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Biomedical subjects

R Narayanan

Publications and source records attributed to R Narayanan.

At least 73 records · Page 4Linked to original sources
PubMed1992

Differential roles of stromal cells, interleukin-7, and kit-ligand in the regulation of B lymphopoiesis.

Newly formed B lymphocytes are a population of rapidly renewed cells in the bone marrow of mammals and their steady state production presumably depends on a cascade of regulatory cells and cytokines. Although considerable information has been forthcoming about the role of interleukin-7 (IL-7) in potentiating pre-B-cell proliferation, few studies have addressed the possibility that multiple cytokines are involved in the progression of early events in cellular differentiation and proliferation in this hematopoietic lineage. Our laboratory previously described pre-B-cell differentiation mediated by the bone marrow stromal cell line S17. In this study, we further delineate the role of stromal cells in differentiation and proliferation of pre-B cells. These experiments show that the stromal cell line S17 potentiates the proliferative effect of IL-7 on B-lineage cells and that this S17-derived potentiator can be replaced with recombinant kit-ligand (KL). Our results further show that pre-B-cell formation from B220-, Ig- progenitor cells and expression of mu heavy chain of immunoglobulin is uniquely dependent on the presence of S17 stromal cells and cannot be reproduced with IL-7, KL, or costimulation with both IL-7 and KL. These data contribute to a rapidly evolving model of stromal cell regulation of B-cell production in the marrow and suggest unique roles for IL-7, KL, and as yet uncharacterized stromal cell-derived lymphokines in this process.

Animals↗
PubMed1992

Regulatory cells and cytokines involved in primary B lymphocyte production.

Based upon the above data, it is now possible to formulate a working model that defines the stages of B cell development on which stromal cells and their products act. During the initial stages of this process, pro-B cells which do not express Ig heavy or light chain protein or other non-Ig B lineage associated molecules develop into B220 and c mu expressing pre-B cells in response to a low (< 10 kD) molecular weight stromal cell derived factor. No defined interleukin or colony stimulating factor, including molecules such as KL and IL-7, can replace stromal cell conditioned medium in mediating this developmental step. There appears to be little cell proliferation associated with the differentiation of pro-B cells into pre-B cells. However, our data indicate that as precursors develop into B220 expressing B cell progenitors, they become sensitive to the proliferation stimulating effects of IL-7 and KL. These results are in accord with findings that progenitor cells that have undergone DJH rearrangements are particularly sensitive to KL and IL-7(18,19). The analysis of pre-B cells present in individual lymphoid colonies indicates that once cells have rearranged and expressed their Ig heavy chain genes, they are no longer sensitive to KL and IL-7. These observations are based on the fact that receptors for these cytokines are not expressed in stromal cell dependent pre-B cells and are consistent with kinetic studies showing that the maturation of pre-B cells into surface Ig expressing B lymphocytes is not dependent upon cell proliferation21.(ABSTRACT TRUNCATED AT 250 WORDS)

B-Lymphocyte Subsets↗
PubMed1992

Functional characterization of the NF-kappa B p65 transcriptional activator and an alternatively spliced derivative.

The NF-kappa B transcription factor complex is composed of two proteins, designated p50 and p65, both having considerable homology to the product of the rel oncogene. We present evidence that the p65 subunit is a potent transcriptional activator in the apparent absence of the p50 subunit, consistent with in vitro results demonstrating that p65 can interact with DNA on its own. To identify the minimal activation domain, chimeric fusion proteins between the DNA binding domain of the yeast transcriptional activator protein GAL4 and regions of the carboxy terminus of p65 were constructed, and their transcriptional activity was assessed by using a GAL4 upstream activation sequence-driven promoter-chloramphenicol acetyltransferase fusion. This analysis suggests that the boundaries of the activation domain lie between amino acids 415 and 550. Moreover, single amino acid changes within residues 435 to 459 greatly diminished activation. Similar to other activation domains, this region contains a leucine zipper-like motif as well as an overall net negative charge. To identify those residues essential for DNA binding, we made use of a naturally occurring derivative of p65, lacking residues 222 to 231 (hereafter referred to as p65 delta), and produced via an alternative splice site. Gel mobility shift analysis using bacterially expressed p65, p65 delta, and various mutants indicates that residues 222 to 231 are important for binding to kappa B DNA. Coimmunoprecipitation analysis suggests that these residues likely contribute to the multimerization function required for homomeric complex formation or heteromeric complex formation with p50 in that no association of p65 delta with itself or with p50 was evident. However, p65 delta was able to form weak heteromeric complexes with p65 that were greatly reduced in their ability to bind DNA. On the basis of these findings, we suggest that subtle changes within the proposed multimerization domain can elicit different effects with the individual Rel-related proteins and that a potential role of p65 delta may be to negatively regulate NF-kappa B function through formation of nonfunctional heteromeric complexes.

