Screening and identification of potassium-dissolving bacteria from different rhizosphere soil of Paris polyphylla var. yunnanensis / 中国中药杂志

The study aiming at exploring the potassium-dissolving capacity of rhizosphere potassium-dissolving bacteria from diffe-rent sources and screen the strains with high potassium-dissolving ability, so as to lay a theoretical foundation for cultivation and quality improvement of Paris polyphylla var. yunnanensis sources. The rhizosphere soil of 10 wild and transplanted species from Yunnan, Sichuan and Guizhou provinces was used as the research object. Potassium-dissolving bacteria were isolated and purified, and their potassium-dissolving capacity was determined by flame spectrophotometry, and identified by physiological, biochemical and molecular biological methods. Twenty-six potassium-dissolving bacteria were purified and 13 were obtained from wild and transplanted strains respectively. It was found through the determination of potassium-dissolving capacity that the potassium-dissolving capacity of 26 strains was significantly different, and the mass concentration of K~+ in the fermentation broth were 1.04-2.75 mg·L~(-1), the mcentration of potassium were 0.01-1.82 mg·L~(-1). The strains were identified as Bacillus, Agrobacterium rhizome and Staphylococcus by physiological, biochemical and 16 S rDNA molecular methods, among them Bacillus amylolyticus(4 strains) was the dominant bacterium of Bacillus. The physiology and biochemistry of rhizosphere potassium-dissolving bacteria in P. polyphylla var. yunnanensis rhizosphere were diffe-rent, and the living environment were different, so the potassium-dissolving capacity also changed. Strain Y4-1 with the highest potassium decomposability was Bacillus amylolytic with a potassium increase of 1.82 mg·L~(-1). The potassium-dissolving ability and the distribution of potassium-dissolving bacteria were different in various habitats. The screening of potassium-dissolving bacteria provided a new strain for the preparation of microbial fertilizer. It is expected that B. amyloidococcus Y4-1 can be used as an ideal strain to cultivate mycorrhizal seedlings of P. polyphylla var. yunnanensis.

Isolation and identification of phosphatolytic bacteria in Paris polyphylla var. yunnanensis / 中国中药杂志

The wild resources of Paris polyphylla var. yunnanensis, a secondary endangered medicinal plant, are severely scarce. Introduction and cultivation can alleviate market demand. To screen phosphatolytic bacteria in the rhizosphere soil of P. polyphylla var. yunnanensis and provide data support for the development of high-efficiency microbial fertilizer, in this study, the dilution plate coating method was used to isolate and screen the phosphorus solubilizing bacteria with the ability of mineralizing organic phosphorus from the rhizosphere soil of wild and transplanted varieties of P. polyphylla var. yunnanensis in 10 different locations in Yunnan, Sichuan and Guizhou. After separation and purification, the phosphatolytic capacity was analyzed by qualitative and quantitative analysis. Combined with physiological and biochemical experiments, the strains were identified using 16 S rDNA sequencing analysis. Forty one strains were selected from the rhizosphere soil of P. polyphylla var. yunnanensis from 10 different habitats. Among them, 21 strains were obtained from the rhizosphere soil of the wild variety P. polyphylla var. yunnanensis and 20 strains were obtained from the rhizosphere soil of the transplanted variety. And significance analysis found that 41 organophosphate solubilizing strains had significant differences in their ability to solubilize phosphorus. The amount of phosphate solubilizing was 0.08-67.61 mg·L~(-1), the pH value was between 4.27 and 6.82. The phosphatolytic amount of strain Y3-5 was 67.61 mg·L~(-1), and the phosphorus increase amount was 57.57 mg·L~(-1). All 41 strains were identified as Gram-positive Bacillus. Combining physiological characteristic and phylogenetic trees, Bacillus mobilis Y3-5 was finally selected as the candidate rhizosphere phosphatolytic bacteria of P. polyphylla var. yunnanensis. The distribution of phosphorus solubilizing bacteria in the rhizosphere soil of P. polyphylla var. yunnanensis was different, and there were significant diffe-rences in phosphorus solubility. Organophosphate-dissolving strain Y3-5 is expected to be a candidate strain of P. polyphylla var. yunnanensis microbial fertilizer.

