Probing the in vitro mechanism of action of cationic lipid/DNA lipoplexes at a nanometric scale.

Cationic lipids are used for delivering nucleic acids (lipoplexes) into cells for both therapeutic and biological applications. A better understanding of the identified key-steps, including endocytosis, endosomal escape and nuclear delivery is required for further developments to improve their efficacy. Here, we developed a labelling protocol using aminated nanoparticles as markers for plasmid DNA to examine the intracellular route of lipoplexes in cell lines using transmission electron microscopy. Morphological changes of lipoplexes, membrane reorganizations and endosomal membrane ruptures were observed allowing the understanding of the lipoplex mechanism until the endosomal escape mediated by cationic lipids. The study carried out on two cationic lipids, bis(guanidinium)-tris(2-aminoethyl)amine-cholesterol (BGTC) and dioleyl succinyl paramomycin (DOSP), showed two pathways of endosomal escape that could explain their different transfection efficiencies. For BGTC, a partial or complete dissociation of DNA from cationic lipids occurred before endosomal escape while for DOSP, lipoplexes remained visible within ruptured vesicles suggesting a more direct pathway for DNA release and endosome escape. In addition, the formation of new multilamellar lipid assemblies was noted, which could result from the interaction between cationic lipids and cellular compounds. These results provide new insights into DNA transfer pathways and possible implications of cationic lipids in lipid metabolism.

Structure-based design, synthesis, and A-site rRNA cocrystal complexes of functionally novel aminoglycoside antibiotics: C2" ether analogues of paromomycin.

A series of 2"-O-substituted ether analogues of paromomycin were prepared based on new site-selective functionalizations. X-ray cocrystal complexes of several such analogues revealed a new mode of binding in the A-site rRNA, whereby rings I and II adopted the familiar orientation and position previously observed with paromomycin, but rings III and IV were oriented differently. With few exceptions, all of the new analogues showed potent inhibitory activity equal or better than paromomycin against a sensitive strain of S. aureus. Single digit microM MIC values were obtained against E. coli, with some of the ether appendages containing polar or basic end groups. Two analogues showed excellent survival rate in a mouse septicemia protection assay. Preliminary histopathological analysis of the kidney showed no overt signs of toxicity, while controls with neomycin and kanamycin were toxic at lower doses.

Inhibition of human tyrosyl-DNA phosphodiesterase by aminoglycoside antibiotics and ribosome inhibitors.

DNA topoisomerase I (Top1) is the target of camptothecin, and novel Top1 inhibitors are in development as anticancer agents. Top1 inhibitors damage DNA by trapping covalent complexes between the Top1 catalytic tyrosine and the 3'-end of the broken DNA. Tyrosyl-DNA phosphodiesterase (Tdp1) can repair Top1-DNA covalent complexes by hydrolyzing the tyrosyl-DNA bond. Inhibiting Tdp1 has the potential to enhance the anticancer activity of Top1 inhibitors (http://discover.nci.nih.gov/pommier/pommier.htm) and to act as antiproliferative agents. In the present study, we report that neomycin inhibits Tdp1 more effectively than the related aminoglycosides paromomycin and lividomycin A. Inhibition of Tdp1 by neomycin is observed both with single- and double-stranded substrates but is slightly stronger with duplex DNA, which is different from aclarubicin, which only inhibits Tdp1 with the double-stranded substrate. Inhibition by neomycin can be overcome with excess Tdp1 and is greatest at low pH. To our knowledge, aminoglycoside antibiotics and the ribosome inhibitors thiostrepton, clindamycin-2-phosphate, and puromycin are the first reported pharmacological Tdp1 inhibitors.

Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system.

The translation machinery recognizes codons that enter the ribosomal A site with remarkable accuracy to ensure that polypeptide synthesis proceeds with a minimum of errors. When a termination codon enters the A site of a eukaryotic ribosome, it is recognized by the release factor eRF1. It has been suggested that the recognition of translation termination signals in these organisms is not limited to a simple trinucleotide codon, but is instead recognized by an extended tetranucleotide termination signal comprised of the stop codon and the first nucleotide that follows. Interestingly, pharmacological agents such as aminoglycoside antibiotics can reduce the efficiency of translation termination by a mechanism that alters this ribosomal proofreading process. This leads to the misincorporation of an amino acid through the pairing of a near-cognate aminoacyl tRNA with the stop codon. To determine whether the sequence context surrounding a stop codon can influence aminoglycoside-mediated suppression of translation termination signals, we developed a series of readthrough constructs that contained different tetranucleotide termination signals, as well as differences in the three bases upstream and downstream of the stop codon. Our results demonstrate that the sequences surrounding a stop codon can play an important role in determining its susceptibility to suppression by aminoglycosides. Furthermore, these distal sequences were found to influence the level of suppression in remarkably distinct ways. These results suggest that the mRNA context influences the suppression of stop codons in response to subtle differences in the conformation of the ribosomal decoding site that result from aminoglycoside binding.

