Fluorescence theranostic PROTACs for real-time visualization of ERα degradation.

Proteolysis targeting chimera (PROTAC) technology, a groundbreaking strategy for degradation of pathogenic proteins by hijacking of the ubiquitin-proteasome-system has become a promising strategy in drug design. However, the real-time monitoring and visualization of protein degradation processes have been long-standing challenges in the realm of drug development. In this research, we sought to amalgamate the highly efficient protein-degrading capabilities of PROTAC technology with the visualization attributes of fluorescent probes, with the potential to pave the path for the design and development of a novel class of visual PROTACs. These novel PROTACs uniquely possess both fluorescence imaging and therapeutic characteristics, all with the goal of enabling real-time observations of protein degradation processes. Our approach involved the utilization of a high ER-targeting fluorescent probe, previously reported in our laboratory, which served as a warhead that specifically binds to the protein of interest (POI). Additionally, a VHL ligand for recruiting E3 ligase and linkers of various lengths were incorporated to synthesize a series of novel ER-inherent fluorescence PROTACs. Among them, compound A3 demonstrated remarkable efficiency in degrading ERα proteins (DC50 = 0.12 µM) and displaying exceptional anti-proliferative activity against MCF-7 cells (IC50 = 0.051 µM). Furthermore, it exhibited impressive fluorescence imaging performance, boasting an emission wavelength of up to 582 nm, a Stokes shift of 116 nm, and consistent optical properties. These attributes make it especially suitable for the real-time, in situ tracking of ERα protein degradation processes, thus may serve as a privileged visual theranostic PROTAC for ERα+ breast cancer. This study not only broadens the application spectrum of PROTAC technology but also introduces a novel approach for real-time visualization of protein degradation processes, ultimately enhancing the diagnostic and treatment efficacy of PROTACs.

Study on fermentation preparation, physicochemical properties and biological activity of carboxymethylpachymaran with different degrees of substitution.

BACKGROUND: Carboxymethylpachymaran (CMP) is created by carboxymethylating pachyman (PM), which increases its water solubility and enhances a number of biological activities. Traditional polysaccharides modified by carboxymethylation employ strong chemical techniques. Carboxymethylcellulose (CMC) has been used previously for liquid fermentation to carboxymethyl modify bacterial polysaccharides. This theory can be applied to fungal polysaccharides because Poria cocos has the ability to naturally utilize cellulose. RESULTS: CMC with different degrees of substitution (DS) (0.7, 0.9 and 1.2) were added to P. cocos fermentation medium, and CMPs with different DS (0.38, 0.56 and 0.78, respectively) were prepared by liquid fermentation. The physical and chemical properties and biological activities of the CMPs were determined. Their structures were confirmed by Fourier transform infrared (FTIR) spectroscopy and monosaccharide composition. With the increase of DS, the viscosity and viscosity-average molecular weight of CMPs decreased, whereas polysaccharide content and water solubility increased, although the triple helix structure was not affected. The results of bioactivity assay showed that the higher the DS of CMPs, the higher the 2,2-diphenyl-1-picrylhydrazyl radical scavenging ability, and the stronger the bacterial inhibition ability. CONCLUSION: The present study has developed a method for producing CMPs by P. cocos liquid fermentation. The results of the study confirm that enhancing the DS of CMP could effectively enhance its potential biological activity. The findings provide safe and reliable raw materials for creating CMP-related foods and encourage CMP application in the functional food industry. © 2024 Society of Chemical Industry.

The physicochemical, antioxidant, hypoglycemic and prebiotic properties of γ-irradiated polysaccharides extracted from Lentinula edodes.

In this study, the influence of γ-irradiation with different dose (0, 4, 8, and 16 kGy) on chemical composition, physicochemical features and bioactivities of polysaccharides extracted from Lentinula edodes (LEP) were investigated. The carbohydrate content (from 59.47 to 70.96%), the solubility, the ⋅OH and DPPH scavenging ability of LEP increased with the increased γ-irradiation dose, while the protein content, the weight-average and number-average molecular weight of LEP were significantly decreased with the increased γ-irradiation dose. Moreover, γ-irradiation treatment caused LEP color changes and surface topography destroyed. γ-Irradiated LEP showed higher hypoglycemic activities in vitro than that of non-irradiated LEP. Moreover, γ-irradiated LEP had better proliferation promoting effects on Lactobacillus rhamnosus and L. plantarum. These results showed that γ-irradiation treatment changes the physicochemical features of LEP, thus affects its antioxidant, hypoglycemic and prebiotic properties, which suggests that γ-irradiated LEP has potential application in the pharmaceutical industries and functional foods. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01234-5.

