Biomedical subjects
Publications and source records attributed to S G Rao.
Prostatic inhibin purified from human seminal plasma (10.7 kDa, 94 amino acids) is very well known for its endocrine action on pituitary to suppress synthesis and secretion of FSH. In the present report we have revealed its antiproliferative action on two fibroblast cell lines, NRK-49F (ED50 = 2.5 ng/ml) and Balb/c 3T3 (ED50 = 24.5 ng/ml) which may mark its emergence as a negative growth regulator.
The haematopoietic microenvironment which is provided by the stromal cells and extracellular matrix elements has been shown to influence the growth and differentiation of haematopoietic cells. However, most of the information thus far available has been derived from experiments performed using Long Term Bone Marrow Cultures and there is as yet no information regarding the haematopoietic microenvironmental status of the developing liver. Stroma were therefore established using 13-day embryonal liver cells, at a point in time when the liver shows peak haematopoietic activity. These stroma when compared to bone marrow stroma showed differences in histochemical staining. 2M NaCl extracts from 13-day embryonal liver were capable of binding pluripotent stem cells and on SDS-PAGE showed a 14 kDa band of increased intensity. The extracts could also induce erythroid colonies in a semisolid medium. However, 13-day embryonal liver stroma were unable to maintain bone marrow stem cells in culture and overall results would suggest differences between the stromal components of bone marrow and embryonal liver.
Haemopoiesis in mammals takes place in yolk-sac and in mouse it can be detected on the 7th day of gestation. Erythropoietin (EPO) responsive cells can be detected from 7th day onwards. However, the cells committed to the myeloid lineage which can respond to the haemopoietic growth factor (viz. granulocyte macrophage colony stimulating factor; GM-CSF) can be demonstrated only on 10th day of gestation. At the same time, the 12-day spleen colony forming cells i.e. the late colony forming unit spleen (CFU-s) which are multipotent stem cells can also be detected. Data suggest that the stem cells seen in the embryo from 7-10 days of gestation may be a primitive population confined only to the yolk-sac. Liver haemopoiesis which begins in the liver of 13-day embryos is due to primitive haemopoietic pluripotent stem cells, arising de novo in the embryo and not in the yolk-sac, since no primitive pluripotent stem cells capable of repopulating lethally irradiated bone-marrow can be detected in the yolk-sac.
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CML, a myeloproliferative clonal disorder of myeloid stem cells, is characterized by the consistent presence of a bcr-c-abl fusion gene which is formed as a result of a translocation of the c-abl gene from chromosome 9 to downstream of the bcr gene on chromosome 22 (ph'). Current approaches to the treatment of CML are chemotherapy (conventional or aggressive with immuno-modulators) and bone marrow transplantation (BMT). Neither of the above treatment modalities results in long-term remission or cure. Hence, an alternative approach which aims at correcting the genetic defect should be considered. Taking advantage of the consistent abnormal presence of the bcr-c-abl gene in the treated and untreated CML patients at all stages, a gene therapy at the level of blocking mRNA might be considered. Such an antisense RNA therapy should include removal of patient's bone marrow, administration of the gene for constitutive expression of an antisense RNA for the bcr-c-abl fusion gene into the myeloid stem cells and reinjecting the engineered marrow into the patient. Such an approach, comparable to autologous BMT, will have the advantages of absence of graft rejection and possibility of 100% remission. The possible nature of the gene construct for such an antisense RNA therapy is discussed.
In this paper the probable mechanism of the haemopoietic pluripotent stem cell (PSC) homing into the bone-marrow is investigated. Experiments were done to understand the role of fibronectin hexapeptide and ubiquitin. Attempts to isolate the PSC adherence molecule were made. A new adherence assay for PSC has been described.
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Urolithiasis, following implantation of Zn discs in urinary bladder (foreign body insertion technique), was examined in albino rats of either sex. Marked variation was observed between sex, regarding the formation of bladder stones. Ethylene glycol (1%) mixed in drinking water for 4 weeks, was unable to augment Zn disc-induced stone deposition. Chemical nature of stones was identified as of magnesium ammonium phosphate type. Neither urinary pH nor infection in the urinary bladder/tract affected chemical nature and quantity of stone formed. There was no significant influence of electrolytes or metabolic products on the uroliths. No correlation could be drawn between the quality and quantity of uroliths formed and the urinary electrolytes concentration. M. Pudica was not effective in either preventing stone deposition or dissolving preformed stones.
Recent findings that retinoic acid (RA) induces terminal granulocytic differentiation of the human promyelocytic leukemia cell line HL-60 in vitro and blast cell maturation in patients suffering from acute non-lymphocytic leukemia (ANLL) prompted an investigation on the ability of this agent to induce terminal maturation in blast cells from ANLL patients in vitro. We tested the ability of RA at 3 x 10(-6) M, 3 x 10(-7) M and 3 x 10(8-) M concentrations to induce differentiation in blastoid cells from 16 patients with ANLL using cytochemical and cytologic parameters, in addition to cytofluorometric methods. Leukemic cells in primary culture from all the patients underwent cytochemical and biochemical changes after treatment with RA. However, the extent of differentiation-positive cell clones (D+ clones) varied from patient to patient. Morphologic maturation was observed in a significant number of bone marrow samples. Leukocyte alkaline phosphatase and NBT reduction ability of cells, which are biochemical markers of granulocytic differentiation, were also significantly increased with a simultaneous decrease in DNA and RNA synthesis (which was estimated using a Phywe ICP-11 impulse flow cytometer).