Sulfato de aminosidina: estudio multicentrico / Aminosidine sulfate: a multicentric study

Se efectua un estudio con 157 enfermos que presentaron como caracteristicas en especial el hecho de tener proceso infeccioso severo de diferente localizacion y distribuidos en 73 hombres y 84 mujeres, con edades que oscilaron de los 10 a mas de 70 anos. Su inclusion requirio al inicio pruebas de funcion renal, estado de hidratacion adecuada y proceso infeccioso en que se justificara el uso de la aminosidina. En siete de nuestros enfermos se ajusto la dosis del antibiotico en el curso de su uso, debido a cambios en las pruebas defuncion renal. Se trata un total de 37 infecciones urinarias, 43 infecciones de aparato respiratorio, 32 infecciones de tejidos blandos, 13 infecciones de heridas quirurgicas, 19 infecciones de origen abdominal, 11 de origen genital, 1 panoftalmitis por estafilococo aureus y 1 infeccion en un individuo con una leucemia aguda sin foco aparente(7 de ellos con infecciones asociadas). En todos los casos se utiliza la via intravenosa, con dilucion en 50 a 100 ml de suero glucosado y administrado en el lapso de 30 minutos. En la mayoria de los casos se obtiene aislamiento del germen causal. La respuesta es muy favorable, del 90% en etapa aguda. Una recaida del 4% en procesos infecciosos cronicos de vida urinarias en control del 15 al 30 dias despues del episodo agudo, y una falla del 7% como consecuencia de agentes del genero Pseudomona,colonizacion por cepas hospitalarias de elevada resistencia y estancia intrahospitaria prolongada. En los casos que lo ameritan se asociaro

[Clinical effects of lividomycin on respiratory tract infections, primarily wet cases of bronchiectasis (author's transl)].

Patients with various respiratory infections, primarily wet cases of bronchiectasis, were treated with lividomycin (LVDM) at a dose of 1 g/day for about a week. The results obtained are as follows: 1. Of the 15 patients with refractory bronchiectasis who had previously been treated with various other antibiotics, 11 patients (73.3%) responded to LVDM. Although there was no patient who responded to LVDM excellently, good cures were achieved in 5 pateints and fair cures in 6 patients. 2. In bronchiectasis patients having infections in association with pulmonary tuberculosis (mixed infections), LVDM did not show any remarkable effects, excepting that fair cures were achieved in 3 patients. 3. In the 7 patients with acute bronchopneumonia, excellent, good and fair cures were achieved in 1, 5 and 1 patient, respectively. Although LVDM was effective in all of the patients with the said infection, the number of the patients responded excellently was meager and the onset of action seemed to be somewhat slow. 4. In one patient with lobar pneumonia, LVDM was not effective. 5. The MIC values of LVDM for clinically isolated various organisms are summarized below: Pseudomonas aeruginosa 50 mcg/ml, Klebsiella pneumoniae 3.13 to 6.25 mcg/ml, Haemophilis influenzae 1.56 to 6.25 mcg/ml, Diplococcus pneumoniae 50 mcg/ml, alpha-Streptococcus 50 to 100 mcg/ml, beta-Streptococcus 100 mcg/ml, and Staphylococcus aureus 1.56 mcg/ml. 6. In spite of the fact that a majority of the patients participated in this study were the aged, LVDM's side effects were limited to an increase of BUN (one patient) and eruption (one patient). Any noticeable changes in hepatic and renal function, hearing acuity, etc. were not observed. As indicated above, LVDM was effective against, respiratory infections as well as infections of the urinary tract on which a considerable number of research has already been reported. Thus, it is considered to be worthwhile to use LVDM for the treatment of respiratory infections.

[Clinical experiences of lividomycin on respiratory tract infections (author's transl)].

Clinical effects of a newly developed aminoglycoside antibiotic lividomycin, were investigated in 13 patients suffering from respiratory infections. 1. A total of 13 patients with the following infectious diseases was treated with 1 g/day of lividomycin for 3 to 11 days (average 6.7 days): Acute pneumonia 3 cases, bronchiectasis 3 cases, acute bronchitis 5 cases, suppurative diseases of the lung 1 case, and pyothorax 1 case. As the results, the antibiotic was effective in 5 patients, moderately effective in 4 patients and ineffective in 4 patients, and thus the global effective rate was 62.9% (9/13). 2. As in the case of kanamycin, lividomycin indicated fairly good sensitivity against a total of 17 strains comprising Staphylococcus aureus (7 strains), alpha-Streptococcus (2 strains), beta-Streptococcus (3 strains), gamma-Streptococcus (1 strain), Neisseria (2 strains) and Haemophilus (2 strains) which were isolated from sputum. 3. No side effects attributable to lividomycin were observed.

