Synthesis and biological activity of [14a-3H]cryptopleurine

[14a-³H]Cryptopleurine was chemically synthesized from the perchlorate salt of 9,11,12,13,14,15-hexahydro-2,3,6-trimethoxyphenanthro(9,10-b)quinolizidinium by reduction with NaB³H₄. The [³H]cryptopleurine was recrystallized from acetone and further purified by chromatography through alumina using benzene as the eluting solvent. Both infrared and ultraviolet spectra of the labeled product were identical to those obtained using either the natural compound or the unlabeled synthetic compound. Thin-layer analysis on various solid supports using several different eluting solvents gave only one radioactive spot with a specific activity of 1438 Ci/mol, which in all cases cochromatographed with the natural sample. The [³H]cryptopleurine was also identical to the unlabeled compound in that it bound strongly to polyribosomes. 80 S ribosomes, and 40 S ribosomal subunits, all isolated from yeast. Binding was less strong using either 60 S ribosomal subunits or Escherichia coli ribosomes.     

Virus-induced alterations of lymphoid tissues. II. Lymphocyte receptors for Newcastle disease virus

Newcastle disease virus (NDV) agglutinates rat, mouse and human lymphocytes. Viral agglutination of rat thoracic duct lymphocytes was specifically inhibited by N-acetylneuraminic acid implying that the receptors terminate in sialic acid. While the attachment of virus to lymphocytes was rapid the reaction was unstable and NDV was shown to elute at 37 °C. Evidence was obtained that the eluting virus cleaved sialic acid from the surface of lymphocytes and concomitantly destroyed this lymphocyte receptor.     

Preparation and purification of pteroic acid from folic acid

Crude pteroic acid, obtained by microbiological degradation of folic acid, has been purified by column chromatography on cellulose CF11, eluting with 0.1 M glycine buffer of pH 10.0, containing 0.15% w/v ascorbic acid and saturated with isoamyl alcohol.     

Multi-step metabolic activation of benzene. Effect of superoxide dismutase on covalent binding to microsomal macromolecules,and identification of glutathione conjugates using high pressure liquid chromatography and field desorption mass spectrometry

Incubation of [¹⁴C]benzene or [¹⁴C]phenol with liver microsomes from untreated rats, in the presence of a NADPH-generating system, gave rise to irreversible binding of metabolites to microsomal macromolecules. For both substrates this binding was inhibited by more than 50% by addition of superoxide dismutase to the incubation mixtures. The decrease in binding was compensated for by accumulation of [¹⁴C]hydroquinone, indicating superoxide-mediated oxidation of hydroquinone as one step in the activation of benzene to metabolites binding to microsomal macromolecules. Since our previous work had shown that binding occurred mainly with protein rather than ribonucleic acid and was virtually completely prevented by glutathione, suggesting identity of metabolite(s) responsible for binding to protein and glutathione, a conjugate was chemically prepared from p-benzoquinone and reduced glutathione (GSH) and identified by field desorption mass spectrometry (FDMS) as 2-(S-glutathionyl) hydroquinone. Microsomal incubations, containing an NADPH-generating system, with benzene, phenol, hydroquinone or p-benzoquinone in the presence of [³H]glutathione or, alternatively, with [¹⁴C]benzene or [¹⁴C]phenol in the presence of unlabeled glutathione, were performed. All of these incubations gave rise to a peak of radioactivity eluting from the high pressure liquid chromatograph (HPLC) at a retention time identical to that of the chemically prepared 2-(S-glutathionyl) hydroquinone, whilst microsomal incubation of catechol in the presence of [³H]glutathione led to a conjugate with a very different retention time which was not observed after incubation of benzene or phenol. The microsomal metabolites of p-benzoquinone, hydroquinone and phenol thus eluting from the HPLC were further identified as the 2-(S-glutathionyl) hydroquinone by field desorption mass spectrometry. The glutathione adduct formed from benzene during microsomal activation eluted from HPLC with the same retention time and its mass spectrum also contained the molecular ion (MH⁺) (m/e 416) of this conjugate as an intense peak, but the fragmentation patterns did not allow definite assignments probably due to the considerably smaller amounts of ultimate reactive metabolites formed from this pre-precursor and thus relatively larger amounts of impurities.The results indicate that rat liver microsomes activate benzene via phenol and hydroquinone to p-benzosemiquinone and/or p-benzoquinone as quantitatively important reactive metabolites.     