Enhancement of antibacterial and growth-promoting effects of Paenibacillus polymyxa by optimizing its fermentation process.

AIMS: We aimed to enhance the antibacterial and growth-promoting effects of Paenibacillus polymyxa by improving the yield of spores, lipopeptides and indole-3-acetic acid (IAA) in the fermentation process. METHODS AND RESULTS: Through medium optimization by the response surface method and feeding fermentation, the number of spores reached 2.37 × 109  cfu ml-1 with an increase of 38%, the content of lipopeptides reached 60.8 mg L-1 with an increase of 89%, and the content of IAA reached 24.3 mg L-1 with an increase of 176%, respectively, comparing with the original (un-optimized) culture conditions. The fermentation culture of P. polymyxa from the optimized medium and feeding fermentation resulted in higher colonization of P. polymyxa in soils than that from the original culture during the 49 days for testing. Comparing with the supernatant of the original culture, the supernatant of the P. polymyxa culture from the optimized medium and feeding fermentation showed enhanced antibacterial effects and plant growth-promoting effects. The enhanced antibacterial effect was shown as the increase of the inhibition zone by 59%, 45% and 26% against Ralstonia solanacearum, Erwinia carotovora and Xanthomonas campestris. The enhanced growth-promoting effects on tomato and strawberry plants were the increase of plant height by 47% and 5%, root length by 23% and 15% and root weight by 65% and 110%. CONCLUSIONS: The combination of medium optimization and feeding fermentation effectively improved the yield of spores, lipopeptides and IAA. Lipopeptides and IAA lead to enhanced antibacterial and plant growth-promoting effects of the P. polymyxa product. SIGNIFICANCE AND IMPACT OF THIS STUDY: The optimized fermentation method significantly improved the yield of spores, lipopeptides and IAA, thus providing theoretical and technical support for enhancing the antibacterial and growth-promoting effects of P. polymyxa products in agriculture.

New technology to improve the thermal stability of botulinum toxin type D by biomimetic mineralization.

The advanced biomimetic mineralization technology was applied to protect the Botulinum neurotoxin type D, and the processing of the mineralization granule of botulinum toxin type D was successfully screened. The loss of activity of the toxin protein at different temperatures and the destructive strength of the gastrointestinal tract against the toxin were determined biologically. The lethal toxicity of the mineralized toxin to wild rodents was determined by median lethal dose. Protective tests at different temperatures showed that the preservation period of botulinum toxin type D mineralized sample 2 was significantly higher than that of the control group at three different temperatures, and its toxicity loss was significantly reduced. The damage intensity of the mineralized toxin to the gastrointestinal contents of plateau zokor and plateau pika was significantly reduced. The minimum lethal doses of the mineralized toxin particles to plateau zokor, plateau pika, and mice were 5200, 8,600,000, and 25,000 MLD/kg. These results showed that biomimetic mineralization could greatly improve the thermal stability of botulinum toxin type D and reduce the damaging effect of the gastrointestinal contents of target animals to botulinum toxin type D. The mineralized toxin could be used to control the population density of urban rodents. This research provides new insights into the protection of toxin protein substances.

Homogenate Extraction of Dihydroartemisinin from Artemisia Hedinii and Its Antifungal Activity.

BACKGROUND: Artemisia hedinii is a well-known traditional Chinese medicine. It can be used to extract dihydroartemisinin (DHA). OBJECTIVE: The purpose of this study was to explore the optimal conditions for the homogenate extraction of DHA from A. hedinii and the antifungal activity of DHA. METHODS: In this study, single-factor experiments and the response surface method were used to determine the optimal extraction conditions of crude extract and DHA. The method of spore germination was used to study the antifungal activity of DHA on Alternaria alternata. RESULTS: The optimal conditions were found as follows: ratio of liquid to material 22 mL/g; extraction time 60 s; and soaking time 34 min. Under these conditions, extraction yield of DHA was (1.76 ± 0.04%). When the concentrations of crude extract were 0.5 and 8 mg/mL, the spore germination inhibition rates of A. alternata were (17.00 ± 2.05%) and (92.56 ± 2.01%), which were 3.34 and 1.15 times that of the DHA standard, respectively. CONCLUSIONS: Homogenate extraction technology is a fast and efficient method for extracting DHA from A. hedinii. The crude extract has significant antifungal activity against A. alternata and is inexpensive, providing possible DHA usage in the prevention and treatment of plant pathogenic fungi. HIGHLIGHTS: The optimum conditions of the extraction of DHA from A. hedinii by homogenate extraction were obtained. DHA has antifungal activity against A. alternata. Compared with pure DHA, the crude extract has stronger antifungal activity against A. alternata.

