Gas chromatographic/mass spectrometric detection of narcotine, papaverine, and thebaine in seeds of Papaver somniferum

In addition to codeine and morphine, three more compounds: narcotine (noscapine), papaverine, and thebaine were found in Indian and Netherlands poppy seeds (Papaver somniferum L). The compounds were detected by a GC/MS technique and the identities were confirmed by comparing retention times and ion ratios with the known references. The concentrations of codeine, morphine, thebaine, papaverine, and narcotine were 44, 167, 41, 67, and 230 micrograms/g in Indian poppy seeds, and were 1.8, 39, 1.0, 0.17, 0.84 micrograms/g in Netherlands poppy seeds, respectively. Because these compounds may be urinary products after poppy seed consumption, the lowest detectable concentrations of codeine, morphine, thebaine, papaverine, and narcotine in urine are of interest and were found to be 4, 4, 5, 0.4, and 4 ng/ml, respectively. The detection of urinary narcotine, papaverine, or thebaine may be utilized to differentiate poppy seed consumption from illicit codeine, morphine, or heroin use.     

Race and clinical outcome in patients with carcinoma of the uterine cervix treated with radiation therapy

Epidemiologic and in vitro data have shown that the association of house-dust mite (HDM) allergy and snail allergy in the same patients was due to cross-reactivity between HDM and snail allergenic components. However, the cross-reacting allergen(s) have not yet been identified. In vitro reactivity of seven patients' sera to the various extracts and hemolymph of four different Helix snail species was analyzed by IgE detection and immunodots and Western blots. Cross-reactivity between snails and Dermatophagoides pteronyssinus was assessed by immunodot and ELISA inhibition in two patients. Heterologous inhibition of the snail immunodot and ELISA was observed in one serum. Western blotting showed a specific binding on all four snail species extracts; molecular weights of snail allergens ranged from < 21 to 200 kDa. Marked individual differences were observed in the seven sera under study; most sera demonstrated IgE recognition of multiple bands, illustrating that no single allergen is responsible for cross-reactivity between snail and mite. These results confirm that cross-reactivity exists between snails of the Helix genus and HDM. This cross-reactivity, involving more than a single allergen, may be of clinical significance in atopic patients allergic to D. pteronyssinus. The identity of the cross-reacting allergens remains to be determined. Potential candidates include the thermostable minor allergens of D. pteronyssinus, tropomyosin and hemocyanin.     

Cloning, expression and isoform classification of a minor oleosin in sesame oil bodies

The oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins termed oleosins. Two distinct oleosins are present in the oil bodies of diverse angiosperms, and classified as high and low Mr isoforms according to their relative molecular masses in each species. In sesame oil bodies, besides the two ubiquitous oleosin isoforms (17 and 15 kDa), an additional minor oleosin (15.5 kDa) was revealed on Tricine SDS-PAGE. A full-length cDNA fragment was cloned, sequenced and deduced to be a putative oleosin of 15,446 Da. The gene was constructed in a fusion or non-fusion vector and then over-expressed with different efficiency in Escherichia coli. All three oleosins purified from sesame oil bodies were subjected to immunoassaying using antibodies raised against the over-expressed oleosin. The results confirmed that this gene encodes the sesame 15.5 kDa oleosin. Sequence comparisons with other known oleosins revealed that sesame 15.5 kDa oleosin does not represent a new oleosin isoform class but may have been derived through gene duplication and truncation of sesame 17 kDa oleosin, and possesses the minimal structure of the high Mr oleosin isoform. A conserved amphipathic alpha-helix is predicted in sesame 15.5 kDa oleosin, which may imply a potential biological function associated with this isoform.     

Identification of three novel unique proteins in seed oil bodies of sesame

Plant seeds store triacylglycerols in discrete organelles called oil bodies. An oil body preserves a matrix of triacylglycerols surrounded by a monolayer of phospholipids embedded with abundant structural proteins termed oleosins and probably some uninvestigated minor proteins of higher molecular mass. Three polypeptides of 27, 37, and 39 kDa (temporarily denominated as Sop1, Sop2, and Sop3) were regularly co-purified with seed oil bodies of sesame. Comparison of amino acid composition indicated that they were substantially less hydrophobic than the known oleosins, and thus should not be aggregated multimers of oleosins. The results of immuno-recognition to sesame proteins extracted from subcellular fractions of mature seeds, various tissues, and oil bodies purified from different stages of seed formation revealed that these three polypeptides were unique proteins gathered in oil bodies, accompanying oleosins and triacylglycerols, during the active assembly of the organelles in maturing seeds. Both in vivo and in intro, immunofluorescence labeling using secondary antibodies conjugated with FITC (fluorescein isothiocyanate) confirmed the localization of these three polypeptides in oil bodies.     

