High-pressure Liquid Chromatography Profiles Research Articles

Fully Automated Radiosynthesis of 2-[18F]Fludarabine for PET Imaging of Low-Grade Lymphoma

An efficient and fully automated radiosynthesis of 2-[(18)F]fluoro-9-β-D-arabinofuranosyl-adenine (2-[(18)F]fludarabine, [(18)F]-5) based on a GE TRACERlab™ FX-FN module has been developed. A 2-nitro purine derivative 3 was developed as precursor for labeling with fluorine-18. The radiosynthesis of [(18)F]-5 was performed in two steps in a single reactor with an intermediary purification on Sep-Pak® silica which involved the addition of a three-way valve on the original module. After hydrolysis, [(18)F]-5 was purified by semi-preparative high-pressure liquid chromatography (HPLC) and a quality control was established. The labeling precursor 3 was obtained in 45% overall yield. Nucleophilic substitution with K(18)F/K2.2.2 afforded protected 2-[(18)F]fludarabine ([(18)F]-4) in 73 ± 4%, radiochemical yield (decay corrected to the end of bombardment (EOB)) and based on the initial [(18)F]F(-) activity. An aqueous ammonia/methanol solution was used for the deprotection reaction and gave the desired [(18)F]-5 in 67 ± 3% yield after 20 min at 70 °C based on HPLC profile. The process afforded pure 2-[(18)F]fludarabine in 48 ± 3% yield (decay corrected to the EOB) in 85 min, with a specific activity of 310 ± 72 GBq/μmol at the end of synthesis (EOS) and a radiochemical purity up to 99%.

Commercial Extract of Ascophyllum nodosum Improves Root Colonization of Alfalfa by Its Bacterial Symbiont Sinorhizobium meliloti

The soil bacterium Sinorhizobium meliloti forms a symbiotic relationship with alfalfa (Medicago sativa) roots, which results in the formation of intracellular root nodules. This symbiosis increases nitrogen (N) in the soil; however, to establish such a synergistic relationship, a complex communication system is required between the bacterium and its legume host. Rhizobacteria are known to respond to plant root exudates and produce signal molecules known as “Nod” factors. Research suggests that the brown seaweed (Ascophyllum nodosum) extract (ANE) stimulates both root nodulation and growth of alfalfa (Khan et al. 2011). To elucidate the mechanism of action, the effects of ANE on the early stages of root–rhizobia interactions were examined. A. nodosum extract (ANE) and its organic fractions were prepared and alfalfa roots were treated. After 2 days, the treated roots were inoculated with S. meliloti. The roots from treated plants were excised and observed for colony-forming units. To verify whether ANE elicited the synthesis and secretion of factors similar to those induced by luteolin, S. meliloti cultures were treated with ANE and the bacterial components were analyzed by high-pressure liquid chromatography (HPLC). To study Nod factor induction by S. meliloti due to ANE treatment, a root hair deformation assay was performed. A translational fusion of S. meliloti NodC:LacZ (strain JM57) was used to observe the effect of ANE on bacterial gene expression. When S. meliloti culture medium was supplemented with ANE, no effect on bacterial growth was observed. However, it was observed that the attachment of S. meliloti to the root hairs was improved. Similarly in vitro ANE root treatments, followed by S. meliloti inoculation, increased bacterial colonies. HPLC profiles and a root hair deformation assay suggested that ANE elicits production of compounds similar to the Nod factor, which are normally induced by the plant signaling molecule luteolin. The results suggest that ANE may contain compound(s) that promote the legume–rhizobia symbiotic relationship and plant signaling.

Identification of Catechin as One of the Flavonoids from Combretum albiflorum Bark Extract That Reduces the Production of Quorum-Sensing-Controlled Virulence Factors in Pseudomonas aeruginosa PAO1

Quorum-sensing (QS) regulates the production of key virulence factors in Pseudomonas aeruginosa and other important pathogenic bacteria. In this report, extracts of leaves and bark of Combretum albiflorum (Tul.) Jongkind (Combretaceae) were found to quench the production of QS-dependent factors in P. aeruginosa PAO1. Chromatographic fractionation of the crude active extract generated several active fractions containing flavonoids, as shown by their typical spectral features. Purification and structural characterization of one of the active compounds led to the identification of the flavan-3-ol catechin [(2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol]. The identity of catechin as one of the active molecules was confirmed by comparing the high-pressure liquid chromatography profiles and the mass spectrometry spectra obtained for a catechin standard and for the active C. albiflorum fraction. Moreover, standard catechin had a significant negative effect on pyocyanin and elastase productions and biofilm formation, as well as on the expression of the QS-regulated genes lasB and rhlA and of the key QS regulatory genes lasI, lasR, rhlI, and rhlR. The use of RhlR- and LasR-based biosensors indicated that catechin might interfere with the perception of the QS signal N-butanoyl-l-homoserine lactone by RhlR, thereby leading to a reduction of the production of QS factors. Hence, catechin, along with other flavonoids produced by higher plants, might constitute a first line of defense against pathogenic attacks by affecting QS mechanisms and thereby virulence factor production.

