Screen of FDA-approved drug library reveals compounds that protect hair cells from aminoglycosides and cisplatin.

Loss of mechanosensory hair cells in the inner ear accounts for many hearing loss and balance disorders. Several beneficial pharmaceutical drugs cause hair cell death as a side effect. These include aminoglycoside antibiotics, such as neomycin, kanamycin and gentamicin, and several cancer chemotherapy drugs, such as cisplatin. Discovering new compounds that protect mammalian hair cells from toxic insults is experimentally difficult because of the inaccessibility of the inner ear. We used the zebrafish lateral line sensory system as an in vivo screening platform to survey a library of FDA-approved pharmaceuticals for compounds that protect hair cells from neomycin, gentamicin, kanamycin and cisplatin. Ten compounds were identified that provide protection from at least two of the four toxins. The resulting compounds fall into several drug classes, including serotonin and dopamine-modulating drugs, adrenergic receptor ligands, and estrogen receptor modulators. The protective compounds show different effects against the different toxins, supporting the idea that each toxin causes hair cell death by distinct, but partially overlapping, mechanisms. Furthermore, some compounds from the same drug classes had different protective properties, suggesting that they might not prevent hair cell death by their known target mechanisms. Some protective compounds blocked gentamicin uptake into hair cells, suggesting that they may block mechanotransduction or other routes of entry. The protective compounds identified in our screen will provide a starting point for studies in mammals as well as further research discovering the cellular signaling pathways that trigger hair cell death.

Intra-cochlear administration of dexamethasone attenuates aminoglycoside ototoxicity in the guinea pig.

This study demonstrates the attenuation of aminoglycoside ototoxicity by cochlear infusion of dexamethasone (Dex) using a microcannulation-osmotic pump delivery system. The results indicate that treating the cochlea with Dex both before and after kanamycin administration was more effective in preventing ototoxicity than Dex treatment only after kanamycin administration. A concentration of 1 ng/ml Dex showed the greatest protective effect on both kanamycin-induced threshold shift of the auditory brainstem response and outer hair cell survival. These results show that the Dex treatment attenuates both functional and structural damage of the inner ear from aminoglycoside toxicity.

Aminoglycoside ototoxicity in adult CBA, C57BL and BALB mice and the Sprague-Dawley rat.

The availability of genetic information, transgenic and knock-out animals make the mouse a primary model in biomedical research. Aminoglycoside ototoxicity, however, has rarely been studied in mature mice because they are considered highly resistant to the drugs. This study presents models for kanamycin ototoxicity in adult CBA/J, C57BL/6 and BALB/c mouse strains and a comparison to Sprague-Dawley rats. Five-week-old mice were injected subcutaneously twice daily with 400-900 mg kanamycin base/kg body weight for 15 days. Kanamycin induced dose-dependent auditory threshold shifts of up to 70 dB at 24 kHz as measured by auditory brain stem-evoked responses. Vestibular function was also affected in all strains. The functional deficits were accompanied by hair cell loss in both cochlear and vestibular neurosensory epithelia. Concomitant administration of the antioxidant 2,3-dihydroxybenzoate significantly attenuated the kanamycin-induced threshold shifts. In adult male Sprague-Dawley rats, doses of 1 x 500 mg or 2 x 300 mg kanamycin base/kg body weight/day x 14 days induced threshold shifts of approximately 50 dB at 20 kHz. These were accompanied by loss of outer hair cells. The order of susceptibility, BALB>CBA>C57, was not due to differences in the pharmacokinetics of kanamycin. It also did not correlate with the presence of Ahl/Ahl2 genes which predispose C57 and BALB strains, respectively, to accelerated age-related hearing loss. Pigmentation, however, paralleled this rank order suggesting an influence of melanin on cochlear antioxidant status.

[The genetic transformation of Lycopersicon peruvianum var. dentatum by using Agrobacterium tumefaciens carrying a plasmid with the human beta-interferon gene]. / Geneticheskaia transformatsiia Lycopersicon peruvianum var. dentatum s pomoshch'iu Agrobacterium tumefaciens, nesushchei plazmidu s genom beta-interferona cheloveka.

Wild tomato plants of Lycopersicon peruvianum var. dentatum were transformed with pG11 plasmid having human beta-interferon (hIFN-beta) and NPTII genes. Transformation was demonstrated by polymerase chain reaction (PCR) and ELISA assay. Expression of hIFN-beta gene was identified by Western blot analysis. Transgenic plants produced seeds and F1 generation inherited integrated traits.