Amino Acid Sequence↗
PubMed1992

Persistent expression of the tumor suppressor gene DCC is essential for neuronal differentiation.

Cell differentiation is associated either with a complete loss of proliferative potential or with a change in growth requirements. Neoplastic transformation may result from the activation of oncogenes that support growth or from inactivation or loss of tumor suppressor genes, which are thought to regulate differentiation. To examine the relationship between tumor suppressor genes and cell differentiation, we chose the gene "deleted in colorectal cancer" (DCC) and studied its role in a pheochromocytoma cell line, PC-12, using antisense RNA as well as antisense oligonucleotides to DCC. When exposed to nerve growth factor for several days, PC-12 cells develop long dendrites. This morphological change follows the transient expression of immediate early genes and is associated with an up-regulation of DCC. Interestingly, if the up-regulation of DCC was counteracted using an antisense RNA technique, the morphological changes were prevented, but the other parameters of the nerve growth factor response were unaffected. Moreover, when DCC expression was inhibited by antisense oligonucleotides to DCC in nerve growth factor-differentiated cells, the neuron-like phenotype was reversed. Our results demonstrate that the gene DCC is involved in a distal segment of neural differentiation and provide the first direct evidence that a tumor suppressor gene plays a role in cell differentiation.

Animals↗
PubMed1992

Antisense RNA to the putative tumor-suppressor gene DCC transforms Rat-1 fibroblasts.

Allelic deletions involving chromosomes 18q occur in a significant number of colorectal cancers. Recently, a highly conserved gene called 'deleted in colorectal cancer' (DCC) has been identified on chromosome 18q. DCC has been postulated to be a colorectal tumor-suppressor gene. In order to understand the role of DCC in cell transformation, we have established a stable Rat-1 cell line expressing dexamethasone-inducible DCC antisense RNA. High levels of dexamethasone-inducible DCC antisense RNA were detected in the Rat-1 transfectants. The antisense DCC-expressing Rat-1 cells showed a faster growth rate, anchorage independence and tumorigenicity in nude mice. Exposure of the parental Rat-1 cells to antisense oligodeoxyribonucleotides to DCC resulted in inhibition of cell adhesion to the substratum which could be abrogated by various extracellular matrices. On the other hand, a bone marrow-derived stromal cell line which does not express DCC showed no detachment from the substratum when treated with the antisense oligo to DCC. These results suggest that the DCC gene is involved in cell adhesion and provide the first direct biological evidence for the possible role of DCC as a tumor-suppressor gene.

Animals↗
PubMed1991

Coordinated expression of intermediate biomarkers for tumorigenic transformation in RAS-transfected mouse mammary epithelial cells.

Deregulated expression of the RAS oncogene is associated with tumorigenic transformation of mammary cells. Because of the complex, multiphasic nature of cancer progression, it is important to systematically identify the biomarkers specific for initiation, promotion, and progression of breast cancer. Mouse mammary epithelial cells (MMEC) were transfected with normal c-Ha-RAS proto oncogene (pH06N) and with mutant c-Ha-RAS oncogene (pH06T). The parental MMEC and the cloned transfectants pH06N1, pH06N2, pH06T1, and pH06T12 were evaluated for the acquisition of transformed characteristics by determining altered cellular metabolism of estradiol, increased ability for anchorage-independent growth, and ability to form tumors at the transplant site in athymic 'nude' mice. Persistent, functional integration of c-Ha-RAS was evidenced by the presence of a 1.2 kb c-Ha-RAS transcript in the four transfectants but not in MMEC. All the transfectants also exhibited a substantial increase in the binding of c-Ha-RAS p21 to [alpha-32P] GTP relative to MMEC (P less than 0.003). The relative extent of estradiol metabolism leading to the formation of 16 alpha-hydroxyestrone was increased (P less than 0.004) in all the four transfectants. These four transfectants also showed a 100-400 fold increase in colony forming efficiency in 0.33% agar, relative to MMEC (P less than 0.0009), and formed rapidly growing tumors within 3-5 weeks of transplantation. Our results demonstrate that i) persistent expression of normal and mutant c-Ha-RAS can bring about tumorigenic transformation of mouse mammary epithelial cells; and ii) alteration in estradiol metabolism and acquisition of anchorage-independent growth precede the emergence of a tumorigenic phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Gastrointestinal candidiasis in a murine model of severe combined immunodeficiency syndrome.

