Antioxidant defense in mitochondria during diapause and postdiapause development of European corn borer (Ostrinia nubilalis, Hubn.)

Antioxidant enzymes (CAT, catalase; GPx, selenium nondependent glutathione peroxidase; GST, glutathione-S-transferase; GR, glutathione reductase; DHAR, dehydroascorbate reductase) were determined in the mitochondria of diapausing and non-diapausing larvae and pupae of both diapausing and non-diapausing larvae of the European corn borer (Ostrinia nubilalis, Hubn., Lepidoptera: Pyralidae). CAT, GST, and DHAR activity in mitochondria of diapausing larvae were reduced compared to non-diapausing larvae. Pupae of diapaused-larvae possessed lower GST, but higher DHAR activities compared to pupae of non-diapaused individuals. Comparison between larvae and pupae revealed lower GPx activity in the mitochondria of pupae. CAT activity in the mitochondria of pupae was higher compared to diapausing larvae, but lower than in non-diapausing ones. Correlation and canonical discriminant analyses revealed different antioxidant enzyme compositions for a particular stage and developmental pattern. Our results show that antioxidant enzymes have a similar role in the regulation of energetics in mitochondria as that in diapause and metamorphosis.     

Protein folding determinants: structural features determining alternative disulfide pairing in alpha- and chi/lambda-conotoxins

Alpha-conotoxins isolated from Conus venoms contain 11-19 residues and preferentially fold into the globular conformation that possesses a specific disulfide pairing pattern (C1-3, C2-4). We and others isolated a new family of chi-conotoxins (also called lambda conotoxins) with the conserved cysteine framework of alpha-conotoxins but with alternative disulfide pairing (C1-4, C2-3) resulting in the ribbon conformation. In both families, disulfide pairing and hence folding are important for their biological potency. By comparing the structural differences, we identified potential structural determinants responsible for the folding tendencies of these conotoxins. We examined the role of conserved proline in the first intercysteine loop and the conserved C-terminal amide on folding patterns of synthetic analogues of ImI conotoxin by comparing the isoforms with the regiospecifically synthesized conformers. Deamidation at the C-terminus and substitution of proline in the first intercysteine loop switch the folding pattern from the globular form of alpha-conotoxins to the ribbon form of chi/lambda-conotoxins. The findings are corroborated by reciprocal folding of CMrVIA chi/lambda-conotoxins. Substitution of Lys-6 from the first intercysteine loop of CMrVIA conotoxin with proline, as well as the inclusion of an amidated C-terminal shifted the folding preference of CMrVIA conotoxin from its native ribbon conformation toward the globular conformation. Binding assays of ImI conotoxin analogues with Aplysia and Bulinus acetylcholine binding protein indicate that both these substitutions and their consequent conformational change substantially impact the binding affinity of ImI conotoxin. These results strongly indicate that the first intercysteine loop proline and C-terminal amidation act as conformational switches in alpha- and chi/lambda-conotoxins.     

Progress since the first international symposium: ‘Genetic aspects of plant mineral nutrition’, Beograd, 1982, and perspectives of future research

Summary The paper discusses the problems of genetic aspects of plant mineral nutrition in the light of the results presented at the First and Second Symposia on ‘Genetic Aspects of Plant Mineral Nutrition’ organized in Beograd in 1982 and Madison in 1985, respectively. On the basis of the results, future directions of research are discussed. The papers deal with the concentration and content of mineral nutrients in different genotypes, physiological and biochemical aspects of the genetic specificity of plant mineral nutrition, relations between plant genotypes and nitrogen fixing micro-organism strains, as well as with some related problems which have been investigated to a lesser extent. Particular attention is paid to papers and problems referring to genetic and breeding research work linked with genetic aspects of plant mineral nutrition as well as the possibilities of developing new cultivars requiring certain soil and mineral nutrition conditions for their cultivation.     

Very-Long-Chain Wax Constituents from Primula veris and P. acaulis: Does the Paradigm of Non-Branched vs. Branched Chain Dominance Universally Hold in all Plant Taxa?

Herein n-, iso- and anteiso-series of very-long-chained (VLC) alkanes (C₂₁–C₃₅), fatty acid benzyl esters (FABEs; C₂₀–C₃₂), and 2-alkanones (C₂₃–C₃₅) were identified in the wax of Primula veris L. and P. acaulis (L.) L. (Primulaceae). For the very first time in a sample of natural origin, the presence of iso- and anteiso-VLC FABEs and 2-alkanones was unequivocally confirmed by synthetic work, derivatization, and NMR. It should be noted that the studied species produced unusually high amounts of branched wax constituents (e. g., >50 % of 2-alkanones were branched isomers). The domination of iso-isomers, probably biosynthesized from leucine-derived starters, is a unique feature in the Plant Kingdom. The plant organ distribution of these VLC compounds in P. acaulis samples (different habitats and phenological phases) pointed to their possible ecological value. This was supported by a eutectic behavior of binary blends of FABEs and alkanes, as well as by high UV-C absorption by FABEs.     

