Design, synthesis, and antifungal activity of inhibitors of brassilexin detoxification in the plant pathogenic fungus Leptosphaeria maculans

Potential inhibitors of Leptosphaeria maculans mediated detoxification of the phytoalexin brassilexin were designed and synthesized based on the planar heteroaromatic structure of isothiazolo[5,4-b]indole. Screening of these compounds for inhibition of brassilexin detoxification in cultures of L. maculans indicated that 4-(2-chlorophenyl)isothiazole had the largest effect on the rate of brassilexin detoxification. However, the most antifungal compound among the potential inhibitors, isothiazolo[5,4-b]quinoline, did not appear to affect the metabolism of brassilexin noticeably, suggesting that growth inhibition is not sufficient to slow down the rate of brassilexin detoxification. Furthermore, it was determined that 4-arylisothiazoles as well as isothiazolo[5,4-b]thianaphthene displayed antifungal activity against L. maculans.     

Bioactive compounds in sweet rowanberry fruits of interspecific Rowan crosses

Sweet rowanberries originated by the crossbreeding of wild rowanberries with other fruit species such as apples, medlars or black chokeberries. They are highly resistant to cold climate. In contrast with wild rowanberries, they have sweet mild taste and show less parasorbic acid toxicity, which can be eliminated, when the consumption is excessive, by heating. The objective of the work was to determine selected antioxidant properties in 6 cultivars. The analyses showed that the contents of total phenolics, total flavonoids and ascorbic acid were high. Similarly, antioxidant capacity (6.58–9.62 g of ascorbic acid equivalents kg^?1) was also high. The work brings novel data, in particular, when comparing the cultivars; moreover, results regarding reactive oxygen and nitrogen species scavenging activity in sweet rowanberries are being published for the first time. The sweet rowanberry extracts (10%) showed inhibitory ability on hydroxyl radical (16.12–24.73%), superoxide anion (26.74–34.02%), nitric oxide (24.75–31.39%), and lipid peroxidation (7.93–13.12%). The values obtained are even many times higher than those found in common commercial fruit species like apples. Therefore, sweet rowanberries appear to be a promising fruit species for human nutrition, especially due to their high content of bioactive substances and ease of cultivation in worse climatic and soil conditions.     

Acute Impact of Selected Pyridoindole Derivatives on Fos Expression in Different Structures of the Rat Brain

The impacts of three pyridoindole derivatives (PDs), designated as PD144, PD143, and PD104, which have previously been shown to have antidepressant (PD144) and anxiolytic (PD143, PD104) properties, were investigated on the Fos expressions in 11 different rat brain areas, including the medial prefrontal cortex, striatum, septum, accumbens nucleus (shell, core), bed nucleus of the stria terminalis, hypothalamic paraventricular nucleus, central amygdala, locus coeruleus, dorsal raphe nucleus, and the solitary tract nucleus. Control rats received vehicle, while the other three groups the PDs in a dose of 25 mg/kg/b.w. The animals were transcardially perfused with a fixative 90 min after the treatments. Coronal sections of 40-µm thickness were processed for Fos-immunostaining by avidin–biotin-peroxidase complex and visualized by nickel-intensified diaminobenzidine complex. Fos-labeled sections were counterstained with neuropeptides including corticoliberine (CRH), oxytocin (OXY), vasopressin (AVP), and vasoactive intestinal polypeptide (VIP) and processed for immunofluorescence staining using Alexa Fluor 555 dye. In all the three groups of animals, the upregulation of PDs-induced Fos expression only in 2 of 11 brain areas was investigated, namely, in the hypothalamic paraventricular nucleus (PVN) and the central amygdaloid nucleus (CeA). The other brain structures studied were devoid of Fos expression. Counterstaining of the Fos-labeled CeA-containing sections with VIP antibody revealed that the Fos expression stimulated by the PDs was upregulated in all the CeA subdivisions (lateral, ventral, capsular), except the medial one. Dual immunoprocessings showed Fos/CRH-labeling in both the PVN and the amygdala and Fos/OXY in the PVN. No Fos/AVP colocalizations were seen in the PVN. The obtained data provide the first view on the intracerebral effects of three new PDs derivatives, which effects were restricted only to the PVN and CeA areas. The present data may help to improve our understanding of the impact of the selected PDs on the brain and to anticipate possible behavioral and neuroendocrine consequences.     