[Prevention of neurosensory hearing disorders in antibiotic-induced ototoxicosis]. / Preduprezhdenie neirosensornykh narushenii slukha pri antibiotikovom ototoksikoze.

On the basis of clinical and audiological examinations of 102 tuberculosis patients who were on a combined treatment which included aminoglycoside antibiotics (streptomycin and canamycin), neurosensory disorders of the hearing function were detected in 75% of them. A clinical trial of obsidan, a beta-blocking agent, was performed in which it was used as a protective drug against ototoxic antibiotics given to 112 patients. Its combined application with antibiotics of the aminoglycoside series decreased their toxic effect from 75.6% to 21.4%.

[Effect of cationic polymers and surface-active substances on bacterial resistance to kanamycin]. / Izuchenie vliianiia kationnykh polimerov i poverkhnostno-aktivnykh veshchestv na ustoichivost' bakterii k kanamitsinu.

The effect of cationic polymers and surface active substances (SAS) on sensitivity of Klebsiella aerogenes 600, Escherichia coli 154 and Proteus vulgaris 7470 to kanamycin was studied. A decrease in the resistance of the above organisms to kanamycin on its use in combination with cationic polymers and SAS was observed. It was shown that such substances inhibited the activity of the enzymes inactivating kanamycin. Their effect was suggested to be due to changes in the lipid surrounding of the enzymes.

Ototoxicity of kanamycin in albino and pigmented guinea pigs. II. A scanning electron microscopic study.

In a previous study we demonstrated an increased ototoxicity in pigmented compared with albino guinea pigs given high doses of kanamycin. In the present investigation using scanning electron microscopy we studied the fine structure of the surface preparation of the organ of Corti to examine whether a qualitative difference in lesion pattern exists between albino and pigmented guinea pigs. Kanamycin was given by daily subcutaneous injections in the amounts of 20, 60, and 200 mg per kilogram of body weight until hearing impairment was noted. No difference in the pattern of degeneration was observed in the albino and pigmented guinea pigs. No giant cilia were seen. The classic gradient of degeneration of the outer hair cells (OHCs), with the most pronounced damage in the first row of OHCs in the basal coil and progressively diminishing lesions in the second and third rows, as observed in both species.

Ototoxicity of kanamycin in albino and pigmented guinea pigs. I. A morphologic and electrophysiologic study.

Ototoxic drugs of the aminoglycoside type have been shown to accumulate to melanin, suggesting a possible mechanism for their ototoxicity. The present study was undertaken by combining electrophysiologic and morphologic methods to investigate whether the ototoxicity of kanamycin is different in pigmented and albino guinea pigs. In pigmented animals a kanamycin dose of 200 mg per kilogram of body weight per day resulted in hearing loss together with loss of both inner and outer hair cells. The albino animals in the same dose group showed significantly less hearing loss and hair cell degeneration. With daily doses of 20 and 60 mg/kg/day, no difference in ototoxicity was found between the pigmented and albino animals. The results support the hypothesis that affinity of kanamycin to inner ear melanin might be responsible for the difference in ototoxicity between albino and pigmented guinea pigs.

[Phenomenon of the amplification of the determinant of kanamycin resistance (Kanr) in constructed hybrid plasmids in a strain of Streptomyces lividans]. / Izuchenie iavleniia amplifikatsii determinanta ustoichivosti k kanamitsinu (Kanr) v sostave skonstruirovannykh gibridnykh plazmid v shtamme Streptomyces lividans.

The restriction maps of the hybrid pSU1, pSU2 ..., pSU13 plasmids were constructed. The plasmids are the derivatives of SLP1.2 plasmid and the kanamycin resistance determinant of S. rimosus. Jt was shown that all the 13 hybrid plasmids are capable of tandem reiteration in the genome (amplification) of S. lividans. The conditions for amplification of the hybrid plasmids by one-stage selection of the variants with increased resistance to kanamycin were developed. It was found that the constructed plasmids could be used for cloning and coamplification of additional DNA fragments in them. All the plasmids determined the synthesis of APH(3')--aminoglycoside phosphotransferase responsible for the in-vivo resistance of the strains containing it to neomycin and paromomycin. It is suggested that kanamycin resistance is determined by some other gene and mechanism. An increase in the number of the copies of gene aph1 on amplification resulted in a 50-fold increase in production of APH(3') by it.

[Action of biologically active compounds on kanamycin inactivation by resistant microorganisms]. / Izuchenie deistviia biologicheski aktivnykh soedinenii na inaktivatsiiu kanamitsina ustoichivymi mikroorganizmami.

