The sensitivity of embryogenic tissue of Picea omorika (Panč.) Purk. to antibiotics

In an attempt to develop a transformation procedure for somatic embryos of Picea omorika (Pan.) Purk. using Agrobacterium, the sensitivity of non-transformed embryogenic tissues of P. omorika to antibiotics was measured. Two groups of antibiotics were tested: antibiotics commonly used to eliminate Agrobacterium from tissue culture (carbenicillin and cefotaxime), and antibiotics for the selection of transformed tissue (kanamycin, paromomycin, neomycin and hygromycin). Carbenicillin (500 mg l^–1) reduced proliferation of P. omorika embryogenic tissue by 50% as compared to the control. Cefotaxime (500 mg l^–1) was less toxic to embryogenic tissue growth (93% of the control) and is thus more suitable for the elimination of A. tumefaciens from embryogenic tissue of P. omorika. Embryogenic tissue showed severe susceptibility to kanamycin and hygromycin. Induction of secondary embryogenesis was blocked by 10 mg l^–1 of kanamycin or 2 mg l^–1 of hygromycin. The aminoglycoside analogs of kanamycin, paromomycin and neomycin inhibited embryogenic tissue induction at concentrations of 5 and 100 mg l^–1, respectively. The higher tolerance of embryogenic tissue to neomycin indicated that of the antibiotics tested neomycin may be most useful for selection of nptII-transformed embryogenic tissue of Picea omorika.     

Ribosomes of the extremely thermophilic eubacterium Thermotoga maritima are uniquely insensitive to the miscoding-inducing action of aminoglycoside antibiotics.

Poly(U)- and poly(UG)-programmed cell-free systems were developed from the extreme thermophilic, anaerobic eubacterium Thermotoga maritima, and their susceptibility to aminoglycoside and other antibiotics was assayed at a temperature (75 degrees C) close to the physiological optimum (80 degrees C) for cell growth and in vitro polypeptide synthesis, using a Bacillus stearothermophilus system as the reference. The synthetic capacity of the Thermotoga assay mixture was abolished by the eubacterium-targeted drugs chloramphenicol, thiostrepton, and kirromycin. However, streptomycin, the disubstituted 2-deoxystreptamines (kanamycin, gentamicin, neomycin, and paromomycin), and the monosubstituted 2-deoxystreptamine (hygromycin) all failed to promote translational misreading of poly(U) on Thermotoga ribosomes; they also failed to block polyphenylalanine synthesis at a low (less than 10(-4) M) concentration and did not inhibit Thermotoga cell growth at a high (10 micrograms/ml) concentration even though Thermotoga ribosomes possess the 16S rRNA sequences required for aminoglycoside action. In contrast to the other eubacteria, Thermotoga elongation factor G was also refractory to the steroid inhibitor of peptidyl-tRNA translocation fusidic acid.     

Characterization of Saccharomyces cerevisiae mutants supersensitive to aminoglycoside antibiotics.

We describe mutants of Saccharomyces cerevisiae that are more sensitive than the wild type to the aminoglycoside antibiotics G418, hygromycin B, destomycin A, and gentamicin X2. In addition, the mutants are sensitive to apramycin, kanamycin B, lividomycin A, neamine, neomycin, paromomycin, and tobramycin--antibiotics which do not inhibit wild-type strains. Mapping studies suggest that supersensitivity is caused by mutations in at least three genes, denoted AGS1, AGS2, and AGS3 (for aminoglycoside antibiotic sensitivity). Mutations in all three genes are required for highest antibiotic sensitivity; ags1 ags2 double mutants have intermediate antibiotic sensitivity. AGS1 was mapped 8 centimorgans distal from LEU2 on chromosome III. Analyses of yeast strains transformed with vectors carrying antibiotic resistance genes revealed that G418, gentamicin X2, kanamycin B, lividomycin A, neamine, and paromomycin are inactivated by the Tn903 phosphotransferase and that destomycin A is inactivated by the hygromycin B phosphotransferase. ags strains are improved host strains for vectors carrying the phosphotransferase genes because a wide spectrum of aminoglycoside antibiotics can be used to select for plasmid maintenance.     