Structural Basis for the Lower Affinity of the Insulin-like Growth Factors for the Insulin Receptor

Insulin and the insulin-like growth factors (IGFs) bind with high affinity to their cognate receptor and with lower affinity to the noncognate receptor. The major structural difference between insulin and the IGFs is that the IGFs are single chain polypeptides containing A-, B-, C-, and D-domains, whereas the insulin molecule contains separate A- and B-chains. The C-domain of IGF-I is critical for high affinity binding to the insulin-like growth factor I receptor, and lack of a C-domain largely explains the low affinity of insulin for the insulin-like growth factor I receptor. It is less clear why the IGFs have lower affinity for the insulin receptor. In this study, 24 insulin analogues and four IGF analogues were expressed and analyzed to explore the role of amino acid differences in the A- and B-domains between insulin and the IGFs in binding affinity for the insulin receptor. Using the information obtained from single substituted analogues, four multiple substituted analogues were produced. A "quadruple insulin" analogue ([Phe(A8), Ser(A10), Thr(B5), Gln(B16)]Ins) showed affinity as IGF-I for the insulin receptor, and a "sextuple insulin" analogue ([Phe(A8), Ser(A10), Thr(A18), Thr(B5), Thr(B14), Gln(B16)]Ins) showed an affinity close to that of IGF-II for the insulin receptor, whereas a "quadruple IGF-I" analogue ([His(4), Tyr(15), Thr(49), Ile(51)]IGF-I) and a "sextuple IGF-II" analogue ([His(7), Ala(16), Tyr(18), Thr(48), Ile(50), Asn(58)]IGF-II) showed affinities similar to that of insulin for the insulin receptor. The mitogenic potency of these analogues correlated well with the binding properties. Thus, a small number of A- and B-domain substitutions that map to the IGF surface equivalent to the classical binding surface of insulin weaken two hotspots that bind to the insulin receptor site 1.

Effects of interaction between pollen coat eluates and pistil at the molecular level in self-compatible and self-incompatible plants ofLolium multiflorum Lam.

Two-dimensional electrophoresis (2-DE) of soluble proteins and enzymes was performed and specific activities of 5 enzymes (esterase, pectinesterase, acid phosphatase, protease and diaphorase) were determined in stigmas of Lolium multiflorum (Italian ryegrass) treated with self or foreign pollen coat eluates (pc). Also, a low-molecular-weight fraction of the treated self-compatible (SC) and self-incompatible (SI) stigmas was analyzed by high-pressure liquid chromatography (HPLC). The treatment of stigmas with foreign pollen induced the loss of 42% of the control sample proteins in SC plants but only of 5.5% in SI plants. In contrast, the treatment of stigmas with foreign pollen induced the loss of 15% proteins in SC plants and of 29% in SI plants. Specific activities of esterase, pectinesterase and diaphorase were higher in SC than in SI stigmas. The 2-DE enzyme patterns indicated qualitative relationships between the presence of some isoforms of acid phosphatase or protease and the treatment with self or foreign pc in SC and SI stigmas. No changes were observed in HPLC profiles of the low-molecular-weight fraction from SC and SI stigmas treated or not with pc. The presented results revealed different reactions of SC and SI stigmas to the treatment with self or foreign pc. Further investigations may explain if any of the observed reactions represent specific reorientations in the style, facilitating cross- or self-pollination.

Invasion bySalmonella typhimuriumInduces Increased Expression of the LMP, MECL, and PA28 Proteasome Genes and Changes in the Peptide Repertoire of HLA-B27

