Technique of intravitreal injection

The polypeptide ubiquitin, up to now almost exclusively discovered in intracellular spaces, was measured immunologically in a total of 187 samples of human seminal plasma. The values were between 1.83 and 19.11 micrograms/ml. In spermatozoa ubiquitin was detected too; the values, however, were significantly lower than in the seminal plasma. The origin and function of ubiquitin in human seminal plasma is still unclear. The possible role of ubiquitin in reproduction is discussed.     

Experimental study of the mutagenic activity of gentamicin sulfate in relation to the causative agent of plague

Gentamicin sulfate was studied with an aim of obtaining mutants of the plague microbe with a changed character of requirements in the growth factors. The antibiotic provided a high yield of auxotrophic mutants and induced streptomycin resistance in some cells. Mutants with single dependence on arginine, leucine, histidine and uracyl were most often. Comparison of the data obtained with gentamicin and those obtained with other chemical mutagens showed that gentamicin by its mutagenic activity was only slightly inferior than the preparations of the group of supermutagens.     

Intratympanic gentamicin in Meniere''s disease

In Chlamydomonas reinhardtii, mutants defective in the cytochrome pathway of respiration lack the capacity to grow under heterotrophic conditions (in darkness on acetate). In the dark- strain duM18, a + 1 T addition in a run of four Ts, located at codon 145 of the mitochondrial cox1 gene encoding subunit I of cytochrome c oxidase, is responsible for the mutant phenotype. A leaky revertant (su11) that grows heterotrophically at a lower rate than wild-type cells was isolated from dum18. Its respiration sensitivity to cyanide was low and its cytochrome c oxidase activity was only 4% of that of the wild-type enzyme. Meiotic progeny obtained from crosses between revertant and wild-type cells inherited the phenotype of the mt- parent, showing that the suppressor mutation, like dum18 itself, is located in the mitochondrial genome. In order to map the su11 mutation relative to dum18, a recombinational analysis was performed on the diploid progeny. It demonstrated that su11 was very closely linked to the dum18 mutation less than 20-30 bp away. The cox1 gene of the su11 revertant was then sequenced. In addition to the + 1 T frameshift mutation still present at codon 145, an A-->C substitution was found at codon 146, leading to the replacement of a glutamic acid by an alanine in the polypeptide chain. No other mutations were detected in the cox1 coding sequence. As the new GCG codon (Ala) created at position 146 is very seldom used in the mitochondrial genome of C. reinhardtii, we suggest that the partial frameshift suppression by the nearby substitution is due to an occasional abnormal translocation of the ribosome (+ 1 base shift) facilitated both by the run of Ts and the low level of weak interaction of alanyl-tRNA.     

Assay of gentamicin in cerebrospinal fluid

A comparison of standard curves obtained from a conventional plate diffusion assay method revealed significant differences when gentamicin standards were made up in different media. Standards made up in distilled water resulted in a curve which differed from that of standards made up in pooled human cerebrospinal fluid by a factor of up to 4. When the assay medium was supplemented with 0-5% sodium chloride, the difference between the two standard curves was reduced to a factor of about 1-5. The curve obtained from standards made up in 150 mM sodium chloride/4-5 mM calcium chloride correlated well with that from standards made up in cerebrospinal fluid. There was no evidence of gentamicin being bound to protein in the cerebrospinal fluid.     

Population pharmacokinetics of gentamicin in horses

OBJECTIVE: To develop and validate a population pharmacokinetic model for gentamicin in horses, using retrospective clinical data. ANIMALS: 62 horses that had been treated IV with multiple doses of gentamicin at our veterinary teaching hospital between 1987 and 1996. Procedure-46 horses were assigned to the study group, and 16 to the validation group. Detailed history of dosage, sample collection times, and selected pathophysiologic variables were recorded for each patient. Samples were analyzed by use of a fluorescence polarization immunoassay method. Pharmacostatistical analysis was conducted, using computer software. The predictive model correlates pharmacokinetic parameters to concomitant pathophysiologic variables and estimates the inter- and intraindividual variability in disposition. RESULTS: A two-compartment model best described the data. Clearance (CI) was linearly correlated to body weight and serum creatinine concentration. Volume of the central compartment (Vd(c)) was linearly related to body weight. Interindividual coefficients of variability for CI and Vd(c) were 24 and 16%, respectively. The residual variability (intraindividual) was 13%; mean prediction error percent (bias) was 2%; and mean absolute prediction error percent (precision) was 29%. CONCLUSIONS: Population pharmacokinetic analysis allows study of the basic features of gentamicin disposition in horses with sparse data per individual. A considerable proportion of the pharmacokinetic variability of gentamicin in our study population was explained by differences in body weight and serum creatinine concentration. CLINICAL RELEVANCE: Population pharmacokinetics can be used to design first-dosage regimens according to the clinical characteristics of individual animals. Population pharmacokinetic models could also be included in Bayesian forecasting strategies to improve plasma concentration predictions in individual patients.     