Aminoglycoside antibiotics preferentially increase permeability in phosphoinositide-containing membranes: a study with carboxyfluorescein in liposomes

The rate of release from multilamellar liposomes of the fluorescent probe carboxyfluorescein was determined as a measure of membrane permeability. Liposomes of phosphatidylcholine and different anionic phospholipids were incubated with low (1 microM) and high (3 mM) concentrations of calcium in the absence or presence of aminoglycoside antibiotics. The leakage of carboxyfluorescein into the medium was not caused by liposomal fusion as no vesicle fusion was observed in experiments with terbium and dipicolinic acid-loaded liposomes. The basal rate of carboxyfluorescein release (in the absence or presence of 1 microM calcium) from all types of liposomes ranged from 0.1 to 0.3% of trapped carboxyfluorescein per hour. The presence of 3 mM calcium caused the greatest increase in the rate of carboxyfluorescein release (about 9-fold) in liposomes containing phosphatidylinositol 4,5-bisphosphate (PIP2) whereas liposomes containing the other anionic phospholipids (phosphatidylserine, phosphatidylinositol and phosphatidylinositol 4-phosphate) showed an approximate 5-fold increase. In the presence of 1 microM calcium, the aminoglycosides neomycin and gentamicin also increased the rate of carboxyfluorescein release, with PIP2-containing liposomes showing a 3-5-times greater response than the other liposomes, releasing up to 4.6% of trapped carboxyfluorescein per hour. This drug-induced release was dose-dependent and antagonized by calcium. In the presence of 3 mM calcium, 0.1 mM gentamicin or neomycin were ineffective while the drug at 1 mM affected carboxyfluorescein release from PIP2-liposomes only. The aminoglycoside antibiotics, neomycin, gentamicin, tobramycin, kanamycin, amikacin, netilmicin, as well as neamine and spectinomycin (all at 0.1 mM) showed a graded effect on the rate of carboxyfluorescein release from PIP2-containing vesicles in the presence of 0.1 mM calcium. The magnitude of the effect correlated well with the ototoxicity of the drugs previously determined directly in cochlear perfusions in the guinea pig. The study demonstrates that aminoglycoside antibiotics are capable of altering membrane permeabilities and that this effect is most pronounced if PIP2 is present in the bilayers. The excellent correlation between this membrane action and the in-situ toxicity of the drugs further establishes the specific role of PIP2 in the molecular mechanism of aminoglycoside-induced hearing loss. Moreover, it confirms the usefulness of such physicochemical models for the screening and prediction of aminoglycoside toxicity.     

Aminoglycoside-3'-phosphotransferase I from aminoglycoside-polyresistant strain E. coli 182]

An aminoglycoside-3'-phosphotransferase I catalyzing phosphorylation of some aminoglycoside antibiotics with the 3'-hydroxyl group has been purified from the cells of aminoglycoside resistant strain E. coli 182 by competitive affinity chromatography on neomycin-Sepharose and gel-filtration on Sephadex G-100. The product of enzymatic phosphorylation of kanamycin A was isolated and identified as kanamycin-3'-phosphate by NMR, thin-layer chromatography and chemical characterization. The kinetic properties of the enzyme were studied. The pH-optimum was between 7,8--8,0; the [S]0.5 values for kanamycin, neomycin and paromomycin were 2.10(-5) M, the energy of activation was 15,9 kcal/mol. The bivalent cations were required for activity of the enzyme, Mg2+ was the most effecient. The relative aminoglycoside antibiotics containing no 3'-hydroxyl group were competitive inhibitors of the enzyme activity with Ki values close to [S]0.5.     