We have analyzed proteasomal adaptation and associated changes in the B27-bound peptide repertoire in response to cellular invasion with Salmonella. The peptide repertoire of HLA-B27 complexes was analyzed by two different methods: (i) high-pressure liquid chromatography (HPLC) profiles of newly synthesized peptides eluted from B27 following metabolic labeling with arginine and (ii) reactivities with two B27 monoclonal antibodies, Ye-2 and B27.M2, sensitive to peptide-induced conformational changes. LMP, MECL, and PA28 expression was analyzed by reverse transcription-PCR (RT-PCR) of mRNA and by Western blot analysis for LMP2. Invasion of HLA-B27-transfected HeLa cells by Salmonella typhimurium induced significant changes in the reactivities of HLA-B27 with these two antibodies, which was accompanied by significant quantitative and qualitative changes in the HPLC profile of peptides eluted from HLA-B27. We also observed increases in the RT-PCR values for the LMP2, LMP7, and MECL proteasome subunit genes, as well as the proteasomal activator PA28alpha and -beta genes, and increased expression of the LMP2 protein by Western blotting. Upregulation of LMP2, but not LMP7, gene expression showed a close correlation with the changes in antibody reactivities observed upon bacterial invasion. We observed similar changes in reactivity with the Ye-2 or the B27.M2 antibody of lymphoblastoid cells upon gamma interferon treatment, which significantly correlated with the increased RT-PCR values for the LMP2 gene. This was accompanied by consistent HPLC profile changes for eluted peptides. Thus, Salmonella invasion leads to serologically recognizable changes in the B27-bound peptide repertoire, which may include peptides of host origin potentially through modulation of proteasome LMP2 subunit expression and, as a consequence, proteasomal activities.

Structural Analyses and Dynamics of Soluble and Cell Wall-bound Phenolics in a Broad Spectrum Resistance to the Powdery Mildew Fungus in Barley

High pressure liquid chromatography profiles of barley leaf epidermal soluble and cell wall-bound phenolics were analyzed in response to challenge with the fungal pathogen Erysiphe graminis f. sp. hordei. Only one soluble phenolic was found to accumulate differentially in a broad spectrum resistance reaction controlled by mlo resistance alleles in comparison to susceptible near isogenic Mlo lines. Structural analysis identified this compound as a novel phenolic conjugate, p-coumaroyl-hydroxyagmatine (p-CHA). p-CHA but not the nonhydroxylated derivative p-coumaroylagmatine exhibited antifungal activity both in vitro and in vivo. The accumulation of p-CHA in epidermal tissue correlated tightly with fungal penetration attempts of attacked host cells. Furthermore, upon penetration, epidermal cell wall-bound phenolics became resistant to saponification at sites of attempted fungal ingress (papilla), indicating a change in, or the addition of, different chemical bonding types. The switch in saponification sensitivity occurred at least 2 h earlier in the mlo-incompatible than in the Mlo-compatible interaction. Our results suggest that p-CHA and the speed of papillae compaction play important roles in non-race-specific powdery mildew defense.

Identification of Transglutaminase-reactive Residues in S100A11

The recent finding that S100A11 is a component of the keratinocyte cornified envelope (CE) (Robinson, N. A., Lapic, S., Welter, J. F., and Eckert, R. L. (1997) J. Biol. Chem. 272, 12035-12046) suggests that S100A11 is a transglutaminase (TG) substrate. In the present study we show that S100A11 forms multimers when cultured keratinocytes are challenged by increased levels of intracellular calcium and that multimer formation is inhibited by the TG inhibitor, cystamine. These S100A11 multimers appear to be incorporated into the CE, as immunoreactive S100A11 is detected in purified envelopes prepared from cultured cells and from foreskin epidermis. To study S100A11 as a transglutaminase substrate, recombinant human S100A11 (rhS100A11) was used in a cell-free cross-linking system. [14C]Putrescine, a primary amine, labels rhS100A11 in a TG-dependent manner. Trypsin digestion of [14C]putrescine-labeled rhS100A11 releases one radiolabeled peptide, Ala98-Lys103. The glutamine residue in this segment, Gln102, is the site of radiolabel incorporation indicating that Gln102 functions as an amine acceptor. The ability of S100A11 to form multimers indicates that it also has a reactive lysine residue that functions as an amine donor. To identify the reactive residue, we compared the high pressure liquid chromatography profile of trypsin-digested rhS100A11 monomer to that of cross-linked rhS100A11. A unique cross-linked peptide was purified and identified as Met-Ala-Lys3-Ilu-Ser-Ser-Pro-Thr-Glu-Thr-Glu-Arg cross-linked via an Lys3-Gln102 isopeptide bond to Ala-Val-Pro-Ser-Gln102-Lys. These studies show that S100A11 is post-translationally modified by transglutaminase, that it can be cross-linked to form multimers, that it is present in CEs from cultured keratinocytes and in vivo epidermis, and that Lys3 and Gln102 are specific sites of cross-link formation.

Vaccinia virus gene H5R encodes a protein that is phosphorylated by the multisubstrate vaccinia virus B1R protein kinase.