Effects of Heat Stress and the Addition of Different Purines on the Synthesis of Eritadenine in Liquid Fermentation of Shiitake Culinary-Medicinal Mushroom, Lentinula edodes (Agaricomycetes).

This work aimed to explore the effects of heat stress and the addition of different purines (adenine, guanine, xanthine, and hypoxanthine) on the synthesis of eritadenine in liquid fermentation of Lentinula edodes. The L. edodes biomass and eritadenine content were determined. The results showed that eritadenine mainly existed in the fermentation broth, and the synthesis of eritadenine might be partially coupled with L. edodes mycelium growth in this study. Heat stress inhibited mycelium growth and the synthesis of eritadenine. The addition of adenine had no effect on the synthesis of eritadenine. However, the addition of guanine and xanthine inhibited the synthesis of eritadenine. When subjected to hypoxanthine, a significant increase in eritadenine production was observed, which was 1.54 times that of the control group.

Anti-intoxication and protective effects of a recombinant serine protease inhibitor from Lentinula edodes against acute alcohol-induced liver injury in mice.

Serine protease inhibitors (serpins) are involved in inflammation, coagulation, fibrinolysis, tumor suppression, molecular chaperone, chromatin densification, and hormone transport. However, their anti-intoxication activity has not been determined. Here, we heterologously expressed the serpin gene from Lentinula edodes in Escherichia coli and purified the recombinant serpin protein from L. edodes (rLeSPI). Then, we administered alcohol and active protein or Haiwangjinzun as a positive control to mice via gavage to evaluate the anti-intoxication activities of rLeSPI in vivo. We also investigated the protective effects of rLeSPI on acute alcohol-induced liver injury in mice by physiological and biochemical assays. The assay results for the anti-intoxication activity revealed that pretreating mice with 5 mg/kg rLeSPI for 0.5 h before gavage with Erguotou liquor (56%V EtOH, 0.15 ml/10 g) significantly prolonged the tolerance time and shortened the intoxication time relative to the results of the control group, thereby proving its anti-intoxication activities. The biochemical analysis showed that rLeSPI improved glutathione peroxidase activity, which was evidently reduced by ethanol. Additionally, rLeSPI significantly improved the activity of aldehyde dehydrogenase, which is important in alcohol metabolism, and reduced the intracellular malondialdehyde content, aspartate amino transferase, and alanine amino transferase activity. We concluded that LeSPI displayed anti-intoxication activity and exerted protective effects against acute alcohol-induced liver injury, providing new insight into the prevention of alcoholism and alcohol-related diseases.Key Points• Anti-intoxication activity of a recombinant serpin protein rLeSPI was assessed.• LeSPI displayed anti-intoxication activity in mice.• LeSPI exerted protective effects against acute alcohol-induced liver injury in mice.

Physicochemical properties and bioactivities of Lentinula edodes polysaccharides at different development stages.

Lentinula edodes polysaccharides from at four different development stages (referred to L1, L2, L3 and L4, respectively) were extracted by hot water method, and graded ethanol precipitation to final concentration of 20%, 50% and 70%, then12 crude polysaccharide fractions (referred to L1P20, L2P20, L3P20; L4P20, L1P50, L2P50, L3P50, L4P50 and L1P70, L2P70, L3P70, L4P70, respectively) were obtained. Physicochemical properties and exoteric bioactivities of the crude polysaccharide fractions were measured. The results of physicochemical properties revealed that extraction yields of P20 fractions were significantly higher than those of P50 and P70 fractions, and the contents of polysaccharide and ß-glucan in L3P50 fractions were higher, and the viscosity-average molecular weight reached a maximum at L2, and high molecular weight polysaccharides could be obtained at a low alcohol concentration in P20 fractions, and the glycosidic bonds were found to exist in all crude polysaccharide fractions. These crude polysaccharide fractions showed different bioactivities, wherein the polysaccharides of higher molecular weight in P20 fractions had greater bioactivity. These results showed that immature stage of Lentinula edodes was the optimal harvest time for obtaining higher bioactivity of crude polysaccharides.

