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[Research on Festuca arundinacea transformation mediated by Agrobacterium tumefaciens].

The embryo-derived calli from four types of tall fescues (Festuca arundinacea Schreb) were transformed with two Agrobactrium tumefaciens strains AGL1 and GV3101. AGL1 harbors an intron-AtNHX1 expression vector pROK2U containing ubiqutin promoter and npt II marker gene. GV3101 harbors an intron-AtNHX1 expression vector pROK2 containing 35S promoter and npt II gene. After infection and co-culture with AGL1 or GV3101, the embyogenic calli were selected with 50-150 mg/L paromomycine and 1126 resistant plants regenerated from the resistant calli. All plants were selected further with 10-20 mg/L Kanamycin and 525 of them remained green. Genome DNA of the resistant plants was checked with specific primers and probe from AtNHX1 gene. The results of PCR assay and Southern blot analysis indicted that exogenous target gene (AtNHX1 gene) had been transferred into different cultivars of Festuca arundinacea. Different transformation frequencies among the four cultivars were obtained.

Agrobacterium tumefaciens↗

[Management of cutaneous leishmaniasis in adults and children].

Cutaneous leishmaniasis can present a variety of clinical features and courses. The causative Leishmania species is an important prognostic factor in immunocompetent patients. Local treatment modalities including topical paromomycin, cryotherapy, localized controlled heat, carbon dioxide laser therapy, or intralesional meglumine antimoniate can be effective against Leishmania major or Leishmania tropica. Oral fluconazole may be a second-line treatment. Parenteral antimonials are useful for persistent or recurrent Old World leishmaniasis. For New World leishmaniasis, parenteral antimonials represent the first-line treatment in all forms except those caused by Leishmania guyanensis in which pentamidine is preferable. Liposomal amphotericin B appears to be effective for treatment of cutaneous leishmaniasis but further study will be needed. Results using oral Miltefosine are promising against Indian kala-azar (Leishmania donovani) but disappointing against South American leishmaniasis.

Administration, Oral↗

Management of visceral leishmaniasis: Indian perspective.

Diagnosis and treatment of Indian visceral leishmaniasis (VL) is extremely unsatisfactory. For diagnosis, demonstration of parasites in splenic/marrow smears remains the gold standard, though k39 rapid strip test is a useful method in regions where access to parasite demonstration is difficult. pentavalent antimony remains the mainstay for the treatment of all forms of leishmaniasis globally; however, development of large-scale antimony resistance in Bihar has necessitated search for alternative drugs. Amphotericin B is the most effective, though toxic, drug for patients with refractory VL. Lipid formulations of amphotericin B, though safe and effective, are too expensive to be useful for poor patients of this region. These hold advantage as large quantity of the drug can safely be given over a short period of time, thus leading to a decrease in the hospital stay to a few days instead of several weeks. Oral miltefosine, an alkyl phospholipid, has recently been approved and marketed in India for the treatment of VL. Miltefosine cures 94% patients with VL if given in a daily dose of 50-100 mg for 28 days. Most common adverse events are mild vomiting and diarrhea. Paromomycin, an amino glycoside, is undergoing a pivotal phase-III clinical trial, and is likely to be approved and available to patients with VL at an affordable cost. To protect the already scarce inventory of antileishmanial drugs, it is time that combination chemotherapy is introduced for the treatment of VL in India.

Antiprotozoal Agents↗

Visceral leishmaniasis - current therapeutic modalities.

