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Biomedical subjects

C Banerjee

Publications and source records attributed to C Banerjee.

At least 19 recordsLinked to original sources
PubMed1997

Runt homology domain proteins in osteoblast differentiation: AML3/CBFA1 is a major component of a bone-specific complex.

The AML/CBFA family of runt homology domain (rhd) transcription factors regulates expression of mammalian genes of the hematopoietic lineage. AML1, AML2 and AML3 are the three AML genes identified to date which influence myeloid cell growth and differentiation. Recently AML-related proteins were identified in an osteoblast-specific promoter binding complex that functionally modulates bone-restricted transcription of the osteocalcin gene. In the present study we demonstrate that in primary rat osteoblasts AML-3 is the AML family member present in the osteoblast-specific complex. Antibody specific for AML-3 completely supershifts this complex, in contrast to antibodies with specificity for AML-1 or AML-2, AML-3 is present as a single 5.4 kb transcript in bone tissues. To establish the functional involvement of AML factors in osteoblast differentiation, we pursued antisense strategies to alter expression of rhd genes. Treatment of osteoblast cultures with rhd antisense oligonucleotides significantly decreased three parameters which are linked to differentiation of normal diploid osteoblasts: the representation of alkaline phosphatase-positive cells, osteocalcin production, and the formation of mineralized nodules. Our findings indicate that AML-3 is a key transcription factor in bone cells and that the activity of rhd proteins is required for completion of osteoblast differentiation.

Animals↗
PubMed1997

Multiple irrigation, debridement, and retention of components in infected total knee arthroplasty.

The results of 24 infected total knee arthroplasties (22 patients) that were treated by irrigation, debridement, and retention of the prosthetic components were prospectively studied. Strict criteria were used for the selection of this method of treatment. Patients had to be less than 30 days after index arthroplasty (postsurgical group) or had to have less than 30 days of knee symptoms (hematogenous group). In addition, there had to be no radiographic signs of osteitis or evidence of a loose prosthetic component. Patients had one to three irrigation and debridement procedures depending on systemic signs, knee symptoms, or the results of knee aspirations. All of the immediate postsurgical infections (10 knees) and 10 of the 14 (71%) late hematogenously infected knees retained the prosthesis without further evidence of infection at the final follow-up visit at 48 months (range, 24-140 months). This study shows that in selected circumstances, irrigation, debridement, and retention of the components can result in low morbidity with high success rates.

Aged↗
PubMed1996

An AML-1 consensus sequence binds an osteoblast-specific complex and transcriptionally activates the osteocalcin gene.

Tissue and cell-type specific expression of the rat osteocalcin (rOC) gene involves the interplay of multiple transcriptional regulatory factors. In this report we demonstrate that AML-1 (acute myeloid leukemia-1), a DNA-binding protein whose genes are disrupted by chromosomal translocations in several human leukemias, interacts with a sequence essential for enhancing tissue-restricted expression of the rOC gene. Deletion analysis of rOC promoter-chloramphenicol acetyltransferase constructs demonstrates that an AML-1-binding sequence within the proximal promoter (-138 to -130 nt) contributes to 75% of the level of osteocalcin gene expression. The activation potential of the AML-1-binding sequence has been established by overexpressing AML-1 in osteoblastic as well as in nonosseous cell lines. Overexpression not only enhances rOC promoter activity in osteoblasts but also mediates OC promoter activity in a nonosseous human fibroblastic cell line. A probe containing this site forms a sequence specific protein-DNA complex with nuclear extracts from osteoblastic cells but not from nonosseous cells. Antisera supershift experiments indicate the presence of AML-1 and its partner protein core-binding factor beta in this osteoblast-restricted complex. Mutations of the critical AML-1-binding nucleotides abrogate formation of the complex and strongly diminish promoter activity. These results indicate that an AML-1 related protein is functional in cells of the osteoblastic lineage and that the AML-1-binding site is a regulatory element important for osteoblast-specific transcriptional activation of the rOC gene.

Animals↗
PubMed1996

Bone tissue-specific transcription of the osteocalcin gene: role of an activator osteoblast-specific complex and suppressor hox proteins that bind the OC box.

