Biomedical subjects

Natasa Anastasov

Publications and source records attributed to Natasa Anastasov.

4 recordsLinked to original sources

PubMed2006

NPM-ALK-dependent expression of the transcription factor CCAAT/enhancer binding protein beta in ALK-positive anaplastic large cell lymphoma.

CCAAT/enhancer binding protein beta (C/EBPbeta) is one of a 6-member family of C/EBPs. These transcription factors are involved in the regulation of various aspects of cellular growth and differentiation. Although C/EBPbeta has important functions in B- and T-cell differentiation, its expression has not been well studied in lymphoid tissues. We, therefore, analyzed its expression by immunohistochemistry and Western blot in normal lymphoid tissues and in 248 well-characterized lymphomas and lymphoma cell lines. Nonneoplastic lymphoid tissues and most B-cell, T-cell, and Hodgkin lymphomas lacked detectable levels of C/EBPbeta. In contrast, most (40 of 45; 88%) cases of ALK-positive anaplastic large cell lymphoma (ALCL) strongly expressed C/EBPbeta. Western blot analysis confirmed C/EBPbeta expression in the ALK-positive ALCLs and demonstrated elevated levels of the LIP isoform, which has been associated with increased proliferation and aggressiveness in carcinomas. Transfection of Ba/F3 and 32D cells with NPM-ALK and a kinase-inhibitable modified NPM-ALK resulted in the induction of C/EBPbeta and demonstrated dependence on NPM-ALK kinase activity. In conclusion, we report the constitutive expression of C/EBPbeta in ALK-positive ALCL and show its relationship to NPM-ALK. We suggest that C/EBPbeta is likely to play an important role in the pathogenesis and unique phenotype of this lymphoma.

Anaplastic Lymphoma Kinase↗

PubMed2006

Real-time quantitative RT-PCR shows variable, assay-dependent sensitivity to formalin fixation: implications for direct comparison of transcript levels in paraffin-embedded tissues.

Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) is a versatile tool for precise quantification of gene expression. Formalin-fixed and paraffin-embedded (FFPE) tissue is well suited for qRT-PCR, if RNA extraction is optimized and small amplicon sizes are used. However, little is known whether individual assays may show variable sensitivity to fixation. This is of great importance, if a direct comparison of different transcripts is performed within the same sample, such as for mRNA splice variants. We established a cell culture model to test for and quantify differences in performance of individual qRT-PCR assays on FFPE as compared with fresh material, using TaqMan methodology. RNA was isolated from 7 different cell lines either directly or after preparation of a FFPE cell block. RNA from both sources was reverse transcribed and gene expression quantified using 13 different TaqMan assays. All assays allowed highly reproducible target quantification, using both fresh and FFPE-derived cDNA. The 13 assays showed an average Ct difference of 3.2 between fresh and FFPE cells, if identical amounts of cDNA were used as template. However, the Ct shifts varied from 1.8 to 5.1 for individual assays, indicating variable resistance to fixation. These Ct shift differences were statistically highly significant in 27/78 (35%) of all possible combinations of assays. Because the Ct shift remained constant for each assay, they could be used for calculation of correction factors which rendered FFPE-derived expression data highly comparable to those obtained from fresh material, and as a consequence among each other. Thus, a standardized assessment of qRT-PCR assay efficiencies in FFPE allows for precise intraindividual comparison of mRNA species, such as splice variants with different biologic functions, in archival tissues.

Biological Specimen Banks↗

PubMed2004

The kgmB gene, encoding ribosomal RNA methylase from Streptomyces tenebrarius, is autogenously regulated.

The KgmB methylase (the kanamycin-gentamicin resistance methylase from Streptomyces tenebrarius) acts at G-1405 of 16S rRNA within the sequence CGUCA that is also found 6 bp in front of ribosomal binding site of the kgmB gene. The kgmBColon, two colonslacZ gene and operon fusions were used in order to test for translational autoregulation of kgmB gene. Overexpression of kgmB either in cis or in trans drastically decreased the level of expression of the fusion protein. However, mutagenesis eliminated any role for the CGUCA sequence in translational autoregulation. Hence, the role of second putative regulatory sequence (CGCCC) that was shown to be involved in regulation of another methylase, Sgm (sisomicin-gentamicin methylase gene from Micromonospora zionensis) was examined. It was shown that the Sgm methylase can also decrease the level of expression of the kgmBColon, two colonslacZ fusion protein.

Artificial Gene Fusion↗

PubMed2003

The AGUAAA motif in cspA1/A2 mRNA is important for adaptation of Yersinia enterocolitica to grow at low temperature.

Acclimatization of the psychrotolerant Yersinia enterocolitica after a cold shock from 30 degrees C to 10 degrees C causes transcription of the major cold shock protein (CSP) bicistronic gene cspA1/A2 to increase by up to 300-fold. Northern blot analysis of cspA1/A2 using four probes that hybridize specifically to different regions of CSP mRNA revealed the appearance of a number of cspA1/A2 transcripts that are smaller than the original transcript and transiently visible at the end of the acclimation period. Primer extension and RNA protection experiments demonstrated that these smaller mRNAs have 5' ends located in the same core sequence (5'-AGUAAA-3') at five different places within the mRNA, indicating preferential cleavage of the CSP mRNA transcripts. A similar result was obtained for cspB of Escherichia coli, containing two such core sequences. Furthermore, this motif is present in the major CSP genes of a variety of Gram-negative and Gram-positive bacteria. We have therefore termed this sequence cold shock cut box (CSC-box). After inserting a CSC-box into a plasmid-bound lacZ gene in Y. enterocolitica, the mRNA of this construct was cleaved within the CSC-box, and a change in this CSC-box from AGUAAA to AGUCCC dramatically reduced cleavage of the mutated lacZ gene. Mutating all CSC-boxes in Y. enterocolitica of a plasmid bound cspA1/A2 dramatically increases the lag time after a cold shock before re-growth occurs. Based on these results, we suggest that the role of the CSC-box is related to downregulation of cspA mRNA after acclimation to low temperature.

Adaptation, Physiological↗