The bone-marrow microenvironment has a decisive role in maintaining haemopoietic stem cells and regulating their differentiation. In diseases of the haemopoietic system, viz. anaemias and leukemias, the microenvironment has been shown to play a key role. In this paper we show that leukemic cell conditioned medium and sera from CML patients, interact with the in vitro bone-marrow environment and bring about changes which affect the maintenance of normal stem cells.
A growth-promoting polypeptide has been purified about 1950-fold from rat abdominal muscle by gel chromatography, isoelectric-focusing (IEF) and reverse phase high pressure liquid chromatographic (RP-HPLC) techniques. This factor is an acidic protein with an isoelectric point (pI) of 3.4 and a mol. wt of 12 kDa when run on SDS-polyacrylamide gels under reducing conditions. It is acid and heat-stable but is sensitive to reducing agent and protease action. It stimulates [3H]thymidine incorporation in NRK-49F and NIH 3T3 cells and induces soft agar colony formation of NIH 3T3 cells. The muscle-derived mitogen appears to differ from other known growth factors (GFs) in its physico-chemical properties.
Peripheral blood lymphocytes (PBL) from chronic myeloid leukemia (CML) patients in remission were stimulated in vitro, in a 3-cell assay with autologous leukemic cells or autologous bone marrow (BM) cells alone, or each in combination with allogeneic PBL. The responder cells were used as effectors in a 4-h 51Cr release cytotoxicity assay using autologous targets such as leukemic cells, BM cells, phytohemagglutinin-induced lymphoblasts, and allogeneic K562 (erythroblastoid leukemic cell line) target cells. Sensitization of lymphocytes from CML patients with either autologous leukemic cells or BM cells generated cytotoxic cells (CTCs) capable of killing both the targets. These results suggested that in CML, the PBL may have been sensitized to myeloid maturation-releated antigens in vivo, which, on secondary stimulation in vitro, may result in differentiation of CTCs cytotoxic to immature myeloid cells, either from autologous leukemic cells or autologous BM. The inability of PBL from patients with oral cancers to lyse autologous BM cells upon in vitro stimulation, supported this possibility. Clonogenic assays conducted to assess the colony forming potential of BM cells which had interacted with CTCs indicated that there was about 37% reduction in committed granulocyte stem cell colony formation without an appreciable change in committed granulocyte/monocyte stem cell units and clusters. Therefore, since the BM toxicity of the CTCs is not very high, these cells may have a potential clinical use in CML.
In serum from chronic myelogenous leukemia (CML) patients an activity has been demonstrated, which recruits more CFU-GM (cells committed to granulocyte-macrophage lineage) from normal human bone marrow into clonal proliferation as shown by standard agar colony forming assay, in the presence of supramaximal levels of colony stimulating activity. All sera from CML patients at diagnosis (before therapy) were tested and found to be significantly (P less than 0.001) positive for the recruitment activity. This observation led us to believe that younger or more pluripotent CFU-GM which were hitherto non-responsive to colony stimulating activity become responsive in the presence of CML sera. We investigated whether more pluripotent stem cells (CFUs) are stimulated to proliferate in the chronic phase of CML than at diagnosis (before therapy). An activity was detected which recruits more spleen colony forming cells (CFUs) from normal Swiss mouse bone marrow into the cell cycle as shown by a significant increase (P less than 0.001) in the thymidine suicide index. However both these activities were either lowered or undetectable in normal (blood groups AB) serum. These results indicate that CML sera may contain enhanced levels of early growth factors which stimulate proliferation of pluripotent stem cells resulting in an increased CFU-GM pool in CML. It is suggested that this activity may be responsible for the myeloid hyperplasia associated with CML in the chronic phase.
Transforming growth factors (TGFs) are implicated in malignancy, therefore qualitative and quantitative differences of these growth factors in chronic myelogenous leukemia (CML) in chronic phase has been investigated in this report. Induction of anchorage independent growth of BALB/c 3T3 and NRK-49F fibroblasts was used as an assay to detect TGF-beta activity in sera, serum free leukocyte and stromal conditioned medium of CML patients as well as normal subjects. The data shows that enhanced levels of transforming growth factor (type beta like activity) are detectable in the sera of chronic myelogenous leukemia patients. We believe that the enhanced levels of TGF-beta type activity may have a role in myeloid hyperplasia characteristic of CML patients in chronic phase of the disease.
A new method has been developed by which human myeloid progenitor cells can be grown on nitrocellulose paper. The method uses human placental conditioned medium for colony-stimulating activity in an agar layer while the cells grow on the overlying nitrocellulose paper in compact colonies containing granulocytic and macrophage cells. The importance of this method is discussed in the light of its usefulness in carrying out molecular studies on differentiating hematopoietic cells.
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