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Biomedical subjects

A Hajra

Publications and source records attributed to A Hajra.

At least 19 recordsLinked to original sources
PubMed1998

Origin of molecular species of diacylglycerol induced by bombesin in smooth muscle cells from rabbit rectosigmoid.

The source of early production of sn-1,2-diacylglycerol (DAG) has for a long time been exclusively linked to hydrolysis of phosphatidylinositol 4,5-diphosphate, which on receptor activation is hydrolyzed into DAG and inositol 1,4,5-trisphosphate. We have investigated the origin of lipid sources of DAG production in smooth muscle cells, in response to contraction induced by peptide agonists. We have performed a quantitative analysis of the molecular species of DAG formed in relation to the known molecular composition of parent phospholipids. The molecular species of phospholipids are sufficiently unique that the phospholipid origin of DAGs and its quantitative contribution to their formation can be measured by HPLC. Cell suspensions (10-15 x 10(6) cells/ml) from the circular muscle of rabbit rectosigmoid were incubated in the presence of the contractile peptide agonist bombesin (BB) at 10(-6) M. Reactions were stopped at different time intervals from 30 s to 4 min. DAGs were extracted, purified by TLC, and benzoylated with benzoic anhydride. The benzoylated DAGs were first purified by TLC and then by normal phase HPLC before they were injected onto a reverse-phase column and eluted isocratically. Furthermore, phospholipids in the lipid extract [phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE)] were purified by TLC and similarly analyzed after hydrolysis to DAGs with phospholipase C (PLC). The DAG molecular species profiles for PI, PC, PS, and PE were all unique. Contraction of cells with BB gave noticeable increases (17-55%) in newly formed DAGs. The major phospholipid source of the newly formed DAGs at 30 s was only approximately 30% from PI, and the remainder was from PC. In contrast, after 4 min of BB stimulation, a decrease was seen in newly formed DAGs in the peak specific for PI hydrolysis. The data suggest that BB-induced contraction by activation of PLCs results in hydrolysis of different phospholipids. The DAGs formed as a result are qualitatively and quantitatively distinct. This could be the basis for the kinetically different pattern of sustained contraction observed with BB.

Animals↗
PubMed1997

Bombesin-stimulated ceramide production and MAP kinase activation in rabbit rectosigmoid smooth muscle cells.

We have investigated the hypotheses that 1) bombesin activation of protein kinase C (PKC) results in the hydrolysis of sphingolipids and the production of ceramide and that 2) ceramide produced on activation by bombesin mediates sustained contraction of smooth muscle cells by activation of PKC and mitogen-activated protein (MAP) kinase. Ceramide production was assessed using a technique that involved benzoylation of purified ceramide extracts, followed by reverse-phase high-performance liquid chromatography. Contraction of smooth muscle cells isolated from the rabbit rectosigmoid and stimulated with bombesin gave a significant increase in newly formed ceramide (38 +/- 3.5%). 12-O-tetradecanoylphorbol-13-acetate also induced production of ceramide, which was blocked by calphostin C. The short-chain permeable C2 ceramide induced a sustained contraction and activation of MAP kinase, which was blocked by calphostin C. The increase in MAP kinase activity was maximal at 30 s and declined at 2 min. The data suggest that stimulation of smooth muscle cells by bombesin results in a functional coupling between sn-1,2-diacylglycerol (DAG)/ PKC and a sphingomyelinase, whereby DAG activates the hydrolysis of sphingomyelin to produce ceramide. Ceramide in turn activates PKC, which then activates MAP kinase. This could be the basis for the sustained contraction observed with bombesin.

Anal Canal↗
PubMed1996

Core binding factor beta-smooth muscle myosin heavy chain chimeric protein involved in acute myeloid leukemia forms unusual nuclear rod-like structures in transformed NIH 3T3 cells.

Patients with the M4Eo subtype of acute myeloid leukemia almost invariably are found to have an inversion of chromosome 16 in their leukemic cells, which results in a gene fusion between the transcription factor called core binding factor beta (CBFbeta) on 16q and a smooth muscle myosin heavy chain (SMMHC) gene on 16p. Subcellular localizations of the wild-type CBFbeta and the CBFbeta-SMMHC fusion protein were determined by immunofluorescence of NIH 3T3 cells that overexpress wild-type or fusion protein. Normal CBFbeta showed an unexpected perinuclear pattern consistent with primary localization in the Golgi complex. The CBFbeta-SMMHC fusion protein had a very different pattern. Nuclear staining included rod-like crystalline structures as long as 11 microm. The heterodimeric partner of CBFbeta, CBFalpha, formed part of this complex. Cytoplasmic staining included stress fibers that colocalized with actin, probably as a consequence of the myosin heavy chain component of the fusion protein. Deletion of different regions of the CBFbeta portion of the fusion protein showed that binding to CBFalpha was not required for nuclear translocation. However, deletion of parts of the SMMHC domain of the fusion protein involved in myosin-mediated filament formation resulted in proteins that did not form rod-like structures. These observations confirm previous indirect evidence that the CBFbeta-SMMHC fusion protein is capable of forming macromolecular nuclear aggregates and suggests possible models for the mechanism of leukemic transformation.

