displays a high activity and stability which allows it to be easily reused for 10 times. Enzyme inhibition assays by known inhibitor glucobay and three candidate traditional Chinese medicines (TCMs) were then investigated using a similar methodology. This assay was able to readily detect the change of the immobilized enzyme activity based on measuring a decrease of product formation using HPLC. The approach is general and offers many attractive advantages including easy product isolation, inexpensive cost, and high efficiency in terms of reagent consumption.     

Isolation of lignans from Schisandra chinensis with anti-proliferative activity in human colorectal carcinoma: Structure–activity relationships

Separate benzocyclooctadiene lignans were isolated from the berries of Schisandra chinensis in milligram quantities on analytical reverse phase (RP) HPLC by an automated repeat-injection method and shown to have anti-proliferative activity against human colorectal cancer cells. Structures of the compounds were determined by a combination of NMR and mass spectrometry. Stereospecific NMR assignments for gomisin-N and deoxyschisandrin, gave more complete and accurate data than previously reported, based on 600 MHz 2D HSQC, DQF-COSY and HMBC data. Comparison of coupling constants and HMBC crosspeak intensities with calculated and X-ray crystal structures confirmed their stereochemistry and conformation. Analysis of structure–activity relationships revealed the importance of key structural determinants. The S-biphenyl configuration of gomisin N, the most active lignan, correlated with increased anti-proliferative activity, while the presence of a hydroxyl group at the C7 position reduced or abolished this activity. Increased activity was also observed when a methylenedioxy group was present between C12 and C13. The percent yield of the most active compounds relative to the starting plant materials was 0.0156% for deoxyschisandrin and 0.0173% for gomisin N. The results of these studies indicate that automated repeat-injection method of analytical HPLC may provide a superior alternative to the standard semi-preparative HPLC techniques for separation of complex mixtures.     

Purification and biochemical characteristics of two distinct human interleukins 1 from the myelomonocytic THP-1 cell line

An effective induction protocol for the production of interleukin 1 (IL 1) by human myelomonocytic cell line (THP-1) cells was developed, and two biochemically distinct human IL 1 peptides were purified. Lipopolysaccharide, silica, and hydroxyurea by themselves did not induce IL 1 production, but these three stimulants in combination had a synergistic effect on the production of IL 1 by THP-1 cells. A 17-kilodalton (kDa) form of human IL 1 with a pI of 7.0 (IL 1-beta) was purified to homogeneity by sequential chromatography on DEAE-Sephacel, Sephacryl S-200, CM high-performance liquid chromatography (HPLC), and hydroxyapatite HPLC. The recovery of IL 1-beta activity was 45%, and the specific activity was 2.3 X 10(7) units/mg. Both IL 1-beta and a second 17-kDa IL 1 moiety with a pI of 5.0 (IL 1-alpha) were also extracted from stimulated THP-1 cells and purified to homogeneity by sequential chromatography on Sephacryl S-200, ion exchange HPLC, and hydroxyapatite HPLC. The recovery of IL 1-beta from cell extracts was 5.6%, and the specific activity was 3 X 10(7) units/mg. In contrast, only 0.85% of IL 1-alpha was recovered with a specific activity of 5.3 X 10(7) units/mg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a factor from the U937 cell line that enhances the toxicity of human eosinophils to Schistosoma mansoni larvae

A factor produced by lipopolysaccharide-stimulated human monocytes, monocyte-derived eosinophil cytotoxicity-enhancing factor (M-ECEF), increases the ability of human eosinophils to kill larvae of Schistosoma mansoni. In order to purify this monokine, a continuous cell line was sought as a generator of source material. It was found that high titers of an ECEF-like activity could be obtained from the U937 cell line cultured in serum-free medium. Production of this activity was optimal when cells were cultured with PMA for 2 days and were further treated with LPS for 2 days. PMA and LPS alone did not enhance eosinophil cytotoxicity and could be separated completely from U937-ECEF activity by reversed-phase HPLC. Thus, the activity was not due to carry-over of these two stimuli. U937-ECEF was compared with M-ECEF by a number of analytical methods. ECEF from both sources was resistant to several denaturing treatments but was sensitive to proteases or to reduction and alkylation. U937-ECEF exhibited activity profiles similar, if not identical, to those of M-ECEF when subjected to molecular sizing HPLC in the presence of 8 M urea, isoelectric focusing, and reversed-phase HPLC. The activity has apparent m.w. of 17,000 and 32,000, isoelectric points ranging from 3.8 to 5.1, and one or more reversed-phase HPLC retention times, depending on the method of sample preparation. These results demonstrate certain physical characteristics of M-ECEF, show that the U937 cell line is an appropriate source for the purification of M-ECEF, and provide information that will allow the design of a purification strategy. Although it appears that tumor necrosis factor (TNF) or a TNF-like molecule is a component of M-ECEF, a major component of M-ECEF is different from TNF as judged by the 1) physical characteristics of M-ECEF, 2) low direct toxicity of M-ECEF to L929 cells, 3) comparative stability of M-ECEF to heat treatment, and 4) inability of an anti-TNF monoclonal antibody to remove M-ECEF activity.     