Bone-specific expression of the osteocalcin gene is transcriptionally controlled. Deletion analysis of osteocalcin promoter sequences by transient transfection of osseous (ROS 17/2.8) and nonosseous (R2 fibroblast) cells revealed that the most proximal 108 nucleotides are sufficient to confer tissue-specific expression. By gel mobility shift assays with wild-type and mutated oligonucleotides and nuclear extracts from several different cell lines we identified a novel transcription factor complex which exhibits sequence-specific interactions with the primary transcriptional element, the OC box (nt -99 to -76). This OC box binding protein (OCBP) is present only in osteoblast-like cells. Methylation interference demonstrated association of the factor with OC box sequences overlapping the Msx homeodomain consensus binding site. By assaying several mutations of the OC box, both in gel shift and transient transfection studies using ROS 17/2.8, we show the following. First, binding of OCBP correlates with osteocalcin promoter activity in ROS 17/2.8 cells. Increased binding leads to a 2-3-fold increase in transcription, while decreased binding results in transcription 30-40% of control. Second, homeodomain protein binding suppresses transcription. However, Msx expression is critical for full development of the bone phenotype as determined by antisense studies. Last, we show that one of the mutations of the OC box permits expression of osteocalcin in non-osseous cell lines. In summary, we demonstrate association of at least two classes of tissue-restricted transcription factors with the OC box element, the OCBP and Msx proteins, supporting the concept that these sequences contribute to defining tissue specificity.

Animals↗
PubMed1996

Developmental expression and activities of specific fos and jun proteins are functionally related to osteoblast maturation: role of Fra-2 and Jun D during differentiation.

Developmental studies of oncogene expression implicate the Fos and Jun family of transcription factors in the regulation of bone growth and differentiation. Promoters of many developmentally regulated genes, including osteocalcin, a marker of osteoblast differentiation, contain AP-1 sites that bind Fos/Jun dimers. Here, we demonstrate that the selective expression of fos- and jun-related genes is functionally related to the stage of osteoblast growth and differentiation in vitro. During osteoblast proliferation, nuclear protein levels of all seven activating protein-1 (AP-1) members are maximal. Subsequently, during the period of extracellular matrix maturation, levels decline. In fully differentiated osteoblasts, Fra-2 and (to a lesser extent) Jun D are the principal AP-1 members detectable by Western blot analysis. AP-1 complex composition and binding activity also exhibit developmental changes. All Fos and Jun family members are involved in AP-1 complex formation in proliferating cells, whereas Fra-2 and Jun D predominate in AP-1 complexes in differentiated osteoblasts. Overexpression of Fos and Jun family members in ROS 17/2.8 cells markedly affects the expression of an osteocalcin promoter-chloramphenicol acetyltransferase construct. Coexpression of only one AP-1 pair, Fra-2 and Jun D, stimulated reporter expression, whereas coexpression of other AP-1 pairs down-regulated expression (i.e. c-jun and any Fos family member) or had no effect (i.e. Fra-1 and Jun B). Promoter deletion analyses indicate that these effects are site specific. Consequential effects of Fra-2 on osteoblast differentiation are further demonstrated by antisense studies in which osteoblast differentiation and the development of a bone tissue-like organization were suppressed. Consistent with recent findings suggesting that AP-1 complex composition can selectively regulate gene transcription, our findings demonstrate that differential expression of Fos and Jun family members could play a role in the developmental regulation of bone-specific gene expression and, as a result, may be functionally significant for osteoblast differentiation.

Animals↗
PubMed1996

Basal and vitamin D-responsive activity of the rat osteocalcin promoter in stably transfected osteosarcoma cells: requirement of upstream sequences for control by the proximal regulatory domain.