Discovery of inhibitors of brassinin oxidase based on the scaffolds of the phytoalexins brassilexin and wasalexin

Inhibitors of brassinin oxidase (BOLm), a unique phytoalexin detoxifying enzyme produced by the plant pathogenic fungus Leptosphaeria maculans (asexual stage Phoma lingam), were designed based on scaffolds of the phytoalexins brassilexin and wasalexin. Evaluation of these compounds using purified BOLm established that the inhibitory effect of brassilexin and derivatives decreased as follows: 6-chlorobrassilexin ≈ 6-bromobrassilexin > 5-bromobrassilexin ≈ 5-chlorobrassilexin ≈ 6-fluorobrassilexin > 8-methylbrassilexin > brassilexin ≈ 5-fluorobrassilexin. 6-Chlorobrassilexin was determined to be the best competitive inhibitor of BOLm discovered to date, with a K_i = 31 μM. Importantly, brassilexin and derivatives did not appear to induce BOLm in fungal cultures. Overall, these results suggest that the brassilexin scaffold is a good lead for further development of paldoxins against L. maculans, as it inhibits competitively BOLm without apparent induction.     

An autoradiographic method of detectingA. laidlawii andM. hyorhinis in cell cultures

Summary The autoradiographic investigation of L cells and Chinese hamster cells for the presence of mycoplasmas (A. laidlawii andM. hyorhinis) using uridine/uracil (UdR/U) testing is a rapid and reliable method suitable for the serial checking of even a small number of cells. It depends on a reduced incorporation of [³H]uridine and an increased uptake of [³H]uracil into the RNA of mycoplasma-infected cells, shown in autoradiograms by the density of the grains and their distribution. Results obtained by the autoradiographic technique correspond approximately to specific activity values of RNA-infected cells after the incorporation of [³H]uridine and [³H]uracil.     

Diurnal behavioral and endocrine effects of chronic shaker stress in mice

Experiments were performed in C57BL/6J male mice to determine 1) light/dark effects of acute and chronic shaker stress on open field behavioral patterns and 2) light/dark effects of chronic stress on plasma corticosterone and oxytocin. Shaker stress was applied acutely (15 min) or chronically (3 or 7 days). Mice were tested in the open field in the light or dark phase of the circadian cycle. For the endocrine study, mice were exposed to 3 days of intermittent shaker stress and sacrificed after the last stress event (09:00 or 19:00 h). Acute or chronic shaker stress had no significant effects on intensity of motor activity and rearing of mice tested under either light condition. Mice tested in the dark phase had higher motor activity and exhibited lower anxiety-like behavior as expressed by central zone activities and had higher emotionality as expressed by increased defecation. Chronic stress increased corticosterone with a greater absolute increase in the dark period. However, the percentage stress-induced increase was not different between the day and night periods. The oxytocin response to stress was observed only during the light phase with no change seen at dark phase. These results show that there is a marked difference in the light/dark pituitary stress response with no alteration in stress induced behavioral changes. They also suggest that there are circadian interactions in the endocrine stress axis that are without consequences for open field behavior.     

A robust and convergent synthesis of dipeptide-DOTAM conjugates as chelators for lanthanide ions: new PARACEST MRI agents

A generally applicable synthetic approach to dipeptide-DOTAM conjugates has been developed which is based on the peralkylation of 1,4,7,10-tetraazacyclododecane (cyclen) with protected N-iodoacetyl dipeptides. Standardized procedures were used for the alkylation, metalation, and purification of the resultant lanthanide complexes. Using this approach, we have been able to rapidly and reliably prepare and screen five different ligands each with up to six lanthanide ions. This preliminary investigation has identified several paramagnetic compounds with strong chemical exchange saturation transfer (PARACEST) properties in water at physiological temperature and pH. Extension of the synthetic approach to a wide variety of amino acids is possible.     

The phytoalexins from cauliflower, caulilexins A, B and C: isolation, structure determination, syntheses and antifungal activity

Our continuous search for phytoalexins from crucifers led us to examine phytoalexin production in florets of cauliflower (Brassica oleracea var. botrytis) under abiotic (UV light) elicitation. Four known (isalexin, S-(-)-spirobrassinin, 1-methoxybrassitin, brassicanal C) and three new (caulilexins A-C) phytoalexins were isolated. The syntheses and antifungal activity of caulilexins A-C against the economically important pathogenic fungi Leptosphaeria maculans, Rhizoctonia solani and Sclerotinia sclerotiorum, and the first synthesis of brassicanal C are reported.