Major therapeutic obstacles in the treatment of visceral leishmaniasis (VL) include the alarming increase in antimonial unresponsiveness especially in Bihar, India and relapses in HIV-Leishmania co-infected patients. The therapeutic armamentarium for VL is currently plagued with several limitations as the available drugs are toxic, majority are effective only parenterally and need to be administered for extended periods. The first orally effective drug, miltefosine has been approved for treating VL. In antimony refractory zones, pentavalent antimony has been largely replaced by amphotericin B deoxycholate, but prolonged hospitalization, toxic effects, and requirement for monitoring greatly hamper its widespread application in endemic regions. Lipid formulations of amphotericin B, a remarkable advance in amphotericin B therapy, have greatly reduced toxicity enabling large doses to be delivered over a short period. Even a single dose treatment with liposomal amphotericin B cures > 90 per cent patients; however, the stumbling block is its prohibitive cost that precludes its widespread accessibility in endemic countries. Studies using paromomycin in VL are encouraging, and judging by the preliminary results of a recently concluded phase III trial, it could be an extremely useful and affordable antileishmanial drug. Other orally effective drugs include the azoles and allopurinol but these have met with limited success owing to either poor efficacy or unacceptable toxicity. Sitamaquine has undergone limited evaluation, and the data suggest effective antileishmanial activity; its role has to be delineated for which additional developmental studies are proposed. This review highlights the progress made in the treatment of VL, including the multiple mechanisms of action of antileishmanial drugs with a view to enable the researcher to undertake the challenge of providing affordable and effective chemotherapy.

Administration, Oral↗

[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.

Aminoglycosides↗

Protein synthesis in yeast. Structural and functional analysis of the gene encoding elongation factor 3.

The yeast translational elongation factor 3 (EF-3) stimulates EF-1 alpha-dependent binding of aminoacyl-tRNA by the ribosome. The requirement for EF-3 is unique to fungi; a functional analog has not been found in prokaryotes or other eukaryotes. We have isolated and characterized the structural gene, YEF3, that encodes EF-3. The YEF3 gene is present in one copy/haploid genome and is essential for vegetative growth. DNA sequence analysis revealed that the YEF3 gene contains an open reading frame of 1044 codons. The deduced amino acid sequence contains two repeats of a nucleotide-binding motif, which is similar to the nucleotide-binding consensus sequences of hydrophilic, membrane-associated ATPases. EF-3 catalyzes ATP hydrolysis in a ribosome-dependent manner. A modified assay procedure has been developed that allows measurement of the ATP hydrolytic activity of EF-3 in cell-free extracts without interference by other nucleotide hydrolyase activities. Using this modified assay, we have shown that the wild-type YEF3 gene restores heat stable EF-3 activity in a yeast strain containing a temperature-sensitive EF-3. Introduction of the YEF3 gene on a high copy number plasmid into yeast strains increases the ribosome-dependent ATPase activity. The level of EF-3 protein is also increased 3-5-fold. Elevated EF-3 protein levels did not cause a significant increase in EF-1 alpha and EF-2 protein. Yeast strains containing elevated EF-3 protein levels are more sensitive to the aminoglycoside antibiotics hygromycin and paromomycin. These drugs are known to increase translational errors. This observation suggests that EF-3 may indirectly affect translational accuracy.

Amino Acid Sequence↗

Campylobacter pylori-associated gastritis: attempts to eradicate the bacteria by various antibiotics and anti-ulcer regimens.

The efficacy of various antimicrobial and anti-ulcer agents on the eradication of Campylobacter pylori in patients with antral gastritis or duodenal ulcers was investigated by several open studies or double-blind, placebo-controlled protocols. Among the anti-ulcer agents, ranitidine, cimetidine or sucraflate had no effect on C. pylori. Colloidal bismuth subcitrate achieved clearance of C. pylori in 40% of treated patients at the end of therapy but a high relapse rate (14/16 patients) was observed after a 6-month follow-up period. The antibacterial agents doxycycline, minocycline, ofloxacin, clindamycin, paromomycin and nifuroxazide failed to eradicate C. pylori in most patients. By contrast, short term elimination of C. pylori could be achieved in more than 90% of patients treated with amoxycillin. However, relapse occurred as a rule in all amoxycillin-treated patients within one month after therapy. Overall, we observed no correlation between the in-vitro activity of the different antibacterial agents and their in vivo efficacy. Development of resistance during therapy does not seem to account for this discrepancy since it occurred only with ofloxacin. On the basis of these results, we conclude that long term eradication of C. pylori from the gastric antrum cannot be achieved after monotherapy either with antibiotics or with bismuth salts.

