Tartrate-resistant acid phosphatase: gene structure and function.
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Acid Phosphatase↗
Biomedical subjects
E Fleckenstein
Publications and source records attributed to E Fleckenstein.
Cloning and characterization of the human tartrate-resistant acid phosphatase (TRAP) gene.
The expression and protein structure of the tartrate-resistant acid phosphatase (TRAP), an iron-containing lysosomal glycoprotein in cells of the mononuclear phagocyte system, have been analyzed extensively in the past. In some diseases, like hairy cell leukemia and Gaucher's disease, cytochemically detected TRAP expression is used as a disease-associated marker. In this paper we describe the isolation of a genomic cosmid clone of the human TRAP gene. Restriction mapping revealed a 22-kb insert and the complete genomic structure of the TRAP gene. A 6-kb HindIII-fragment harboring the entire TRAP gene was subcloned and the 5'-flanking region of 3026 bp was sequenced. Analysis of the sequence data showed the presence of potential transcription factor binding sites. Two transcriptional start sites were identified in the untranslated exon 1 at positions -349 and -347 bp relative to the translational start codon. Linked to a luciferase-encoding reporter gene the 5'-flanking region was sufficient to direct transcription in the heterologous cell line BHK-21. Treatment of the transfected cells with different modulators of the intracellular iron content showed that regulation of TRAP expression is dependent on iron. In summary, these data imply a possible functional role of the TRAP gene product either in the storage or the transport of iron.
Acid Phosphatase↗
Treatment of mycoplasma contamination in a large panel of cell cultures.
Mycoplasmal contamination remains a significant impediment to the culture of eukaryotic cells. For certain cultures, attempts to eliminate the infection are feasible alternatives to the normally recommended disposal of the contaminated culture. Here, three antibiotic regimens for mycoplasmal decontamination were compared in a large panel of naturally infected cultures: a 1-wk treatment with the fluoroquinolone mycoplasma removal agent (MRA), a 2-wk treatment with the fluoroquinolone ciprofloxacin, and three rounds of a sequential 1-wk treatment with BM-Cyclin containing tiamulin and minocyclin. These antibiotic treatments had a high efficiency of permanent cure: MRA 69%, ciprofloxacin 75%, BM-Cyclin 87%. Resistance to mycoplasma eradication was observed in some cell cultures: BM-Cyclin 0%, MRA 20%, ciprofloxacin 20%. Nearly all resistant contaminants that could be identified belonged to the species Mycoplasma arginini and M. orale. Detrimental effects of the antibiotics were seen in the form of culture death caused by cytotoxicity (in 5 to 13% of the cultures). Alterations of the cellular phenotypic features or selective clonal outgrowth might represent further untoward side effects of exposure to these antibiotics. Overall, antibiotic decontamination of mycoplasmas is an efficient, inexpensive, reliable, and simple method: 150/200 (75%) chronically and heavily contaminated cultures were cured and 50/200 (25%) cultures could not be cleansed and were either lost or remained infected. It is concluded that eukaryotic cell cultures containing mycoplasmas are amenable to antibiotic treatment and that a cure rate of three-quarters is a reasonable expectation.
Cells, Cultured↗
Effective treatment of mycoplasma contamination in cell lines with enrofloxacin (Baytril).
Continuous cell lines are frequently contaminated with microorganisms, mycoplasmas being the most prominent and cumbersome. In our experience, of the 300 cell lines examined more than one third was infected with mycoplasmas. Mycoplasma contamination can affect virtually every parameter and functional activity of a cultured cell. An alternative to the recommended disposal of infected cultures is an attempt to eliminate the contaminants. Adding antibiotics with strong activity against mycoplasmas to the culture medium is a simple, inexpensive and efficient decontamination method. Here, we studied the effectiveness of the new antibiotic enrofloxacin (Baytril) developed specifically for use against mycoplasmas. Baytril is a new synthetic agent from the group of quinolone derivatives that are DNA gyrase inhibitors. Thirty-two chronically infected cell lines (27 human leukemia-lymphoma cell lines) were treated with Baytril in a prospective study in direct comparison with three other well-established anti-mycoplasma regimens, the antibiotics BM-Cyclin, Ciprobay and MRA (Mycoplasma Removal Agent). Mycoplasmas were detected by DNA staining, agar colony growth, DNA-RNA hybridization, polymerase chain reaction, and monoclonal antibody staining. Treatment with Baytril eliminated the contaminants in 30/32 cultures (94%). The cure rates for Ciprobay, BM-Cyclin and MRA were 91%, 81%, and 75%, respectively. The IC50 values of Baytril for cell lines varied over a wide range depending on the type of hematopoietic cell lineage with T- and B-cell lines being more sensitive targets. Baytril-treated cell lines remained mycoplasma-negative over a 12-week antibiotic-free culture period. Low levels of mycoplasma infection were shown not to persist by repeat testing after growth without antibiotics. A retrospective analysis of anti-mycoplasma treatments with BM-Cyclin, Ciprobay, MRA or Baytril showed that 265/351 cultures (75%) were immediately cured of mycoplasma; however, all of the remaining, mycoplasma-positive cultures harboring mycoplasms resistant to the first antibiotic could be cleaned up by a second round with a different antibiotic. Baytril is an efficient anti-mycoplasma antibiotic and based on its high cure rate might be the treatment of first choice.
Anti-Bacterial Agents↗