The inhibition effect on methicillin resistant staphylococcus aureus N31 5 of human βdefensin 3 combined with antibiotics / 预防医学

Objective To analyze the inhibition effect of antimicrobial peptide human βdefensin 3 (hBD3 )against methicillin resistant Staphylococcus aureus (MRSA)N31 5.Methods Antimicrobial peptide hBD3 was synthesized.The direct inhibition on N31 5 was detected by the MIC (minimal inhibitory concentration,MIC)test,antibacterial activity test and the analysis on some important functional genes.The peptide was combined with penicillin,tetracycline,cephradine, rifampin,vancomycin,cefuroxime and chloramphenicol to evaluate the effects on antibiotic MIC values.Results The MIC of hBD3 against N31 5 was 8 μg/mL and the bactericidal activity was observed while the hBD3 concentration reached 2 μg/mL.The peptide hBD3 could improve the expression of agr and reduce the expression of icaA.With 5 μg/mL peptide hBD3,the MIC values against N31 5 of penicillin,cephradine,vancomycin,cefuroxime and chloramphenicol were dropped down.Conclusion There is considerable bactericidal activity of antimicrobial peptide hBD3 against MRSA N31 5. Antibiotic peptide hBD3 could be used to prevent the spread of antibiotic resistant bacteria in the future.

Activation of nuclear factor kappaB signaling pathway in human osteoblasts responses to Staphylococcus aureus in vitro / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate whether S. aureus could activate NF-κB signaling pathway in human osteoblasts.</p><p><b>METHODS</b>Immunoblot and electrophoretic mobility shift assay were used to detect the degradation of I-κBα and activation of NF-κB in human osteoblasts following infection with S.aureus, respectively, and there were investigated the activated state of NF-κB signaling pathway in human osteoblasts. In addition, enzyme-linked immunosorbent assay was used to measure the secretion of IL-6 in culture supernatants, which was represented as one of important cytokines in osteomyelitis, and an inhibitor of NF-κB, SN50, which was added to human osteoblasts culture prior to 1 hour at 50 µmol/L before the infection of S.aureus, was used to determine whether S.aureus-activated NF-κB signaling pathway regulates IL-6 secretion of human osteoblasts.</p><p><b>RESULTS</b>S.aureus could induce the degradation of I-κBα (I-κBα(15 min)/I-κBα(0 min) = 0.409 ± 0.245 and I-κBα(30 min)/I-κBα(0 min) = 0.061 ± 0.010) and activation of NF-κB in human osteoblasts in a time and dose-dependent manner following infection. In addition, the secretion of IL-6 in the supernatants of human osteoblasts ((2.17 ± 0.11) µg/L) was suppressed by 50 µmol/L SN50 compared to without the addition of SN50 ((3.58 ± 0.31) µg/L) (F = 174.25, P < 0.05).</p><p><b>CONCLUSIONS</b>S.aureus could activate NF-κB signaling pathway in human osteoblasts, which could regulate cytokines secretions of human osteoblasts.</p>

Co-location of ACh-sensitive BK channels and L-type calcium channels in type II vestibular hair cells of guinea pig / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore the mechanisms of the influx of calcium ions during the activation of ACh-sensitive BK channel (big conductance, calcium-dependent potassium channel) in type II vestibular hair cells of guinea pigs.</p><p><b>METHODS</b>Type II vestibular hair cells were isolated by collagenase type IA. Under the whole-cell patch mode, the sensitivity of ACh-sensitive BK current to the calcium channels blockers was investigated, the pharmacological property of L-type calcium channel activator-sensitive current and ACh-sensitive BK current was compared.</p><p><b>RESULTS</b>Following application of ACh, type II vestibular hair cells displayed a sustained outward potassium current, with a reversal potential of (-70.5 +/- 10.6) mV (x +/- s, n = 10). At the holding potential of -50 mV, the current amplitude of ACh-sensitive potassium current activated by 100 micromol/L ACh was (267 +/- 106) pA (n = 11). ACh-sensitive potassium current was potently sensitive to the BK current blocker, IBTX (iberiotoxin, 200 nmol/L). Apamin, the well-known small conductance, calcium-dependent potassium current blocker, failed to inhibit the amplitude of ACh-sensitive potassium current at a dose of 1 micromol/L. ACh-sensitive BK current was sensitive to NiCl2 and potently inhibited by CdCl2. NiCl2 and CdCl2 showed a dose-dependent blocking effect with a half inhibition-maximal response of (135.5 +/- 18.5) micromol/L (n = 7) and (23.4 +/- 2.6) micromol/L (n = 7). The L-type calcium channel activator, (-) -Bay-K 8644 (10 micromol /L), mimicked the role of ACh and activated the IBTX-sensitive outward current.</p><p><b>CONCLUSION</b>ACh-sensitive BK and L-type calcium channels are co-located in type II vestibular hair cells of guinea pigs.</p>