Buffer induced modification of 6-sulfated disaccharide in chondroitin lyase digestions

High-performance liquid chromatographic analyses of chondroitin lyase AC or ABC hydrolysates revealed unexpected high content of material coeluting with the nonsulfated disaccharide 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyl uronic acid)-d-galactose. Incubation of a commercial preparation of the 6-sulfated disaccharide, 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyl uronic acid)-6-O-sulfo-d-galactose with “enriched Tris buffer” generated material coeluting with nonsulfated disaccharide. The amount of material exhibiting this anomalous chromatographic behavior was proportional to the amount of 6-sulfated disaccharide added to the incubation mixture. This suggested a precursor/product relationship between the 6-sulfated disaccharide and the anomalous peak. The result was specific for the 6-sulfated disaccharide: incubation of the 4-sulfated disaccharide, 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyl uronic acid)-4-O-sulfo-d-galactose, with enriched Tris buffer did not generate material with anomalous chromatographic properties. When [³⁵S]sulfate labeled cartilage glycosaminoglycans were hydrolyzed with chondroitin lyases, some of the radioactivity coeluted with the nonsulfated disaccharide. Thus, buffer-induced modification of 6-sulfated disaccharide was not caused by hydrolysis of ester sulfate. Although the proportion of the 6-sulfated disaccharide which was recovered in the anomalous peak was constant for incubations done simultaneously, incubations done at different times gave variable results. Thus, control incubations of 6-sulfated disaccharide with chondroitinase buffer must be included with each reaction series to allow correction for the proportion of the material eluting with nonsulfated disaccharide which is actually 6-sulfated.     

Transfer Ribonucleic Acid in KB Cells Infected with Adenovirus Type 2

Populations of transfer ribonucleic acid (tRNA) extracted from control and type 2 adenovirus (Ad2)-infected KB cells were compared. No consistent differences in acceptor activity for 11 amino acids were observed. Comparison of methylated albumin-kieselguhr (MAK) elution profiles of arginyl-tRNA from control and infected cells revealed a minor modification in that the proportion of arginyl-tRNA eluting at high salt concentration was somewhat greater in infected cells. No similar differences were observed in MAK elution profiles of aspartyl-, isoleucyl-, leucyl-, phenylalanyl-, seryl-, tyrosyl-, and valyl-tRNA. Hybridization of 4S RNA from infected cells labeled by incorporation of ³H-uridine with Ad2 deoxyribonucleic acid revealed the presence of a complementary species of RNA in this preparation. Hybridization of ³H-arginyl-tRNA and of ³H-aminoacyl-tRNA labeled by charging with ³H-arginine or a ³H-mixture of amino acids, respectively, failed to detect the presence of virus-specific tRNA in Ad2-infected cells.     

Immunity repressor of bacteriophage P2: Identification and DNA-binding activity

The product of gene C of the temperate bacteriophage P2, the immunity repressor, can be detected as a unique band eluting from phosphocellulose columns at 0.12 m-potassium phosphate when differentially labelled with a radioactive amino acid: the band is absent when phages that either have lost gene C through deletion or carry a suppressor-sensitive mutation in the gene are used. The repressor in its monomeric form is about 11,000 in molecular weight. At near physiological salt concentrations, the form predominantly recovered is the dimer.In filter-binding assays, the partially purified repressor binds wild-type P2 DNA strongly. It does not bind DNA of P2 vir94, a deletion that removes all the genetic elements involved in the regulation of lysogeny; it also does not bind, or binds inefficiently, DNA of P2 vir3, a mutation in the operator that controls the early replicative functions of P2. At the concentrations employed, the dimer is the active form in binding.The P2 repressor clearly differs in several features from the well-studied immunity repressor of bacteriophage lambda.     

Fractionation of plant Polyadenylated RNA on the basis of poly(A) size

Polyadenylated RNA from Vicia faba meristematic root cells was fractionated on the basis of mean poly(A) size by a thermal stepwise elution from poly(U) Sepharose. Such a procedure allowed the elimination of contaminating RNA at 30° and the collection of two populations of purified polyadenylated RNA at 40° and 50°, respectively. RNA eluting at the higher temperature carried a poly(A) segment (mean size of 100 nucleotides), twice as large as the RNA eluting at the lower temperature.     