Effects of insulin on calcium metabolism and platelet aggregation

Poppy seeds from seven different origins (Dutch, Australian, Hungarian, Spanish, Czech, and two Turkish) were analyzed for the amount of opiates present. Four grams of each kind of seeds, equivalent to the amount of seeds on two bagels, were ingested by volunteers. One volunteer also ingested four times the same amount of poppy seeds from the same origin (Spanish). During 24 hours urine samples were obtained and screened for the presence of morphine and codeine using the FPIA technique (cut-off = 200 ng/mL) and a GC/MS confirmation with a limit of detection (LOD) of 25 ng/mL for codeine and morphine. Poppy seeds from different origins contain a wide variation of morphine (2-251 micro g/g) and codeine (0.4-57.1 micro g/g) content. No other opiate could be detected. After ingestion a large interindividual variation of excretion of opiates exists. The testing results from the same kind of seeds ingested four times with a one week interval by the same volunteer also show a poor reproduceability. Several kinds of poppy seeds can give positive testing results (Australian, Hungarian, Spanish and one kind of Turkish seeds). Within 24 hours all testing results became negative.     

Pathological gambling: a review of the literature

The aim of this study was to characterize human anti-Rhesus monoclonal antibodies cross-reacting with tissue antigens. Of the 155 monoclonal alloantibodies tested, 49 also reacted with intracellular antigens, as demonstrated by immunofluorescence assay on cryostat sections of animal and human tissues. This cross-reactivity was mainly a property of monoclonal alloantibodies belonging to the IgM isotype (among the 49 cross-reacting Mabs, 37 were IgM). The results confirm that during an immune response against a foreign antigen (alloantigen), B cells that produce polyreactive antibodies are not excluded from the pool of responding cells.     

A simple method for producing a monospecific antiserum against IgM

To avoid the chemical purification of mastomys IgM, a reliable alternative method for producing a specific anti-mastomys IgM antiserum was developed. It employs the strong cross-reactivity of antisera against mouse serum proteins with mastomys serum proteins. By the immunoelectrophoresis technique, precipitates of mastomys IgM and cross-reacting rabbit anti-mouse IgM were obtained. A rabbit was immunized with these precipitates. Activity against the light chains was removed from the antiserum by passage over an immunoadsorbent column of mastomys IgG. The adsorbed antiserum was found to be specific for determinants on mastomys IgM.     

Low nutritional quality of unconventional tropical crop seeds in rats

As the search for alternative sources of food to alleviate hunger continues, this study was undertaken to determine the biological value in growing rats (BV) of proteins of some lesser known tropical seeds gathered in Nigeria. Antinutritional factors (trypsin inhibitors, phytic acid, oxalate, tannin, alkaloids) and amino acid compositions were also determined, and protein digestibility-corrected amino acid score (PDCAAS) was calculated using the amino acid requirement pattern of the preschool child and individual seed-specific correction factors for crude protein. A rat growth and balance study was conducted to determine digestibility, nitrogen-, and energy balance by feeding as the only unsupplemented protein source milled and heat-treated seeds of Adansonia digitata (Bombacaceae) and Prosopis africana, Lonchocarpus sericeus, Enterolobium cyclocarpium, Sesbania pachycarpa and Pterocarpus osun (Leguminosae) in comparison to casein fortified with methionine (control). Diets containing P. africana and L. sericeus seeds caused poor feed intake and weight loss in rats and were excluded from the nitrogen-balance test. Among the seed samples, S. pachycarpa followed by A. digitata showed the most advantageous nutritional quality [amino acid composition, digestibility, BV and net protein utilization (NPU)]. True digestibility was 82.9 and 74.5 vs. 98.5, BV was 64.6 and 70.0 vs. 90.4, and NPU was 53.5 and 52.1 vs. 89.0 for S. pachycarpa and A. digitata vs. casein (control), respectively. In terms of PDCAAS, lysine was the first limiting amino acid for S. pachycarpa (88%) and for A. digitata (58%). The PDCAAS of all essential amino acids was below 100% for E. cyclocarpium (e.g., cysteine + methionine: 37%) and for P. africana (e.g., threonine: 46%, except valine and a very high content of cysteine and methionine). In conclusion, all seeds tested in the rat balance trial were of inferior quality compared to casein. Before these tropical seeds could be used as food components or feed supplements, safety studies and proper processing to remove antinutritional factors and possible toxic constituents were required.     