Osteocalcin (OC) is a bone-specific vitamin D- responsive protein that is developmentally expressed during osteoblast differentiation. In transient transfection assays, as little as approximately 0.1 kilobase (kb) of the OC proximal promoter is sufficient for basal expression. Because eukaryotic genes are packaged as nucleosomes that contribute to both chromatin organization and transcriptional control, we functionally examined the activity of OC promoter constructs within a chromatin context. ROS 17/2.8 osteosarcoma cells were stably transfected with a series of rat OC promoter-reporter constructs, containing progressive 5'-deletions. The results demonstrate that in contrast to transient transfection assays, the proximal 0.11-kb promoter is no longer active when integrated in the genome. Progressive gain of basal expression with 0.35-, 0.53-, and 0.72-kb promoters suggests that upstream sequences facilitate the formation of an appropriate higher order nuclear structure, thereby potentiating the activity of the chromosomally integrated proximal promoter elements. This is consistent with location of both deoxyribonuclease I (DNase I)-hypersensitive sites and nuclear matrix protein-DNA interaction sites in the osteocalcin promoter. Vitamin D responsiveness in the stably transfected cells is obtained with the inclusion of 0.53 kb or additional upstream promoter sequences. Therefore, these sequences satisfy the requirements for binding of basal and enhancer transcription factors as well as interactions between them within a chromatin context. Both maximal basal expression and maximal vitamin D responsiveness are obtained with cells carrying either the 0.72-kb or the 1.1-kb promoter fragment. Cells carrying the 1.1-kb promoter show DNase I hypersensitivity at both the basal promoter and the vitamin D response element-containing domains, locations that also exhibit DNase I hypersensitivity in the endogenous OC promoter. In addition, we have documented changes in the basal activity and vitamin D responsiveness of the stably integrated 1.1 kb promoter as a function of cell density-mediated growth inhibition, which is accompanied by up-regulation of bone phenotypic genes. Thus, important aspects of OC gene transcriptional regulation that cannot be investigated in transient transfection assays can be addressed using ROS 17/2.8 cells stably transfected with OC promoter-reporter constructs.

Animals↗
PubMed1996

Transforming growth factor-beta 1 responsiveness of the rat osteocalcin gene is mediated by an activator protein-1 binding site.

Osteocalcin (OC), a bone specific protein expressed during differentiation and mineralization of the bone extracellular matrix, is down-regulated upon treatment with transforming growth factor (TGF)-beta 1. To address the potential role of OC gene expression in relation to TGF-beta 1 regulation of bone formation and resorption, we examined the transcriptional activity of the rat OC promoter after TGF-beta 1 treatment. 5' deletion analysis of rat OC promoter-chloramphenicol acetyltransferase constructs demonstrated that TGF-beta 1 treatment repressed chloramphenicol acetyltransferase activity by 2.4-fold in transient transfections of ROS 17/2.8 cells. A 29-bp region between -162 and -134 identified as the TGF-beta 1 response domain, conferred TGF-beta 1 responsiveness to the -108 to +24 rat OC basal promoter in an orientation dependent manner. Mutation of an activator protein-1/cAMP-response element-like motif (- 146 to -139) abolished TGF-beta 1 responsiveness of the construct. In vitro gel-mobility shift and competition assays using wild-type and mutated oligonucleotides and antibodies indicate that Fra-2, a Fos related transcription factor, binds to this motif. We show that Fra-2 is an activator of the OC promoter, and TGF-beta 1 inhibits this activation. Our results demonstrate that Fra-2 is hyperphosphorylated upon TGF-beta 1 treatment of ROS 17/2.8 cells. Additionally, treatment of cells with a staurosporine protein kinase C inhibitor abrogates TGF-beta 1 mediated down-regulation of the OC promoter activity. Together, these results demonstrate that TGF-beta 1 responsiveness of the rat osteocalcin gene in ROS 17/2.8 cells is mediated through an activator protein-1 like cis-acting element that interacts with Fra-2. Furthermore, our results are consistent with a critical role for TGF-beta 1 induced phosphorylation of Fra-2 in the repression of OC gene transcription.

Animals↗
PubMed1996

The osteocalcin gene promoter provides a molecular blueprint for regulatory mechanisms controlling bone tissue formation: role of transcription factors involved in development.

