Synthesis of kanamycin A derivatives by regioselective masking drug resistant enzymes targeting hydroxyl groups

The 3＇-OH, 4＇-OH and 2＂-OH of kanamycin A were modified in search of new aminoglycosides to overcome resistant enzymes, ANTs and APHs. The key intermediate was a dibenzylidene-protected derivative of kanamycin A. The aimed sites were masked by benzyl, methyl and allyl groups. Multi-step reactions gave the desired aminoglycoside derivatives but showed less antibiotic activity than kanamycin A.

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.

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.

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.

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.

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.

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 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.

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 （shRNAs） on adult CD-1 mouse cochlea damaged by aminoglycoside anti-biotic kanamycin. METHODS： Three different shRNAs were designed （p27^Kip1, p53 and p27^Kip1＋p53） and tested in COS cells. A total of 20 adult CD-1 mice were used in the experiment. Mice were divided into fve 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-p27^Kip1-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. Cotransductionof Cotransduction of individual AAV2/2-shRNAs with AAV2/2-EGFP resulted in EGFP expression in the organ of Corti.Kanamycin Kanamycin treatment had no effect on the expression pattern of the EGFP. AAV2/2-shRNA treated mice （either with p53 or p27Kip1and p53 together） showed fewer apop-totic 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 kana-mycin treated ears may decrease cell death in the organ of Corti.

Field released transgenic papaya effect on soil microbial communities and enzyme activities

Soil properties, microbial communities and enzyme activities were studied in soil amended with replicase （RP）-transgenic or non-transgenic papaya under field conditions. Compared with non-transgenic papaya, significant differences （P〈0.05） were observed in total nitrogen in soils grown with transgenic papaya. There were also significant differences （P〈0.05） in the total number of colony forming units （CFUs） of bacteria, actinomycetes and fungi between soils amended with RP-transgenic plants and non-transgenic plants. Compared with non-transgenic papaya, the total CFUs of bacteria, actinomycetes and fungi in soil with transgenic papaya increased by 0.43-1.1, 0.21-0.80 and 0.46-0.73 times respectively. Significantly higher （P〈0.05） CFUs of bacteria, actinomycetes and fungi resistant to kanamycin （Km） were obtained in soils with RP-transgenic papaya than those with non-transgenic papaya in all concentrations of Km. Higher resistance quotients for Km＇ （kanamycin resistant） bacteria, actinomycetes and fungi were found in soil planted with RP-transgenic papaya, and the resistance quotients for Km＇ bacteria, actinomycetes and fungi in soils with transgenic papaya increased 1.6-4.46, 0.63-2.5 and 0.75-2.30 times. RP-transgenic papaya and non-transgenic papaya produced significantly different enzyme activities in arylsulfatase （5.4-5.9x）, polyphenol oxidase （0.7-1.4x）, invertase （0.5-0.79x）, cellulase （0.23-0.35x） and phosphodiesterase （0.16-0.2x）. The former three soil enzymes appeared to be more sensitive to the transgenic papaya than the others, and could be useful parameters in assessing the effects of transgenic papaya. Transgenic papaya could alter soil chemical properties, enzyme activities and microbial communities.

Pharmacodynamics of aminoglycosides and tetracycline derivatives against Japanese encephalitis virus

Objective:To explore the antiviral activity of antibiotic compounds,mainly aminoglycosides and tetracyclines against Japanese encephalitis virus(JEV) induced infection in vitro.Methods:Antiviral activity were evaluated against JEV using cytopathic effect inhibition assay,virus yield reduction assay,caspase 3 level,extracellular viral detection by antigen capture ELISA and viral RNA levels.Roults:JEV induced cytopathic effect along with reduction of viral progeny plaque formation indicated antiviral potential of the compounds suggesting that antibiotics had broad spectrum activity.Doxycycline and kanamycin administration in dose dependent manner declined viral RNA replication.Conclusions:The present study shows kanamycin and doxycyclinc can affect virion structure and alter replication causing inhibition of JEV induced pathogenesis in vitro.