Correlation between the activities of alpha-helical antimicrobial peptides and hydrophobicities represented as RP HPLC retention times

PTP7 is a 13-amino acid residue peptide designed from gaegurin 6, an antimicrobial peptide isolated from skin secretions of Rana rugosa. In order to examine the effect of hydrophobicity on antimicrobial activity, a series of PTP7 derivatives were constructed and analyzed the activity against bacteria and artificial membrane. We found that the mean hydrophobicity by simple summation of hydrophobicity of each constituent amino acid did not necessarily describe the hydrophobic property of antimicrobial peptides. The mean hydrophobicity did not show close correlation with the observed hydrophobicity by measuring reverse phase high performance liquid chromatography (RP HPLC) retention time. The observed hydrophobicity represented as RP HPLC retention time correlated well with the activity against artificial membrane and Gram positive bacterial species, such as Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus, rather than mean hydrophobicity. However, antimicrobial activity against Gram negative bacteria, such as Escherichia coli, did not show correlation with RP HPLC retention time. These data indicate that the RP HPLC retention time should be exploited rather than the mean hydrophobicity in the analysis of the relationship between hydrophobicity and antimicrobial activity.     

Assay of human platelet guanylate cyclase activity by high-performance liquid chromatography with fluorescence derivatization

A selective and sensitive high-performance liquid chromatography (HPLC) method with fluorescence derivatization for the assay of guanylate cyclase (GC) activity is described. GTP and cGMP, which are the substrate and the product of GC, respectively, and other guanine-containing compounds are selectively converted by the reaction with (3,4-dimethoxyphenyl)glyoxal to the fluorescent derivatives. The derivatives were separated by reversed-phase HPLC. The limit of detection at a signal-to-noise ratio of 3 for cGMP was 10 fmol on the column. The sensitivity of this method was less than that of the conventional radioisotopic method, but this method is simple and convenient. Human platelet GC activity was measured, and the effects of some compounds were investigated.     

Detection of metabolic conversions of ascorbate-2-monophosphate and ascorbate-2-sulfate to ascorbic acid in tiger prawn (Penaeus monodon) using high-performance liquid chromatography and colorimetry

Biochemical conversions of ascorbate-2-monophosphate and ascorbate-2-sulfate to ascorbic acid by acid phosphatase and ascorbate-2-sulfate sulfohydrolase, respectively, were found in extracts of a hepatopancreas of Penaeus monodon, bovine liver and tilapia liver. Both enzymes were assayed with high-performance liquid chromatography (HPLC) and colorimetry. Colorimetry was based on the reduction of a color of 2,6-dichlorophenolindophenol (DCIP) when ascorbic acid was released from enzymatic activity. Assay of acid phosphatase either with HPLC or with colorimetry was found to be equally reliable. However, sensitivity of the HPLC assay was slightly higher than that of colorimetry; HPLC was able to detect activity as little as 1 nmol ascorbic acid released per min, whereas colorimetry was limited at 6–7 nmol/min. Assay of ascorbate-2-sulfate sulfohydrolase in crude extracts with the HPLC technique was found to be more specific than that with the colorimetric assay. The excess reduction of DCIP color not related to the sulfohydrolase activity was observed in the colorimetric technique. An accumulation of ascorbic acid in a hepatopancreas of P. monodon fed with feeds supplemented with phosphorylated or sulfated ascorbic acid was higher than that of the prawn fed with feed without ascorbic acid. The accumulated ascorbic acid was possibly from the activity of acid phosphatase or the sulfohydrolase that hydrolyzed phosphorylated or sulfated derivatives in vivo, respectively. Metabolism of the ascorbate derivatives in the prawn is discussed.     

Identification by high-performance liquid chromatography of tyrosine ammonia-lyase activity in purified fractions of Phaseolus vulgaris phenylalanine ammonia-lyase

Activities of phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) were assessed at each stage of a three-step purification of PAL. Assays were performed by high-performance liquid chromatographic (HPLC) separation and ultraviolet detection of reaction products. Use of HPLC permitted assay of low activities of PAL and TAL for periods up to approximately four and two days, respectively. HPLC also facilitated the accurate quantitation of the product of the TAL reaction, trans-p-coumaric acid, which was observed to isomerize readily under experimental conditions. PAL and TAL were associated throughout the purification procedure, with TAL activity at 0.6–1.3% of PAL activity. It was concluded that, contrary to previous reports, TAL and PAL activities are mediated by the same enzyme, or else by chromatographically very similar enzymes.     