Multiple forms of hydroxycinnamate : CoA ligase in etiolated pea seedlings

A survey of a range of plant tissues showed that the hydroxycinnamate CoA ligase in crude extracts of pea shoots had a high relative activity towards sinapic and other methoxycinnamic acids, together with high activity with p-coumaric acid. The pea enzyme has been resolved by chromatography on DEAE-cellulose into two peaks which differ in their substrate specificity. The form which elutes at relatively low salt concentrations has a ratio activity towards p-coumaric and sinapic acids of about 1.8:1 while the form eluting at higher salt concentrations, although showing very high activity with p-coumaric acid, is inactive towards sinapic acid. The pattern of elution of these forms following gel filtration on Ultragel AcA 34 and Sephadex G100 suggests that these two isoenzymes which differ in ionic properties and substrate specificity can exist in two or three molecular weight forms and there is evidence that these forms are under certain circumstances interconvertible.     

Polyamine metabolism in ripening tomato fruit : I. Identification of metabolites of putrescine and spermidine

The metabolism of [1,4-¹⁴C]putrescine and [terminal methylene-³H]spermidine was studied in the fruit pericarp (breaker stage) discs of tomato (Lycopersicon esculentum Mill.) cv Rutgers, and the metabolites identified by high performance liquid chromatography and gas chromatography-mass spectrometry. The metabolism of both putrescine and spermidine was relatively slow; in 24 hours about 25% of each amine was metabolized. The ¹⁴C label from putrescine was incorporated into spermidine, γ-aminobutyric acid (GABA), glutamic acid, and a polar fraction eluting with sugars and organic acids. In the presence of gabaculine, a specific inhibitor of GABA:pyruvate transaminase, the label going into glutamic acid, sugars and organic acids decreased by 80% while that in GABA increased about twofold, indicating that the transamination reaction is probably a major fate of GABA produced from putrescine in vivo. [³H]Spermidine was catabolized into putrescine and β-alanine. The conversion of putrescine into GABA, and that of spermidine into putrescine, suggests the presence of polyamine oxidizing enzymes in tomato pericarp tissues. The possible pathways of putrescine and spermidine metabolism are discussed.     

Affinity chromatographic purification of morphine antibody.

Morphine-6-hemisuccinate was synthesized and linked to agarose affinity beads by either direct amide bond formation or by an N-hydroxysuccinimide ester intermediate using various conditions. The various preparative routes resulted in differing ampunts of covalently bound ligand. Affinity chromatography of morphine antisera with a variety of eluting solvents indicated that 0.5 M acetic acid and 1 M propionic acid were most efficacious for eluting the bound antibody. Affinity isolation of a papain digest of purified antibody yielded fragments with reactivity and other characteristics consistent with their being designated as morphine antibody Fab fragments.     

New internal standards for basic amino acid analyses

Eight derivatives of cysteine and penicillamine with 2- and 4-vinyl-pyridine, p-nitrobenzyl bromide, and p-nitrostyrene were evaluated as potential internal standards for the short and long (physiological) basic columns in amino acid analysis by ion-exchange chromatography. S-β-(4-pyridylethyl)-dl-penicillamine (4-PEP) was found to have an advantage over the previously proposed S-β-(4-pyridylethyl)-l-cysteine (4-PEC) since the elution position of 4-PEP on the short basic column is insensitive to minor changes in pH of the eluting citrate buffer. 4-PEP was found to be stable to acid hydrolysis as used for proteins and its recovery from protein hydrolysates was unaffected by the presence of starch during hydrolysis. However, an extra 14 min is required to elute 4-PEP on the short column.Of the eight compounds studied, six appcar suitable as internal standards on the physiological (long) column. These elute in widely differing positions between histidine and arginine, thus offering a choice of internal standards for special analysis on the basic long column.     

Drug-eluting stents in the treatment of proximal vertebral artery stenosis

The objective of the study was to evaluate the efficacy of endovascular revascularization treatment using drug-eluting stents in patients with atherosclerotic proximal vertebral artery (VA) stenosis. Thirty-two patients (61 ± 10 years old) were implanted with 35 sirolimus and tacrolimus eluting stents (3 patients had them from two sides). 27 patients (84%) had vertebrobasilar symptoms at enrollment. All patients were pretreated with dual antiplatelet therapy. The intervention was technically successful in 89% cases. No stroke, myocardial infarction, or death occurred in perioperative period. On duplex scanning the stents remained completely functional. In the late postoperative period 29 (91%) patients, with 32 implanted stents were followed- up. The mean follow-up was 9.5 months. No stroke occurred in patients during this period. Recurrence of vertebrobasilar insufficiency symptoms was noted in 3 patients. VA renarrowing was detected in 16 (50%) arteries in 15 patients, and 12 (80%) of them were asymptomatic. Restenosis ≥50% (n = 13) and reocclusion (n = 3) were more frequent in those with implantation of tacrolimus eluting stents compared to those with sirolimus eluting stents: 10 (71%) of 14 observations to 6 (33%) of 18 cases (p = 0.1794), respectively. Stent fracture was observed in 2 cases (6%), followed by restenosis. Restenosis rate prevailed in men (p = 0.0173). Thus, stenting of VA extracranial portion is reasonably safe procedure with a good clinical effect. The use of drug-eluting stents looks promising but does not solve the problem of high restenosis rate in the late postoperative period.     