3T3 Cells↗
PubMed1996

Identification of the chimeric protein product of the CBFB-MYH11 fusion gene in inv(16) leukemia cells.

An expressed gene formed by fusion between the CBFB transcription factor gene and the smooth muscle myosin heavy chain gene MYH11 is consistently detected by reverse transcription polymerase chain reaction (RT-PCR) in patients who have acute myeloid leukemia (AML) subtype M4Eo with an inversion of chromosome 16. We have previously shown that a CBFB-MYH11 cDNA construct can produce a chimeric protein and transform NIH 3T3 cells. However, the presence of the chimeric protein in patient cells has not been demonstrated previously. Here, we show that such chimeric proteins can be identified in vivo, primarily in the nuclei of the leukemic cells, by use of antibodies against the C-terminus of the smooth muscle myosin heavy chain and the fusion junction peptide. A very high molecular weight protein/DNA complex is generated when nuclear extracts from patient cells are used in electrophoretic mobility shift assays, as seen in NIH 3T3 cells transfected with the CBFB-MYH11 cDNA. Immunofluorescence staining shows that the proteins are organized in vivo into novel structures within cell nuclei. One isoform of the transcript of the CBFB-MYH11 fusion gene, containing the MHC204 C-terminus, was the predominant from in all five cases studied.

3T3 Cells↗
PubMed1995

Structure of the leukemia-associated human CBFB gene.

We have determined the structure of the human CBFB gene, which encodes the beta subunit of the heterodimeric transcription factor core binding factor (CBF). This gene becomes fused to the MYH11 gene encoding smooth muscle myosin heavy chain by an inversion of chromosome 16 that occurs in the M4Eo subtype of acute myeloid leukemia. The CBFB gene contains 6 exons and spans 50 kb. The gene is highly conserved in animal species as distant as Drosophila, and the exon boundaries are in locations identical to those of the murine Cbfb homologue. The CBFB promoter region has typical features of a housekeeping gene, including high G+C content, high frequency of CpG dinucleotides, and lack of canonical TATA and CCAAT boxes. This gene has a single transcriptional start site, 345 nucleotides upstream of the beginning of the coding region. The human and mouse CBFB promoters show conservation of several transcriptional regulatory sequence motifs, including binding sites for Sp1, Ets family members, and Myc, but do not contain any CBF binding sites. The 5' end of the human CBFB gene also contains a highly polymorphic, transcribed CGG repeat that is not present in the murine homologue.

Acute Disease↗
PubMed1995

The leukemic core binding factor beta-smooth muscle myosin heavy chain (CBF beta-SMMHC) chimeric protein requires both CBF beta and myosin heavy chain domains for transformation of NIH 3T3 cells.

An inversion of chromosome 16 associated with the M4Eo subtype of acute myeloid leukemia produces a chimeric protein fusing the beta subunit of the transcription factor core binding factor (CBF beta) to the tail region of smooth muscle myosin heavy chain (SMMHC). We investigated the oncogenic properties of this CBF beta-SMMHC chimeric protein using a 3T3 transformation assay. NIH 3T3 cells expressing CBF beta-SMMHC acquired a transformed phenotype, as indicated by their ability to form foci, grow in soft agarose, and form tumors in nude mice. Cells expressing normal CBF beta or the SMMHC tail domain did not become transformed. Electrophoretic mobility-shift assays showed that extracts from cells transformed by CBF beta-SMMHC no longer formed the normal CBF/DNA complex but instead formed a much larger complex that did not migrate into the gel. Analysis of CBF beta-SMMHC deletion mutants demonstrated that the chimeric protein was transforming only if two domains were both present: (i) CBF beta sequences necessary for association with the CBF alpha subunit, and (ii) SMMHC sequences important for the formation of multimeric filaments. These results are direct evidence that CBF beta-SMMHC can function as an oncoprotein.

3T3 Cells↗
PubMed1995

Overexpression of core-binding factor alpha (CBF alpha) reverses cellular transformation by the CBF beta-smooth muscle myosin heavy chain chimeric oncoprotein.

