Incorporation of L-[U-14C]leucine into ergosine by cell-free extracts of Clavicepspurpurea (FR.) tul

A cell-free system of $\text{Claviceps}\text{purpurea}$ has been described which incorporates [¹⁴C] leucine into the peptide-type ergot alkaloid ergosine. Cell-free extracts may be prepared either from protoplasts or lyophilized mycelium. Production was markedly stimulated by addition of agroclavine compared with d-lysergic acid and by agitation of the incubation mixture. The synthesis of ergosine is strongly dependent on the presence of ATP in the reaction mixture.     

Critical role of RelB serine 368 for dimerization and p100 stabilization

In mature B cells RelB-containing complexes are constitutively present in the nucleus, and they are less susceptible to inhibitory kappaB proteins. In most other cell types inhibitory kappaB proteins prevent nuclear translocation and activation of NFkappaB. We reasoned that this characteristic might be because of post-translational modifications of RelB. In Drosophila, signal-dependent phosphorylation of the Rel homologue Dorsal at serine 317 has been shown to be critical for nuclear import. The evolutionary conservation of this serine prompted us to analyze the function of the corresponding site in RelB. As a model system we used the murine S107 plasmacytoma cell line, which lacks endogenous RelB expression. Analysis of S107 cells expressing wild type RelB and serine 368 mutants reveals that serine 368 is not required for nuclear import but that it is critical for RelB dimerization with other members of the NFkappaB family. Similar effects were obtained when the conserved serine in RelA was mutated. We further demonstrate that expression of functional RelB, but not of serine 368 mutants, severely reduces p52 generation and strongly increases expression of the p52 precursor, p100. Wild type RelB, but not mutant RelB, prolonged p100 half-life. We therefore suggest an inhibitory effect of RelB on p100 processing, which is possibly regulated in a signal-dependent manner.     

Apoplastic peroxidases and ascorbate are involved in manganese toxicity and tolerance of Vigna unguiculata

Excessive manganese (Mn) supply induced the formation of brown spots on leaves as typical Mn toxicity symptoms in cowpea ( Vigna unguiculata L. Walp.) grown in hydroponics. Differences in Mn resistance between cv. TVu 91 (Mn-sensitive) and cv. TVu 1987 (Mn-tolerant) expressed in the density of brown spots in older leaves were due to higher Mn tissue tolerance. Apoplastic water-soluble peroxidase (POD) in the apoplastic washing fluid (AWF) was enhanced by increasing Mn leaf content and generally significantly higher in leaves of cv. TVu 91 than in cv. TVu 1987. Electrophoresis of AWF revealed the presence of several water-soluble POD isoenzymes. At toxic Mn supply, the activities of these and additional POD isoenzymes increased more in the Mn-sensitive cultivar. Levels of ascorbic acid in the apoplast and cytoplasm of the Mn-sensitive cv. TVu 91 decreased with increasing leaf Mn contents, whereas Mn-tolerant cv. TVu 1987 was not affected. Mn treatment lead to a stimulation of the enzymes of the ascorbic acid regeneration system (monodehydroascorbic acid reductase and glutathione reductase) in both cultivars, but the activation of glutathione reductase was clearly more enhanced in the Mn-tolerant cultivar TVu 1987. The results provide circumstantial evidence that apoplastic ascorbate and peroxidases are involved in the expression of Mn toxicity and genotypic Mn tolerance.     

A Comparative pO2 Probe and [18F]-Fluoro-Azomycinarabino-Furanoside ([18F]FAZA) PET Study Reveals Anesthesia-Induced Impairment of Oxygenation and Perfusion in Tumor and Muscle

Tumor hypoxia can be identified by [¹⁸F]FAZA positron emission tomography, or invasively using oxygen probes. The impact of anesthetics on tumor hypoxia remains controversial. The aim of this comprehensive study was to investigate the impact of isoflurane and ketamine/xylazine anesthesia on [¹⁸F]FAZA uptake and partial oxygen pressure (pO₂) in carcinoma and muscle tissue of air- and oxygen-breathing mice.

Methods

CT26 colon carcinoma-bearing mice were anesthetized with isoflurane (IF) or ketamine/xylazine (KX) while breathing air or oxygen (O₂). We performed 10 min static PET scans 1 h, 2 h and 3 h after [¹⁸F]FAZA injection and calculated the [¹⁸F]FAZA-uptake and tumor-to-muscle ratios (T/M). In another experimental group, we placed a pO₂ probe in the tumor as well as in the gastrocnemius muscle to measure the pO₂ and perfusion.