Structural origins of gentamicin antibiotic action

Aminoglycoside antibiotics that bind to the ribosomal A site cause misreading of the genetic code and inhibit translocation. The clinically important aminoglycoside, gentamicin C, is a mixture of three components. Binding of each gentamicin component to the ribosome and to a model RNA oligonucleotide was studied biochemically and the structure of the RNA complexed to gentamicin C1a was solved using magnetic resonance nuclear spectroscopy. Gentamicin C1a binds in the major groove of the RNA. Rings I and II of gentamicin direct specific RNA-drug interactions. Ring III of gentamicin, which distinguishes this subclass of aminoglycosides, also directs specific RNA interactions with conserved base pairs. The structure leads to a general model for specific ribosome recognition by aminoglycoside antibiotics and a possible mechanism for translational inhibition and miscoding. This study provides a structural rationale for chemical synthesis of novel aminoglycosides.     

Systemic absorption of gentamicin ear drops

Aminoglycoside-containing topical preparations are commonly used in the treatment of chronic suppurative otitis media and otitis externa. We report a case where systemic absorption of topical Gentisone HC occurred producing a serum gentamicin level of 6.2 micrograms/ml.     

Renal toxicity. Tobramycin and gentamicin

To see if urinary 2-methyl-4-chlorophenoxyacetic acid (MCPA) excretion could be used to estimate MCPA exposure, four healthy males ingested 5 mg MCPA. The MCPA in the urine was extracted and anlyzed by high pressure liquid chromatography. About 50% of the ingested dose was detected in the urine within 48 h. On the fifth day after intake the MCPA concentration in the urine was below the level of detection, 0.2 microgram/ml. The MCPA did not increase those serum enzymes indicating liver cell damage (S-alanine-aminotransferase, S-alkaline-phosphate). Some creatine kinase (CK) and S-aspartate-aminotransferase (ASAT) values were pathological, but, as all CK values were normal in two persons and all ASAT values were normal in three persons, it not likely that MCPA had a toxic effect on muscle cells. MCPA in urine seems to be a useful indicator of MCPA intake in humans. All the urine passed within 48 h of MCPA exposure must be collected.     

Histopathological findings in clinical gentamicin ototoxicity

The temporal bone histopathological findings in a case of gentamicin sulfate-induced hearing loss and vertigo in an anephric patient undergoing hemodialysis are presented. A study of the sensory neuroepithelium of the cristae and maculae disclosed the presence of vacuoles with clubbing of the sensory cells. In the cochlea, loss of the innermost row of outer hair cells in the basal turn was the most prominent feature. These findings are discussed in light of reports of similar morphological changes in laboratory studies of gentamicin ototoxicity.     

Severe intravitreal fibrovascular growth after trauma

This report describes an unusual case of penetrating ocular trauma resulting in the growth of an intravitreal fibrovascular frond, which eventually attached to the lens. Associated features were a retinal dialysis and the attachment of a pars plana tear to the lens. The possible pathogenesis of the fibrovascular frond is discussed.     

Rapid serum gentamicin assay by enzymatic adenylation

An improved, rapid and accurate enzymatic adenylation assay for gentamicin has been developed. It can be completed in one hour with an accuracy of +/- 2.7% (+/- 1 SD) for the range of gentamicin concentrations of 6-12 microgram/ml. Concentrated enzyme was used to prepare a complete adenylation mixture, which was stored frozen in aliquots suitable for daily assays. A special apparatus designed for efficient washing of phosphocellulose disks was used in the assay.     