A murine model of severe combined immunodeficiency syndrome (scid mice) affords an opportunity to study the interaction of Candida albicans with a host lacking functional B- and T-cell mechanisms. We have previously reported no significant difference in yeast recovery after intravenous challenge of BALB/c mice and scid mice with C. albicans (S. Mahanty, R.A. Greenfield, W.A. Joyce, and P.W. Kincade, Infect. Immun. 56:3162-3166, 1988). In this study, we evaluate the course of gastrointestinal candidiasis after a single oral challenge with C. albicans. BALB/c and scid mice received H2O containing 10(6) C. albicans per ml for 16 h. Half the mice of each strain continuously received H2O containing 1 mg of tetracycline per ml. Stool samples were cultured for yeast twice weekly until they were negative three consecutive times or positive for 8 weeks. Mice were then sacrificed for quantitative cultures of liver, spleen, and kidneys. At eight weeks postinoculation, 2 of 13 BALB/c mice, 0 of 14 BALB/c mice receiving tetracycline, 6 of 12 scid mice, and 8 of 13 scid mice receiving tetracycline had positive stool cultures (P less than 0.05, likelihood ratio chi-square). Quantitative recovery of yeasts from stools was also higher in the scid mice. Cultures of liver, spleen, and kidneys wer negative in all BALB/c mice and essentially all negative in scid mice; a single colony was isolated from the kidney of one scid mouse and the liver of another scid mouse. We conclude that B cells and/or T cells and their products are important in gastrointestinal colonization with C. albicans but that even in their absence, dissemination of infection from the gastrointestinal tract does not consistently occur. Thus, other aspects of host defense must be critical in containing gastrointestinal Candida colonization.

Administration, Oral↗
PubMed1991

Ankle-foot prosthesis with articulated human bone endoskeleton: force-deflection and fatigue study.

The durability and function of three ankle-foot prostheses fabricated using a naturally articulated, fresh cadaveric human bone endoskeleton set in a fiber reinforced rubber shell were studied. Radiographic and force-deflection analyses before and after cyclic dorsiflexion for 5,000, 100,000, and 3 million cycles revealed no structural or functional changes. The endoskeleton foot compared favorably with the Jaipur ankle-foot prosthesis. It is concluded that formalin-fixed fresh foot bones with intact articulations obtained from cadaveric or surgically amputated limbs are suitable for use in ankle-foot prostheses because they withstand prolonged use without functional or structural deterioration.

Amputation, Surgical↗
PubMed1991

Equipment and methods for laboratory testing of ankle-foot prostheses as exemplified by the Jaipur foot.

A load deflection and a cyclic loading apparatus capable of measuring dorsiflexion/plantarflexion, pronation/supination, internal/external rotation, and cyclic dorsiflexion (60 cycles per minute) of ankle-foot prostheses are described. A test protocol was developed to assess the functional parameters of the Jaipur ankle-foot prosthesis before and after prolonged cyclic loading, with the simultaneous aim of evaluating these machines. The results on 26 Jaipur ankle-foot prostheses revealed that: 1) the prosthesis enjoys considerable mobility in three planes, confirming its known versatility; 2) the prosthesis is robust; and, 3) the testing machines deliver reproducible results and are suitable for in-house testing of ankle-foot prostheses.

Amputation, Surgical↗
PubMed1990

Evaluation of the role of extracellular matrix proteins, polyunsaturated fatty acids and c-myc expression in the inhibition of the serum-free growth of epithelial cells by TGF-beta 1.

Novel or modified serum-free media were developed for the anchorage-dependent growth of nontransformed murine mammary epithelial cells (MMEC) and Balb/MK murine keratinocytes respectively. Growth rates for both cell lines were similar in serum-containing and serum-free media. The serum-free media were used to evaluate potential mechanisms of epithelial cell growth regulation by type 1 transforming growth factor beta (TGF-beta 1). The growth of MMEC and Balb/MK cells was reversibly inhibited 40-65% in a time- and dose-dependent fashion by TGF-beta 1 under both serum-containing and serum-free conditions. Constitutive over-expression of a transfected c-myc oncogene in MMEC did not result in loss of sensitivity to growth inhibition by TGF-beta 1. In addition, Balb/MK and MMEC growth inhibition by TGF-beta 1 was not potentiated by polyunsaturated fatty acids or reversed by vitamin E. Exogenous type V collagen was able to mimic the inhibitory effects of TGF-beta 1 on the serum-free growth of Balb/MK and MMEC. In contrast, collagen types I and IV, fibronectin and laminin did not inhibit the growth of these cells. The type V collagen used was not contaminated with TGF-beta, and subsaturating, but not saturating concentrations of type V collagen and TGF-beta 1 were additive with respect to Balb/MK and MMEC growth inhibition. These results demonstrate that nontransformed epithelial cell growth inhibition by TGF-beta 1 is mediated by mechanisms distinct from those observed with certain carcinoma and melanoma cells. Our results also suggest the possible involvement of type V collagen in Balb/MK and MMEC growth inhibition by TGF-beta 1.