Interaction of aminoglycoside antibiotics with surface Asp and Glu residues of phosphatidylinositol-specific phospholipase C

The aminoglycoside antibiotics such as neomycin, gentamicin, kanamycin and streptomycin stimulated the purified enzyme phosphatidylinositol-specific phospholipases C from Bacillus thuringiensis at pH 5.5. The involvement of net positive charge of aminoglycoside antibiotics (AA) on phosphatidylinositol-specific phospholipases C activation was probed by modifying the carboxyl group of Asp and Glu present in the enzyme by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC). Intrinsic Trp fluorescence of EDAC modified and unmodified PI-PLC in the presence of AA confirmed the interaction of AA with side chain carboxyl group of aspartic and glutamic acid of the enzyme. Thus, the possible interaction of aminoglycoside antibiotics with phosphatidylinositol-specific phospholipases C is predicted to be mediated through the aspartic and glutamic acid residue(s) of the protein.     

不同抗生素对雪莲愈伤组织生长的影响

以雪莲叶片为外植体,探讨了卡那霉素(kanamycin,Kan)、潮霉素(hygromycin B,Hyg)、羧苄青霉素(car-benidillin,Car)3种抗生素对雪莲愈伤组织诱导、生长及分化的影响,以确定农杆菌介导的遗传转化研究中筛选剂和抑菌剂的最适浓度。结果表明:40mg/L的卡那霉素已抑制雪莲愈伤组织生长,当卡那霉素为50mg/L时,愈伤组织的生长基本停止;8.0mg/L潮霉素能够有效抑制雪莲愈伤组织的生长,当潮霉素为20mg/L时则生长的愈伤组织块较小、褐化、甚至死亡。同时,低浓度(0.5～2.0mg/L)的潮霉素可以提高雪莲愈伤组织的分化率;作为农杆菌抑菌剂,不同浓度羧苄青霉素对雪莲愈伤组织生长的影响差异极显著,当羧苄青霉素的浓度超过400mg/L时对雪莲愈伤组织的出愈及生长均有明显的抑制作用。     

Transgenic plants of coffee Coffea canephora from embryogenic callus via Agrobacterium tumefaciens-mediated transformation

 Embryogenic calli were induced from leaf explants of coffee (Coffea canephora) on McCown's woody plant medium (WPM) supplemented with 5 μM N⁶–(2-isopentenyl)-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pIG121-Hm, containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransferase II genes. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentration (5, 50, 100 mg/l). The embryogenic calli surviving on medium containing 100 mg/l hygromycin showed a strong GUS-positive reaction with X-Gluc solution. Somatic embryos were formed from these putative transgenic calli and germinated on WPM medium with 5 μM 2-iP. Regenerated small plantlets with shoots and roots were transferred to medium containing both 100 mg/l hygromycin and 100 mg/l kanamycin for final selection of transgenic plants. The selected plantlets exhibited strong GUS activity in leaves and roots as indicated by a deep blue color. GUS and HPT genes were confirmed to be stably integrated into the genome of the coffee plants by the polymerase chain reaction. Received: 14 December 1998 / Revision received: 12 March 1999 / Accepted: 24 March 1999     

The effect of aminoglycoside antibiotics on the adventitious regeneration from apricot leaves and selection of <Emphasis Type="Italic">npt</Emphasis>II-transformed leaf tissues

The effect of different concentrations of the aminoglycoside antibiotics, geneticin, paromomycin and streptomycin on adventitious regeneration from leaf explants of apricot was tested to design an alternative procedure for selecting transgenic shoots. Streptomycin and paromomycin reduced shoot regeneration percentage with increasing concentration of antibiotics. Almost a complete inhibition of regeneration was reached when 20M paromomycin was used, although up to 40M streptomycin was necessary to completely inhibit regeneration. Geneticin had a very toxic effect on apricot leaves and regeneration was inhibited at almost all concentrations tested. Addition of kanamycin hastened the development of adventitious buds although silver thiosulfate and not kanamycin was responsible for the observed increase in the consistency of the results from independent experiments. Kanamycin and paromomycin at the concentrations tested improved selection of transformed cells and resulted in a larger number of gfp-expressing regions. Paromomycin at 40 and 25.7M kanamycin improved proliferation of transformed tissues as compared with the other antibiotics used and non-selected controls.     