Effects of Tween 80 on the liquid fermentation of Lentinus edodes.

This paper explored the effects of Tween 80 on the biomass, intracellular polysaccharide (IPS) content, fermentation parameters, the pellets size of mycelium, and the antioxidant activity of IPS in Lentinus edodes liquid fermentation. With adding to Tween 80, the outputs of biomass and IPS increased during the L. edodes fermentation, respectively, while the reducing sugar content was decreased, as well as, the time courses of pH value were different. It was also shown that the addition of Tween 80 could protect the intact of pellets from breaking down. The effects of Tween 80 on the main structure of IPS were no obvious, and the IPS were revealed similar infrared spectrum, as was indicated by the infrared spectrum analysis. Improvements in the scavenging capacity of DPPH radicals of IPS were observed in Tween 80 treated group compared with the control group. Tween 80 exerts impacts on the liquid fermentation of L. edodes.

Ultra-high performance liquid chromatography tandem mass spectrometry for the determination of five glycopeptide antibiotics in food and biological samples using solid-phase extraction.

This paper demonstrated the development and validation of an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of five glycopeptide antibiotics in food and biological samples. The target glycopeptide antibiotics were isolated from the samples by solvent extraction, and the extracts were cleaned with a tandem solid-phase extraction step using mixed strong cation exchange and hydrophilic/lipophilic balance cartridges. Subsequently, the analytes were eluted with different solvents, and then quantified by UHPLC-MS/MS in the positive ionization mode with multiple reaction monitoring. Under optimal conditions, good linear correlations were obtained for the five glycopeptide antibiotics in the concentration range of 1.0 µg/L to 20.0 µg/L, and with linear correlation coefficients >0.998. Employing this method, the target glycopeptide antibiotics in food and biological samples were identified with a recovery of 83.0-102%, and a low quantitation limit of 1.0 µg/kg in food and 2.0 µg/L in biological samples with low matrix effects.

Comparison of the effects of DC031050, a class III antiarrhythmic agent, on hERG channel and three neuronal potassium channels.

AIM: This study was conducted to test the selectivity of DC031050 on cardiac and neuronal potassium channels. METHODS: Human ether-à-go-go related gene (hERG), KCNQ and Kv1.2 channels were expressed in CHO cells. The delayed rectifier potassium current (I(K)) was recorded from dissociated hippocampal pyramidal neurons of neonatal rats. Whole-cell voltage patch clamp was used to record the voltage-activated potassium currents. Drug-containing solution was delivered using a RSC-100 Rapid Solution Changer. RESULTS: Both DC031050 and dofetilide potently inhibited hERG currents with IC(50) values of 2.3 ± 1.0 and 17.9 ± 1.2 nmol/L, respectively. DC031050 inhibited the I(K) current with an IC(50) value of 2.7 ± 1.5 µmol/L, which was >1000 times the concentration required to inhibit hERG current. DC031050 at 3 µmol/L did not significantly affect the voltage-dependence of the steady activation, steady inactivation of I(K), or the rate of I(K) from inactivation. Intracellular application of DC031050 (5 µmol/L) was insufficient to inhibit I(K). DC031050 up to 10 µmol/L had no effects on KCNQ2 and Kv1.2 channel currents. CONCLUSION: DC031050 is a highly selective hERG potassium channel blocker with a substantial safety margin of activity over neuronal potassium channels, thus holds significant potential for therapeutic application as a class III antiarrhythmic agent.

SKF83959 suppresses excitatory synaptic transmission in rat hippocampus via a dopamine receptor-independent mechanism.

