Geneticin sulfate

The first complex species detected by potentiometry and spectroscopy at low pH is CuH₂L. A CuH3L complex, with {NH₂, OH} coordination, detected by EPR for geneticin, is not present here, apparently due to the higher basicity of kanamycin A. Among the aminoglycosides studied by us, geneticin is the closest analogue of kanamycin A. The only differences between these two molecules are the position of the amino group in ring A (at A2 for geneticin, and at A6 for kanamycin A) and the methylation of the amino group of ring C in geneticin. By analogy with other unmodified aminoglycosides, the chelate in the CuH₂L complex is thought to involve the deprotonated C3 amine and C4 hydroxyl donors of the kanosamine moiety (ring C). All other amino groups are protonated in this species.

Interestingly, the relative efficiency of cleavage induced in the presence of Cu(Ⅱ)–antibiotic complexes at Y37 strongly depended on the number of amino functions in the aminoglycoside molecules. Stronger cleavage occurred in the presence of kanamycin B and tobramycin, each of them containing five amino groups, than in the presence of amikacin and kanamycin A, containing four amino functions (Fig. 1). In order to confirm this observation we also studied some other antibiotics, such as kasugamycin, geneticin, sisomicin and neomycin B. Although the cleavage patterns were very similar to the pattern presented in Fig. 1, the cleavage intensities at Y37 differed (data not shown). The two-ring kasugamycin, containing one primary and two secondary amines, carried the lowest positive charge at pH 7.4 and it caused the least cleavage of tRNA. On the other hand, the strongest cleavage occurred in the presence of neomycin B, which contains six amino functions. The intensities of cleavages induced by geneticin and sisomicin place them between kasugamycin and neomycin, with respect to their positive charge.

Geneticin (0.4–0.8 mg ml^-1) was included to maintain selection of antibiotic resistant cells, and in the case of the double stable cell lines K^V7.1 + KCNE1 and K^V7.2/3 both subunits were selected for with a combination of geneticin and hygromycin (0.2 mg ml^-1). Cryopreserved cell batches wereproduced by first dissociating cells from the culture flasks and then resuspending in 90% FBS and 10% DMSO. Cell vials were then placed in a controlled rate freezer, and stored at -140 ℃. On the day prior to recording continuously cultured cells were dissociated or frozen cell vials defrosted and then added to T175 culture flasks containing warmed culture media. Immediately before use cells were washed with versene and then incubated at 37 ℃ in 2 ml versene for 6 min to gently lift the cells. The resulting cell suspension was centrifuged at 700 rpm for 2 min, and then resuspended in the external recording solution with gentle trituration.

Cells were transfected with 17 mg of pcDNA3.1(+) vector containing the target 5-HT_{7<./sub> DNA sequence, as per standard protocols by using transfection Reagent in Opti-MEM medium without serum. Vector-expressing cells were selected using geneticin (G418). After transfection, cells were cultured in normal growth medium. One day later, cells were detached and replated into growth medium containing geneticin (800 mg mL^-1) and cultured for 20 days. Surviving cell clones were picked and propagated separately in 60 mm culture dishes in the same medium, with 800 mg mL^-1 geneticin to maintain selection.}