Products of kanamycin inactivation were identified chromatographically and radiometrically in E. coli 154, K. aerogenes 600 and P. vulgaris 7470. It was shown that the efficiency of kanamycin inactivation in the membrane fraction was 2 times higher than that in cytosol. A decrease in the culture resistance to kanamycin was observed, when the antibiotic was used in combination with the main proteins or phosphonites: a 16-64-fold decrease in the kanamycin MIC. Comparison of the data on the efficiency of the inactivation inhibition by intact cells and in acellular extracts suggests that the effect of protamine on this process is mediated by the cell membrane, whereas phosphonites can also directly interact with the enzymes inactivating the antibiotic.

[Antitoxic properties of pantothenic acid derivatives, precursors of coenzyme A biosynthesis, with regard to kanamycin]. / Antitoksicheskie svoistva proizvodnykh pantotenovoi kisloty, predshestvennikov biosinteza kofermenta A v otnoshenii kanamitsina.

The effect of calcium pantothenate (CPN)B 4'-phospho-CPN (PCP), pantetheine (PT) and calcium S-sulfopantetheine (SPN) on acute toxicity of kanamycin sulfate was studied on albino mice. The above derivatives of pantothenic acid except PT lowered the antibiotic toxicity. The coefficient of the antitoxic effect (LD50/ED50) of SPN and PCP was 1.3-1.4 times higher than that of CPN. The combined use of kanamycin (1/5 of the LD50) with CPN, PCP or PT (30 mg/kg bw was equivalent to CPN) for 15 days prevented the increase in the total content of CoA and in the content of the fraction of free CoA and the precursors of its biosynthesis participating in the reaction of N-acetylation in the liver and brain. The contents of these substances were within the normal during the whole experiment. A certain increase in the activity of pantothenate kinase in the liver cytosol due to the use of kanamycin was eliminated by the simultaneous use of PCP and PT. The vitamin-containing compounds PCP and SPN were recommended for the clinical trials as agents preventing complications of kanamycin therapy.

[Selection, analysis and mapping of mutations in the gene for resistance to kanamycin in plasmid RP4]. / Selektivnyi otbor, analiz i kartirovanie mutatsii v gene ustoichivosti k kanamitsinu plazmidy RP4.

We have tested possibilities of Escherichia coli strains dependent on drugs streptomycin and paromomycin for selection of spontaneous mutations in the RP4 kan gene specifying resistance to aminoglycosids--kanamycin, neomycin and paromycin. A set of kan gene mutations were obtained, classified ad mapped.

[Transfer of kanamycin resistance among familial strains of Enterobacteriaceae isolated from a chronic bacterial carrier of Salmonella schottmuelleri]. / Peredacha ustoichivosti k kanamitsinu sredi shtammov semeistva Enterobacteriaceae, vydelennykh ot khronicheskogo bakterionositelia Salmonella schottmuelleri.

Escherichia coli, Enterobacter aerogenes and S. schottmuelleri were isolated from the large intestine of a bacteriocarrier. E. coli and E. aerogenes strains proved to be resistant to a number of antibiotics. Plasmids were detected in DNA preparations obtained from E. coli strains. After the hybridization of these E. coli strains with E. coli C600 5K and S. schottmuelleri at 28 degrees C the transfer of resistance to kanamycin was found to occur. From some of the transconjugates thus obtained resistance to kanamycin was transferred to E. aerogenes. This resistance was found to be controlled by the plasmid with a molecular weight exceeding 2 Md. The fact that S. schottmuelleri in the carrier's body retained their sensitivity to antibiotics can be explained by the absence of the transfer of plasmid Kmr at a temperature exceeding 28 degrees C and by the existence of the infective agent in an ecological niche other than that of E. coli.

[DNA cloning in actinomycetes: the creation of vector systems]. / Klonirovanie DNK u aktinomitsetov: sozdanie vektornykh sistem.