Vaccinia virus gene B1R encodes a protein kinase, the previously identified substrates of which include the proteins S2 and Sa of 40S ribosomal subunits. This work characterizes another substrate of the B1R kinase: a 36-kDa protein induced at the early stage of infection. Partially purified 36-kDa protein, eluted from a single-stranded DNA-cellulose column by 0.5 M NaCl, was separated by two-dimensional gel electrophoresis. Phosphorylation in vitro yielded multiple forms of the 36-kDa protein with approximate isoelectric points (pI) of 5.5, 5.7, 5.9, and 6.3, in addition to the apparently unphosphorylated form with a pI of approximately 6.8. The tryptic peptides derived from 36-kDa proteins with pI values of 5.7, 5.9, and 6.3 yielded almost identical high-pressure liquid chromatography profiles, strongly suggesting that the 36-kDa protein was modified by the phosphorylation of at least four sites, which were characterized as threonine residues. The amino acid sequence of several tryptic peptides derived from the 36-kDa protein showed that the 36-kDa protein was encoded by gene H5R of vaccinia virus. Consistent with this, the B1R kinase--either expressed in Escherichia coli or highly purified from HeLa cells--phosphorylated a recombinant trpE-H5R fusion protein in vitro. Fingerprints of the trpE-H5R and 36-kDa proteins phosphorylated by recombinant B1R kinase revealed common sites of phosphorylation, although some tryptic peptides were specific to either protein. Comparison was made of fingerprints of tryptic phosphopeptides derived from 36-kDa single-stranded DNA-binding protein labelled in vivo or in vitro. A common subset of peptides was observed, suggesting that some sites on H5R protein are phosphorylated by the B1R kinase in infected cells. These results suggest that some of the multiple threonine sites in the H5R protein are phosphorylated in vivo by the B1R protein kinase.

Separation of 32P-Postlabeled DNA Adducts of Polycyclic Aromatic Hydrocarbons and Nitrated Polycyclic Aromatic Hydrocarbons by HPLC

The 32P-postlabeling assay, thin-layer chromatography, and reverse-phase high-pressure liquid chromatography (HPLC) were used to separate DNA adducts formed from 10 polycyclic aromatic hydrocarbons (PAHs) and 6 nitrated polycyclic aromatic hydrocarbons (NO2-PAHs). The PAHs included benzo[j]fluoranthene, benzo[k]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[a]pyrene, chrysene, 6-methylchrysene, 5-methylchrysene, and benz[a]anthracene. The NO2-PAHs included 1-nitropyrene, 2-nitrofluoranthene, 3-nitrofluoranthene, 1,6-dinitropyrene, 1,3-dinitropyrene, and 1,8-dinitropyrene. Separation of seven of the major PAH-DNA adducts was achieved by an initial PAH HPLC gradient system. The major NO2-PAH-DNA adducts were not all separated from each other using the initial PAH HPLC gradient but were clearly separated from the PAH-DNA adducts. A second NO2-PAH HPLC gradient system was developed to separate NO2-PAH-DNA adducts following one-dimensional TLC and HPLC analysis. HPLC profiles of NO2-PAH-DNA adducts were compared using both adduct enhancement versions of the 32P-postlabeling assay to evaluate the use of this technique on HPLC to screen for the presence of NO2-PAH-DNA adducts. To demonstrate the application of these separation methods to a complex mixture of DNA adducts, the chromatographic mobilities of the 32P-postlabeled DNA adduct standards (PAHs and NO2-PAHs) were compared with those produced by a complex mixture of polycyclic organic matter (POM) extracted from diesel emission particles. The diesel-derived adducts did not elute with the identical retention time of any of the PAH or NO2-PAH standards used in this study. HPLC analyses of the NO2-PAH-derived adducts (butanol extracted) revealed the presence of multiple DNA adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

The Effect of Pretreatment on the Biliary Excretion of 2,3,7,8-Tetrachlorodibenzo-p-dioxin, 2,3,7,8-Tetrachlorodibenzofuran, and 3,3′,4,4′-Tetrachlorobiphenyl in the Rat

