Comparative HILIC/ESI-QTOF-MS and HPTLC studies of pyrrolizidine alkaloids in flowers of Tussilago farfara and roots of Arnebia euchroma

The utility of HPTLC and HILIC/ESI-QTOF-MS for the determination of pyrrolizidine alkaloids (PAs) and their N-oxides (PANOs) was compared in the selected plant species: Tussilago farfara L. (TF, flower) and Arnebia euchroma (Royle) I.M. Johnst. (AE, root). HPTLC confirmed the postulated presence of PAs (saturated and unsaturated) or PANOs in the tested extracts. In accordance with previous studies, HILIC/ESI-Q-TOF-MS confirmed the presence of the toxic PA senkirkine and the saturated otonecine-type PAs, tussilagine and isotussilagine in the TF extract and 7-angeloylretronecine and 9-angeloylretronecine in AE extract. Moreover, the following alkaloids were identified in AE root: intermedine, intermedine-N-oxide, leptanthine-N-oxide, echimidine-N-oxide (or their corresponding stereoisomers) and traces of 7-angeloylretronecine and 9-angeloylretronecine-N-oxide. The study demonstrates the HILIC/ESI-Q-TOF-MS method to be a very useful tool for monitoring PAs and PANOs in the test samples, even when not all of the necessary standards are available. Quantitative analysis of senkirkine in TF flower by HILIC/ESI-QTOF-MS featured high resolution, high precision, high mass accuracy, and very high sensitivity with limit-of-detection (LOD) of 27.50 fg/μL and limit-of-quantitation (LOQ) of 91.60 fg/μL. The results from both methods may be used for the development or rejection of European Pharmacopoeia (X) monographs of both investigated species.     

Detection of cyanobacterial sxt genes and paralytic shellfish toxins in freshwater lakes and brackish waters on Åland Islands,Finland

Harmful cyanobacteria are a globally growing concern. They produce a large variety of toxic compounds, including saxitoxin and its many structural variants, a group of potent neurotoxins collectively called paralytic shellfish toxins or PST. Nucleic acid based detection methods, such as qPCR, have been proposed as potential screening and monitoring tools for toxic cyanobacteria, but it is not clear how well the presence and quantity of saxitoxin biosynthesis (sxt) genes can be used to predict the production of PST in the environment. In this study, the prevalence of three sxt genes and their co-occurrence with paralytic shellfish toxins in the environment was investigated. The sxtA, sxtG and sxtB genes were present on average in 31% of the samples collected from lakes and brackish coastal waters on Åland Islands, Finland, during the three-year monitoring period. PST detection frequency varied from 13% to 59% from year to year, and concentrations were generally low. On average higher sxtB copy numbers were associated with PST detection, and although a positive correlation between gene copy numbers and toxin concentrations was observed (Spearman rank correlation, ρ = 0.53, P = 0.012), sxt gene presence or quantity didn’t reliably predict PST production. Sequencing of sxtA fragments and identification of main cyanobacteria indicated that the likely candidate responsible for PST production in the samples belonged to the genus Anabaena.     

Quantitative trait locus mapping using sets of near-isogenic lines: relative power comparisons and technical considerations

 The power to detect QTL using near-isogenic line (NIL) comparisons versus recombinant inbred (RI) populations was assessed. The power to detect QTL was found to be generally greater when using RI populations than when using NIL contrasts. Power to detect QTL with NIL contrasts never exceeded that of RI populations when the number of RI lines is maximized relative to replication of lines for a given number of experimental units. The relative power of NIL contrasts is highest for traits with high heritability and when a gain in precision is realized due to increased replication of entries. Although NIL populations are generally less powerful than RI populations of similar size, some practical considerations may enhance the value of these materials. Availability of NILs allows the genetic effect of a specific chromosome region to be determined by comparing two lines; all RI lines in a population need to be rescored for each new trait even if the effect of a specific chromosome region is suspected. NIL comparisons may allow genetic differences to be detected by visual inspection; genetic effects can only be expressed as means and variances with recombinant inbred populations. In summary, RI populations generally, and in some cases, substantially, provide better power for QTL detection than NIL comparisons. Practical considerations, however, indicate that many factors need to be considered when choosing a population structure to meet an experimental objective. Received: 12 December 1996 / Accepted: 21 March 1997     

Measurement and Selection of Parasitoid Quality for Mass-Reared Trichogramma minutum Riley Used in Inundative Release