A new vector type was constructed on the basis of SLP 1.2 plasmid and the Kanr determinant of S. rimosus P3. It was shown that the determinant was capable of amplifying in the chromosomes of S. rimosus during its improvement for increasing the level of resistance to kanamycin. Cloning of the Kanr determinant was performed on the Pst I-A fragment of the SLP 1.2 plasmid in S. lividans 66. The Kanr determinant was expressed in the resulting hybrid plasmids, e. g. pSU 3, thus providing resistance of S. lividans to 20 micrograms/ml of kanamycin. As a result of repeated passages variants capable of growing in the presence of 50 000 micrograms/ml of the antibiotic were selected. The electrophoretic analysis of the total DNA fragments obtained after exposure to different endonucleases showed that they were identical to the respective fragments of the hybrid pSU 3 plasmid and amounted to approximately 40 per cent of the total DNA. The presence of the unique sites for the Bam HI, ClaI and SacI restriction endonucleases on the pSU 3 plasmid in an insignificant area and the relative stability (30-100 per cent) of the amplified variants allowed using it for cloning and amplification of DNA in Streptomyces. Plasmids were identified with the genetic and physical methods in 13 strains of the blue systematic group among the 72 strains studied. A multicopy plasmid with a molecular weight of 5.6 MD designated as pSB 24.1 was identified in the family of the plasmids of S. cyanogenus. The deletion and insertion variants of the pSB 24.1 plasmid were obtained. The comparative study of the properties of these plasmids revealed the areas insignificant for replication and maintenance of the pSB 24.1 plasmid and the area determining the Ltz+-phenotype. It was suggested that formation of the deletion and insertion variants of the pSB 24.1 plasmid was provided by the site specific recombination mechanisms. The presence of the unique sites for a number of the restriction endonucleases located in the insignificant area, the presence of the selective marker and the high transformation frequency allowed one to consider the multicopy pSB 24.1 plasmid an acceptable vector for cloning of DNA in Streptomyces.

[Genetic activity of base analogs in Salmonella typhimurium and Escherichia coli and Saccharomyces cerevisiae]. / Geneticheskaia aktivnost' analogov osnovanii u bakterii Salmonella typhimurium, Escherichia coli i drozhzhei Saccharomyces cerevisiae.

The study of 6-N-hydroxylaminopurine (HAP) and 2-amino-6-N-hydroxylaminopurine (AHAP) activity in bacteria and the yeast was undertaken. AHAP was found to be more effective as a mutagen in bacteria and HAP--in the yeast. Mutagenic and lethal effects or analogues were independent of excision and mutagenic repair both in bacteria and the yeast. Deletion in uvrB region of Salmonella genome leads to hypersensitivity to lethal and mutagenic action of analogues. Both of the latter only cause reversions of base-substitution but not frameshift mutations. Considering the data obtained and the information from published papers, we proposed that HAP and AHAP exert their mutagenic action, like classical analogues, by means of incorporation into DNA and disturbing the regular replication laws.

Cloning of the kanamycin resistance gene from a kanamycin-producing Streptomyces species.

A kanamycin-producing strain, Streptomyces kanamyceticus ISP5500, is resistant to kanamycin. A kanamycin resistance determinant was cloned from S. kanamyceticus into Streptomyces lividans 1326, using the plasmid vector pIJ702. The resulting plasmid, pMCP5, could also transform Streptomyces lavendulae S985 and Streptomyces parvulus 2283 to kanamycin resistance. Transformants carrying pMCP5 were markedly more resistant than S. kanamyceticus to the aminoglycoside antibiotics sisomicin, tobramycin, amikacin, and gentamicin. Studies in vitro polyphenylalanine synthesis showed that strains carrying pMCP5 contained kanamycin-resistant ribosomes. However, growing S. kanamyceticus contained kanamycin-sensitive ribosomes. Ribosomes from S. kanamyceticus grown under kanamycin-producing conditions were kanamycin resistant.

[Induced resistance to kanamycin and tetracycline in Rhizobium japonicum]. / Indutsirovannaia ustoichivost' k kanamitsinu i tetratsiklinu u Rhizobium japonicum.

Spontaneous mutants Km-R and Tc-R of R. japonicum with various levels of resistance to kanamycin (0.8-20 mg/ml) and tetracycline (130-210 micrograms/ml) were isolated. No cross resistance in the mutants was observed. Plasmid R68.45 transferred to the wild strains resistance to 210 micrograms/ml of tetracycline and to 20 mg/ml of kanamycin. This plasmid did not practically increase the resistance to tetracycline in mutants Tc-R. At the same time it markedly increased the resistance to kanamycin in mutants Km-R.

Mechanism of antibiotic resistance in Mycobacterium intracellulare.

The mechanism of resistance of Mycobacterium intracellulare strain 103 and other clinical isolates to a variety of drugs including aminoglycoside and peptide antibiotics was investigated. Enzymatic inactivation of aminoglycoside and peptide antibiotics could not be demonstrated. Ribosomes of the strain were found to be sensitive to the antibiotics. The levels of resistance of strain 103 and other clinical isolates decreased dramatically when the culture medium was changed from Dubos agar to Tween 80-containing agar. These results suggest that a permeability barrier is the reason for naturally occurring resistance in M. intracellulare.