Animals↗
PubMed1990

Neoplastic transformation of mouse mammary epithelial cells by deregulated myc expression.

A spontaneously immortalized, nontumorigenic mouse mammary epithelial cell line (MMEC) was transfected with an activated myc construct by electroporation. Constitutive expression of myc in MMEC resulted in anchorage independence in soft agar and tumorigenicity in nude mice. The myc-expressing MMEC showed higher saturation density, faster growth rate, and partial abrogation of serum-derived growth factor(s) requirement compared with parent MMEC. Epidermal growth factor or transforming growth factor alpha stimulated the anchorage-independent growth, but not the anchorage-dependent growth, of MMEC-myc cells. Type 1 transforming growth factor beta, on the other hand, inhibited both the anchorage-independent and anchorage-dependent growth of MMEC-myc cells. These results demonstrate that deregulated expression of myc results in neoplastic transformation iin mammary epithelial cells. Accompanying the transformation is altered sensitivity to polypeptide growth factors.

Animals↗
PubMed1990

Transcriptional regulation of early growth response genes in FOS-expressing PC-12 cells.

Deregulated c-fos expression in the rat pheochromocytoma cell line, PC-12, causes pronounced downregulation of nerve growth factor (NGF)-induced c-fos and c-jun activation, accompanied by a block in NGF-induced differentiation of PC-12 cells. The FOS-expressing PC-12 cells were exposed to diverse agents such as NGF, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), dibutyryl cyclic adenosine 3',5' monophosphate (db cAMP), and Ca-ionophore; and the expression of egr-1, c-fos, c-jun, jun-B, and jun-D was analyzed. Pronounced downregulation of c-fos, c-jun, and--to a lesser extent--jun-B was observed on treatment with NGF, bFGF, db cAMP, and Ca-ionophore, whereas EGF-induced activation of these early response genes was not inhibited in FOS-expressing PC-12 cells. Ca-ionophore- and db cAMP-induced egr-1 activation in PC-12 fos cells was completely inhibited. Both parent and PC-12 fos cells expressed similar high basal levels of jun-D, whose expression was the least regulatable by all of these agents. Transfection of fos promoter-chloramphenicol acetyltransferase (promoter-CAT) plasmid into these stable FOS-expressing PC-12 cells revealed that these effects are exerted at the fos promoter level.

Adrenal Gland Neoplasms↗
PubMed1990

Inhibition of PC-12 cell differentiation by the immediate early gene fra-1.

The rat pheochromocytoma cell line (PC-12) offers a powerful in vitro model to study the mechanism of growth factor-induced differentiation and proliferation. Within minutes of addition, agents such as nerve growth factor (NGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and dibutyryl cyclic AMP (db cAMP) rapidly activate cellular immediate early genes such as c-fos, c-jun, jun-B, and egr-1. fra-1, a member of the immediate early gene family, follows a distinctly later time course of induction than c-fos, c-jun, jun-B, and egr-1, suggesting that fra-1 may attenuate the action of genes induced earlier. We demonstrate that constitutive expression of fra-1 in PC-12 cells results in pronounced inhibition of NGF-induced differentiation. Transcriptional activation of c-fos, c-jun, jun-B, and egr-1 by NGF, EGF, and db cAMP was down-regulated to a varying extent whereas NGF-induced ornithine decarboxylase (ODC) was not affected. Expression of jun-D was not affected in PC-12 fra-1 cells. Transfection of fos and egr-1 promoter-chloramphenicol acetyl transferase (CAT) plasmid into these stable fra-1-expressing PC-12 cells revealed that repression of fos and egr-1 was exerted at the promoter level. Thus deregulated fra-1 expression may inhibit PC-12 cell differentiation by altering the patterns of immediate early gene expression.

Adrenal Gland Neoplasms↗