Effects of kanamycin on tissue culture and somatic embryogenesis in cotton

The aminoglycoside antibiotic kanamycin was evaluated for its effects on callus initiation from hypocotyl and cotyledon explants, proliferation of non-embryogenic and embryogenic calli, initiation and development of somatic embryos in cotton (Gossypium hirsutum L.). On this basis, the potential use of kanamycin as a selective agent in genetic transformation with the neomycin phosphotransferase II gene as the selective marker gene was evaluated. Cotton cotyledon and hypocotyl explants, and embryogenic calluses were highly sensitive to kanamycin. Kanamycin at 10 mg/L or higher concentrations reduced callus formation, with complete inhibition at 60 mg/L. Kanamycin inhibited embryogenic callus growth and proliferation, as well as the initiation and development of cotton somatic embryos. The sensitivity of embryogenic callus and somatic embryos to kanamycin was different during the initiation and development stages. Kanamycin was considered as a suitable selective agent for transformed callus formation and growth of non-embryogenic callus. Forty to sixty mg/L was the optimal kanamycin concentration for the induction and proliferation of transformed callus. The concentration of kanamycin must be increased (from 50 to 200 mg/L) for the selection of transformation embryogenic callus and somatic embryos. A scheme for selection of transgenic cotton plants when kanamycin is used as the selection agent is discussed.     

The mechanism of cobalt toxicity in mung beans

The effects of cobalt on the growth and nutrient balance of mung beans were investigated. Inhibition of seedling growth occurred at 5 μ M Co and was associated with chlorosis of the younger leaves. Analysis of nutrient concentrations in root and leaf tissue of mung beans treated with 5 μ M Co showed that none of the macronutrients and only two of the micronutrients, Mn and Fe, were significantly affected. The Mn concentration in roots was reduced by 55% and the Fe concentration in the leaves by 80%. Uptake of Fe into roots was not inhibited by Co but transport of Fe to the shoot was greatly reduced. It was shown that the effect of Co on growth was additive to that of Fe deficiency, which argues against Co-induced Fe deficiency as the primary cause of growth inhibition by Co. Rather, it was considered that the high concentrations of Co in the roots and leaves compared with essential micronutrient cations can disrupt a range of metabolic processes due to competitive interactions. Comparison of the toxic effects of Co with those of other toxic trace metals Cd, Cu, Ni and Hg showed that at an applied concentration of 5 μ M , there were obvious differences in both the visual symptoms and in nutrient concentrations. The main difference between Co and the other metals was that only Co stimulated the uptake of S into the plant and its transport to the shoots, where the S concentration in the leaves was increased 2-fold. The common feature of all the trace metals examined was the strong inhibition of Fe transport to the shoot. A possible mechanism for the interaction of other trace metals with Fe transport is discussed.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of niger [<Emphasis Type="Italic">Guizotia abyssinica</Emphasis> (L. f.) Cass.] using seedling explants

A protocol was developed for Agrobacterium-mediated genetic transformation of niger [ Guizotia abyssinica (L.f.) Cass.] using hypocotyl and cotyledon explants. Hypocotyls and cotyledons obtained from 7-day-old seedlings were co-cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm that harbored genes for beta-glucuronidase (GUS), kanamycin, and hygromycin resistance. Following co-cultivation, the hypocotyl and cotyledon explants were cultivated on MS medium containing 1 mg/l 6-benzylaminopurine (BA) for 3 days in darkness. Subsequently, hypocotyl and cotyledon explants were transferred to selective MS medium containing 1 mg/l BA, 10 mg/l hygromycin, 10 mg/l kanamycin, and 500 mg/l cefotaxime. After 6 weeks, hypocotyls and cotyledons produced multiple adventitious shoot buds, and these explants were subcultured to MS medium containing 1 mg/l BA, 30 mg/l hygromycin, and 30 mg/l kanamycin. After a further 3 weeks, the explants (along with developing shoot buds) were subcultured to MS medium containing 1 mg/l BA, 50 mg/l kanamycin, and 50 mg/l hygromycin for further selection. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 0.1 mg/l alpha-naphthaleneacetic acid, 50 mg/l kanamycin, and 50 mg/l hygromycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis. Genomic Southern blot hybridization confirmed the incorporation of the neomycin phosphotransferase II gene into the host genome.     