Electrophysiological findings for 106 cases with auditory neuropathy / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To analyses the clinical characteristics and electrophysiological finding of 106 patients with auditory neuropathy (AN). Investigate the differential curve type of pure tone audiogram and the abnormal ABR.</p><p><b>METHODS</b>Review the history of patients, pure tone audiometry, middle ear acoustic reflexes, auditory brainstem response, distortion product otoacoustic emission and radiologic imaging studies of the brain of 106 patients with AN during December 2001 to May 2007 in retrospect.</p><p><b>RESULTS</b>The 106 patients with AN were of both genders. The age were between 11-37 years old, and the average age was 17.5 years old. The most patients were adolescence (70.8%). Twelve cases of the 106 patients had evidence of other peripheral neuropathy in addition to hearing loss. Another 94 patients were idiopathic origins. The pure tone audiogram showed a minimal to moderate sensorineural hearing loss at low frequencies 0.5 kHz and 0.25 kHz in 209 ears (98.6%). The average hearing threshold (WHO 1997) in 23.2% of disordered ears at less than 25 dB in the "normal" range. Auditory brainstem response could not be recorded in 124 ears (58.5%) at maximum stimulus. The other 88 ears showed 1 or 2 wave, but the wave were small. There were 23 patients which one side ear ABR was 1 or 2 small waves presented, but the contralateral side were all waves absent. In 3 cases of AN with other peripheral neuropathy which ABR were both ears 1 or 2 small wave ear recorded. However, 1 patient in our sample could not be detected distortion product otoacoustic emission at 3-6 kHz (left ear) and 5-6 kHz (right ear).</p><p><b>CONCLUSIONS</b>AN was not rare in adolescence. The average hearing threshold for AN should be discussed.</p>

Feature of ACh-sensitive potassium current in outer hair cells of guinea pig cochlea / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore the feature of the ACh-sensitive potassium current in guinea pig cochlear outer hair cells.</p><p><b>METHODS</b>Cochlear outer hair cells of guinea pigs (n=38) were isolated by collagenase type IV. Under the whole-cell patch mode, the ions nature and the pharmacological properties of the ACh-sensitive potassium current were investigated by applying the inhibitors of calcium-dependent potassium currents and the inhibitors of nicotinic ACh receptor.</p><p><b>RESULTS</b>Following application of ACh, cochlear outer hair cells displayed a rapidly activating outward potassium current with a fast desensitized kinetic and a reversal (x +/- s) potential of (-67.3 +/- 8.2) mV (n=10). At the holding potential of -50 mV, the current amplitude of ACh-sensitive potassium current activated by 100 micronmol/L ACh was (506.6 +/- 186.3) pA (n=9). ACh-sensitive potassium current was sensitive to TEA (tetraethylammonium chloride, 10 mmol/L) and potently inhibited by the small conductance calcium-dependent potassium current (SK) blocker, apamin (1 micromol/L). Iberiotoxin (IBTX), the well-known blocker of big conductance calcium-dependent potassium current (BK), failed to inhibit the amplitude of the ACh-sensitive potassium current at the dose of 200 nmol/L. The dose for half-maximal response (EC50) of the ACh-sensitive potassium current was (33.5 +/- 5.7) micromol/L (n=7). The ACh-sensitive potassium current was sensitive to the GABA (gamma-aminobutyric acid)-A receptor blocker, bicuculline, and strongly inhibited by the selective blocker of the alpha 9-nicotinic ACh receptor, strychnine. Strychnine and bicuculline showed the dose-dependent blocking effect with a half inhibition-maximal response (IC50) of (22.3 +/- 2.6) nmol/L (n=7) and (1.2 +/- 0.4) micromol/L (n=6), respectively.</p><p><b>CONCLUSIONS</b>This work provides direct evidences that the ACh-sensitive SK current was present on guinea pig cochlear outer hair cells. The activation of the ACh-sensitive SK current was most possibly mediated by a alpha 9-nicotinic ACh receptor.</p>

