ABSTRACT
Hemsleya chinensis is a Chinese traditional medicinal plant, containing cucurbitacin IIa (CuIIa) and cucurbitacin IIb (CuIIb), both of which have a wide range of pharmacological effects, including antiallergic, anti-inflammatory, and anticancer properties. However, few studies have been explored on the key enzymes that are involved in cucurbitacins biosynthesis in H. chinensis. Oxidosqualene cyclase (OSC) is a vital enzyme for cyclizing 2,3-oxidosqualene and its analogues. Here, a gene encoding the oxidosqualene cyclase of H. chinensis (HcOSC6), catalyzing to produce cucurbitadienol, was used as a template of mutagenesis. With the assistance of AlphaFold2 and molecular docking, we have proposed for the first time to our knowledge the 3D structure of HcOSC6 and its binding features to 2,3-oxidosqualene. Mutagenesis experiments on HcOSC6 generated seventeen different single-point mutants, showing that single-residue changes could affect its activity. Three key amino acid residues of HcOSC6, E246, M261 and D490, were identified as a prominent role in controlling cyclization ability. Our findings not only comprehensively characterize three key residues that are potentially useful for producing cucurbitacins, but also provide insights into the significant role they could play in metabolic engineering.
ABSTRACT
The aim of the present study was to observe whether protein tyrosine kinase (PTK) within the nucleus tractus solitarius (NTS) was involved in the regulation of ventilatory responses of peripheral chemoreflex. The experiments were performed on anesthetized, immobilized and artificially ventilated rabbits. Peripheral chemoreflex was elicited by ventilating the animal with 10% O2-balance 90% N2. Changes in the peak amplitude and frequency of integrated phrenic nerve activity were observed. The ventilatory responses of peripheral chemoreflex following 0.1 microl microinjection within the NTS of either PTK inhibitor genistein (10 mol/L), AMPA glutamate receptor inhibitor CNQX (10 mol/L),or inactive PTK inhibitor daidzein (10 mol/L) were recorded. The results are as follows: Both genistein and CNQX attenuated the ventilatory responses of peripheral chemoreflex, while no changes occurred following daidzein. The amplitude of integrated phrenic nerve discharge and the phrenic burst frequency were decreased by (-21.77+/-6.93)% and (-24.70+/-7.61)% respectively after administration of genistein. CNQX resulted in similar decreases in the amplitude of phrenic nerve discharge (-27.13+/-7.63)% and the burst frequency (-21.34+/-4.88)%. In addition, the inhibitory effects of CNQX and genistein were the same whether they were applied alone or one after another, indicating that they had no cooperative effects. The results obtained suggest that PTK within the NTS regulates the peripheral chemoreflex control of respiration and that this regulation of PTK may be mediated through the phosphorylation of AMPA receptors in NTS neurons.
Subject(s)
Animals , Female , Male , Rabbits , Brain Stem , Physiology , Chemoreceptor Cells , Physiology , Protein-Tyrosine Kinases , Physiology , Receptors, AMPA , Physiology , Respiration , Solitary NucleusABSTRACT
<p><b>AIM</b>To observe the effects of sustained electrical stimulation at Bötzinger complex (Böt. c) on phrenic nerve discharges.</p><p><b>METHODS</b>Sustained electrical stimulation (10--50 microA, 40-100 Hz, 0.3 ms, for 15-30 s) of Böt. C on 30 urethane anaesthetized, vagotomized, paralyzed and artificially ventilated rabbits.</p><p><b>RESULTS</b>Sustained electrical stimulation of Bot. C produced the inhibition or "inspiratory off-switch" of phrenic discharge during the stimulation. The inhibition of the phrenic discharges showed intensity and frequency dependence. Habituation was shown during the stimulation, showing the magnitude of the phrenic nerve discharge increased gradually. Post-stimulus rebound exhibited upon the cessation of the stimulation, showing the magnitude of the phrenic activity increased significantly. Short-term memory was shown in the habituation of the phrenic activity.</p><p><b>CONCLUSION</b>Non-associative learning is involved in the central control of respiratory modulation in the Böt. C and synaptic plasticity may exist in the respiratory neurons of Böt. C.</p>