不同抗生素对金发草愈伤组织生长分化的影响

该研究以金发草愈伤组织为材料,通过分析比较不同抗生素种类(卡那霉素、潮霉素、头孢噻呋钠和氨苄青霉素)和浓度对金发草愈伤组织生长分化的影响,来确定适用于金发草遗传转化体系中的抗性筛选剂和抑菌剂。结果表明：(1)金发草愈伤组织对卡那霉素很敏感,且其分化率随着卡那霉素浓度的增加显著减少( P＝0.01)。当卡那霉素浓度为10 mg·L-1时,金发草愈伤组织的生长分化受到明显抑制,且有大量的白化苗形成,但分化率仍有36.56％;当卡那霉素浓度为15 mg·L-1时,金发草愈伤组织的分化率为11.94％,只有很少部分的愈伤分化出绿色的丛生苗;当卡那霉素浓度为20 mg·L-1时,金发草愈伤组织基本褐化死亡,分化率仅为2.26％。因此,浓度为15 mg·L-1的卡那霉素适合作为金发草遗传转化体系中的抗性筛选剂。(2)金发草愈伤组织对潮霉素的敏感性要比卡那霉素弱,且潮霉素对金发草愈伤组织分化率的影响小,但毒害作用大。因此,潮霉素不适合作为金发草遗传转化体系中的抗性筛选剂。(3)300 mg·L-1的头孢霉素和氨苄青霉素对金发草愈伤组织生长分化影响很小且能有效抑制杂菌的生长,较高浓度的氨苄青霉素对金发草愈伤组织的抑制作用不太明显。因此,300 mg·L-1的头孢霉素和较高浓度的氨苄青霉素均可作为金发草遗传转化体系中的抑菌剂。该研究确定了适用于农杆菌介导的金发草遗传转化体系中的抗性筛选剂和抑菌剂,为金发草的遗传改良及功能性基因的研究奠定了基础。     

Aminoglycoside antibiotics: structure, functions and effects on in vitro plant culture and genetic transformation protocols

Plant transformation protocols generally involve the use of selectable marker genes for the screening of transgenic material. The bacterial gene nptII, coding for a neomycin phosphotransferase, and the hpt gene, coding for a hygromycin phosphotransferase, are frequently used. These enzymes detoxify aminoglycoside antibiotics by phosphorylation, thereby permitting cell growth in the presence of antibiotics. Nevertheless, the screening for transgenic regenerated shoots is often partial and difficult due to regeneration of escapes and chimeras. These difficulties can be caused, in part, by an incorrect assumption about the mode of action of antibiotics in bacterial and eukaryotic cells and in in vitro tissue culture. The information contained in this review could be useful to establish better selection strategies by taking into account factors such as explant complexity, transformation and selection protocols that allow better accessibility to cells of Agrobacterium and antibiotics, and faster regeneration methods that avoid collateral effects of antibiotics on recovered, putative transgenic shoots.     

Effects of aminoglycoside antibiotics on the coupling of protein and RNA syntheses in Escherichia coli

The interdependency of protein and RNA syntheses was studied comparatively in bacteria confronted with amino acid starvation or treated separately with various aminoglycoside antibiotics. By contrast with the concomitant inhibition of macromolecular syntheses in cells deprived of an essential amino acid, RNA production was found to continue in drug-treated cells while protein synthesis was arrested. Such uncoupling process was also observed in bacteria subjected simultaneously to amino acid starvation and treatment with certain antibiotics (neomycin, gentamicin, spectinomycin and kasugamycin) but not with others (streptomycin and kanamycin). These results were related to the intracellular concentration of guanosine polyphosphates, ppGpp and pppGpp. They were discussed in terms of interaction of aminoglycosides with ribosomes.     