Molecular characterization of berberine bridge enzyme genes from opium poppy

In Papaver somniferum (opium poppy) and related species, (S)-reticuline serves as a branch-point intermediate in the biosynthesis of numerous isoquinoline alkaloids. The berberine bridge enzyme (BBE) ([S]-reticuline:oxygen oxidoreductase [methylene bridge forming], EC 1.5.3.9) catalyzes the stereospecific conversion of the N-methyl moiety of (S)-reticuline into the berberine bridge carbon of (S)-scoulerine and represents the first committed step in the pathway leading to the antimicrobial alkaloid sanguinarine. Three unique genomic clones (bbe1, bbe2, and bbe3) similar to a BBE cDNA from Eschscholtzia californica (California poppy) were isolated from opium poppy. Two clones (bbe2 and bbe3) contained frame-shift mutations of which bbe2 was identified as a putative, nonexpressed pseudogene by RNA blot hybridization using a gene-specific probe and by the lack of transient expression of a chimeric gene fusion between the bbe2 5' flanking region and a beta-glucuronidase reporter gene. Similarly, bbe1 was shown to be expressed in opium poppy plants and cultured cells. Genomic DNA blot-hybridization data were consistent with a limited number of bbe homologs. RNA blot hybridization showed that bbe genes are expressed in roots and stems of mature plants and in seedlings within 3 d after germination. Rapid and transient BBE mRNA accumulation also occurred after treatment with a fungal elicitor or with methyl jasmonate. However, sanguinarine was found only in roots, seedlings, and fungal elicitor-treated cell cultures.     

A new method for seed oil body purification and examination of oil body integrity following germination

Plant seeds store triacylglycerols as energy sources for germination and postgerminative growth of seedlings. The triacylglycerols are preserved in small, discrete, intracellular organelles called oil bodies. A new method was developed to purify seed oil bodies. The method included extraction, flotation by centrifugation, detergent washing, ionic elution, treatment with a chaotropic agent, and integrity testing by use of hexane. These processes subsequently removed non-specifically associated or trapped proteins within the oil bodies. Oil bodies purified by this method maintained their integrity and displayed electrostatic repulsion and steric hindrance on their surface. Compared with the previous procedure, this method allowed higher purification of oil bodies, as demonstrated by SDS-PAGE using five species of oilseeds. Oil bodies purified from sesame were further analyzed by two-dimensional gel electrophoresis and revealed two potential oleosin isoforms. The integrity of oil bodies in germinating sesame seedlings was examined by hexane extraction. Our results indicated that consumption of triacylglycerols reduced gradually the total amount of oil bodies in seedlings, whereas no alteration was observed in the integrity of remaining oil bodies. This observation implies that oil bodies in germinating seeds are not degraded simultaneously. It is suggested that glyoxisomes, with the assistance of mitochondria, fuse and digest oil bodies one at a time, while the remaining oil bodies are preserved intact during the whole period of germination.     

A probarley lectin processing enzyme purified from Arabidopsis thaliana seeds

An aspartic proteinase was purified from the seeds of Arabidopsis thaliana (ecotype RLD) using affinity chromatography on pepstatin-agarose and ion exchange chromatography. The purified enzyme is optimally active at pH 3.5 and completely inhibited by pepstatin A. The purified Arabidopsis aspartic proteinase contains four subunits (apparent molecular weights 31, 28.5, 15 and 6 kDa), two of which are probably linked by disulfide bridges. These properties are similar to the aspartic proteinase previously isolated from barley seeds. The amino acid sequence of the peptide subunits corresponds exactly with the sequence of the previously isolated cDNA for the Arabidopsis aspartic proteinase. The Arabidopsis enzyme processed probarley lectin in vitro at the carboxy-terminus between phenylalanine and alanine, the same place where the barley enzyme cleaves the lectin in vitro. The aspartic proteinase appears to be the major enzyme processing the lectin in seeds as pepstatin A inhibited this activity in a crude seed extract.     