A fusion between the transcription factor core-binding factor beta (CBF beta; also known as PEBP2 beta) and the tail region of smooth muscle myosin heavy chain (SMMHC) is generated by an inversion of chromosome 16 [inv(16) (p13q22)] associated with the M4Eo subtype of acute myeloid leukemia. We have previously shown that this CBF beta-SMMHC chimeric protein can transform NIH 3T3 cells and that this process requires regions of the chimeric protein necessary for association with the CBF alpha subunit. In this study, we show that NIH 3T3 cells overexpressing murine Cbf alpha 2 (also known as Aml1) cannot be transformed by CBF beta-SMMHC and that overexpression of Cbf alpha 2 in cells previously transformed by CBF beta-SMMHC reverts the cells to a less transformed phenotype. Cbf alpha 2 overexpression does not cause any gross morphological changes to NIH 3T3 cells but does result in increased CBF activity, as indicated by electrophoretic mobility shift assays and transactivation of reporter constructs. Cells transformed by CBF beta-SMMHC lack normal CBF-DNA complexes and have decreased levels of transactivation. Reversion of CBF beta-SMMHC transformation by Cbf alpha 2 is associated with a restoration of normal CBF-DNA complexes and transactivation activity. A Cbf alpha 2 mutant lacking transactivation properties does not transform cells when overexpressed, nor does it protect cells from CBF beta-SMMHC transformation. These results suggest that CBF beta-SMMHC interferes with the normal function of CBF and that this interference is necessary but not sufficient for cellular transformation.

3T3 Cells↗
PubMed1994

DNA sequences in the promoter region of the NF1 gene are highly conserved between human and mouse.

The gene for type 1 neurofibromatosis (NF1) is most highly expressed in brain and spinal cord, although low levels of mRNA can be found in nearly all tissues. As a first step in investigating the regulation of NF1 gene expression, we have cloned and sequenced the promoter regions of the human and mouse NF1 genes and mapped the transcriptional start sites in both species. We report here that the 5' ends of the human and murine NF1 genes are highly conserved. While no discernable TATA or CCAAT box sequences are seen, transcription initiates at identical sites in both species, 484 nucleotides upstream of the ATG initiation codon in the human gene. The human and mouse NF1 genes share particularly high sequence homology (95%) between nucleotides -33 and +261 and contain several perfectly conserved transcription factor binding site motifs, including a cAMP response element, several AP2 consensus binding sites, and a serum response element. The high conservation of these sequences indicates that they are likely to be significant in the regulation of NF1 gene expression.

Animals↗
PubMed1993

Fusion between transcription factor CBF beta/PEBP2 beta and a myosin heavy chain in acute myeloid leukemia.

The pericentric inversion of chromosome 16 [inv(16)(p13q22)] is a characteristic karyotypic abnormality associated with acute myeloid leukemia, most commonly of the M4Eo subtype. The 16p and 16q breakpoints were pinpointed by yeast artificial chromosome and cosmid cloning, and the two genes involved in this inversion were identified. On 16q the inversion occurred near the end of the coding region for CBF beta, also known as PEBP2 beta, a subunit of a heterodimeric transcription factor regulating genes expressed in T cells; on 16p a smooth muscle myosin heavy chain (SMMHC) gene (MYH11) was interrupted. In six of six inv(16) patient samples tested, an in-frame fusion messenger RNA was demonstrated that connected the first 165 amino acids of CBF beta with the tail region of SMMHC. The repeated coiled coil of SMMHC may result in dimerization of the CBF beta fusion protein, which in turn would lead to alterations in transcriptional regulation and contribute to leukemic transformation.

Amino Acid Sequence↗
PubMed1993

Identification of yeast artificial chromosomes containing the inversion 16 p-arm breakpoint associated with acute myelomonocytic leukemia.

We report the cloning of the chromosome 16 p-arm breakpoint involved in inversion 16(p13;q22) associated with subtype of acute myelomonocytic leukemia (AMML) M4Eo. Inter-Alu polymerase chain reaction (PCR) products from a series of interspecific somatic cell hybrids that contain only small portions of the human chromosome 16 p-arm were generated for use as fluorescent in-situ hybridization (FISH) probes. When applied to patient cells, rapid and unambiguous identification of the inversion resulted. Using FISH analysis, cosmid clones associated with the hybrids were identified that bracketed the p-arm breakpoint. A repeat-free fragment of one of these cosmids (35B11) when used as probe on Southern blots from pulsed-field gels identified rearranged macrorestriction fragments in patient DNA. Yeast artificial chromosomes (YACs) were isolated using sequences derived from cosmids flanking 35B11 in a cosmid contig. Of 4 YACs so identified, 3 were shown by FISH to cross the inversion-16 p-arm breakpoint. Therefore, the breakpoint has been molecularly cloned, and identified as being within these 3 YACs. These clones will facilitate the unraveling of the genetic events associated with inversion-16 and are available tools with immediate clinical application.

Base Sequence↗