Characterization of regulatory sequences and their cognate binding factors in the bone-specific osteocalcin (OC) gene promoter has provided insight into mechanisms that control expression of the gene under diverse biological conditions. We present evidence for AP-1 motifs and two multipartite conserved regulatory sequences, the OC Box I (nt-99 to-76) and a site designated OC Box II (nt-136 to-130) in contributing to developmental and tissue-specific expression of osteocalcin. OC Box I is characterized by a homeodomain binding site and OC Box II is a recognition sequence for AML-1 (also called PEBP2 alpha), a runt homology-related DNA binding protein. Functional activity of the elements was established in osseous and non-osseous cell lines and is in part related to the binding of osteoblast-specific complexes which enhance OC transcription. The contribution of several elements and binding of multiple classes of transcription factors to independent elements in both these domains serve to illustrate the complexity of control required for tissue-specific OC expression.

Animals↗
PubMed1995

Contributions of distal and proximal promoter elements to glucocorticoid regulation of osteocalcin gene transcription.

Previous studies identified several glucocorticoid response elements (GREs) in the 5'-promoter region of the rat osteocalcin (OC) gene by purified receptor binding. The present study addresses functionality of the GRE sequences in the proximal promoter at nucleotide (nt) -16 to -1 downstream of the TATA element together with the GRE half-element in the OC box at nt -86 to -81. This was done by assaying glucocorticoid responsiveness [at 10(-6) M dexamethasone (DEX)], and in combination with 10(-8) M 1,25-dihydroxyvitamin D3, of a series of deleted and mutated OC promoter reporter constructs (OCCAT) in osteoblast-like cells, the ROS 17/2.8 rat osteosarcoma line. Promoter deletion analysis revealed an additional GRE in the distal promoter at nt -697 to -683 that functions to suppress OC transcription. In the absence of this upstream negative GRE (nGRE), the -531 OCCAT construct exhibited enhanced promoter activity in response to DEX (1.8-fold DEX/Control), but further deletion (-348 and -108 OCCAT constructs) restored DEX suppression to OC promoter activity (0.6- and 0.8-fold DEX/Control, respectively). Mutations introduced in both the proximal GRE (nt -16 to -1) and the half-GRE in the OC box, or in the proximal GRE alone, nearly abrogated DEX responsiveness of OC promoter activity. Both distal and proximal GREs specifically bound glucocorticoid receptor present in ROS 17/2.8 nuclear extracts as shown by competition with wild type and mutated oligonucleotides and antibody inhibition of binding. Furthermore, both GREs, independently, conferred DEX-responsive transcriptional repression to the heterologous thymidine kinase basal promoter. We also report that glucocorticoid suppression of 1,25-dihydroxyvitamin D3-stimulated transcription occurs independently of distal or proximal GREs. Taken together, these results demonstrate that in vivo responsiveness of OC to DEX involves the integrative activities of several functional promoter elements.

Animals↗
PubMed1994

Parathyroid-responsive modifications in the nuclear matrix of ROS 17/2.8 rat osteosarcoma cells.

PTH is a mediator of skeletal development and remodeling that influences gene expression in osteoblastic cells. It is well established that PTH modulates the activity of membrane-associated second messenger signal transduction pathways. In these studies we have addressed the potential contribution of components of cell structure to the integration of PTH-related regulatory signals that influence the expression of bone cell genes. Chronic treatment of ROS 17/2.8 rat osteosarcoma cells with PTH is accompanied by changes in gene expression that are at least in part transcriptionally controlled. To explore the involvement of nuclear architecture in PTH-responsive modifications in gene expression, we investigated changes in the nuclear matrix after PTH treatment. Consistent with a role for the nuclear matrix in determining spatial organization and topology of chromatin as well as in the localization and targeting of transcription factors, we observed PTH-associated changes in a 200-kilodalton nuclear matrix protein in response to PTH. A significant down-regulation of synthesis was observed when nuclear matrix proteins were resolved electrophoretically in two-dimensional gels. This protein was restricted to the nuclear matrix and was not detected in the chromatin or cytoskeletal cellular fractions. These alterations in nuclear matrix proteins that occur after PTH treatment in osteosarcoma cells were phenotype related. They did not occur in UMR-106 POL or H4 hepatoma cells. Our findings support a role for the nuclear matrix in transducing PTH-mediated regulatory signals to facilitate the extent to which genes in osteoblasts are transcribed.