[Therapeutic effect of lividomycin on infections of the respiratory tract (author's transl)].

Therapeutic effect of lividomycin was observed in patients with infections of the respiratory tract (infections in association with bronchiectasis, mixed infections of cavities after negative conversion of tubercle bacilli, pneumonia, and so forth). Significant improvement of clinical symptoms was observed in 15 cases and no improvement in 4 cases. Lividomycin was administered at a dose of 1 g/day for 3 to 15 days. Staphylococcus aureus (9 cases), Klebsiella pneumoniae (1 case), alpha-hemolytic Streptococcus (3 cases), Aerococcus (4 cases) and Pseudomonas aeruginosa (1 case) were suggested to be causative organisms in the above-mentioned patients. (In one case, however, causative organism was not determined.) Lividomycin seemed to be effective in cases of infections with Staphylococcus aureus, alpha-hemolytic Streptococcus and Klebsiella pneumoniae, whereas not effective in a case of infection with Pseudomonas aeruginosa. In order to differentiate causative organisms from the organisms constantly existing on the upper respiratory tract, alkali treatment was employed as an aid for the determination of the causative organisms. However, further studies are desirable for the evaluation of this procedure.

[Studies on the therapeutic effects of lividomycin in respiratory infections (author's transl)].

Therapeutic effects of lividomycin (LVDM) were studied in 33 patients with respiratory infections including pneumonia, lung abscess, chronic bronchitis, etc. LVDM was intramuscularly administered at the dose of 1 or 2 g per day for consecutive 4 to 25 days. The results obtained are summarized below: 1. At the end of the first week of the treatment, rate of improvement in such parameters as cough, sputum, rales, fever and blood sedimentation rate were 69%, 56.7%, 60%, 79.2% and 70% respectively. Also, in 16% of the patients, abnormal shadow noted in X-ray film of the chest was disappeared and in 20% of the patients, size of the same was reduced during the first week of treatment. 2. Therapeutic effects of LVDM were evaluated synthetically and were graded as excellent, good, fair and ineffective. LVDM was effective in about 70 per cent of the patients, that is, excellent results were obtained in 4 patients, good in 12 patients, fair in 6 patients, and in 10 patients this antibiotic was ineffective. 3. In one patient with slight loss of high frequency perception was observed on the audiogram, but no other ototoxic effects such as subjective hearing loss, tinnitus, etc. In addition, no untoward effects on renal and hepatic function were observed. 4. The MIC values of LVDM for clinically isolated 20 strains of Pseudomonas aeruginosa were examined, using kanamycin for comparison. The MIC values of LVDM for many strains were superior to those of kanamycin. In view of the test results mentioned above, LVDM would appear to be useful medication for the treatment of some of respiratory infections.

Phosphorylation of kanamycin, lividomycin A, amd butirosin B by Providencia stuartii.

The isolation of Providencia stuartii resistant to multiple aminoglycoside antibiotics prompted an investigation into the mechanism of their resistance. Crude enzyme extracts of a strain of P. stuartii inactivated kanamycin, lividomycin A, and butirosin B in the presence of adenosine 5'-triphosphate (ATP), as measured by a microbiological assay. The occurrence of inhibitory concentrations of 500 mug or greater per ml against kanamycin, lividomycin A, and butirosin B, coupled with the inactivation of these antibiotics in the presence of ATP, suggested enzymatic phosphorylation. This was documented by the transfer of the gamma-phosphate of [gamma-(32)P]ATP. In contrast, the inability to inactivate gentamicin or tobramycin by the crude enzyme extracts in the presence of ATP suggests another enzymatic mechanism of resistance for these antibiotics, such as adenylation or acetylation. Of importance is the fact that amikacin, a semisynthetic analogue of kanamycin A which is resistant to inactivation by most resistance transfer factor enzymes, was found to inhibit the growth of P. stuartii at low concentrations.

Antibacterial activity of lividomycin toward R factor-resistant strains of Escherichia coli.

Forty-eight R factors conferring resistance to both kanamycin (KM) and streptomycin (SM) were demonstrated from 1,270 Escherichia coli strains of clinical origin. Among these R factors isolated, 42 also conferred resistance to the new antibiotic lividomycin (LV). Extracts of E. coli ML1410 carrying one such factor (R(M81)) inactivated LV as well as KM and SM. But extracts of E. coli ML1410 carrying an R factor (R(M82) or R(M83)) sensitive to LV could not inactivate LV. LV inactivation required both adenosinetriphosphate (ATP) and Mg(2+). Inactivated LV was reactivable by alkaline phosphatase. Isotopic studies with labeled ATP and elemental analysis of the reaction product indicate that it is a monophosphorylated derivative of LV.