Dopamine (DA) profoundly modulates excitatory synaptic transmission and synaptic plasticity in the brain. In the present study the effects of SKF83959, the selective agonist of phosphatidylinositol (PI)-linked D(1) -like receptor, on the excitatory synaptic transmission were investigated in rat hippocampus. SKF83959 (10-100 µM) reversibly suppressed the field excitatory postsynaptic potential (fEPSP) elicited by stimulating the Schaffer's collateral-commissural fibers in CA1 area of hippocampal slices. However, the inhibition was not blocked by the D(1) receptor antagonist SCH23390, the D(2) receptor antagonist raclopride, the 5-HT(2A/2C) receptor antagonist mesulergine, or the α(1) -adrenoceptor antagonist prazosin. In addition, SKF83959 inhibited the afferent volley and significantly reduced the paired-pulse facilitation ratios. In dissociated hippocampal CA1 pyramidal neurons, SKF83959 had no detectable effect on glutamate-induced currents but potently inhibited voltage-activated Na(+) current (IC50 value = 26.9 ± 1.0 µM), which was not blocked by SCH23390 or by intracellular dialysis of GDP-ß-S. These results demonstrate that SKF83959 suppressed the excitatory synaptic transmission in hippocampal CA1 area, which was independent of D(1) -like receptor. The mechanism underlying the effect could be mainly inhibition of Na(+) channel in the afferent fibers. The suppression of excitatory synaptic transmission and the Na(+) channel by SKF83959 may contribute to its therapeutic benefits in Parkinson's disease.

Electrophysiological effects of SKF83959 on hippocampal CA1 pyramidal neurons: potential mechanisms for the drug's neuroprotective effects.

Although the potent anti-parkinsonian action of the atypical D1-like receptor agonist SKF83959 has been attributed to the selective activation of phosphoinositol(PI)-linked D1 receptor, whereas the mechanism underlying its potent neuroprotective effect is not fully understood. In the present study, the actions of SKF83959 on neuronal membrane potential and neuronal excitability were investigated in CA1 pyramidal neurons of rat hippocampal slices. SKF83959 (10-100 µM) caused a concentration-dependent depolarization, associated with a reduction of input resistance in CA1 pyramidal neurons. The depolarization was blocked neither by antagonists for D1, D2, 5-HT(2A/2C) receptors and α1-adrenoceptor, nor by intracellular dialysis of GDP-ß-S. However, the specific HCN channel blocker ZD7288 (10 µM) antagonized both the depolarization and reduction of input resistance caused by SKF83959. In voltage-clamp experiments, SKF83959 (10-100 µM) caused a concentration-dependent increase of Ih current in CA1 pyramidal neurons, which was independent of D1 receptor activation. Moreover, SKF83959 (50 µM) caused a 6 mV positive shift in the activation curve of Ih and significantly accelerated the activation of Ih current. In addition, SKF83959 also reduced the neuronal excitability of CA1 pyramidal neurons, which was manifested by the decrease in the number and amplitude of action potentials evoked by depolarizing currents, and by the increase of firing threshold and rhoebase current. The above results suggest that SKF83959 increased Ih current through a D1 receptor-independent mechanism, which led to the depolarization of hippocampal CA1 pyramidal neurons. These findings provide a novel mechanism for the drug's neuroprotective effects, which may contributes to its therapeutic benefits in Parkinson's disease.

Incompatibility behavior of a symbiotic plasmid pMH7653Rb in Mesorhizobium huakuii 7653R.

Mesorhizobium huakuii strain 7653R harbored two indigenous plasmids named pMH7653Ra and pMH7653Rb. The larger plasmid pMH7653Rb (symbiotic plasmid) was transferred to M. huakuii HN308SR harboring three plasmids: pMHHN308a, pMHHN308b and pMHHN308c, and HN3015SR harboring three plasmids: pMHHN3015a, pMHHN3015b and pMHHN3015c by tri-parent mating. Two stable indigenous plasmids, pMHHN308b and pMHHN308c of HN308SR, were co-eliminated due to the introduction of pMH7653Rb, and the transconjugant was named HN308SRN14. The results implied that pMH7653Rb and pMHHN308b, pMHHN308c were incompatible and might have been ascribed to the same incompatible group. The plasmid profiles of transconjugant HN3015SRN14 showed that the second largest plasmid pMHHN3015b of HN3015SR was cured due to the introduction of pMH7653Rb. The results also implied that pMH7653Rb and pMHHN3015b were incompatible. Results from plant nodulation tests showed that pMH7653Rb could only maintain the nodulation ability in transconjugant HN308SRN14 and its nodule number was more than that of wild strain HN308SR, but could not replace the nitrogen fixation effect of pMHHN308b and pMHHN308c. The plasmid cured mutant HN308SRN14D harboring only pMHHN308a formed null nodules that demonstrated pMHHN308a was relevant to nodulation ability. HN3015SRN14 harboring pMH7653Rb, pMHHN3015a and pMHHN3015c formed null nodules while HN3015SRN14D containing pMHHN3015a and pMHHN3015c lost the nodulation ability. The plasmid replication repC-like gene sequences were detected by a polymerase chain reaction from 7653R, HN308, HN3015, HN308SRN14 and HN3015SRN14. The repC gene sequence similarities of the strains tested attained 99%.