Dependence of Ca2+ on the acetylcholine-sensitive current in guinea pig type II vestibular hair cells / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore the dependence of Ca2+ on the acetylcholine (ACh)-sensitive potassium current in guinea pig type II vestibular hair cells.</p><p><b>METHODS</b>Under the whole-cell patch mode, the current amplitude of the ACh-sensitive potassium current was recorded in response to the concentration change of the extracellular or intracellular Ca2+.</p><p><b>RESULTS</b>Following application of ACh, type II vestibular hair cells displayed the sustained potassium current, which was inhibited by tetraethylammonium chloride (TEA), but not inhibited by 4-aminopyrine (4-AP). The activation of the ACh-sensitive potassium current was strongly affected by the concentration of the extracellular Ca2+. The current amplitude of the ACh-sensitive potassium increased following the increase of Ca2+ concentration from 0 mmol/L to 4 mmol/L At the concentration of 4 mmol/L Ca2+, the current amplitude of the ACh-sensitive potassium current reached the maximal response. Lowering the Ca2 concentration in the external solution from 4 mmol/L to 0. 5 mmol/L, the current amplitude of the ACh-sensitive potassium current was inhibited to (36.5 +/- 6.5)%. However, no difference was found in the presence and in the absence of the intracellular heparin, which was a well-known blocker of the inositol trisphosphate-dependent calcium release channels. In addition, the calcium channel blocker, Cd2+, inhibited the ACh-sensitive potassium current.</p><p><b>CONCLUSIONS</b>The activation of the ACh-sensitive potassium current in guinea pig type II vestibular hair cells was dependent on the extracellular Ca2+ influx through the calcium channel. The application of ACh would stimulate membrane Ca2+ channels; the influx of Ca2+ will then activate the calcium-dependent potassium current in guinea pig type II hair cells to mediate the hyperpolarization effect.</p>

Cation ions modulate the ACh-sensitive current in type II vestibular hair cells of guinea pigs / 生理学报

Molecular biological studies and electrophysiological data have demonstrated that acetylcholine (ACh) is the principal cochlear and vestibular efferent neurotransmitter among mammalians. However, the functional roles of ACh in type II vestibular hair cells among mammalians are still unclear, with the exception of the well-known alpha9-containing nicotinic ACh receptor (alpha9-nAChR) in cochlear hair cells and frog saccular hair cells. In this study, the properties of the ACh-sensitive current were investigated by whole-cell patch clamp technique in isolated type II vestibular hair cells of guinea pigs. The direct effect of extracellular ACh was to induce a hyperpolarization effect in type II vestibular hair cells. Type II vestibular hair cells displayed a sustained outward current in response to the perfusion of ACh. It took about 60 s for the ACh-sensitive current to get a complete re-activation. The reversal potential of the ACh-sensitive current was (-66 +/- 8) mV, which indicated that potassium ion was the main carrier of this current. The blocking effect by the submillimolar concentration of tetraethylammonium (TEA) further indicated that extracellular ACh stimulated the calcium-dependent potassium current. Following replacement of the compartment of NaCl in the normal external solution with TrisCl, LiCl or saccharose respectively, the amplitude of the ACh-sensitive current was not affected. Blocking of the release of intracellular Ca(2+) stores by intracellular application of heparin failed to inhibit the ACh-sensitive current. Therefore, extracellular Na(+)and the inositol 1,4,5-trisphosphate (IP(3))-dependent intracellular Ca(2+)release were not involved in the activation of the ACh-sensitive current. However, the ACh-sensitive current was strongly affected by the concentration of the extracellular K(+), extracellular Ca(2+) and intracellular Mg(2+). The amplitude of the ACh- sensitive current was strongly inhibited by high concentration of extracellular K(+). In the Ca(2+)-free external solution, ACh only activated a very small current; however, the ACh-sensitive current demonstrated a Ca(2+)-dependent inhibition effect in high concentration of Ca(2+)solution. In addition, the ACh-sensitive current was inhibited by increasing of the concentration of intracellular Mg(2+). In conclusion, the present results demonstrate that ACh plays an important role in the vestibular efferent system. The fact that Na(+) is not involved in the ACh-sensitive current will not favor the well-known profile of alpha9-nAChR, which is reported to display a small but important permeability to Na(+). It is also suggested that, in vivo, the amplitude of the ACh-induced hyperpolarization may strongly depend on the concentration of extracellular Ca(2+)and intracellular Mg(2+).