The Effect of Pretreatment on the Biliary Excretion of 2,3,7,8-Tetrachlorodibenzo-p-dioxin, 2,3,7,8-Tetrachlorodibenzofuran, and 3,3′,4,4′-Tetrachlorobiphenyl in the rat. McKinley, M. K., Kedderis, L. B., and Birnbaum, L. S. (1993). Fundam. Appl. Toxicol. 21, 425-432.The laterally halogenated chemicals 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 3,3′,4,4′-tetrachlorobiphenyl (TCB) exhibit the same spectrum of toxic effects as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototype and most toxic member of the halogenated aromatic hydrocarbon family. Metabolism of all three compounds appears to be the rate-limiting step for excretion, which is primarily via the bile into the feces. Therefore, the biliary elimination of TCDF, TCDD, and TCB was examined as an indirect measure of metabolism. Male F344 rats were anesthetized with pentobarbital, the bile duct was cannulated, and 0.1 μmol [3H]TCDD, [14C]TCDF, or [14C]TCB/kg body wt was administered iv. Bile was collected for 0-8 hr while the animals were kept under anesthesia. To determine if TCDF was able to induce its own metabolism in vivo, a single dose of 1.0 μmol TCDF/kg was administered to rats by oral garage 3 days prior to iv injection of 0.1 or 0.3 μmol [14C]TCDF/kg. Biliary excretion and hepatic concentrations of [14C]TCDF were significantly increased in the pretreated animals. These results suggest an autoinduction of TCDF metabolism. Essentially all biliary [14C]TCDF radioactivity was attributable to metabolites. High-pressure liquid chromatography profiles of biliary radioactivity from 0 to 4 hr were qualitatively different between naive and pretreated rats. To determine if pretreatment with TCDD altered the metabolism of TCDF and vice versa, a single dose of 1.0 μmol TCDF/kg or 0.1 μmol TCDD/kg was administered by oral gavage 3 days prior to iv injection of 0.1 μmol [3H]TCDD or [14C]TCDF/kg, respectively. TCDD pretreatment increased the metabolism and hepatic concentrations of [14C]TCDF in pretreated rats, while biliary elimination and hepatic concentrations of [3H]TCDD were unaffected by pretreatment with TCDF. In a fourth experiment, the ability of TCDD to alter the metabolism of TCB, a laterally substituted polychlorinated biphenyl (PCB), was examined. Pretreatment with TCDD increased the metabolism of [14C]TCB by approximately twofold, but no differences in hepatic concentrations were seen due to the rapid elimination of TCB. Rats pretreated with Aroclor 1254, a commercial mixture of PCBs, demonstrated significantly increased metabolism of [14C]TCB compared to the naive group. Therefore, under these experimental conditions, induction of TCDF metabolism occurred in the rat upon pretreatment with either TCDD or TCDF at doses which also elicited enhanced hepatic uptake. TCDD and Aroclor 1254 induced the metabolism of TCB. These findings suggest that repeated exposure to a chemical or to a mixture of these halogenated aromatic hydrocarbons can result in more rapid metabolism and elimination from the body than following a single, acute exposure.

The Effect to Pretreatment on the Biliary Excretion of 2,3,7,8-Tetrachlorodibenzo-p-dioxin, 2,3,7,8-Tetrachlorodibenzofuran, and 3,3',4,4'-Tetrachlorobiphenyl in the Rat

The Effect of Pretreatment on the Biliary Excretion of 2,3,7,8-Tetrachlorodibenzo-p-dioxin, 2,3,7,8-Tetrachlorodibenzofuran, and 3,3′,4,4′-Tetrachlorobiphenyl in the rat. MCKINLEY, M. K., KEDDERIS, L. B., and BIRNBAUM, L. S. (1993). Fundam. Appl. Toxicol. 21, 425–432. The laterally halogenated chemicals 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 3,3′,4,4′-tetrachlorobiphenyl (TCB) exhibit the same spectrum of toxic effects as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototype and most toxic member of the halogenated aromatic hydrocarbon family. Metabolism of all three compounds appears to be the rate-limiting step for excretion, which is primarily via the bile into the feces. Therefore, the biliary elimination of TCDF, TCDD, and TCB was examined as an indirect measure of metabolism. Male F344 rats were anesthetized with pentobarbital, the bile duct was cannulated, and 0.1 μmol [3H]TCDD, [14C]TCDF, or [14C]TCB/kg body wt was administered iv. Bile was collected for 0–8 hr while the animals were kept under anesthesia. To determine if TCDF was able to induce its own metabolism in vivo, a single dose of 1.0 μmol TCDF/kg was administered to rats by oral gavage 3 days prior to iv injection of 0.1 or 0.3 μmol [14C]TCDF/kg. Biliary excretion and hepatic concentrations of [14C]TCDF were significantly increased in the pretreated animals. These results suggest an autoinduction of TCDF metabolism. Essentially all biliary [14C]TCDF radioactivity was attributable to metabolites. High-pressure liquid chromatography profiles of biliary radioactivity from 0 to 4 hr were qualitatively different between naive and pretreated rats. To determine if pretreatment with TCDD altered the metabolism of TCDF and vice versa, a single dose of 1.0 μmol TCDF/kg or 0.1 μmol TCDD/kg was administered by oral gavage 3 days prior to iv injection of 0.1 μmol [3H]TCDD or [14C]TCDF/kg, respectively. TCDD pretreatment increased the metabolism and hepatic concentrations of [14C]TCDF in pretreated rats, while biliary elimination and hepatic concentrations of [3H]TCDD were unaffected by pretreatment with TCDF. In a fourth experiment, the ability of TCDD to alter the metabolism of TCB, a laterally substituted polychlorinated biphenyl (PCB), was examined. Pretreatment with TCDD increased the metabolism of [14C]TCB by approximately twofold, but no differences in hepatic concentrations were seen due to the rapid elimination of TCB. Rats pretreated with Aroclor 1254, a commercial mixture of PCBs, demonstrated significantly increased metabolism of [14C]TCB compared to the naive group. Therefore, under these experimental conditions, induction of TCDF metabolism occurred in the rat upon pretreatment with either TCDD or TCDF at doses which also elicited enhanced hepatic uptake. TCDD and Aroclor 1254 induced the metabolism of TCB. These findings suggest that repeated exposure to a chemical or to a mixture of these halogenated aromatic hydrocarbons can result in more rapid metabolism and elimination from the body than following a single, acute exposure.