Lysine: arginine ratio of a protein influences cholesterol metabolism. Part 1--Studies on sesame protein having low lysine: arginine ratio

The effect of globulin fraction with a lysine: arginine (lys:arg) ratio 0.67, isolated from sesame (Sesamum Indicum) seeds on cholesterol metabolism was studied in rats fed cholesterol free and cholesterol containing diet and compared with casein (lys:arg ratio-2.0). Rats fed sesame seed globulin showed significantly lower concentrations of cholesterol in the serum and aorta. The decrease in serum was manifested in both HDL and LDL + VLDL fractions. There was increased cholesterogenesis in the liver as was evident from increased incorporation of labeled acetate into cholesterol and increased activity of 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Increased hepatic diversion of cholesterol to bile acid synthesis and increased fecal excretion of bile acids and sterols were also observed in rats fed sesame seed globulins. Rats fed sesame globulins also showed significantly higher activity of lipoprotein lipase in the heart and adipose tissue and that of plasma Lecithin: cholesterol acyltransferase (LCAT). These studies suggest that low lysine: arginine ratios of a protein exert hypocholesterolemic effects.     

Sesame seed and sesame seed oil contain masked allergens of growing importance

Sesame seed and sesame seed oil have been thought of as rare causes of food allergy, representing less than 1% of all food allergy cases. We now report nine cases of IgE-dependent allergy to sesame seed and/or sesame seed oil, six of which were diagnosed in 1995 alone. Our skin test results draw attention to the poor quality of a commercial sesame seed extract and the good sensitivity of skin prick tests made with a freshly prepared sesame seed flour extract. The diagnosis of this food allergy was established by double-blind oral provocation tests, with doses of sesame seed flour ranging from 100 mg to 10 g. Allergy to sesame seed oil was also demonstrated in some cases. The sensitivity of the Pharmacia Phadebas CAP System for the detection of sesame seed-specific IgE was only mediocre. We draw attention to the important use of sesame seed in modern cooking, a fact which may explain the growing frequency of this allergy. We underline the particular risk with sesame seed oil. Sesame seed should also be considered a cause of allergic reactions to drug products and cosmetics.     

Cross-reactivity of the NPa and NPb idiotypic responses of BALB/c and C57BL/6 mice to (4-hydroxy-3-nitrophenyl)acetyl (NP)

A comparative antigenic analysis was carried out to determine whether cross-reactivity exists between the major idiotypic responses to (4-hydroxy-3-nitrophenyl)acetyl (NP) in BALB/c and C57BL/6 mice. Extensive cross-reactivity exists between the NPa (BALB/c) and NPb (C57BL/6) allotype-linked idiotypic responses to NP. The cross-reactive determinants of the NPb idiotype are confined to one particular group of NPb-positive monoclonal antibodies. The extent of cross-reactivity between this group of C57BL/6 antibodies and idiotype-positive monoclonal antibodies of BALB/c is so great that they cannot be readily distinguished as NPb- or NPa-positive antibodies with polyclonal anti-idiotypic reagents. That this cross-reactivity is not unique to monoclonal antibodies was confirmed by the demonstration of these cross-reactive determinants in the immunesera of individual BALB/c and C57BL/6 mice. Additionally, evidence was obtained from these experiments and from earlier ones from this laboratory which suggests that the BALB/c idiotypic response to NP-protein conjugate is more homogeneous than the C57BL/6 idiotypic responses.     

Rice bifunctional alpha-amylase/subtilisin inhibitor: characterization, localization, and changes in developing and germinating seeds

A bifunctional alpha-amylase/subtilisin inhibitor (RASI) was purified to electrophoretic homogeneity from rice (Oryza sativa L.) bran. Its molecular mass was 21 kDa by SDS-PAGE and its isoelectric point was 9.05. Purified RASI inhibited subtilisin Carlsberg strongly and inhibited alpha-amylase from germinating rice seeds weakly. It inhibited rice alpha-amylase more than barley alpha-amylase, and the inhibition of rice alpha-amylase was greater at higher pHs. RASI did not inhibit trypsin, chymotrypsin, cucumisin, or mammalian alpha-amylase. The RASI was in the outermost part of the rice grain and its subcellular site seemed to be aleurone particles in aleurone cells. SDS-PAGE and western blotting showed that RASI was synthesized in the late milky stage in developing seeds, and it remained fairly constant during the first 7 days of germination.     