Identification of selective basophil chemoattractants in human nasal polyps as insulin-like growth factor-1 and insulin-like growth factor-2

In a search for novel leukocyte chemoattractants at sites of allergic inflammation, we found basophil-selective chemoattractant activity in extracts of human nasal polyps. The extracts were fractionated by reverse phase HPLC, and the resulting fractions were tested for leukocyte-stimulating activity using sensitive shape change assays. The basophil-selective activity detected was not depleted by a poxvirus CC-chemokine-binding protein affinity column. This activity was further purified by HPLC, and proteins in the bioactive fractions were analyzed by tandem electrospray mass spectrometry. Insulin-like growth factor-2 (IGF-2) was identified in these HPLC fractions, and the basophil-stimulating activity was inhibited by an anti-IGF-2-neutralizing Ab. Recombinant IGF-2 induced a substantial shape change response in basophils, but not eosinophils, neutrophils, or monocytes. IGF-2 stimulated chemokinesis of basophils, but not eosinophils or neutrophils, and synergized with eotaxin-1/CCL11 in basophil chemotaxis. IGF-2 also caused up-regulation of basophil CD11b expression and inhibited apoptosis, but did not stimulate degranulation or Ca(2+) flux. Recombinant IGF-1 exhibited similar basophil-selective effects as IGF-2, and both growth factors were detected in nasal polyp extracts by ELISA. This is the first demonstration of chemokinetic factors that increase the motility of basophils, but do not act on other granulocytes or monocytes. IGF-1 and IGF-2 could play a role in the selective recruitment of basophils in vivo.     

A highly sensitive method for measurement of myosin ATPase activity by reversed-phase high-performance liquid chromatography

A new method for measurement of myosin ATPase activity has been developed utilizing reversed-phase high-performance liquid chromatography (HPLC), which detects as low as 0.05 nmol of ADP hydrolyzed from ATP. After termination of the ATPase reaction by addition of perchloric acid, the hydrolysate ADP and substrate ATP were separated by reversed-phase HPLC. The absorbance of ADP was monitored at 259 nm, and the amount of ADP was quantified from its peak area on the chromatogram by use of the NIH Image computer software. Our method showed linearity over a wide range from 0.05 to 10 nmol of ADP per 20 microl with a coefficient of determination (r(2)) of 0.99. Myosin ATPase activities determined by the HPLC method were almost identical to those determined by the malachite green method, a widely used spectrophotometric method with range of detection from 1 to 8 nmol of phosphate. Because our method requires only a small volume of reaction solution, it will be a powerful tool for measuring ATPase activity of motor proteins, which are difficult to obtain in large amount.     

HPLC quantification of uncarine D and the anti-plasmodial activity of alkaloids from leaves of Mitragyna inermis (Willd.) O. Kuntze

An efficient system for the analysis of the total alkaloids extracted from leaves of Mitragyna inermis (Willd.) O. Kuntze (Rubiaceae) by HPLC using a reversed-phase column is described. The chromatographic conditions allowed the separation of indole and oxindole alkaloids in leaf extracts, and the quantification of uncarine D in samples collected in Burkina Faso and Mali. The HPLC method described was validated for its specificity, linearity and precision using an internal standard (naphthalene). The concentrations of uncarine D in various extracts were compared with their in vitro anti-plasmodial activity. The anti-proliferative activity on chloroquine-resistant strain (W2) of Plasmodium falciparum was not correlated with the concentration of uncarine D in leaves.     

Appearance of uridine 5'-diphospho-N-acetylglucosamine-4-epimerase during sporulation of Bacillus megaterium

In a biosynthetic study of the spore coat of Bacillus megaterium ATCC 12872 spore with galactosamine phosphate as a major component of the outer coat, high-performance liquid chromatography (HPLC) and enzyme immunoassay were applied for the measurement of UDP-N-acetylglucosamine-4-epimerase [EC 5.1.3.7] activity and the enzyme protein concentration, respectively. The new HPLC system using an ion-pair (or anion-exchange) column allowed us to determine successfully the enzyme activity and its application, proving that the specific activity of the enzyme in the cells increased at the later stage of sporulation. This increase in activity was parallel to the induction of enzyme protein synthesis, which was detected by sandwich enzyme immunoassay using antiserum to the purified enzyme. These results suggested that the regulation of this enzyme is at the genetic level and it plays an important role in the outer coat synthesis in the later sporulation stage of B. megaterium.