Sex differences in the response of rats to drugs affecting GABAergic transmission

The administration of diazepam 1.0 mg/kg decreased the level of plasma corticosterone in female but not in male Wistar rats. Picrotoxin, another drug affecting GABAergic transmission, also brought about an increase of plasma corticosterone in both sexes. However, in order to achieve a plasma corticosterone increase of similar magnitude (more than 500%) a threefold higher dose of picrotoxin had to be given to males. When the convulsive properties of picrotoxin were tested, it became evident that the dose of picrotoxin (2.5 mg/kg) which was subconvulsive in male was almost 100% convulsive in female rats. The existing sex differences in the response of rats to drugs affecting GABAergic transmission might have possible implications in the treatment of GABA system dysfunction.     

Chemical Diversity of the Natural Populations of Dalmatian Pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch.Bip.) in Croatia

Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir. ) Sch.Bip. ) is a plant species endemic to the east Adriatic coast. The bioactive substance of Dalmatian pyrethrum is a natural insecticide, pyrethrin, a mixture of six active components (pyrethrins I and II, cinerins I and II, and jasmolins I and II). The insecticidal potential of pyrethrin was recognized decades ago, and dried and ground flowers have traditionally been used in Croatian agriculture and households. A total of 25 Dalmatian pyrethrum populations from Croatia were studied to determine the pyrethrin content and composition, and to identify distinct chemotypes. The total pyrethrin content ranged from 0.36 to 1.30% (dry flower weight; DW) and the pyrethrin I/pyrethrin II ratio ranged from 0.64 to 3.33%. The statistical analyses revealed that the correlations between the percentage of pyrethrin I and of all the other components were significant and negative. The total pyrethrin content was positively correlated with the percentage of pyrethrin I and negatively correlated with cinerin II. The multivariate analysis of the chemical variability enabled the identification of five chemotypes among 25 Dalmatian pyrethrum populations. The chemical characterization of indigenous Dalmatian pyrethrum populations may serve as a good background for future breeding and agricultural exploitation.     

Design,synthesis and cholinesterase inhibitory properties of new oxazole benzylamine derivatives

Abstract

The enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are primary targets in attenuating the symptoms of neurodegenerative diseases. Their inhibition results in elevated concentrations of the neurotransmitter acetylcholine which supports communication among nerve cells. It was previously shown for trans-4/5-arylethenyloxazole compounds to have moderate AChE and BChE inhibitory properties. A preliminary docking study showed that elongating oxazole molecules and adding a new NH group could make them more prone to bind to the active site of both enzymes. Therefore, new trans-amino-4-/5-arylethenyl-oxazoles were designed and synthesised by the Buchwald-Hartwig amination of a previously synthesised trans-chloro-arylethenyloxazole derivative. Additionally, naphthoxazole benzylamine photoproducts were obtained by efficient photochemical electrocyclization reaction. Novel compounds were tested as inhibitors of both AChE and BChE. All of the compounds exhibited binding preference for BChE over AChE, especially for trans-amino-4-/5-arylethenyl-oxazole derivatives which inhibited BChE potently (IC₅₀ in µM range) and AChE poorly (IC₅₀?100?µM). Therefore, due to the selectivity of all of the tested compounds for binding to BChE, these compounds could be applied for further development of cholinesterase selective inhibitors.

• HIGHLIGHTS

• Series of oxazole benzylamines were designed and synthesised

• The tested compounds showed binding selectivity for BChE

• Naphthoxazoles were more potent AChE inhibitors

     

Characterisation of alpha3beta1 and alpha(v)beta3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of beta1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, alpha3beta1 and alpha(v)beta3, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with beta1,6-branches and short polylactosamine chains. In WM9 cells, alpha3beta1 integrin was more variously glycosylated than alpha(v)beta3; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and alpha3beta1 integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of alpha(v)beta3 integrin glycans in melanoma or in any cancer cells.     

SARS-CoV-2 RNA screening in routine pathology specimens

Virus detection methods are important to cope with the SARS-CoV-2 pandemics. Apart from the lung, SARS-CoV-2 was detected in multiple organs in severe cases. Less is known on organ tropism in patients developing mild or no symptoms, and some of such patients might be missed in symptom-indicated swab testing. Here, we tested and validated several approaches and selected the most reliable RT-PCR protocol for the detection of SARS-CoV-2 RNA in patients’ routine diagnostic formalin-fixed and paraffin-embedded (FFPE) specimens available in pathology, to assess (i) organ tropism in samples from COVID-19-positive patients, (ii) unrecognized cases in selected tissues from negative or not-tested patients during a pandemic peak, and (iii) retrospectively, pre-pandemic lung samples. We identified SARS-CoV-2 RNA in seven samples from confirmed COVID-19 patients, in two gastric biopsies, one small bowel and one colon resection, one lung biopsy, one pleural resection and one pleural effusion specimen, while all other specimens were negative. In the pandemic peak cohort, we identified one previously unrecognized COVID-19 case in tonsillectomy samples. All pre-pandemic lung samples were negative. In conclusion, SARS-CoV-2 RNA detection in FFPE pathology specimens can potentially improve surveillance of COVID-19, allow retrospective studies, and advance our understanding of SARS-CoV-2 organ tropism and effects.