Enzyme-based high-performance liquid chromatography supports as probes of enzyme activity and inhibition: the immobilization of trypsin and alpha-chymotrypsin on an immobilized artificial membrane high-performance liquid chromatography support.

Immobilized artificial membrane (IAM) HPLC supports have been used to immobilize the enzymes alpha-chymotrypsin and trypsin. The enzymes were trapped in hydrophobic cavities on the support and were not covalently attached to the IAM surface. The resulting IAM-enzyme supports retained the hydrolytic activity of the immobilized enzymes: the IAM-trypsin support catalyzed the hydrolysis of N alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA), and the IAM-alpha-chymotrypsin support (IAM-ACHT) catalyzed the hydrolysis of a number of substrates, including tryptophan methyl ester. The activities of both supports were decreased by known enzyme inhibitors and the activity of the IAM-ACHT was affected by changes in pH and temperature. When a substrate was chromatographed on an IAM-ACHT HPLC, the hydrolytic activity of the immobilized enzyme could be determined from the resulting substrate/product ratios. These data were obtained either directly from the IAM-ACHT chromatogram or from the chromatogram produced by a coupled column system. The results of this study indicate that IAM-immobilized alpha-chymotrypsin and trypsin can be used as chromatographic probes for the qualitative determination of enzyme/substrate and enzyme/inhibitor interactions.     

Distribution of the enantiomers of hydroxychloroquine and its metabolites in ocular tissues of the rabbit after oral administration of racemic-hydroxychloroquine

The disposition of the enantiomers of hydroxychloroquine (HCQ) and its major metabolites in ocular tissues of rabbits has been studied. Both albino, New Zealand White (NZW), and pigmented animals were administered daily oral doses of rac-HCQ, (S)-HCQ or (R)-HCQ (20 mg/kg) over 1, 6, or 8 day periods or for 8 days followed by a 7-day washout period. At the end of the study periods, plasma and whole blood samples were collected and the rabbits were sacrificed. The eyes were collected, the aqueous humor removed with a syringe, and the eyes separated into the cornea, lens, vitreous body, iris, choroid-retina, sclera, and conjunctiva. The concentrations of (R)-HCQ, (S)-HCQ, and their respective metabolites were determined using a validated enantioselective liquid chromatographic assay. The data from these studies indicate that HCQ accumulated in both pigmented and nonpigmented ocular tissues. In the pigmented tissues, HCQ and its metabolites were bound to melanin and the binding was not enantiospecific. In the nonpigmented tissues and in the iris and retina-choroid of the NZW rabbits, the accumulation appeared to be the result of a reversible and enantioselective binding of HCQ and its metabolites to an unidentified biopolymer present in these ocular tissues. © 1994 Wiley-liss, Inc.     

Observaciones de campo sobre la biologia deMetriona elatior (Col.: Chrysomelidae) enSolanum elaeagnifolium (Solanaceae) del uruguay

Field observations on the biology ofMetriona elatior (Col.: Chrysomelidae) onSolanum elaeagnifolium (Solanaceae) from Uruguay. Specimens ofMetriona elatior were found feeding on leaves of the silverleaf nightshade,Solanum elaeagnifolium, from the eastern of Uruguay since 1984. According to a field study the young leaves were skeletonized since spring to early autumn by larvae and adults. Oothecae and pupae were found mainly on old leaves. The egg mean volume was of 0.614 mm³. Young plants of the weed were always killed after massive natural infections ofM. elatior. Therefore, this tortoise leaf-beetle may be studied as a potential biocontrol agent on populations ofS. elaeagnifolium.

     

An Artifact Of Liquid Emulsion Autoradiography

The localization of ³H-opiatcs in the myenteric plexus of the guinea pig ileum is subject to systematic artifact when stretch preparations of the myenteric plexus are dipped into liquid Kodak NTB-3 emulsion for autoradiography. The cause of the artifact was determined to be a discontinuous distribution, or retraction, of emulsion over plexuses. The apposition of frozen freeze-dried ilial sections to dried photographic emulsion avoids this source of error.     