Safety of the bolus administration of gentamicin

A study was done to assess the safety of gentamicin administration by a bolus method. A total of 63 patients were randomly treated with intravenous gentamicin by bolus administration (3 to 5 min) and by slow infusion (2 h). Serum gentamicin levels were measured. Renal and audiovestibular function were monitored. Pure-tone audiometry was performed at the beginning, on day 3, and at the end of therapy. The study revealed that the bolus administration was safe and nontoxic.     

Estimation of serum gentamicin by quenching fluoroimmunoassay

A new type of non-isotopic immunoassay, applied to the determination of serum gentamicin, is reported. The method is based on partial quenching of fluorescence observed when fluorescein-labelled gentamicin is bound by anti-gentamicin serum. The fluorescence intensity of the labelled gentamicin in an unseparated immunoassay incubation mixture therefore serves to indicate the extent of binding, which is related to the amount of competing unlabelled gentamicin present. Precision and accuracy are shown to be similar to those of the best existing methods for gentamicin, while the new assay is more rapid and technically simpler, and avoids the use of expensive radio-chemicals with their attendant health hazard. Assays of patient samples correlate with established bioassay and polarisation fluoroimmunoassay methods.     

Renal extraction of gentamicin in anesthetized dogs

Follow-up examinations, ranging from four to more than 20 years, were performed on 100 patients with chronic cyclitis whose ages at onset were from 4 to 58 years. Cataracts were found in 42% of eyes and macular disease secondary to macular edema in 28% of eyes. Band keratopathy, glaucoma, retinal detachment, retinoschisis, vitreous hemorrhage, retinal hemorrhage, and vessels leaving the disk margin were also noted. The complications resulting in decreased vision in chronic cyclitis were macular edema in active cases and macular degenerative changes in the late inactive stages. Of all eyes with final visual acuity of 6/12 (20/40) or less, 74% had permanent, late macular changes secondary to earlier cystoid macular edema. Vitreous opacities or cells, or both, caused decreased visual acuity in the early active stages of chronic cyclitis but were not major factors in the ultimate visual prognosis in the late inactive stages. At the final examination, vitreous opacities caused a visual loss in only 9% of the eyes that had visual acuity of 6/12 (20/40) or less. It was difficult to determine whether corticosteroids caused cataract formation and glaucoma.     

Gene transfer, gentamicin resistance and enterococci

1. Intravenous administration of the ganglionic nicotinic receptor agonist DMPP (1,1-dimethyl-4-phenylpiperazinium iodide) into urethane-anaesthetized rats evoked dose-dependent increases in mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). 2. The ganglionic nicotinic receptor antagonists pentolinium and hexamethonium either alone or combined did not inhibit the increase in RSNA and MAP evoked by 50 to 200 micrograms kg-1 doses of DMPP. The increase in renal sympathetic nerve activity evoked by DMPP occurred as a brief burst in firing. 3. The increase in MAP, but not RSNA, evoked by DMPP in the presence of pentolinium was inhibited by the selective alpha 1-adrenergic receptor antagonist prazosin. 4. The non-selective alpha-adrenoceptor and NPY receptor antagonist benextramine also inhibited the increase in MAP without inhibiting the increase in RSNA. Surprisingly, the combination of benextramine and pentolinium, or benextramine and hexamethonium, completely blocked the DMPP-evoked increase in RSNA and thus the increase in MAP. 5. The uptake1 antagonist desipramine combined with pentolinium did not affect the DMPP-evoked increases in MAP or RSNA when compared to the responses evoked in the presence of pentolinium alone. 6. Adding the selective M1 muscarinic receptor antagonist telenzepine to pentolinium and prazosin did not inhibit the increase in RSNA evoked by a 100 micrograms kg-1 dose of DMPP. 7. While the DMPP-evoked increase in MAP in the presence of ganglionic nicotinic receptor antagonists is primarily dependent upon activation of alpha 1-adrenoceptors, the increase in RSNA occurs via activation of ganglionic nicotinic receptors and activation of a mechanism susceptible to blockade by benextramine.     