Primary colonic epithelial cell culture of the rabbit producing prostaglandins

We have established primary colonic epithelial cell culture from adult rabbits and examined effects of anti-inflammatory drugs on prostaglandin (PG) E 2 production. Colonic epithelium of adult rabbits was scraped and minced into small pieces. They were incubated for isolation in Hanks' balanced salt solution with 0.35 % collagenase and Earle's solution with 1 mM EDTA. Isolated cells were cultured in Coon's modified Ham's F-12 medium with 10 % fetal bovine serum and antibiotics on collagen coated cell wells. The medium was refed twice a week. The production of PGs was assessed by high pressure liquid chromatography (HPLC). PGE 2 and PGF 2α were measured by radioimmunoassay. Within 24 hours after inoculation, the cell clumps attached to the surface of the wells and cells began to spread out and grow. Monolayer cultures became confluent in 4 days. Phase contrast microscopy showed that these cells consisted of a homogeneous population of epithelial cells with large oval nuclei, polyhedral shape, and organized sheet-like growth pattern. HPLC profile showed synthesis of 6-keto-PGF 1α, thromboxane B 2, PGF 2α, PGE 2, and PGD 2 by cultured cells. Quantitatively, 117±7 ng/mg-protein/hour PGE 2 by 7.4±0.7 ng/mg-protein/hour PGF 2α were produced. While hydrocortisone (10 −4-10 −2 M) did not show a significant effect on PGE 2 production, indomethacin (10 −8-10 −6 M), and 5-aminosalicylic acid (2×10 −4-5×10 −3 M) inhibited PGE 2 production. We have established relatively convenient procedure for primary culture of colonic epithelial cells from adult rabbits. Different actions of anti-inflammatory drugs on PGE 2 synthesis suggest that these cultured cells might be a good tool for the various cellular functional studies of normal colonic epithelial cells.

Processing of TRH precursor peptides in rat brain and pituitary is zinc dependent

The enzymes responsible for the posttranslational processing of precursor proteins to form alpha-amidated peptide hormones require the availability of several cofactors, including zinc, copper and ascorbate ions. Major changes in the availability of these cofactors, as well as the rate of hormone precursor conversion to active hormone, occur during neonatal development, aging and caloric restriction. The effects of 6 weeks of a zinc-deficient (ZD1) diet, pair feeding (PF) and partial zinc deficiency (ZD6) compared to a control diet on the enzymatic cleavage and processing of prepro-TRH to form TRH have been studied in the hypothalamus, brain, and pituitary of young adult male Sprague-Dawley rats. Reverse phase high pressure liquid chromatography (HPLC) revealed that TRH was the major TRH-IR component of the hypothalamus, brain and pituitary. The effect of zinc deficiency on the TRH-Gly-IR HPLC profile of rat brain was to reduce selectively the area of the peaks for TRH-Gly and other low molecular weight pro-TRH peptide fragments with a C-terminal Gly compared to the corresponding TRH-Gly-IR peaks of the control group. We conclude that the processing of prepro-TRH to form TRH is zinc dependent via posttranslational processing enzymes such as carboxypeptidase H.

