卡那霉素（Kanamycin）对大豆种子发芽的影响

利用４个大豆品种，设置４种卡那霉素浓度，研究了卡那霉素对大豆种子发芽的影响。结果表明：卡那霉素对大豆种子的发芽势和发芽率没有影响，也未引起幼苗黄化，但能明显抑制幼苗的生长，引起幼苗和主根变短、侧根数减少。在３００μｇ／ｍＬ浓度下，大豆幼苗主根长度为１３ｃｍ，且没有侧根长出。

露草卡那霉素(Kanamycin)抗性筛选研究

露草种子卡那霉素(Km)抑制发芽试验结果显示,在含有Km的MS固体培养基上露草种子萌发苗芽长、主根长、侧根数明显减小,子叶颜色由绿转黄;培养皿浸种发芽,当Km浓度达到100mg/L时,萌发苗子叶颜色为黄色。由此确立了卡那霉素筛选转基因露草种子的适宜方法。

Mechanism of alpha-lipoic acid in attenuating kanamycin-induced ototoxicity

In view of the theory that alpha-lipoic acid effectively prevents cochlear cells from injury caused by various factors such as cisplatin and noise, this study examined whether alpha-lipoic acid can prevent kanamycin-induced ototoxicity. To this end, healthy BALB/c mice were injected subcutaneously with alpha-lipoic acid and kanamycin for 14 days. Auditory brainstem response test showed that increased auditory brainstem response threshold shifts caused by kanamycin were significantly inhibited. Immunohistochemical staining and western blot analysis showed that the expression of phosphorylated p38 mitogen-activated protein kinase and phosphorylated c-Jun N-terminal kinase in mouse cochlea was significantly decreased. The experimental findings suggest that phosphorylated p38 and phosphorylated c-Jun N-terminal kinase mediated kanamycin-induced ototoxic injury in BALB/c mice. AIpha-lipoic acid effectively attenuated kanamycin ototoxicity by inhibiting the kanamycin-induced high expression of phosphorylated p38 and phosphorylated c-Jun N-terminal kinase.

Screening of Transgenic Soybean Transformed by Means of Pollen-tube Using Kanamycin

Kanamycin was used to screen To seeds of the variety Dongnong 46 transformed by means of pollen-tube method. The results showed that 400 mg·L^-1 kanamycin could inhibit growth of non-transgenic plants, and 2 positive plants were gotten combined with Gus dyeing and PCR detection. It is proved that this method is economic and effective in preliminary screening the transgenic plants.

Ototoxicity induced by kanamycin in iron-deficient young rats

Using auditory electrophysiological,histopathological,enzymehistochemical and scanning electron microscopic techniques,we observed thechanges of function and structure of cochlea in iron-deficient growing rats receiv-ing kanamycin intramuscularly.The results showed that at an equal dose ofkanamycin,hearing loss and hair cell damage were more serious,and incidence ofhearing loss was much higher in iron-deficient rats than in normal rats.Theinhibition of succinate dehydrogenase activity of cochlea induced by interaction ofkanamycin and iron deficiency was more obvious than that by either one of them.Iron deficiency can increase the sensitivity of cochlea to kanamycin.The resultsindicated that iron deficiency may be one of the reasons for high incidence ofototoxic deafness in children,and it should be cautious when using such drugs inchildren with iron-deficient anemia.

Effects of combined administration of furosemide and kanamycin on rat auditory nerve

Objective To determine the effects of combined administration of furosemide and kanamycin on inner ear structures and the auditory nerve in rats. Methods The rats in the treatment group received intravenous injections of combined furosemide and kanamycin sulfate, and the rats in the normal control group received no treatment. The auditory brainstem response （ABR） test was carried out 7 days after drug administration to determine the effects of drug administration on hearing. Cochlear slice and cochlear wholcmount were prepared after 7 days of drug treatment. Results After 7 days of drug administration, ABR thresholds were significantly higher in the treatment group than in the control group and neurofilaments were significantly reduced, although the number of spiral ganglia showed no decrease and there were no signs of supporting cell injury. Conclusions Combined administration of furosemide and kanamyein sulfate has an apparent synergistic ototoxic effect. Although spiral ganglion damage may not be apparent within a short time period of drug administration, damage to auditory nerve fibers is obvious.