Anti-Bacterial Agents↗

Enzymatic modification of aminoglycoside antibiotics by Branhamella catarrhalis carrying an R factor.

Fifteen out of 89 clinical strains of Branhamella catarrhalis isolated from patients at the University Hospital of Zaragoza were resistant to aminoglycosides and other antimicrobials. In two strains, B. catarrhalis 220 and B. catarrhalis 115, the resistance to aminoglycosides was associated with synthesis of aminoglycoside-modifying enzymes, namely 3"-O-phosphotransferase [APH(3")] and 3'-O-phosphotransferase [APH(3')]. B. catarrhalis 115 was resistant to ampicillin, streptomycin, kanamycin, neomycin, butirosin, lividomycin, ribostamycin, paromomycin and trimethoprim-sulfamethoxazole and harboured a 32 megadalton (Md) plasmid. The resistance determinants of the latter were transferred to Neisseria subflava by conjugation and to Escherichia coli by transformation. The transconjugant strain presented an antibiotic resistance pattern similar to the donor strain and carried the same plasmid. The transformant strain acquired the 32 Md plasmid but presented, besides the resistance pattern already mentioned, resistance to tetracycline, gentamicin and tobramycin. Resistance to gentamicin and tobramycin was mediated by the synthesis of a 3-N-acetyltransferase. This resistance and the related enzyme were expressed neither in the donor B. catarrhalis strain nor in the transconjugant N. subflava strain.

Aminoglycosides↗

[Phosphate group donors for neomycin inactivation by resistant microorganism strains].

The substrate specificity of aminoglycoside phosphotransferases isolated from 3 strain of E. coli and purified was studied. All pure enzymes phosphorilated neomycin, paromomycin, lividomycin, neamine, ribostamycin, kanamycins A and B. Only ATP was the donor of the phosphate groups in these reactions, while in the non-purified extracts GTP but not UTP or CTP served as the donor of the phosphate group for inactivation of neomycin. The substrate specificity indicated that the above enzymes were aminoglycoside-3(1)-phosphotransferases. Inactivation of neomycin with the use of the phosphate group of phosphoenolpiruvate as the donor in the non-purified enzymatic preparations of the neomycin-resistant strains of E. coli and Pseudomonas was not observed.

Adenosine Triphosphate↗

Physical mapping of the yeast mitochondrial genome: derivation of the fine structure and gene map of strain D273-10B and comparison with a strain (MH41-7B) differing in genome size.

(1) We have derived a fine-structure map of the 70 kb mitochondrial genome of the yeast S. cerevisiae, strain D273-10B, and compared it with our previous maps for strain MH41-7B. Restriction fragment maps for 56 enzyme recognition sites for 13 endonucleases, Eco RI, Hpa I, Bam HI, Hha I, Hinc II, Xba I, Hind III, Bgl II, Pvu II, Sal I, Pst I, Sst I, and Xho I, have been derived. We have used several methods to obtain these maps: (a) Four enzymes (Sal I, Sst I, Xho I, Pst I), each of which cuts D273-10B mtDNA at a single site, were employed to localize and orient fragments from multi-site enzyme digests that are cleaved by the single-site enzyme. (b) Radioactively labeled probes (rRNA or copy RNA [cRNA] transcribed from simple-sequence petite mtDNA) were hybridized to restriction fragments from different digests for identification of fragments which share common sequences. (c) The products of double or triple enzyme digests were identified for mapping and confirmation of the localization of restriction sites. (2) The antibiotic-resistant (antR) loci for erythromycin (E), chloramphenicol (C), paromomycin (P), and oligomycin (OI, OII) were positioned on the physical restriction map by hybridization of 3H-labeled cRNA transcribed from simple-sequence petite mtDNAs that retain a single genetic antR marker to appropriate restriction fragments bound to nitrocellulose filters. (3) Mitochondrial transcripts (21s rRNA, 14s rRNA, and tRNAs) labeled with 125I were hybridized to restriction fragments for identification of the corresponding coding sequence. (4) The gene order and localization of the antR loci and mitochondrial transcripts are as follows: C(0-1.5u)-tRNA I(0-21.5u)-P(29-36.6u)-tRNA II(29-46.4u)-14s rRNA(36-38.3u)-OII(60.3-62.5u) - tRNA III(73-76u) - OI(78.6-83.0u) - tRNA IV(82.5-83.0u) - E(94.2-98.6u) - 21s rRNA (94.2-99.4u). (5) The DNA fine structure and gene map of the 70 kb D273-10B mtDNA were compared to the map of the larger MH41-7B (76 kb) mtDNA. There are 56 restriction sites on D273-10B and 67 sites on MH41-7B for the 13 enzymes studied. The additional restriction sites are largely accounted for by the presence, in MH41-7B, of two sets of sequences, "A" (2.7 kb) and "B" (3.0 kb), located on either side of the OII marker. The remainder of the fragments map is remarkably similar for the two strains. The distances separating the antR loci and the mitochondrial transcripts are very similar except in the two regions surrounding OII.