Eicosanoid synthesis by alveolar macrophages in rats with malignant mammary tumors: Differences in rats treated with and without carrageenan implants

Eicosanoid synthesis by alveolar macrophages (AM), harvested from tumor bearing animals, was measured after tumor inoculation in rats treated with or without carrageenan (carra), an immunomodulating agent. After incubaticn of the cells with [14]C-arachidonic acid and the Ca-ionophore A23187, samples were measured by high pressure liquid chromatography (HPLC). From the HPLC profiles the lypoxygenase products, 12-hydroxyeicosatetraenoic acid (12-HETE), 15-HETE, and leukotriene-B 4 (LTB 4) were determined as well as the cyclooxygenase products, prostaglandin (PG)E 2, PGF 2α and TXB 2. After tumor inoculation AM-synthesis of lipoxygenase products tended to increase to values twice those of the base line values, whereas cyclooxygenase products showed subnormal values. In the non treated animals, 10 days after tumor inoculation, statistically significant increases in 12- and 15-HETE, LTB 4 and PGE 2 were observed when compared with carra treated animals. Later measurements did not show these differences in AM metabolism. AM metabolism was (negatively) correlated with the number of macrophages, which was particularly evident in the correlation with 12-HETE synthesis.

HPLC profiles of mutagens in lean ground pork fried at different temperatures.

Ground lean pork was formed into patties and fried under ordinary conditions making sure that the crust was not charred. No fat was added when frying. The meat was fried at pan temperatures of 200 degrees C, 250 degrees C, and 300 degrees C until the temperature at the centre of the patties was either 65 degrees C or 70 degrees C. The crust was extracted with aqueous acid followed by concentration of the mutagens on an XAD-2 column and elution with acetone. The total mutagenic activity and high-pressure liquid chromatography (HPLC) analysis of the mutagenic components ("mutagrams") in the eluates were determined for the different frying procedures using the Salmonella/mammalian microsome test strain TA 98. Each 50 degrees C increase in the pan temperature (from 200 degrees C to 250 degrees C and from 250 degrees C to 300 degrees C) resulted in a doubling of the total mutagenic activity. The HPLC profiles of the mutagens were quite similar for the different frying temperatures, although a strong increase in the relative amount of more apolar mutagens was seen at 300 degrees C (the highest temperature). The major mutagenic activity of the HPLC fractions was confined to seven regions (mutagenic peaks) and a comparison of the HPLC profiles of the mutagens in fried beef and pork patties showed identical profiles. It is therefore concluded that the mutagenic compounds formed in fried beef and pork are similar in structure.

Metabolism of 7,12-dimethylbenz[ a]anthracene by mouse mammary epithelial cells cultured serum-free inside collagen gels

Mammary epithelial cells from 3–4-month-old BALB c virgin mice were cultured inside collagen gels in the following serum-free media: Dulbecco's Modified Eagle's Medium/Ham's F-12 (1:1) supplemented with (A) insulin, bovine serum albumin, epidermal growth factor; (B) insulin, bovine serum albumin, progesterone, prolactin; (C) insulin, bovine serum albumin, progesterone, prolactin, linoleic acid. Cell number increased with all media used. At day 7 of culture, [ 3H]dimethylbenz[ a]anthracene (DMBA) was added to the cultures and its metabolism to water soluble and organic soluble compounds was determined. Mouse mammary epithelial cells were able to metabolize [ 3H]DMBA to water and organosoluble metabolites. By 72 h, 77– 94% of the added DMBA had been metabolized by the epithelial cells in the three media to water and organosoluble metabolites in equivalent amounts. The distribution between water soluble and organosoluble metabolites was approximately equivalent. The high pressure liquid chromatography profiles of organosoluble fractions from the media indicated that the major products appeared to be the phenols, 2-, 3-, or 4-hydroxydimethylbenz[ a] anthracene, the hydroxymethyl derivatives, 7-methylbenz[ a]anthracene and 7-hydroxymethylbenz[ a]anthracene, trans-3,4-dihydro-3,4-dihydroxydimethylbenz[ a] anthracene and one or two major fractions eluting just behind the marker cis-5,6-dihydro-5,6-dihydroxydimethylbenz[ a]anthracene. The major fraction eluting just ahead of the cis-5,6-dihydro-5,6-dihydroxydimethylbenz[ a] anthracene was most likely trans-8,9-dihydro 8,9-dihydroxydimethylbenz[ a] anthracene. The profiles were similar for the cells cultured in all three serum-free media. The results demonstrate that mouse mammary epithelial cells cultured inside collagen gels with serum-free media can metabolize DMBA to putative carcinogenic forms.

Analysis of oligosaccharides from heparin by reversed-phase ion-pairing high-performance liquid chromatography

An ion-pairing high-pressure liquid chromatography procedure was developed for analysis of mixtures of oligosaccharides generated by nitrous acid cleavage of heparin. Oligosaccharides were eluted from a Hi-Chrom 5S ODS (C18) column using mixtures of acetonitrile and buffers containing 40 m m ammonium phosphate and 1 m m tetrabutylammonium phosphate. Isocratic conditions were developed for optimal separation of a number of individual disaccharides and tetrasaccharides that were characterized previously ( M. J. Bienkowski and H. E. Conrad (1985) J. Biol. Chem. 260, 356–365 ). These isocratic conditions were then coupled to obtain gradient elution conditions for the ion-pairing separations of mixtures of disaccharides and mixtures of tetrasaccharides. A comparison of the elution profiles obtained in the ion-pairing chromatography procedure with profiles obtained by anion-exchange high-pressure liquid chromatography profiles showed markedly better overall resolution by the ion-pairing procedure. As a result of this improved resolution, the new procedure showed the presence of previously unidentified products in the heparin oligosaccharide mixtures.