Acute respiratory failure due to gentamicin aerosolization

OBJECTIVE: Evoked otoacoustic emissions (OEAE) are an excellent evaluation method of the hearing organ and are principally useful in newborn screening. During the performance of this technique, doubtful cases sometimes exist and they are customarily considered as failures of the test, increasing the group of children to be re-evaluated. The program IL088 has the possibility of increasing the stimulus intensity (gain). We applied this gain to all children with doubtful OEAE in attempts to give a pass or fail score. PATIENTS AND METHODS: The record of OEAE was accomplished with the system IL088 v3.5. It was applied to 70 ears of healthy newborns, a gain of +15 dB initially and/or +21 dB (if a pass score was not obtained). Some normal ears were included in this group as controls. Of the explored ears, 23 had a normal OEAE, 17 presented a doubtful response and in 30 cases the answers were negative (fail). RESULTS: This study demonstrates that in doubtful cases, the usefulness of increasing the gain is very high permitting that these cases accomplish pass criteria (15/17). In the ears with a lack of response there is an increased response, but it usually does not reach a passing level (6/30). In children with normal response the pass rate does not vary. CONCLUSIONS: The increase in gain in ears with a doubtful response offers a high a rentability, reducing the the initial failure rate of OEAE without altering the real failure rate or affecting those that initially pass allowing a reduction in the number of second evaluations.     

Production of gentamicin antibiotics by Micromonospora pupurea

The role of sodium concentration, of alpha- and beta-adrenergic receptors, and of a microtubular inhibtor (vincristine) on renin release was studied in rat kidney slices in vitro. Renin release was an active, linear, and temperature-dependent process. Kidneys from young rats released much more renin than those from adults. Lowering sodium concentration inhibited renin release by one-half, even when osmolality was kept constant. Isoproterenol (10(-8) to 10(-5) M) stimulated renin release significantly in a partially dose-related manner. dl-propranolol inhibited this stimulation. Significant (P less than 0.05) inhibition of renin release was induced by l-epinephrine or l-norepinephrine (10(-5) M). In the presence of an alpha-receptor blocking drug, phenoxybenzamine (10(-5) M), inhibition no longer occurred with epinephrine and stimulation was observed with l-norepinephrine. Vincristine (10(-5) M) did not affect renin release when slices from the kidneys of normal rats or adrenalectomized, sodium-depleted rats were incubated, but significantly inhibited (P less than 0.01) release that had been stimulated in vitro by isoproterenol. These results suggest to us that there may be (1) a direct or indirect (mediated through the macula densa) effect of sodium on juxtaglomerular cells, (2) an inhibitory role for alpha-adrenergic receptors on renin release, in addition to the stimulatory role of beta-receptors, (3) possible participation of microtubules in isoproterenol-stimulated renin release, and (4) an alternative mode of secretion of renin under stimulation by adrenalectomy and salt depletion.     

Surface action of gentamicin on Pseudomonas aeruginosa

The mode of action of gentamicin has traditionally been considered to be at the 30S ribosomal level. However, the inhibition of bacterial protein synthesis alone appears to be insufficient to entirely explain the bactericidal effects. Bacteriolysis is also mediated through perturbation of the cell surface by gentamicin (J.L. Kadurugamuwa, J.S. Lam, and T.J. Beveridge, Antimicrob. Agents Chemother. 37:715-721, 1993). In order to separate the surface effect from protein synthesis in Pseudomonas aeruginosa PAO1, we chemically conjugated bovine serum albumin (BSA) to gentamicin, making the antibiotic too large to penetrate through the cell envelope to interact with the ribosomes of the cytoplasm. Furthermore, this BSA-gentamicin conjugate was also used to coat colloidal gold particles as a probe for electron microscopy to study the surface effect during antibiotic exposure. High-performance liquid chromatography confirmed the conjugation of the protein to the antibiotic. The conjugated gentamicin and BSA retained bactericidal activity and inhibited protein synthesis on isolated ribosomes in vitro but not on intact cells in vivo because of its exclusion from the cytoplasm. When reacted against the bacteria, numerous gentamicin-BSA-gold particles were clearly seen on the cell surfaces of whole mounts and thin sections of cells, while the cytoplasm was devoid of such particles. Disruption of the cell envelope was also observed since gentamicin-BSA and gentamicin-BSA-gold destabilized the outer membrane, evolved outer membrane blebs and vesicles, and formed holes in the cell surface. The morphological evidence suggests that the initial binding of the antibiotic disrupts the packing order of lipopolysaccharide of the outer membrane, which ultimately forms holes in the cell envelope and can lead to cell lysis. It is apparent that gentamicin has two potentially lethal effects on gram-negative cells, that resulting from inhibition of protein synthesis and that resulting from surface perturbation; the two effects in concert make aminoglycoside drugs particularly effective antibiotics.