Abundance of Cysteine Endopeptidase Dionain in Digestive Fluid of Venus Flytrap (Dionaea muscipula Ellis) Is Regulated by Different Stimuli from Prey through Jasmonates

The trap of the carnivorous plant Venus flytrap (Dionaea muscipula) catches prey by very rapid closure of its modified leaves. After the rapid closure secures the prey, repeated mechanical stimulation of trigger hairs by struggling prey and the generation of action potentials (APs) result in secretion of digestive fluid. Once the prey''s movement stops, the secretion is maintained by chemical stimuli released from digested prey. We investigated the effect of mechanical and chemical stimulation (NH₄Cl, KH₂PO₄, further N(Cl) and P(K) stimulation) on enzyme activities in digestive fluid. Activities of β-D-glucosidases and N-acetyl-β-D-glucosaminidases were not detected. Acid phosphatase activity was higher in N(Cl) stimulated traps while proteolytic activity was higher in both chemically induced traps in comparison to mechanical stimulation. This is in accordance with higher abundance of recently described enzyme cysteine endopeptidase dionain in digestive fluid of chemically induced traps. Mechanical stimulation induced high levels of cis-12-oxophytodienoic acid (cis-OPDA) but jasmonic acid (JA) and its isoleucine conjugate (JA-Ile) accumulated to higher level after chemical stimulation. The concentration of indole-3-acetic acid (IAA), salicylic acid (SA) and abscisic acid (ABA) did not change significantly. The external application of JA bypassed the mechanical and chemical stimulation and induced a high abundance of dionain and proteolytic activity in digestive fluid. These results document the role of jasmonates in regulation of proteolytic activity in response to different stimuli from captured prey. The double trigger mechanism in protein digestion is proposed.     

Two N-glycosylation Sites in the GluN1 Subunit Are Essential for Releasing N-methyl-d-aspartate (NMDA) Receptors from the Endoplasmic Reticulum

NMDA receptors (NMDARs) comprise a subclass of neurotransmitter receptors whose surface expression is regulated at multiple levels, including processing in the endoplasmic reticulum (ER), intracellular trafficking via the Golgi apparatus, internalization, recycling, and degradation. With respect to early processing, NMDARs are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications, including phosphorylation and palmitoylation. However, the role of N-glycosylation, one of the most common posttranslational modifications, in regulating NMDAR processing has not been studied in detail. Using biochemistry, confocal and electron microscopy, and electrophysiology in conjunction with a lentivirus-based molecular replacement strategy, we found that NMDARs are released from the ER only when two asparagine residues in the GluN1 subunit (Asn-203 and Asn-368) are N-glycosylated. Although the GluN2A and GluN2B subunits are also N-glycosylated, their N-glycosylation sites do not appear to be essential for surface delivery of NMDARs. Furthermore, we found that removing N-glycans from native NMDARs altered the receptor affinity for glutamate. Our results suggest a novel mechanism by which neurons ensure that postsynaptic membranes contain sufficient numbers of functional NMDARs.     

Maternal control of the fetal and neonatal rat suprachiasmatic nucleus

The molecular clockwork underlying the generation of circadian rhythmicity within the suprachiasmatic nucleus (SCN) develops gradually during ontogenesis. The authors' previous work has shown that rhythms in clock gene expression in the rat SCN are not detectable at embryonic day (E) 19, start to form at E20 and develop further via increasing amplitude until postnatal day (P) 10. The aim of the present work was to elucidate whether and how swiftly the immature fetal and neonatal molecular SCN clocks can be reset by maternal cues. Pregnant rats maintained under a light-dark (LD) regimen with 12 h of light and 12 h of darkness were exposed to a 6-h delay of the dark period and released into constant darkness at different stages of the fetal SCN development. Adult rats maintained under the same LD regimen were exposed to an identical shifting procedure. Daily rhythms in spontaneous c-fos, Avp, Per1, and Per2 expression were examined within the adult and newborn SCN by in situ hybridization. Exposure of adult rats to the shifting procedure induced a significant phase delay of locomotor activity within 3 days after the phase shift as well as a delay in the rhythms of c-fos and Avp expression within 3 days and Per1 and Per2 expression within 5 days. Exposure of pregnant rats to the shifting procedure at E18, but not at E20, delayed the rhythm in c-fos and Avp expression in the SCN of newborn pups at P0-1. The shifting procedure at E20 did, however, induce a phase delay of Per1 and Per2 expression rhythms at P3 and P6. Hence, 5 days were necessary for phase-shifting the pups' SCN clock by maternal cues, be it the interval between E18 and P0-1 or the interval between E20 and P3, while only 3 days were necessary for phase-shifting the maternal SCN by photic cues. These results demonstrate that the SCN clock is capable of significant phase shifts at fetal developmental stages when no or very faint molecular oscillations can be detected.     

Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells

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Gas-liquid dispersion with dual Rushton turbine impellers

Aerated and unaerated power consumption and flow patterns in a 0.56 m diameter agitated vessel containing water with dual Rushton turbines have been studied. Under unaerated conditions with a liquid height-to-diameter ratio of 2, an impeller spacing of 2 to 3 times the impeller is required for each to draw an amount of power equal to a single impeller. For aerated conditions, if a similar spacing is used, equations for the flooding-loading transition and for power consumption for a single Rushton impeller can be extended relatively easily to dual systems. All results for this spacing are explained by reference to bulk flow patterns and gassed-filled cavity structures and the proportion of sparged gas flowing through the upper impeller is also estimated. Such a spacing is generally recommended since it maximizes the power draw and hence the potential for oxygen mass transfer. Data are presented for other spacings but the results do not fit in easily with single agitator studies because strong impeller-impeller flow pattern interactions occur.