Magnesium lithospermate B dilates mesenteric arteries by activating BKCa currents and contracts arteries by inhibiting K(V) currents.

AIM: To examine the involvement of K(+) channels and endothelium in the vascular effects of magnesium lithospermate B (MLB), a hydrophilic active component of Salviae miltiorrhiza Radix. METHODS: Isolated rat mesenteric artery rings were employed to investigate the effects of MLB on KCl- or norepinephrine-induced contractions. Conventional whole-cell patch-clamp technique was used to study the effects of MLB on K(+) currents in single isolated mesenteric artery myocytes. RESULTS: MLB produced a concentration-dependent relaxation in mesenteric artery rings precontracted by norepinephrine (1 micromol/L) with an EC(50) of 111.3 micromol/L. MLB-induced relaxation was reduced in denuded artery rings with an EC(50) of 224.4 micromol/L. MLB caused contractions in KCl-precontracted artery rings in the presence of N-nitro-L-arginine methyl ester (L-NAME) with a maximal value of 130.3%. The vasodilatory effect of MLB was inhibited by tetraethylammonium (TEA) in both intact and denuded artery rings. In single smooth muscle cells, MLB activated BK(Ca) currents (EC(50) 156.3 micromol/L) but inhibited K(V) currents (IC(50) 26.1 micromol/L) in a voltage- and concentration-dependent manner. CONCLUSION: MLB dilated arteries by activating BK(Ca) channels in smooth muscle cells and increasing NO release from endothelium, but it also contracted arteries precontracted with KCl in the presence of L-NAME.

Activation of phosphatidylinositol-linked D1-like receptors increases spontaneous glutamate release in rat somatosensory cortical neurons in vitro.

Central dopaminergic system exerts profound modulation on spontaneous glutamate release in various brain regions mainly through D(1) receptor/cAMP/PKA pathway. It remains unclear whether the phosphatidylinositol (PI)-linked D(1)-like receptors are also involved in such modulatory actions. The identification of substituted phenylbenzazepine SKF83959 as the selective agonist for the atypical D(1)-like receptors has given impetus to study their influence on the spontaneous glutamate release in the brain. In the present study the effects of SKF83959 on the spontaneous excitatory postsynaptic currents (sEPSCs) were investigated through whole-cell recording from layer V-VI pyramidal neurons in rat somatosensory cortical slices. Perfusion with SKF83959 (10-100 microM) considerably increased the frequency of sEPSCs, while had no significant effect on the amplitude of sEPSCs. The increase of sEPSC frequency by SKF83959 was blocked by SCH23390, a D(1)-like receptor antagonist, but not by the antagonists for D(2) receptor, alpha(1)-adrenoceptor and 5-HT(2A/2C) receptor. U-73122 (PLCbeta inhibitor), 2-APB (IP(3) receptor antagonist), chelerythrine chloride (PKC inhibitor) and capsazepine (TRPV1 antagonist) could block the effects of SKF83959, whereas H-89 (PKA inhibitor) and forskolin (adenylyl cyclase activator) had no effect. Taken together, sensitization of TRPV1 channels by PKC after activation of D(1) receptor/PLCbeta signaling pathway mediated SKF83959-induced increase in the sEPSC frequency. To our knowledge, this is the first pharmacological evidence that PI-linked D(1)-like dopamine receptors do exist in presynaptic terminals of cortical neurons and play an important role in controlling the spontaneous glutamate release.

Arylbenzazepines are potent modulators for the delayed rectifier K+ channel: a potential mechanism for their neuroprotective effects.