不同浓度卡那霉素、潮霉素对楸树试管苗生长的影响

目的:将不同质量浓度的卡那霉素、潮霉素加入楸树培养基中,研究卡那霉素、潮霉素对楸树组培苗生长的影响,以确定抗生素对楸树茎段分化与生根的敏感质量浓度。方法:待楸树继代、生根培养基灭菌后温度降至30~50℃,将不同质量浓度的卡那霉素、潮霉素经抽滤式灭菌加入培养基中,在培养基中接入楸树组培无菌茎段培养,观测茎段继代(增殖芽数、芽长、叶数等)、生根(发根数、根长、芽长等)生长指标并统计分析。结果:楸树组培继代培养基添加卡那霉素质量浓度为100 mg/L时组培瓶苗生长缓慢,浓度为150 mg/L时叶片大部分发白并干枯,茎段基部无愈伤组织形成,瓶苗基本停止生长,楸树继代瓶苗对卡那霉素耐受性范围为100~150 mg/L;添加潮霉素质量浓度为5 mg/L时瓶苗生长较为缓慢,浓度为10 mg/L时叶片开始干枯,茎段基部愈伤组织较小,瓶苗基本停止生长,楸树继代瓶苗对潮霉素耐受性范围为10 mg/L左右。楸树组培生根培养基添加卡那霉素质量浓度为100 mg/L时大部分茎段干枯,少部分为绿但未分化芽与根,浓度为150 mg/L时大部分茎段干枯,极少上部为绿,基部干枯,但未分化芽与根,楸树组培瓶苗生根培养苗对卡那霉素耐受性范围为100~150 mg/L;添加潮霉素质量浓度为5 mg/L时少部分茎段干枯,浓度为10 mg/L时大部分茎段干枯,少部分为绿,茎段未出现芽的分化与根的萌发现象,楸树组培瓶苗生根培养苗对潮霉素耐受性范围为5~10 mg/L。结论:卡那霉素、潮霉素对楸树组培苗生长有明显的抑制作用且与抗生素浓度呈负相关,但低质量浓度(1 mg/L)的潮霉素对楸树继代分化芽数有促进作用;同一抗生素对楸树不同无性系间组培苗生长的影响无显著差异。     

Aminoglycoside and aminocyclitol antibiotics: hygromycin B is an atypical bactericidal compound that exerts effects on cells of Escherichia coli characteristics for bacteriostatic aminocyclitols.

The effects of aminoglycoside and aminocyclitol antibiotics on intact cells of Escherichia coli were compared. The aminoglycosides streptomycin, gentamicin, kanamycin and neomycin had similar, but not identical, effects. They all caused misreading during protein synthesis, permeabilization of the cell membrane, inhibition of the initiation of DNA replication, and loss of cell viability. Cells treated with these antibiotics continued to synthesize two proteins (apparent molecular masses 72 and 60 kDa) that were not made by cells treated with the aminocyclitol hygromycin B, which did not cause misreading. Cells treated with the aminoglycosides regained their membrane tightness after residual protein synthesis in these cells had been inhibited by chloramphenicol, suggesting that under these conditions the mistranslated membrane proteins were rapidly degraded. The bacteriostatic aminocyclitols spectinomycin and kasugamycin did not cause membrane permeabilization, suggesting that these compounds do not cause misreading. Hygromycin B resembled these aminocyclitols in that it inhibited protein synthesis without causing misreading, membrane permeabilization or inhibition of initiation of DNA synthesis. However, hygromycin B also decreased cell viability. In minimal medium this lethal effect began late in comparison to the process of inhibition of protein synthesis. It is concluded that hygromycin B is an atypical bactericidal antibiotic that strongly resembles the bacteriostatic aminocyclitols spectinomycin and kasugamycin in its action.     