Animals↗
PubMed1993

Position and orientation-selective silencer in protein-coding sequences of the rat osteocalcin gene.

Osteocalcin (OC) is a bone-specific protein which is expressed postproliferatively by osteoblasts during late stages of differentiation. We have found that a silencer element is present within the rat OC gene (between nt +39 and +104), overlapping the OC signal prepropeptide-coding sequence. The presence of this sequence in OC promoter-CAT reporter constructs suppresses promoter activity in transiently transfected proliferating osteoblasts, which do not express OC, by up to 50-fold. This is the first demonstration of contribution from protein-coding sequences to silencing of animal genes. The element appears to be bipartite; silencer activity requires both the protein-coding sequence +39 to +63 and the +93 to +104 exon 1/intron 1 border region. Both of these domains contain sequences highly similar to silencer motifs in several other genes, including chicken lysozyme as well as rat collagen type II, insulin, and growth hormone. OC silencer activity is fully retained when the element is placed outside the RNA-coding region, 3' but not 5' of the OC-CAT fusion gene. Repression activity is orientation independent in the native position but requires the native orientation when located in 3' extragenic positions. The silencer does not inhibit the activity of the heterologous SV40 early promoter. These results suggest interaction between the transcribed silencer and specific OC promoter element(s) residing farther upstream. The OC transcribed silencer may contribute to developmental control of OC expression.

Animals↗
PubMed1993

Identification and characterization of two proximal elements in the rat osteocalcin gene promoter that may confer species-specific regulation.

The rat osteocalcin gene encodes a 6-kD osteoblast-specific protein that is expressed post-proliferatively. The developmental and steroid hormone responsive expression of the osteocalcin gene is transcriptionally regulated by a promoter with multiple basal and enhancer elements that exhibit activity controlled by a series of physiological mediators (e.g., 1.25(OH)2D3, glucocorticoids). In this study, we established the contribution of the rat osteocalcin (OC) box domain (-99 to -76), a proximal basal element with a CCAAT motif as a central core, to transcriptional activity of the rat osteocalcin gene with in vivo co-transfection assays. By this same assay, however, the highly homologous (22 of 24 nt) human OC box element was unable to compete for transcription factor binding with the rat OC promoter. In vitro protein/DNA interaction studies confirm the presence of two protein binding sites in the OC box region, one of which overlaps the CCAAT motif and, at least in part, accounts for species-specific expression. Competition analysis established that the single nucleotide substitution of adenine for thymine, which converts the core motif of the rat OC box (CCAAT) to the core motif of the human OC box (CCAAA), accounts for observed species differences in transcription factor interactions. The CCAAT-specific protein/DNA interactions are heat stable and insensitive to phosphatase treatment. At second protein/DNA interaction located upstream of the CCAAT motif includes two steroid-like half-elements. These interactions are heat labile and sensitive to phosphatase treatment in contrast to the CCAAT-specific interactions. The human OC promoter contains only a single steroid-like half-element, while two steroid half-elements with an 11 nucleotide spacer are present in the rat OC promoter. These observed variations in sequence organization and transactivation factor binding in analogous proximal basal regulatory regions of the OC gene promoter may provide a basis for species-restricted variations in responsiveness to physiological mediators of OC gene expression at the transcriptional level.

Animals↗
PubMed1993

Burns in elderly patients.

Admissions in the burn unit of IPGME & R, Calcutta over a period of 5 years from 1985 to 1990 have been reviewed. There were 1980 patients who had been treated and 84 of them were of 60 years of age or above. Mortality rate in elderly patients with burns had been found to be much higher. High mortality rate might be due to pre-existing medical problems and low general condition due to aging. Most of them were treated with conservative surgery. Early excision and grafting was the surgical approach in few cases with discouraging results.

Age Factors↗
PubMed1992

Isolation and characterization of a cyclic nucleotide-independent protein kinase from Leishmania donovani.