PCR Targeting of Antibiotic Resistant Bacteria in Public Drinking Water of Lahore Metropolitan,Pakistan

Objective To investigate the prevalence of kanamycin （kan） and ampicillin （amp） resistant bacteria in public drinking water. Methods Bacteria containing kan and amp resistant genes were amplified by PCR and further characterized by colony hybridization and transformation studies. The genus of kan and amp resistant bacteria was determined with standard methods. Results Among the 625 drinking water samples, 400 contained kan and amp resistant bacteria and the percentage was 42.5% and 57.5%, respectively, which was further confirmed by the amplification of a 810 bp kan resistant gene and a 850 bp amp resistant gene. Of the 170 kan resistant bacteria, 90 were Gram negative and 80 were Gram positive. Of the 230 amp resistant bacteria, 160 were Gram negative while 70 were Gram positive. Salmonella, Shigella, Staphylococcus, Streptococcus, and E.coli were detected as 13%, 11%, 17%, 30%, and 29%, respectively. Bacterial strain DH5α transformed with plasmids isolated from kan and amp resistant bacteria confirmed that the antibiotic resistant genes were mediated by plasmids. Conclusion Drinking water is contaminated with kan and amp resistant bacteria due to poor sanitary conditions.

An efficient strategy for establishing a model of sensorineural deafness in rats

Ototoxic drugs can be used to produce a loss of cochlear hair cells to create animal models of deafness. However, to the best of our knowledge, there is no report on the establishment of a rat deafness model through the combined application of aminoglycosides and loop diuretics. The aim of this study was to use single or combined administration of furosemide and kanamycin sulfate to establish rat models of deafness. The rats received intravenous injections of different doses of furosemide and/or intramuscular injections of kanamycin sulfate. The auditory brainstem response was measured to determine the hearing threshold after drug application. Immunocytochemistry and confocal microscopy were performed to evaluate inner ear morphology. In the group receiving combined administration of furosemide and kanamycin, the auditory brainstem response threshold showed significant elevation 3 days after administration, higher than that produced by furosemide or kanamycin alone. The hair cells showed varying degrees of injury, from the apical turn to the basal turn of the cochlea and from the outer hair cells to the inner hair cells. The spiral ganglion cells maintained a normal morphology during the first week after the hair cells completely disappeared, and then gradually degenerated. After 2 months, the majority of spiral ganglion cells disappeared, but a few remained. These findings demonstrate that the combined administration of furosemide and kanamycin has a synergistic ototoxic effect, and that these drugs can produce hair cell loss and hearing loss in rats. These findings suggest that even in patients with severe deafness, electronic cochlear implants may partially restore hearing.

COMBINATIONAL ADMINISTRATION OF AMINOGLYCOSIDES AND LOOP DIURETICS AS AN EFFICIENT STRATEGY TO ESTABLISH DEAFNESS MODELS IN RATS

It is known that aminoglycoside antibiotics can damage the vestibular and auditory sensory epithelia, and the loop diuretics can enhance the ototoxic effect of aminoglycosides. Previous studies on the synergistic effect of these two types of drugs have used mice, guinea pigs and cats, but not rats. The aim of this study was to determine this synergistic effects in rat cochleae. Rats received intravenous injections of different doses of furosemide and/or intramuscular injections of kanamycin sulfate. Au- ditory brainstem response （ABR）, scanning electron microscopy （SEM） and immunocytochemistry were used to determine the effects of drug administration. In the group receiving combined administration of furosemide and kanamycin, the ABR thresh- old showed significant elevation 3 days after drug administration, greater than single drug administration. The hair cells showed various degrees of injury from the apical turn to the basal turn of the cochlea and from the outer hair cells to the inner hair cells. Neuron fibers of the hair cells showed significant loss 7 days after the drug administration, but the number of spiral ganglia did not decrease and supporting cells showed no signs of injury. Our study suggest that combined administration of fu- rosemide and kanamycin has an synergistic ototoxic effect, and can result in hair cell loss and hearing loss in rats.