Results

Ketamine/xylazine-anesthetized mice yielded up to 3.5-fold higher T/M-ratios compared to their isoflurane-anesthetized littermates 1 h, 2 h and 3 h after [¹⁸F]FAZA injection regardless of whether the mice breathed air or oxygen (3 h, KX-air: 7.1 vs. IF-air: 1.8, p = 0.0001, KX-O₂: 4.4 vs. IF-O₂: 1.4, p < 0.0001). The enhanced T/M-ratios in ketamine/xylazine-anesthetized mice were mainly caused by an increased [¹⁸F]FAZA uptake in the carcinomas. Invasive pO₂ probe measurements yielded enhanced intra-tumoral pO₂ values in air- and oxygen-breathing ketamine/xylazine-anesthetized mice compared to isoflurane-anesthetized mice (KX-air: 1.01 mmHg, IF-air: 0.45 mmHg; KX-O₂ 9.73 mmHg, IF-O₂: 6.25 mmHg). Muscle oxygenation was significantly higher in air-breathing isoflurane-anesthetized (56.9 mmHg) than in ketamine/xylazine-anesthetized mice (33.8 mmHg, p = 0.0003).

Conclusion

[¹⁸F]FAZA tumor uptake was highest in ketamine/xylazine-anesthetized mice regardless of whether the mice breathed air or oxygen. The generally lower [¹⁸F]FAZA whole-body uptake in isoflurane-anesthetized mice could be due to the higher muscle pO₂-values in these mice compared to ketamine/xylazine-anesthetized mice. When performing preclinical in vivo hypoxia PET studies, oxygen should be avoided, and ketamine/xylazine-anesthesia might alleviate the identification of tumor hypoxia areals.     

Arfophilins are dual Arf/Rab 11 binding proteins that regulate recycling endosome distribution and are related to Drosophila nuclear fallout

Arfophilin is an ADP ribosylation factor (Arf) binding protein of unknown function. It is identical to the Rab11 binding protein eferin/Rab11-FIP3, and we show it binds both Arf5 and Rab11. We describe a related protein, arfophilin-2, that interacts with Arf5 in a nucleotide-dependent manner, but not Arf1, 4, or 6 and also binds Rab11. Arfophilin-2 localized to a perinuclear compartment, the centrosomal area, and focal adhesions. The localization of arfophilin-2 to the perinuclear compartment was selectively blocked by overexpression of Arf5-T31N. In contrast, a green fluorescent protein-arfophilin-2 chimera or arfophilin-2 deletions were localized around the centrosome in a region that was also enriched for transferrin receptors and Rab11 but not early endosome markers, suggesting that the distribution of the endosomal recycling compartment was altered. The arfophilins belong to a conserved family that includes Drosophila melanogaster nuclear fallout, a centrosomal protein required for cellularization. Expression of green fluorescent protein-nuclear fallout in HeLa cells resulted in a similar phenotype, indicative of functional homology and thus implicating the arfophilins in mitosis/cytokinesis. We suggest that the novel dual GTPase-binding capacity of the arfophilins could serve as an interface of signals from Rab and Arf GTPases to regulate membrane traffic and integrate distinct signals in the late endosomal recycling compartment.     

Synthesis and acetylcholinesterase inhibitory activity of polyhydroxylated sulfated steroids: Structure/activity studies

Disulfated and trisulfated steroids have been synthesized from cholesterol and their acetylcholinesterase inhibitory activity has been evaluated. In our studies we have found that the activity was not only dependent on the location of the sulfate groups but on their configurations. 2β,3α,6α-trihydroxy-5α-cholestan-6-one trisulfate (18) was the most active steroid with an IC₅₀ value of 15.48 μM comparable to that of 2β,3α-dihydroxy-5α-cholestan-6-one disulfate (1). Both compounds were found to be less active than the reference compound eserine. The butyrylcholinesterase activity of 1 and 18 was one magnitude lower than that against acetylcholinesterase revealing a selective inhibitor profile.     

Metabolic changes and improved growth in micropropagated red raspberry “Indian summer” are tied to improved mineral nutrition

Mineral nutrition is directly involved in plant metabolism and greatly affects growth and development. An initial study modeling Murashige and Skoog (MS) medium mineral components revealed that the quality of red raspberry shoot cultures was significantly affected by CaCl₂, MgSO₄, and KH₂PO₄ (mesos components). This study investigated the effects of increased mesos components on shoot growth and metabolism. Rubus idaeus L. “Indian summer” shoots grown on standard MS medium (1.0× MS mesos components) were compared to shoots grown with 1.5× and 2.5× MS mesos components. After 9 wk, shoots were evaluated for shoot quality, multiplication, elongation, and metabolic changes. Metabolic changes were determined by liquid chromatography (LC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). Shoots grown on increased mesos components had improved quality, shoot length, and leaf color compared to shoots grown on MS medium. Metabolomic analysis indicated that shoots grown on high mesos component medium had reduced amounts of some free amino acids (glutamine, arginine, histidine, and proline) and some secondary metabolites (epicatechin, quercetin, and ellagic acid) compared to shoots on MS medium, which indicated reduced stress. Shoots grown on high mesos component also had increases in fructose 1-phosphate and glutathione associated with biosynthetic pathways, plant defense mechanisms, and redox homeostasis. Another factor involved in improved growth responses may be that increased glutamine was also found in high mesos component treatments, possibly influenced by ammonium accumulated from photorespiration. These metabolic changes provide initial insights into medium optimization and in vitro mineral nutrition, and the impact of nutrients on plant growth and development in micropropagated red raspberry shoots.