Effect of o-Benzyl- p-chlorophenol on drug-metabolizing enzymes in rats

o-Benzyl- p-chlorophenol (BCP) is widely used as a broad spectrum disinfectant. Treatment of male Fischer 344 rats with BCP resulted in an increase in cytochrome P-450 content and an accompanying decrease in aryl hydrocarbon hydroxylase (AHH) activity in both liver and kidney microsomes. Several other drug-metabolizing enzymes were not affected by BCP treatment. However, in kidney, BCP induced NADPH-cytochrome c reductase and uridine diphosphate glucuronyl transferase activities and caused a small increase in total cytochrome P-450 content and glutathione concentration. The cytochrome P-450 isozymes induced by BCP were fractionated by high pressure liquid chromatography (HPLC). The HPLC profile following BCP treatment most closely resembled that seen after phenobarbital. Using an immunoblotting procedure and a radioimmunoassay, it was shown that the increase in cytochrome P-450 content in the liver after BCP treatment was, in part, due to an increase in the phenobarbital-inducible isozymes, P-450b + e. In the kidney, the increase in total cytochrome P-450 content after BCP exposure was not due to an increase in P-450b + e. The decrease in AHH activity appeared to be caused by noncompetitive inhibition of constitutive AHH activity by BCP. BCP also inhibited benzphetamine demethylation, although to a lesser extent. The failure to observe an increase in benzphetamine demethylase activity in vivo, despite the induction of P-450b, was probably due to the concomitant induction and inhibition of drug-metabolizing enzymes by BCP.

Utilization of anion-exchange chromatography and monoclonal antibodies to characterize multiple pilus types on a uropathogenic Escherichia coli O6 isolate

Multiple pilus types from a uropathogenic strain of Escherichia coli O6, strain 6260, were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high-pressure liquid chromatography, binding assays, and erythrocyte adsorption. In addition, monoclonal antibodies were raised against purified pili of E. coli 6260 and used for immunological characterization. SDS-PAGE analysis of the purified pili showed at least three different subunits with molecular weights of 15,700, 17,800, and 19,300. SDS-PAGE analysis of four protein peaks from anion-exchange chromatography of intact pili showed polypeptides with molecular weights of 19,300 (fraction 1), 15,700 (fraction 2), and 17,800 and 15,700 (both fractions 3 and 4). Erythrocyte adsorption of the whole-pilus preparation removed the 17,800-molecular-weight subunit (17.8K subunit) and reduced the 15.7K subunit. Pili from an isogenic hemagglutination-negative variant of E. coli 6260, showing only the 15.7K and 19.3K subunits by SDS-PAGE, lacked the 17.8K subunit of fractions 3 and 4 present in the parent high-pressure liquid chromatography profile. Our data suggest that two of the pilus subunits, the 15.7K and 17.8K subunits, mediate mannose-resistant agglutination of human erythrocytes. Pili in fractions 1 and 2 from the parent strain bound specifically to mannose residues, while pili in fraction 4 bound to P-coated horse erythrocytes; no receptor specificity was identified for pili in fraction 3. Immunological analysis by the immunoblot technique showed that monoclonal antibody 11-2 reacted with the 19.3K subunit, monoclonal antibodies 34-3 and 73-3 reacted with the 15.7K subunit, and monoclonal antibodies 81-1, 82-1, and 91-1 reacted with polymers of subunits retained in the stacking gel. Intact pili precipitated by any of the six monoclonal antibodies showed two polypeptides by SDS-PAGE: 15.7K and 19.3K polypeptides for monoclonal antibody 11-2, and 15.7K and 17.8K polypeptides for monoclonal antibodies 34-3, 73-3, 81-1, 82-1, and 91-1. The cross-reactivity of the monoclonal antibodies with purified pili from other E. coli strains was determined by enzyme-linked immunosorbent assay. Monoclonal antibody 11-2 showed no significant cross-reactivity with heterogeneous pili. In contrast, the other monoclonal antibodies showed equivalent or greater reactivity with P pili from heterologous strains as compared with reactivity with E. coli 6260 pili.(ABSTRACT TRUNCATED AT 400 WORDS)