Parasitoid quality, subject to both genetic and environmental influences, is critical to the success of any biological control program, however, its measurement and improvement is poorly understood. In this study, a classic genetic approach is taken to develop two indices, namely a character index and a fitness index, for the measurement and selection of high quality parasitoids used in inundative release. Six life-history traits and corresponding fitness components in 33 inbred strains of Trichogramma minutum were used to generate both genotypic and phenotypic variance-covariance matrices that then allowed for the construction of the indices. Most traits and their fitness components were positively correlated, both phenotypically and genotypically, with lifetime fecundity and the number of female offspring appearing to have an important influence. Selection of the top three strains showed that parasitoid quality could be improved by 36% using the character index and possibly up to 150% using the fitness index. The two indices were linearly correlated suggesting that either could be used to measure quality. The character index is recommended because it requires information on only three life-history traits (fecundity, number of female offspring, and number of male offspring) and has highly correlated responses of fitness components. Our work demonstrates that the best quality T. minutum will be obtained by using the character index to select for inbred strains which have high fecundity and number of female offspring.     

The lateral cervical nucleus in the cat

Summary Profiles of 14 neurons all sectioned through the nucleolar plane and 87 isolated dendritic profiles have been analyzed with respect to the surface area covered by boutons and astroglial processes. This analysis has revealed two different types of neurons within the lateral cervical nucleus (LCN) of the cat. The cell types also differ in other ultrastructural respects. One type, which probably consists of projection neurons, is characterized by a rather large size, a relatively small nucleus, numerous mitochondria, well developed granular and agranular endoplasmic reticulum. The cell membrane of these cells shows somatic spines and the perikaryon is covered with boutons to a mean extent of 42%. The other cell type, which probably is internuncial, is smaller, has a proportionally larger nucleus, few mitochondria and a poorly developed granular and agranular endoplasmic reticulum. These cells show no somatic spines and the perikaryal membrane is covered with boutons to an extent of about 10%. Also the bouton populations contacting the two cell types differ from one another. The proportion of internuncial neurons within the LCN has been estimated to about 8%. The internuncial neurons seem to have no preferential localization.The primary dendrites of the projection neurons have a bouton covering of about 48%. No proportional differences in covering could be revealed between different sizes of dendrites.The results are discussed in relation to what is known about the anatomical and physiological organization of the LCN, and also compared with the results obtained in other similar investigations on other parts of the central nervous system.The author is grateful to fil.kand. Göran Engholm for his help with the statistical considerations.This work was supported by grants from Anders Otto Swärds Stiftelse, Stiftelsen Lars Hiertas minne, Åhlén och Holms stiftelse, Åke Wibergs stiftelse and the Swedish Medical Research Council (Project No B70-12X-2710).     

Pharmacological and Physicochemical Properties of Pre-Versus Postsynaptic 5-Hydroxytryptamine1A Receptor Binding Sites in the Rat Brain: A Quantitative Autoradiographic Study

Numerous data suggested that the pharmacological and biochemical properties of 5-hydroxytryptamine1A (5-HT1A) receptors exhibit some regional differences in the CNS, notably within the raphe nuclei compared with various forebrain areas (such as the hippocampus). This possibility has been further investigated in the dorsal raphe nucleus and two areas within the hippocampus, the dentate gyrus and the CA1 area, using the quantitative autoradiographic technique. The potencies of 5'-guanylylimidodiphosphate to inhibit the specific binding of 125I-Bolton-Hunter-8-methoxy-2-(N-propyl-N-propylamino)tetralin (125I-BH-8-MeO-N-PAT) to 5-HT1A sites and of N-ethylmaleimide to block these sites irreversibly were identical in the dorsal raphe nucleus and the hippocampal areas in rat brain sections. In contrast, slight but significant differences were noted in the pH dependence and pharmacological properties of 5-HT1A sites labeled by 125I-BH-8-MeO-N-PAT in these three regions. Similarly, heat denaturation experiments and tissue exposure to either phospholipase A2 or the alkylating agent 8-methoxy-2-(N-2'-chloropropyl,N-propyl)aminotetraline revealed regional differences in the properties of 5-HT1A sites. However, in most cases, the observed variations were of greater amplitude between the CA1 area and the dentate gyrus, where 5-HT1A sites are located postsynaptically, than between any one of these areas and the dorsal raphe nucleus where they act as (presynaptic) somatodendritic autoreceptors. These data further support that subtypes of 5-HT1A receptors probably exist in the rat brain, but this heterogeneity seems unrelated to the pre- or post-synaptic location of these receptors.     