(+/-) SKF83959, like many other arylbenzazepines, elicits powerful neuroprotection in vitro and in vivo. The neuroprotective action of the compound was found to partially depend on its D(1)-like dopamine receptor agonistic activity. The precise mechanism for the (+/-) SKF83959-mediated neuroprotection remains elusive. We report here that (+/-) SKF83959 is a potent blocker for delayed rectifier K(+) channel. (+/-) SKF83959 inhibited the delayed rectifier K(+) current (I(K)) dose-dependently in rat hippocampal neurons. The IC(50) value for inhibition of I(K) was 41.9+/-2.3 microM (Hill coefficient = 1.81+/-0.13, n = 6), whereas that for inhibition of I(A) was 307.9+/-38.5 microM (Hill coefficient = 1.37+/-0.08, n = 6). Thus, (+/-) SKF83959 is 7.3-fold more potent in suppressing I(K) than I(A). Moreover, the inhibition of I(K) by (+/-) SKF83959 was voltage-dependent and not related to dopamine receptors. The rapidly onset of inhibition and recovery suggests that the inhibition resulted from a direct interaction of (+/-) SKF83959 with the K(+) channel. The intracellular application of (+/-) SKF83959 had no effects of on I(K), indicating that the compound most likely acts at the outer mouth of the pore of K(+) channel. We also tested the enantiomers of (+/-) SKF83959, R-(+) SKF83959 (MCL-201), and S-(-) SKF83959 (MCL-202), as well as SKF38393; all these compounds inhibited I(K). However, (+/-) SKF83959, at either 0.1 or 1 mM, exhibited the strongest inhibition on the currents among all tested drug. The present findings not only revealed a new potent blocker of I(K) , but also provided a novel mechanism for the neuroprotective action of arylbenzazepines such as (+/-) SKF83959.

[Cloning and identification of the plasmid replication gene repC in Mesorhizobium huakuii].

OBJECTIVE: The repC gene is the principal initiation protein gene for plasmid replication. We identified the repC-like sequences from Mesorhizobium huakuii strain HN3015 and its derivatives of plasmid curing. METHODS: Primers of RC1 and RC3 were used to amplify the repC-like sequences by a polymerase chain reaction, the PCR products were obtained and cloned into plasmid vector pMD-18T and then sequenced. Location of the repC sequences on different plasmids in the strains tested was carried out by Southern blotting. The nucleotides homology analysis of the repC gene was carried out using the BLAST. Amino acid sequences were deduced using the ExPASy. Multiple sequences alignments were performed using the ClustalW. Analysis of protein secondary structure was carried out using the PredictProtein. RESULTS: The repC-like sequences were obtained from the strains tested. The sizes of the PCR products were about 750 bp. The results of Southern blotting showed that the repC-like sequences were only associated with a plasmid in the stains tested. CONCLUSIONS: The repC sequences of the strains tested showed 100% sequence similarity, but were obviously different from that of other rhizobia strains.

Electrophysiological characterization of a novel Kv channel blocker N,N'-[oxybis(2,1-ethanediyloxy-2,1-ethanediyl) ]bis(4-methyl)-benzenesulfonamide found in virtual screening.

AIM: N,No-[oxybis(2,1-ethanediyloxy-2,1-ethanediyl)]bis(4-methyl)- benzenesulfonamide (OMBSA) is a hit compound with potent voltage-gated K+ (Kv) channel-blocking activities that was found while searching the MDL Available Chemicals Directory with a virtual screening approach. In the present study, the blocking actions of OMBSA on Kv channels and relevant mechanisms were characterized. METHODS: Whole-cell voltage-clamp recording was made in acutely dissociated hippocampal CA1 pyramidal neurons of newborn rats. RESULTS: Superfusion of OMBSA reversibly inhibited both the delayed rectifier (I(K)) and fast transient K+ currents (I(A)) with IC50 values of 2.1+/-1.1 micromol/L and 27.8+/-1.5 micromol/L, respectively. The inhibition was voltage independent. OMBSA markedly accelerated the decay time course of IK, without a significant effect on that of I(A). OMBSA did not change the activation, steady-state inactivation of IK, and its recovery from inactivation, but the compound caused a significant hyperpolarizing shift of the voltage dependence of the steady-state inactivation of I(A) and slowed down its recovery from inactivation. Intracellular dialysis of OMBSA had no effect on both I(K) and I(A). CONCLUSION: The results demonstrate that OMBSA blocks both I(K) and I(A) through binding to the outer mouth of the channel pore, as predicted by the molecular docking model used in the virtual screening. In addition, the compound differentially moderates the inactivation kinetics of the K+ channels through allosteric mechanisms.