The Stimulatory Effects of the Antimicrobial Agents Bavistin, Cefotaxime and Kanamycin on <i>In Vitro</i>Plant Regeneration of <i>Centella asiatica</i>(L.)—An Important Antijaundice Medicinal Plant

Antimicrobial agents such as bavistin, cefotaxime and kanamycin were evaluated for their effects on the rapid shoot regeneration from nodal explants of Centella asiatica (L.). Filter sterilized bavistin (250 mg/L) was augmented alone and in combination with cytokinins such as BAP and TDZ into the media to trace the effect on regeneration. On this basis, the potential use of bavistin (150 mg/L) along with BAP (2.0 mg/L) was evaluated which showed the maximum shoot number (6.6) and shoot length (4.4 cm) respectively. Cefotaxime at the concentration of 100 μM/L was found to be effective to obtain the maximum shoot number formation (5.8) with the regeneration frequency (90%). Kanamycin at the concentration of 80 μM/L induced maximum shoot regeneration (5.12). Kanamycin at 100 μM/L or at higher concentrations reduced the shoot regeneration. The best rooting response was noticed when in vitro regenerated microshoots were transferred to the rooting medim which was supplemented with IBA (2.0 mg/L). This combination generates 90% of regeneration frequency and maximum number of roots per shoot (14.2) and high root length (4.2 cm). The rooted plants were acclimatized and transferred to field for survivalance. The addition of antibiotics was found to be more effective and safer for using since their effects on regeneration were found to be negligible.

The effect of Some Boron Derivatives on Kanamycin Resistance and Survival of E.coli and P.aeruginosa in Lake Water

Objective To study MIC value of 7 boron derivatives namely [Boric acid （H3BO3）, Anhydrous Borax （Na2B407）, Sodium Borate （NaBO2）, Diammonium Tetraborate （NH4）2B4O7, Sodium Perborate （NaBO3）, Boron Trioxide （B203）, Potassium Tetraborate （K2B407）] on E. coil and P. aeruginosa and their effects on survival of bacteria in lake water and resistance against kanamycin antibiotic. Methods MIC values of Boron derivatives and antibiotic were studied by broth microdilution method. The effect of boron derivatives on survival of bacteria in lake water were also determined with plate count. Results Sodium perborate was determined as the substances. Effectiveness increased as temperature most effective substance among the studied increased. E. coil was more affected from P. aeruginosa in 8 mg/mL sodium perborate concentration in lake water. Moreover, it was determined that MIC value of kanamycin antibiotic decreased 200 times by especially treating P. aeruginosa with sodium perborate in lake water. However, it can be stated that this change in resistance did not arise from microorganisms. Conclusion Sodium perborate solution can be used supportedly in kanamycin antibiotic applications for P. aeruginosa. Future studies are necessary to explore the relation between sodium perborate and kanamycin which is effective on P. aeruginosa in lake water.

Surveillance of Kanamycin Resistance to Escherichia coll from Swine by Digoxigenin-labled Plasmid Probe

A 4.34kb EcoR I fragment of kanamycin resistance plasmid from pET - 9a was purified by a DNA purification kit. The fragment was labeled with digoxigenin-dUTP with a commercial kit. A dot-blot hybridization and a colony hybridization test with the probe were successfully developed for the surveillance of Kanamycin resistance to E.coli from swine. It was shown that the methods obtained 100% concordance in a positive tate. It was indicated that the method was available for the surveillance of kanamycin resistance to ? coli from swine.

Influence of Bavistin, Cefotoxime, Kanamycin and Silver Thiosulphate on Plant Regeneration of <i>Solanum viarum</i>(Dunal)—An Important Anticancer Medicinal Plant

The effects of bavistin, cefotoxime and kanamycin along with the ethylene inhibitor silver thiosulphate on in vitro regeneration using the axillary buds of Solanum viarum (D.) have been investigated. Axillary bud explants when cultured on MS medium supplemented with bavistin (100 mg/l) maximum number of shoots (4.21 ± 0.83) were observed. In a separate experiment, bavistin along with growth regulators such as BAP and TDZ was added to MS medium containing sucrose (3%). Maximum number of shoots was obtained (5.55 ± 0.28) in MS medium supplemented with bavistin (100 mg/l) and BAP (2.0 mg/l). Cefotoxime and kanamycin (10-150 μM/L) were added to MS medium supplemented with BAP (2.0 mg/l) with sucrose (3%) having shown the maximum shoot regeneration (4.38 ± 0.45 and 3.4 ± 0.48) at 80 μM/L and 50 μM/L respectively. Similarly the regeneration medium was supplemented with (5.0-100 μM/L) of STS. Maximum number of shoots (4.75 ± 0.43) was seen with 40 μM/L. These plantlets were further maintained for root emergence on a rooting medium supplemented with growth regulators such as (0.5-2.0 mg/l) IAA, IBA and NAA. In MS medium supplemented with IBA (1.0 mg/l), maximum number of roots (24.3 ± 0.31) was seen with 100% regeneration. The rooted plants were acclimatized and transferred to field plots with (95%) of plant surviving in the field.