Detection of nonenzymatic browning products in the human lens

The nonenzymatic browning or Maillard reaction is an aging process in stored foods. The initial stage of this reaction, nonenzymatic glycosylation, has been shown to occur in the human lens. The possible occurrence of further steps of the Maillard reaction involving lysine residues and glucose has been investigated. A lipid-free protein extract from a pool of human cataractous lenses was reduced, alkylated, and digested with pronase. The digest was reduced with [³H]borohydride, acid hydrolyzed and fractionated by Sephadex G-15 chromatography. The fractions eluting ahead of ?-1-deoxyglucitolyllysine were pooled and separated with an amino acid analyzer. Four fluorescent, yellow, and radioactive peaks were obtained. One of these, which co-eluted with tyrosine, was isolated, acetylated, and further analyzed by reverse phase chromatography using HPLC. Two new peaks were separated which co-chromatographed with lysine derivatives isolated from the nonenzymatic browning reaction of α-tert-butyloxycarbonyllysine with glucose. Control experiments showed that they were not artifacts due to acid hydrolysis of ?-glucitolyllysine. These results suggest that dehydration and rearrangement of the Amadori product, ?-fructosyllysine, has occured in vivo, thus leading to the formation of at least two nonenzymatic browning products.     

Protein Kinase Activities in Neurospora crassa

Several protein kinase activities have been found in 105,000g supernatant of Neurospora crassa mycelia grown up to the logarithmic phase. By chromatography on DEAE-cellulose the following enzyme activities have been resolved: (i) a cyclic AMP-dependent protein kinase (peak I kinase) eluting at 0.20 m NaCl, more active with histone than with phosvitin (it was inhibited by both a thermolabile fraction having cyclic AMP-binding activity and a thermostable inhibitor isolated from 105,0005g mycelial supernates), (ii) a cyclic nucleotide-independent protein kinase (peak II kinase) eluting at 0.35 m NaCl, also more active with histone than with phosvitin (this kinase was not inhibited by the fraction having cyclic AMP-binding activity but it was sensitive to the thermostable inhibitor); and finally, (iii) a protein kinase eluting at 0.43 m NaCl (peak II kinase), with similar activity toward histone and phosvitin, insensitive to cyclic nucleotides and to fractions carrying cyclic AMP-binding capacity (this kinase activity also resulted insensitive to the thermostable inhibiting factor).     

Isolation of gibberellin precursors from heavily pigmented tissues

The kauranoid precursors of gibberellins are difficult to isolate from heavily pigmented plant tissues. In this paper, we describe relatively simple and efficient procedures for the purification of these compounds from tissues containing chlorophyll and other high molecular weight pigments. Extracts of shoots from Thlaspi arvense L. were subjected first to size exclusion chromatography using ethyl acetate as the eluting solvent. This procedure resulted in the separation of kauranoids as a class of compounds from chlorophyll. Typically, a 90% reduction in mass of the kauranoid enriched-fraction was observed. This fraction was subjected to reverse phase high performance liquid chromatography and individual fractions analyzed by combined gas chromatography-mass spectrometry. Five kauranoids were identified in shoot extracts of T. arvense: ent-kaur-16-ene, ent-kaur-16-en-19-ol, ent-kaur-16-en-19-oic acid, trachylobanoic acid, and 7β, 13-dihydroxykaurenolide. The metabolic relationships of these compounds to the gibberellins previously identified in this species (JD Metzger, MC Mardaus [1986] Plant Physiol 80: 396-402) are discussed. In addition, the utility of size exclusion chromatography in preparative situations is demonstrated by the purification of ent-kaurenoic acid in milligram quantities from the florets of Helianthus annuus L.     

Separation of naturally occurring 3',5'-cyclic ribonucleotides using high pressure liquid chromatography.

A simple and fairly rapid procedure is described for separation of a mixture of five 3′, 5′-cyclic ribonucleotides. The separation was achieved by high pressure liquid chromatography on a Vydac pellicular anion exchange column using a linear gradient of 0.5 to 1.0 m acetic acid as the eluting solvent.     