Efficient Regeneration System for Genetic Transformation of Mulberry (<i>Morus indica</i>L. Cultivar S-36) Using <i>in Vitro</i>Derived Shoot Meristems

Shoot meristems used for the study were exercised from the in vitro regenerated shoots cultured on MS medium supplemented with 0.5 mg/L of BAP for multiplication. The sensitivity of the in vitro regenerated was studied using shoot meristems of 0.5 cm. Shoot meristems were cultured on medium containing 10-100 mg/l kanamycin to determine the concentration that was lethal for multiple shoot induction and root induction. The response of shoot multiplication decreased (66.2%-6.2%) as the concentration of kanamycin increased (10.0-70.0 mg/L) with complete inhibition of shoot proliferation at 100 mg/L kanamycin. The rooting phase was very sensitive to kanamycin compared to shoot multiplication. The percentage of shoots that rooted decreased (53.8%-4.8%) with increase in the concentration of kanamycin (10.0-70.0 mg/l) on IBA and 2,4-D supplemented medium. For transformation studies, the shoot tips that were infected with Agrobacterium strain were placed on selection medium containing MS medium with 0.5 mg/L BAP and 100 mg/L kanamycin and scored for the putative transformed shoots. An average of 62.2% of shoot tips developed shoot buds from the base and the shoots reached a length of 0.5-1.0 cm at the end of 30 days of culture on the selective medium in comparison to control which showed no response. An average of 66.7% of the regenerated plants showed GUS expression on selection medium where 43.2% and 65% of GUS expression was recorded in the leaves and callus. Leaves and callus induced from the controls did not show GUS activity. Stable integration of nptII gene with the genomic DNA from these transformed plants was confirmed through PCR analysis. Our result presents an efficient regeneration system using in vitro derived shoot meristems for Agrobacterium mediated gene transfer.

Correlations between Gene Resistant Markers and Second-Line Anti-TB Drug Resistance in Pre-XDR and XDR-TB Patients

Background: Extensively drug resistant tuberculosis (XDR-TB) is a serious problem in public health and XDR-TB patients usually develop from multi-drug resistance tuberculosis (MDR-TB) and pre-XDR-TB. The rapid molecular test for drug susceptibility testing (DST) can be used for early detection to prevent XDR-TB. Methods: We examined 34 clinical Mycobacterium tuberculosis (M. tuberculosis) isolates from MDR/XDR-TB patients in the upper north of Thailand that were identified with drug susceptibility profiles by indirect agar proportion method from 2005-2012. Our study investigated the genetic mutations in gyrA for ofloxacin resistance and rrs for kanamycin resistance. The genetic mutations and drug susceptibility test results were analyzed using the exact test. Results: The majority of the ofloxacin resistance was detected in gyrA 21, gyrA 70, gyrA 87, gyrA 102, gyrA 162, and gyrA 187 were at 0%, 12.5%, 37.5%, 0%, 50.0% and 25.0% sensitivity, respectively, and at 96.2, 96.2%, 20.1%, 96.2%, 57.7% and 61.5% specificity, respectively. Kanamycin resistance was found in rrs 512, rrs 241, rrs 223, rrs 414 and rrs 408 at 16.7%, 0%, 0%, 16.7% and 16.7% sensitivity, respectively, and at 96.4%, 92.9%, 82.1%, 82.1% and 71.4% specificity, respectively. This study found no significant correlation between gyrA mutations and ofloxacin resistance and also no correlation between the rrs gene and kanamycin resistance. Conclusion: These primer sequences and PCR products in our study such as gyrA and rrs might be unsuitable to detect ofloxacin and kanamycin resistance in the upper north of Thailand.