Multiple Marker Detection in Peripheral Blood for NSCLC Diagnosis

Background

Non-invasive early detection of lung cancer could reduce the number of patients diagnosed with advanced disease, which is associated with a poor prognosis. We analyzed the diagnostic accuracy of a panel of peripheral blood markers in detecting non small cell lung cancer (NSCLC).

Methods

100 healthy donors and 100 patients with NSCLC were enrolled onto this study. Free circulating DNA, circulating mRNA expression of peptidylarginine deiminase type 4 (PAD4/PADI4), pro-platelet basic protein (PPBP) and haptoglobin were evaluated using a Real-Time PCR-based method.

Results

Free circulating DNA, PADI4, PPBP and haptoglobin levels were significantly higher in NSCLC patients than in healthy donors (p<0.0001, p<0.0001, p = 0.0002 and p = 0.0001, respectively). The fitted logistic regression model demonstrated a significant direct association between marker expression and lung cancer risk. The odds ratios of individual markers were 6.93 (95% CI 4.15–11.58; p<0.0001) for free DNA, 6.99 (95% CI 3.75–13.03; p<0.0001) for PADI4, 2.85 (95% CI 1.71–4.75; p<0.0001) for PPBP and 1.16 (95% CI 1.01–1.33; p = 0.031) for haptoglobin. Free DNA in combination with PPBP and PADI4 gave an area under the ROC curve of 0.93, 95% CI = 0.90–0.97, with sensitivity and specificity over 90%.

Conclusions

Free circulating DNA analysis combined with PPBP and PADI4 expression determination appears to accurately discriminate between healthy donors and NSCLC patients. This non-invasive multimarker approach warrants further research to assess its potential role in the diagnostic or screening workup of subjects with suspected lung cancer.     

Enantioselective separation of cyclic chiral ketones and their corresponding diastereomeric alcohols by HPLC on chiral and chiral/chiral coupled stationary phases

Enantioselective HPLC methods have been developed for the resolution of (RS)-2-phenylcyclohexanone (compound 1) and (RS)-2-phenyltetrahydropyran-4-one (compound 4) and the diastereoselective and enantioselective separations of their respective cis- and trans-alcohols; reduction of compound 1 yields trans- and cis-2-phenyl-1-cyclohexanol (compounds 2 and 3, respectively) and reduction of compound 4 yields trans- and cis-2-phenyl-tetrahydropyran-4-ol (compounds 5 and 6, respectively). Compounds 1, 2, and 3 were stereochemically resolved using a chiral stationary phase (CSP) based upon amylose tris(3,5-dimethylphenyl carbamate) coated on 10 μm silica-gel (Chiralpak AD-CSP). Compounds 4, 5, and 6 were stereochemically resolved on a coupled column system where a column containing a CSP based upon cellulose tris(3,5-dimethylphenyl carbamate) coated on 5 μm silica (Chiralcel OD-H-CSP) was coupled in series to the AD-CSP. The strategy employed in the identification of the peaks in the respective chromatograms is also discussed in this presentation. Chirality 8:551–555, 1996. © 1997 Wiley-Liss, Inc.     

Synthesis and characterization of an immobilized phenylethanolamine N-methyltransferase liquid chromatographic stationary phase

Norepinephrine is N-methylated to epinephrine by the catalytic effect of the terminal enzyme in catecholamine biosynthesis, phenylethanolamine N-methyltransferase (PNMT). PNMT has been covalently immobilized onto a silica-based liquid chromatographic support, glutaraldehyde-P (Glut-P). The resulting PNMT-Glut-P stationary phase (PNMT-SP) was enzymatically active, stable, and reusable. Standard Michaelis-Menten kinetic studies were performed with both free and immobilized PNMT and known substrates and inhibitors were examined. The results demonstrate that the PNMT-SP can be utilized for the rapid screening of potential PNMT substrates as well as the screening of compounds for PNMT inhibitory activity.