DNA Restriction Enzymes↗

Transcription, processing, and mapping of mitochondrial RNA from grande and petite yeast.

Mitochondrial RNA (mtRNA) from petite yeast strains was analyzed by electrophoresis in agarose-urea, acrylamide-urea, and agarose-methyl mercuric hydroxide gels, and by transfer to diazobenzyloxy-methyl paper and hybridization to labeled mitochondrial DNA (mtDNA). Petites contain numerous mitochondrial transcripts, including processed species like 21 S and 14 S rRNA. Petite transcripts were found to fall into three classes: 1) bands that comigrate with grande mtRNA species; 2) "group-specific" new bands found in multiple strains and coinciding with specific regions of the mitochondrial genome; and 3) "strain-specific" new bands found only in individual petite strains. A deletion map was constructed in which we used the presence or absence of the first two types of mtRNA bands in specific strains, and the restriction endonuclease map of these strains. This map confirmed the localization of 21 S and 14 S rRNA, which were mapped previously by hybridization, and also localized more than 20 additional mtRNA species. The mtRNA species were grouped in regions of the genome in a fashion that strongly suggests that many of them are precursors to fully processed mtRNA species. Hybridization experiments with grande mtRNA and cloned mtDNA fragments have shown the same kind of transcript grouping. Other hybridization experiments have demonstrated two apparent precursors to 21 S rRNA (3700 nucleotides) measuring 5500 and 4500 nucleotides. Processed tRNAs are found only in petites that contain a specific region of the genome near the P (paromomycin resistance) locus. When this region is absent, processed tRNAs are not detected, even for tRNA genes quite distant from the P locus. Since this phenotype is expressed in petites that lack mitochondrial protein synthesis, and since it maps to a specific location in the mitochondrial genome, there appears to be a mtRNA species which has a role in processing of mitochondrial tRNA.

DNA, Mitochondrial↗

[Phenomenon of the amplification of the determinant of kanamycin resistance (Kanr) in constructed hybrid plasmids in a strain of 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.

Chemical Phenomena↗

Determination of neomycin in animal tissues, using ion-pair liquid chromatography with fluorometric detection.

A liquid chromatographic (LC) method is described for the determination of neomycin in animal tissues. Tissues are homogenized in 0.2M potassium phosphate buffer (pH 8.0); the homogenate is centrifuged, and the supernate is heated to precipitate the protein. The heat-deproteinated extract is acidified to pH 3.5-4 and directly analyzed by LC. The LC method consists of an ion-pairing mobile phase, a reverse phase ODS column, post-column derivatization with o-phthalaldehyde reagent, and fluorometric detection. The LC method uses paromomycin as an internal standard, and separates neomycin from streptomycin or dihydrostreptomycin because they have different retention times. The LC column separates neomycin in 25 min; the detection limit is about 3.5 ng neomycin. The overall recovery of neomycin from kidney tissues spiked at 1-30 ppm was 96% with a 9.0% coefficient of variation. The method was also applied to muscle tissue.