Improving the affinity and the fine specificity of an anti-cortisol antibody by parsimonious mutagenesis and phage display

Immunoassays are widely used to determine steroid concentrations. However, they are limited by the specificity of antisteroid mAbs. We used the phage display system combined with molecular modeling and site-specific randomization to improve the affinity and the fine specificity of an anti-cortisol mAb. Using parsimonious mutagenesis, we have generated a library of mutant Ab fragments (scFv) derived from this Ab by randomizing five amino acids chosen by molecular modeling and Ab-hapten contact structural analysis. Anti-cortisol Ab fragments were selected from the library in the presence of steroid analogues to block cross-reacting binders. Specific elution with free cortisol allowed the recovery of clones with up to eightfold better affinity and fivefold less cross-reactivity than the wild-type scFv. This approach can be applied to any anti-hapten Ab and represents a useful approach for obtaining highly specific Abs for use in steroid immunoassays.     

Malignant tumors invading chest wall: treatment with CT-directed implantation of radioactive seeds

Computed tomography (CT) was used to direct permanent implantation of radioactive iodine-125 seeds in two patients with unresectable lung cancer and in one with recurrent breast cancer invading the chest wall. An average of 60 seeds were implanted, with a mean total radioactivity of 35.6 mCi (1,317 MBq). Tumor coverage was adequate and pain relief was good in all patients. One patient had histologically documented complete response and another had CT-documented partial response.     

Aspects of microbiological and chemical quality of turmus, lupin seeds debittered by soaking in water

Eleven species of spherical lactic acid bacteria (LAB) belonging to the genera Leuconostoc, Lactococcus, Enterococcus and Pediococcus were the predominant microorganisms in 40 samples of turmus, ready-to-eat lupin seeds debittered by boiling and soaking in water. The average counts of the LAB in the 20 winter samples and the 20 summer samples were 7.4 and 8.7 log CFU/g, respectively. The averages of the Enterobacteriaceae counts were 5.1 and 6.6 log CFU/g, respectively, and the 11 species isolated belonged to the genera Enterobacter, Citrobacter, Escherichia and Klebsiella. The average yeast counts in winter and summer samples were 3 and 3.2 log CFU/g, respectively, and the 5 species isolated were in the genera Saccharomyces, Cryptococcus, Rhodotorula and Candida. Although Salmonella was not isolated from any sample and the Staphylococcus aureus count in all samples was < 1 log CFU/g, microbial hazards could be associated with the high Enterobacteriaceae counts and the presence of Escherichia coli. Total alkaloid concentration in 30% of the samples examined was higher than 0.02%, thus making the seeds a potential chemical hazard. Boiling the turmus directly before consumption and discarding the seeds with a bitter taste may help in avoiding some of the microbial and chemical hazards which could be associated with turmus consumption.     

Remarkable preservation of biomolecules in ancient radish seeds

Desiccated seeds from a 6th century AD storage vessel recovered from Qasr Ibr?m, Egypt, were examined for the presence of lipids and nucleic acids. A remarkable degree of lipid preservation was discovered, the fatty acid and sterol profiles being very similar to those of modern radish seeds. The only significant differences were hydrolysis of triacylglycerols and depletion of the polyunsaturated fatty acids (C18:2 and C18:3). The delta 13 C values of the principal fatty acids were in the range -25.4 to -29.2/1000, which is congruent with modern radish (C3 seeds) taking account of isotopic shifts caused by recent changes in atmospheric CO2. Deoxyribonucleosides and nucleic acid bases were detected by direct chemical analysis, and polymerase chain reactions gave products with sequences comparable to those from modern radish. The degree of lipid preservation, which was much greater than that reported for other archaeological remains, suggests that the microenvironment within desiccated seeds retards biomolecular decay. The results illustrate the utility of combined lipid-nucleic acid analysis in chemotaxonomic and genotypic studies of archaeobotanical remains.