用PEG法把外源基因导入甘蓝型油菜原生质体再生转基因植株

通过PEG处理把外源基因导入甘蓝型油菜原生质体。转化介质中二价阳离子的种类和浓度、携带DNA及PEG溶液的pH值都会影响基因导入效率。以潮霉素抗性和卡那霉素抗性作标记,均成功地筛选到了抗性愈伤组织。相对转化频率分别为1.3%和0.2%。前者明显高于后者。把抗性愈伤组织转到分化培养基上,分化出芽。诱导生根后移栽到土壤中,生长状况良好。酶活性测定和Southern blotting分析表明外源基因已插入到植物细胞基因组中。该遗传转化系统存在的主要问题是抗性愈伤组织分化频率较低。本文对其原因作了初步分析。     

Aminoglycoside antibiotics block voltage-dependent calcium channels in intact vertebrate nerve terminals

Intrinsic and extrinsic optical signals recorded from the intact nerve terminals of vertebrate neurohypophyses were used to investigate the anatomical site and physiological mechanism of the antagonistic effects of aminoglycoside antibiotics on neurotransmission. Aminoglycoside antibiotics blocked the intrinsic light scattering signal closely associated with neurosecretion in the mouse neurohypophysis in a concentration-dependent manner with an IC50 of approximately 60 microM and the block was relieved by increasing [Ca2+]o. The rank order potency of different aminoglycoside antibiotics for blocking neurosecretion in this preparation was determined to be: neomycin greater than gentamicin = kanamycin greater than streptomycin. Optical recordings of rapid changes in membrane potential using voltage-sensitive dyes revealed that aminoglycoside antibiotics decreased the Ca(2+)-dependent after-hyperpolarization of the normal action potential and both the magnitude and after-hyperpolarization of the regenerative Ca2+ spike. The after-hyperpolarization results from a Ca-activated potassium conductance whose block by aminoglycoside antibiotics was also reversed by increased [Ca2+]o. These studies demonstrate that the capacity of aminoglycoside antibiotics to antagonize neurotransmission can be attributed to the block of Ca channels in the nerve terminal.     

Molecular determinants of antibiotic recognition and resistance by aminoglycoside phosphotransferase (3')-IIIa: a calorimetric and mutational analysis

The growing threat from the emergence of multidrug resistant pathogens highlights a critical need to expand our currently available arsenal of broad-spectrum antibiotics. In this connection, new antibiotics must be developed that exhibit the abilities to circumvent known resistance pathways. An important step toward achieving this goal is to define the key molecular interactions that govern antibiotic resistance. Here, we use site-specific mutagenesis, coupled with calorimetric, NMR, and enzymological techniques, to define the key interactions that govern the binding of the aminoglycoside antibiotics neomycin and kanamycin B to APH(3')-IIIa (an antibiotic phosphorylating enzyme that confers resistance). Our mutational analyses identify the D261, E262, and C-terminal F264 residues of the enzyme as being critical for recognition of the two drugs as well as for the manifestation of the resistance phenotype. In addition, the E160 residue is more important for recognition of kanamycin B than neomycin, with mutation of this residue partially restoring sensitivity to kanamycin B but not to neomycin. By contrast, the D193 residue partially restores sensitivity to neomycin but not to kanamycin B, with the origins of this differential effect being due to the importance of D193 for catalyzing the phosphorylation of neomycin. These collective mutational results, coupled with (15)N NMR-derived pK(a) and calorimetrically derived binding-linked drug protonation data, identify the 1-, 3-, and 2'-amino groups of both neomycin and kanamycin B as being critical functionalities for binding to APH(3')-IIIa. These drug amino functionalities represent potential sites of modification in the design of next-generation compounds that can overcome APH(3')-IIIa-induced resistance.