Relationships between rhizoplane and rhizosphere bacteria and verticillium wilt resistance in potato

Six cultivars and breeding lines of potato (Solanum tuberosum) differing in susceptibility to verticillium wilt caused by Verticillium dahliae were studied with respect to quantitative and qualitative differences in the bacterial flora of their soil and rhizosphere-rhizoplane. Although, no association was observed between the types of bacteria that inhabited the soil or roots of wilt resistant and susceptible cultivars, quantitative differences were evident. These differences provide the first direct evidence that potato genotypes can influence bacterial populations. Bacterial populations were 9–25-fold higher on roots than in the adjacent soil. As the plants aged, the total number of rootcolonizing bacteria increased between 15 and 245%. Pseudomonas spp. were the most abundant microbes in the soil and rhizosphere-rhizoplane. The bacteria antagonistic to V. dahliae in vitro were identified as members of the genera Bacillus, Pseudomonas, Flavobacterium, and Gluconobacter. A statistically significant trend was evident toward the association of antagonistic bacteria with the more resistant potato cultivars.     

Studien über die endokrinen Zellen des Magendarmtraktes

Zusammenfassung Die serotoninhaltigen, nach Formaldehydbedampfung im UV-Licht gelbfluoreszierenden EC-Zellen im Magendarmepithel wurden bei normalen und bei tryptophanfrei ernährten Ratten untersucht. Bei Kontrolltieren ist die Zahl der EC-Zellen, bezogen auf die Schleimhautfläche, im Duodenum größer als im Pylorus. Im Duodenum sind die EC-Zellen etwa gleichmäßig auf Oberflächen- und Drüsenepithel verteilt, im Pylorus ganz überwiegend im Drüsenepithel lokalisiert. Die Brunnerschen Drüsen besitzen keine EC-Zellen. Tryptophanfreie Diät führt zu starker Abnahme des Serotoningehalts der EC-Zellen, nach Umsetzung auf Normalkost steigt er wieder an. — Die Ergebnisse sprechen weiterhin dafür, daß die EC-Zelle sowohl Speicher- als auch Syntheseort von Serotonin ist. Dabei kommt Serotonin in der EC-Zelle wahrscheinlich in zwei Fraktionen vor, nämlich zytoplasmatisch gelöst und granulagebunden. Die histochemischen Nachweismethoden für Serotonin, Möglichkeiten des Eingriffs in die verschiedenen Syntheseschritte des Serotonins und deren Spezifität in bezug auf die EC-Zelle als physiologischem Ort der Serotoninsynthese werden diskutiert.

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Studies on the endocrine cells of the gastrointestinal tractII. Fluorescence microscopy of the EC-cells after tryptophan-free diet

Summary The serotonin-containing EC-cells of the gastro-intestinal mucosa, which show yellow fluorescence after treatment with gaseous formaldehyde, were investigated in the rat after normal and tryptophan-free nourishment. In control animals the number of EC-cells, related to epithelium area is higher in the duodenum than in the pyloric region. In the duodenum the EC-cells show about the same distribution in surface epithelium and gland epithelium. In the pyloric region EC-cells are localized predominantly in the gland epithelium. No EC-cells were found in the Brunner glands. After tryptophan-free diet the serotonin-level of the EC-cells strongly decreases; serotonin-level increases after return to normal nourishment. The results also suggest, that serotonin is synthesized as well as stored in the EC-cell and that it occurs in two fractions: bound to granules and dissolved in the cytoplasm. Discussion deals with the histochemical methods for determination of serotonin, the possibilities of influencing the pathways of serotonin metabolism and the specificity of these possibilities concerning the EC-cell as the physiological site of serotonin synthesis.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft, Antrag Fo 77, 1–4.     

Sizes of granular vesicles in the rat median eminence,with special reference to the zona granulosa

Summary A quantitative evaluation has been made of the sizes of neuronal granular vesicles (GV) in different (sub-)zones of the rat median eminence. On the basis of the frequency distribution of the mean sizes of the GV in the individual nerve profiles, at least 4 tentative categories of nerve profiles may be distinguished. However, available data do not permit characterization of nerve fibre categories in terms of neurohormone and/or neurotransmitter storage. From dorsal to ventral the palisade zone shows a decrease in mean size of GV; furthermore, the relative number of nerve profiles characterized by small GV (mean size smaller than 112 nm) increases. In contrast, the GV in the zona granulosa, that part of the external zone of the median eminence ventral to the palisade zone, are of relatively large size. Qualitative observations indicate that the zona granulosa may be regarded as an area for both storage and release of biologically active substances. We are grateful to Mrs. R.M.Y. Hartsteen for technical assistance, to Miss P.C. Delfos and Mr. W. van den Oudenalder for photographic assistance and to Prof. J. Moll for helpful criticism.