Isolation of Fasciola hepatica genus-specific antigens

Santiago de Weil N., Hillyer G. V. and Pacheco E. 1984. Isolation of Fasciola hepatica genus-specific antigens. International Journal for Parasitology14: 197–206. The Fasciola hepatica antigens which induce antibody formation in acute fascioliasis were isolated by acid elution after reacting an F. hepatica tegument antigen extract with a CNBr-Sepharose 4B column coupled with IgG obtained from the serum of rabbits infected with fascioliasis for 6–10 weeks. These isolated antigens were further separated by gel filtration using a column packed with Sephacryl S-200. In this manner three major peaks were obtained. The best serologic antigens were found in peak 2 which had a mol. wt range of 14,000–43,000. This peak contains genus-specific F. hepatica antigens which are highly reactive with fascioliasis serum. These antigens do not cross-react with either Schistosoma mansoni or with bovine serum albumin by gel diffusion. Monitoring by ELISA and gel diffusion with heterologous and homologous antisera showed that as purification by antibody affinity chromatography proceeded, cross reactivity with S. mansoni was eliminated. The rabbit antiserum obtained against peak 2, when tested by immunoelectrophoresis with a crude F. hepatica extract shows one main band identical to the main band observed with serum from acutely infected rabbits. Up to two other minor bands can be detected using concentrated homologous antisera. Fractions obtained from preparative iso-electric focusing of the F. hepatica tegument extract were reacted with sera from rabbits with acute fascioliasis. Two main bands were observed in immunodiffusion with antigens eluting in a pH range of 7.4–8.7. When these fractions were monitored with anti peak 2 antisera, two precipitin bands appeared with antigens eluting in a pH range of 7.4–7.9. The F. hepatica genus-specific antigen pool was applied to ELISA to evaluate its ability to detect antibody in a primary F. hepatica infection in rabbits. A rise in absorbance values could be detected by 2 weeks of infection, reached high levels by 6 weeks and remained high through 28 weeks of infection.     

Characterization of the low mol. wt zinc-binding ligand from rat small intestine by comparison to the organic zinc-binding ligands

1. 65Zn complexes of picolinate (PA), citrate (CA), L-histidine (L-his), arachidonic acid (AA) or low mol. wt zinc-binding ligand from rat intestine (LMW-ZBL) gave 65Zn eluting peak fraction numbers of 53, 53, 56, 59 and 59 respectively, in a Sephadex G-75 column chromatography. 2. The 65Zn eluting peak fraction numbers with CA, L-his, PA, prostaglandin (PG)E2, AA, no ligand, arachidonate (AT) or LMW-ZBL were 49, 50, 54, 55, 58, 64, 75 and 76 respectively in a Sephadex G-25 column chromatography. 3. In a Sephadex G-15 column chromatography, the 65Zn eluting peak fraction numbers with CA, PGE2, AA, L-his, LMW-ZBL or PA were 49, 50, 51, 52, 52 and 55 respectively. 4. The LMW-ZBL in rat small intestine appears to be an AA-like substance.     

Affinity purification of immunoglobulin M using a novel synthetic ligand

While monoclonal antibodies of the G class can be conveniently purified by affinity chromatography using immobilized protein A or G, even on a large scale, scaling up IgM purification still presents several problems, since specific and cost-effective ligands for IgM are not available. A synthetic peptide (TG19318), deduced from the screening of a combinatorial peptide library, was characterized previously by our group for its binding properties for immunoglobulins of the G class and its applicability as a synthetic ligand for polyclonal and monoclonal IgG purification, from sera or cell culture supernatants. In this study, we have examined the ligand recognition properties for IgM, immobilizing the synthetic peptide on different affinity supports and examining its ability to purify IgMs from serum, ascitic fluid and cell culture supernatants. TG19318 affinity columns proved useful for a very convenient one-step purification of monoclonal IgMs directly from crude sources, loading the samples on the columns equilibrated with saline buffers at pH values ranging from 5 to 7, and eluting adsorbed IgM by a buffer change to 0.1 M acetic acid or 0.05–0.1 M sodium bicarbonate, pH 9.0. Antibody purity after affinity purification was very high, close to 85–95%, as determined by densitometric scanning of sodium dodecyl sulfate–polyacrylamide gels of purified fractions, and by gel permeation analysis. Antibody activity was fully recovered after purification, as determined by immunoassays. Column capacity was related to the type of support used for ligand immobilization, and ranged from 2 to 8 mg of IgM/ml of support.