We describe here a protein kinase from the promastigote form of the parasitic protozoan, Leishmania donovani, purified to near homogeneity to a single-subunit, 34-kDa protein. This enzyme does not require a cofactor, and has several characteristics in common with the catalytic subunit of mammalian cAMP-dependent protein kinase, for example, preference for kemptide as a substrate, phosphorylation of serine residues of protamine and inhibition by the mammalian heat-stable inhibitor. The leishmanial enzyme can associate with the regulatory subunit of mammalian cAMP-dependent protein kinase to form an inactive holoenzyme that is activated by cAMP and is protected from inhibition by thiol reagents. From these results it is concluded that L. donovani promastigotes possess a protein kinase which has similar characteristics with the mammalian catalytic subunit of cAMP-dependent protein kinase.

Animals↗
PubMed1990

Partial purification and characterization of a soluble protein kinase from Leishmania donovani promastigotes.

A soluble protein kinase from the promastigote form of the parasitic protozoon Leishmania donovani was partially purified using DEAE-cellulose, Sephadex G-200 and phosphocellulose columns. The enzyme preferentially utilized protamine as exogenous phosphate acceptor. The native molecular mass of the enzyme was about 85 kDa. Mg2+ ions were essential for enzyme activity; other metal ions, e.g. Ca2+, Co2+, Zn2+ and Mn2+, could not substitute for Mg2+. cAMP, cGMP, Ca2+/calmodulin and Ca2+/phospholipid did not stimulate enzyme activity. The pH optimum of the enzyme was 7.0-7.5, and the temperature optimum 37 degrees C. The apparent Km for ATP was 60 microM. Phosphoamino acid analysis revealed that the protein kinase transferred the gamma-phosphate of ATP to serine residues in protamine. The thiol reagents p-hydroxymercuribenzoic acid, 5-5'-dithio-bis(2-nitrobenzoic acid) and N-ethylmaleimide inhibited enzyme activity; the inhibition by p-hydroxymercuribenzoic acid and 5-5'-dithio-bis(2-nitrobenzoic acid) was reversed by dithiothreitol.

Adenosine Triphosphate↗
PubMed1990

Histomorphological criteria for prognosis in breast cancer.

One hundred cases of carcinoma breast were analysed with regard to histological features and clinical follow-up. Bad prognostic factors included tumour size larger than 5cms; absent or minimal intraduct component in the tumour and absent or minimal periductal elastosis. Conversely significant intraduct component in the tumour and periductal elastosis were associated with better survival. Degree of lymph nodal involvement did not influence the outcome. One reason for this unexpected finding could be that patients with more than 3 lymph node involvement were given additional chemotherapy.

Breast Neoplasms↗
PubMed1988

Bacillus infections in patients with cancer.

Eighteen febrile patients experienced 24 episodes of Bacillus bacteremias from January 1978 to June 1986. Bacillus species isolated included Bacillus cereus (eight cases), Bacillus circulans (three), Bacillus subtilis (two), Bacillus pumilus (two), Bacillus licheniformis (one), Bacillus sphaericus (one), Bacillus coagulans (one), and six that could not be speciated. Fifteen patients had lymphoma or leukemia and three had breast cancer. Nine patients were neutropenic (polymorphonuclear neutrophil count, less than 1.0 x 10(9)/L), seven patients had a Hickman catheter in place, and 14 had recently received chemotherapy. Twelve of the bacteremic episodes were clinically significant, and four of these 12 involved Hickman catheters. Catheter removal was ultimately necessary in all four patients. Scanning and transmission electron microscopy was performed on one of the removed Hickman catheters and showed Bacillus organisms embedded in a biofilm composed of gram-positive cocci and glycocalyx. Bacillus species were uniformly susceptible to vancomycin, imipenem, and aminoglycosides, with penicillin susceptibilities being variable. Bacillus appears to be another gram-positive organism now being recognized as a bacterial pathogen for compromised hosts. When such infections involve long-term indwelling venous access devices, treatment should include immediate catheter removal as well as antibiotic therapy.

Adolescent↗