Screening Method for Transgenic Maize with Kanamycin Resistance

[Objective]The paper was to establish simple and effective method to screen marker gene in maize with kanamycin resistance.[Method]Using inbred line "Chang 7-2" and hybrid "Zhengdan 958" of maize as test materials,their seeds were soaked with different concentrations and volumes of kanamycin for3 and 4 d,respectively,the rate of albino seedlings and average seedling height after sowing for10 d were investigated.[Result]The rate of albino seedlings not only was related to kanamycin concentration,but also had relationship with solution volume during soaking process.The difference between inbred line and hybrid was no significant.When 100 ml of kanamycin solution with concentration of 200 mg/L was used to soak seeds for 3 d,the rate of albino seedlings basically could reach 100%.When 100 ml of kanamycin solution with concentration of 100 mg/L was used to soak 20 seeds for 3 d to carry out resistance screening,the accuracy was up to 84.8% after verifying the screening test of T1 transgenic maize plants.[Conclusion]The method was feasible,which could be used as a simple method for screening transgenic gene maize with kanamycin resistance.

AN EXPERIMENTAL STUDY OF ELECTROACUPUNCTURE ON AUDITORY IMPAIRMENT CAUSED BY KANAMYCIN IN GUINEA PIGS

Frequency following response(FFR)and auditory brain stem evoked potential response(ABR)were used to determine the auditory acuity in evaluating the effect of electro-acupuncture treatment of kanamycin-induced auditory impairment in guinea pigs.Thesuccinate dehydrogenase(SDH)activity and morphological changes of the inner earreceptors were examined under the light and scanning electron microscope in cochlearspread preparations.The results showed that 1)electro-acupuncture was effective but nosignificant differences were found among the stimulating wave forms;2)Tinggong(SI19),Yifeng(SJ 17),Shenshu(UB 23),Sanyinjiao(Sp 6),Zhubin(K 9)and Waiguan(SJ 5)are all effective acupoints,especially the combination of Tinggong(SI 19),Sanyinjiao(Sp6)and Zhubin(K 9)acupoints;3)improvement in the cochlear function and excitabilityof the cortical and lower auditory center and increase of the mitochondrial SDH activityand energy supply in hair cells might contribute to the mechanism of the treatment.

Effects of p27^(Kip1)- and p53- shRNAs on kanamycin damaged mouse cochlea

AIM: To study the effects of adeno-associated virus(AAV) delivered short hairpin RNAs(shR NAs) on adult CD-1 mouse cochlea damaged by aminoglycoside antibiotic kanamycin. METHODS: Three different sh RNAs were designed(p27Kip1, p53 and p27Kip1+p53) and tested in COS cells. A total of 20 adult CD-1 mice were used in the experiment. Mice were divided into five different groups(four animals/group) depending on the AAV-shRNA construct they received and whether they received kanamycin or not. Saline and AAV-EGFP injected animals were used as controls. All constructs were injected through the round window membrane(RWM) into the cochlea. Cochleae were harvested after 1 mo. Apoptosis was detected with Tunel labeling from paraffin-embedded cochlear tissue sections.RESULTS: AAV2/2-p27Kip1-shRNA and AAV2/2-p53-shRNA were tested in COS cells. Western blotting analysis confirmed that both constructs silenced their target genes effectively in the cell culture. AAV2/2-shRNA constructs were injected into the cochlea of CD-1 mice through the intact RWM. Cotransductionn off individual AAV2/2-sh RNAs with AAV2/2-EGFP resulted in EGFP expression in the organ of Corti. Kaannaammyycciinn treatment had no effect on the expression pattern of the EGFP. AAV2/2-sh RNA treated mice(either with p53 or p27Kip1 and p53 together) showed fewer apoptotic hair cells in the cochlea than the control group(P < 0.05; AAV2/2-p53-shRNA vs saline P = 0.00014; AAV2/2-p27+p53-shRNA vs saline P = 0.0011). AAV2/2-p27-shRNA injected cochleae showed no significant difference in the number of apoptotic cells when compared to the saline injected cochleae.CONCLUSION: Silencing of p53 protein in the kanamycin treated ears may decrease cell death in the organ of Corti.