Aminoglycosides↗

Drug therapy reviews: Antimicrobial spectrum, pharmacology and therapeutic use of antibiotics--part 4: aminoglycosides.

The aminoglycoside antibiotics are reviewed with regard to mechanism of action, bacterial resistance, antimicrobial spectrum, combinations with other agents, pharmacology, dosages in patients with normal and impaired renal function, adverse reactions, therapeutic use, prophylatic use and selection. Streptomycin is suggested in the therapy of tuberculosis, brucellosis, tularemia and yersinia infections; several of these require the coadministration of another agent. The choice between streptomycin and gentamicin for combination therapy of enterococcal endocarditis may be simplified by knowledge of the prevalence of high-level streptomycin-resistant strains in the hospital or by use of an in vitro screening test. Neomycin is the agent used orally in the treatment of hepatic encephalopathy. Paromomycin is indicated only for the treatment of amebic infections. The major difference among gentamicin, tobramycin and amikacin lies in the low but increasing prevalence of gram-negative bacilli which are resistant to gentamicin and tobramycin and susceptible to amikacin. In those institutions in which gentamicin-resistant strains are of concern, amikacin is the aminoglycoside of choice in high-risk patients until the infecting bacterium has been determined.

Aminoglycosides↗

Naturally occurring drug resistance plasmids in hospital strains of Staphylococcus aureus.

A genetic analysis of multiply inorganic salts and antibiotic--resistant strains of Staphylococcus aureus was performed. Experiments designed to show reversion of organisms to antibiotic and inorganic salt susceptibility, as well as studies on the influence of ultraviolet irradiation of phage on the transduction frequencies of the resistance markers, indicated that determinants of chloramphenicol, tetracycline, aminoglycoside antibiotics, inorganic salts, and penicillin resistance in hospital strain are present on separate plasmids. Transduced by us plasmids pN742 and pN794 determined resistance to neomycin, kanamycin, paromomycin, lividomycin and streptomycin.

Anti-Bacterial Agents↗

Enteric diseases.

This paper focuses on the growing incidence of the "new" sexually transmitted parasitic enteric diseases: amebiasis and giardiasis. Two major behavioral factors influence transmission of these diseases in the gay community: 1) oral-rectal and oral-genital sexual contact and 2) multiple sexual partners. Pathogenesis, clinical signs and symptoms and complications associated with these diseases are also discussed. Although many patients present with severe symptoms, approximately 50 percent of infected patients are asymptomatic. The diagnostic procedures include a fresh purged stool examination, nonpurged warm stool examinations, and/or cold stool specimens. The serologic tests (serum precipitin and hemagglutination) are of value only in severely symptomatic invasive disease. Different treatment regimens and their side effects are discussed. Drugs used in the treatment of the enteric diseases include diiodohydroxyquin, metronidazole, furamide, and paromomycin. Only 60 to 70 percent of patients with the disease are cured, and 30 to 40 percent of patients require multiple courses of therapy. Test-of-cure examinations, ideally consisting of one or two purged stool specimens, are necessary in follow-up management.

Amebiasis↗

[Systemic antibiotic prophylaxis in elective colon surgery. Results of a controlled study].

A prospective controlled, randomized trial was carried out among 70 patients undergoing elective colonic surgery; 31 patients received a systemic prophylaxis with cefotaxime (3 h) before surgery for 4 days and 29 patients were controls. The large bowel preparation consisted of whole gut irrigation with 0.9% NaCl and Paromomycin 1 g/l. Both groups were well matched after randomization. The rate wound infections in the control group was 28% and, in the test group, 6.4% (p less than 0.05, chi2-test). A trend toward a lower rate of urinary tract infections, an astomosic dehiscences and septicemic complications was recognizable in the test group, but not significant.

Aged↗