Ototoxic destruction by co-administration of kanamycin and ethacrynic acid in rats

It is well known that ethacrynic acid (EA) can potentiate the ototoxicity of aminoglycoside antibiotics (AmAn) such as kanamycin (KM),if they were applied at the same time.Currently,to create the model of EA-KMinduced cochlear lesion in rats,adult rats received a single injection of EA (75 mg/kg,intravenous injection),or followed immediately by KM (500 mg/kg,intramuscular injection).The hearing function was assessed by auditory brainstem response (ABR) measurement in response to click and/or tone bursts at 4,8,12,16,20,24,and 32 kHz.The static microcirculation status in the stria vascularis after a single EA injection was evaluated with eosin staining.The pathological changes in cochlear and vestibular hair cells were also quantified after co-administration of EA and KM.After a single EA injection,blood flow in vessels supplying the stria vascularis rapidly diminished.However,the blood supply to the cochlear lateral wall partially recovered 5 h after EA treatment.Threshold changes in ABR were basically parallel to the microcirculation changes in stria vascularis after single EA treatment.Importantly,disposable co-administration of EA and KM resulted in a permanent hearing loss and severe damage to the cochlear hair cells,but spared the vestibular hair cells.Since the cochlear lateral wall is the important part of the blood-cochlea barrier,EA-induced anoxic damage to the epithelium of stria vascularis may enhance the entry of KM to the cochlea.Thus,experimental animal model of selective cochlear damage with normal vestibular systems can be reliably created through co-administration of EA and KM.

Preparation of Polyclonal Antibody and Development of a Biotin-streptavidin-based ELISA Method for Detecting Kanamycin in Milk and Honey

Kanamycin is an aminoglycoside antibiotic used increasingly in human and veterinary medicine. However, kanamycin residues in food can cause serious side effects, Here we reported the preparation of polyclonal antibody and the development of an indirect competitive biotin-streptavidin-amplified-based enzyme-linked immunosorbent assay（BA-ELISA） that can sensitively and specifically detect kanamycin residues in milk and honey. The immtmogen and coating antigen were synthesized by covalently linking kanamycin to carrier proteins using the carbodiimide method. The anti-kanamycin polyclonal antibodies were obtained from immunized rabbits. The key assay parameters were investigated and optimized. The results show that under optimum conditions, the limit of detection for kanamycin is 0.07 ng/mL and the ICs0 is 6.48 ng/mL. Cross-reactivity values of the antibody with four kanamycin analogues are all 〈1%. Trace amounts of kanamycin in milk and honey samples can be detected by this novel BA-ELISA method successfully with satisfactory recoveries of 91.0%-103.3%. The developed protocol was also validated against liquid chromatography-mass spectrometry, returning a significant correlation. These results indicate that BA-ELISA is a viable option for monitoring kanamycin residues in milk and honey.

A Label-free and Functional Fluorescent Oligonucleotide Probe Based on a G-Quadruplex Molecular Beacon for the Detection of Kanamycin

A label-free and turn-off fluorescent method for the quantitative detection of kanamycin based on a funtional molecular beacon was developed. The molecular beacon consists of two hairpin structures with a split G-rich oligonucleotide in the middle. The kanamycin＇s aptamer formed the loops portion for recognizing kanamycin, and the G-quadruplex bound by Thioflavin T（ThT） was employed as the reporter. In the absence of target, the molecular beacon folded into double stem-loops and the splited G-rich oligonucleotid came close to form a G-quadruplex. When ThT bound to the G-quadruplex, the fluorescence intensity of the solution increased. Upon the addition of kanamycin, the function between kanamycin and aptamer unfolded the hairpin and disassembled the G-quadraplex structure, resulting in a significant decrease in the fluorescence intensity. A good linear relationship ranging from 0.7 nmol/L to 10 nmol/L was achieved and the limit of detection was 0.37 nmol/L. Besides, it could efficiently recognize kanamycin in real samples.

Antagonistic Effect of Poria Cocos on Ototoxicity of Kanamycin in Guinea－Pigs

In order to prevent ototoxicity of kanamycin ( KM) , the effects of Poria Cocos (PC) on the o-totoxicity induced by KM in guinea-pigs was observed by giving the PC decoction to guinea-pigs with compar-ing the difference in the general intoxicating symptom, Prayer's retlex (PR) threshold, brainstem auditory e-voked potentials (BAEPs) and the absence rate of outer hair cells in the first turn of cochlea. The results sug-gested that the ototoxicity od KM was retarded by PC decoction. On the 7th day after KM was given, PRthreshold of 4, 6, 8 kHz in the medicated group and control group were 4. 7 1 . 5 dB, 5. 7 2. 4 dB, 2. 7 1 . 2 dB and 10. 5 3 . 2 dB, 12 . 1 3 . 7 dB, 8. 5 2 . 7 dB respectively. BAEPs threshold on the 13th day afterKM was given, raised 22 . 7 9 . 7 dB in the PC group and 51 . 3 14. 4 dB in the control group. The absencerate of outer hair cells in the first turn of cochlea were 39 . 4% and 67 . 4% respectively. The results suggestedthat PC might be antagonistic to the ototoxicity of KM in guinea-pigs.

基于表面修饰的高灵敏度免疫分析方法检测牛奶中的卡那霉素

目的为了提高卡那霉素的检测灵敏度,将卡那霉素分子直接共价固化在酶标板上,由此开发一种直接免疫分析方法检测卡那霉素。方法本研究采用戊二醛以及聚乙二胺(polyamidoamine,PAMAM)-戊二醛两种方式,对酶标板进行表面修饰,同时通过酶联免疫吸附实验(enzyme-linked immunosorbent assays,ELISA)比较两种方式的差异,并对牛奶样本中卡那霉素进行检测。结果基于戊二醛表面修饰的ELISA检出限为0.219 ng/mL,线性范围为2.251~87.791 ng/mL;基于PAMAM-戊二醛表面修饰的ELISA检出限为0.119 ng/mL,线性范围为1.348~20.414 ng/mL。此两种方法比常规ELISA方法灵敏度分别提高5倍和10倍。将基于PAMAM-戊二醛表面修饰的ELISA应用于牛奶样本的检测,发现卡那霉素的检出率为12.5%,最高检出质量浓度为1.12 ng/mL。结论本研究采用的戊二醛以及PAMAM-戊二醛两种方式修饰酶标板后大大提高了免疫检测的灵敏度,为食品中小分子污染物的快速筛查提供了新的思路。

Graphene-Gold Nanoparticle-modified Electrochemical Sensor for Detection of Kanamycin Based on Target-induced Aptamer Displacement

A highly sensitive and selective label-free electrochemical sensor was developed for the determination of kanamycin. To improve the sensitivity of the electrochemical sensor, graphene-gold nanoparticles were prepared by a one-step electrochemical coreduction process and were modified on the surface of a glassy carbon electrode. The double-stranded DNA（ds-DNA） duplex probe was immobilized onto the graphene-gold nanoparticle-modified elec-trode. The introduction of target kanamycin induced the displacement of aptamer from the ds-DNA duplex into the solution. Methylene blue（MB） as a redox indicator monitored the current change using differential pulse voltammetry Under optimal conditions, the designed electrochemical aptasensor exhibited a wide linear range from 0. i pmol/L to 10 pmol/L with a detection limit of 0.03 pmol/L for kanamycin. The experimental strategy enabled the direct analysis of milk samples, and the results showed high sensitivity and good selectivity.

Effect of Ligustrazine on Kanamycin Ototoxicity

Objective: To evaluate the effect of ligustrazine on kanamycin ototoxicity. Methods: By measuring superoxide dismutase (SOD) and malondialdehyde (MDA) in cochlear tissues, compound action potentials (CAP) and cochlear microphonics (CM) in guinea-pigs which were treated with kanamycin and ligustrazine. Results : Kanamycin produced a severe hearing loss and significant increase in SOD and MDA. Ligustrazine could reduce these changes significantly and improve hearing of animals. Conclusion: Lipid peroxidativereaction is correlated with kanamycin ototoxicity and ligustrazine could prevent and treat kanamycin ototoxicity by antioxidation.