Biomedical subjects

J Ochieng

Publications and source records attributed to J Ochieng.

25 records · Page 2Linked to original sources

PubMed1993

Structure-function relationship of a recombinant human galactoside-binding protein.

A galactoside-binding lectin (hL-31) containing a collagen-like sequence was identified in human tumor cells. It was found to be the homologue of the IgE-binding protein, the macrophage cell-surface Mac-2 antigen, and the murine CBP35, RL-29, and mL-34 lectins. Here we report on the expression in Escherichia coli and functional analysis of recombinant hL-31 (rhL-31). The rhL-31 was purified in one step through an asialofetuin affinity column. The rhL-31 was reactive to anti-lectin antibodies and retained its lactose-dependent hemagglutination of trypsin-treated glutaraldehyde-fixed rabbit erythrocytes. The rhL-31 elutes from an affinity column as a 31-kDa monomer and undergoes homodimerization at relatively high protein concentrations, comparable to those used to mediate hemagglutination. Electron microscopy showed that the rhL-31 appears as a Y-shaped structure. Lactoperoxidase-catalyzed iodination of murine tumor cell-surface proteins followed by collagenase treatment revealed that the lectin is probably a peripheral membrane protein whereby both the amino and the carboxy termini are exposed on the outer cell membrane. These results point to the membrane disposition and orientation of the lectin and suggest a mechanism for a structure-function relationship of lectin activity.

Amino Acid Sequence↗

PubMed1992

Dichotomy in the laminin-binding properties of soluble and membrane-bound human galactoside-binding protein.

Recent studies indicate that galactoside-binding proteins may bind the poly-N-acetyllactosamine sequences of laminin. We questioned whether human galactoside-binding protein (hL-31) binds to laminin and whether cells that express hL-31 on their surface use it as a laminin receptor to promote cellular attachment. The data show that both lectin and cells bind to immobilized laminin. The binding of soluble lectin to laminin is inhibited by lactose, while cell adhesion to it is not. The results indicate that laminin may be a ligand for soluble galactoside-binding proteins.

Binding Sites↗

PubMed1991

Buffering of intracellular calcium in response to increased extracellular levels in mortal, immortal, and transformed human breast epithelial cells.

Extracellular levels of calcium at 1.05 mM or higher induce terminal differentiation and senescence in the mortal (MCF-10M) line of human breast epithelial cells, but does not retard the growth or induce differentiation in the immortal (MCF-10A) and oncogene transformed (MCF-10AneoT) lines. Intracellular levels of calcium and inositol triphosphate were determined in MCF-10M, MCF-10A, and MCF-10AneoT, under conditions of low and high extracellular calcium. We hereby report that increases in extracellular calcium is translated into significant increases in intracellular levels of calcium and inositol triphosphate in MCF-10M, but not in MCF-10A and MCF-10AneoT. This difference in the apparent calcium buffering capacity between the mortal and the immortalized human breast epithelial cells could account for the latter's unperturbed growth potential in high extracellular calcium environment.

Breast↗

PubMed1991

Increased invasive, chemotactic and locomotive abilities of c-Ha-ras-transformed human breast epithelial cells.

Transfection of the immortalized human breast epithelial cells MCF-10A with the mutated ras oncogene resulted in cell transformation (MCF-10A-neoT). Since the transformed state is usually associated with enhanced migratory activity, increased capability to invade basement membranes and to grow in a three-dimensional basement membrane gel (growth in matrigel), we compared these properties in MCF-10A-neoT cells with those of MCF-10A cells transfected with either the neomycin resistance gene alone (MCF-10A-neo cells) or with the normal ras proto-oncogene (MCF-10A-neoN cells). MCF-10A-neoT cells exhibited enhanced migratory activity, as assessed by chemotaxis and chemokinesis assays. and increased capability to invade the basement membrane. These cells also formed large colonies in matrigel. MCF-10A-neo and MCF-10A-neoN cells, on the other hand, showed only marginal migratory, invasive and semisolid medium growth properties. These results indicate that the mutated ras oncogene induces in human breast epithelial cells phenotypic characteristics of malignant transformation.

Adenocarcinoma↗

PubMed1990

Calcium-mediated modulation of microtubule assembly in human breast epithelial cells.

Normal human breast epithelial cells obtained from a reduction mammoplasty (S130) have been maintained in culture for up to a year in Ham's F12:Dulbecco's medium, with 5% equine serum and a low calcium concentration (0.04 mM). These cells undergo senescence and terminal differentiation if they are switched to high Ca2+ medium (1.05 mM). To clarify the mechanism by which Ca2+ regulates the growth of these cells, we studied the role of tubulin assembly-disassembly and the morphologic changes subsequent to high Ca2+ switch. An early Passage (9) of S130 breast epithelial cells growing in low Ca2+ medium was analyzed. Of a total of 785 counted cells, 720 (92%) were rounded and 65 (8%) were flat, elongated, and fibroblastlike. When the cells were switched to high Ca2+ medium, out of 553 cells, only 111 (20%) were rounded and the remaining 442 (80%) were elongated and fibroblastlike. Immunocytochemical localization of tubulin, using the immunogold silver enhancement technique, showed that the majority of low Ca2(+)-grown cells did not display a network of tubulin fibers, whereas high Ca2(+)-grown cells revealed extensive cytoplasmic network of polymerized tubulin, which seemed to stretch out the cells. Experiments designed to determine the mechanisms of tubulin polymerization in these cells revealed that: a) Cells grown in high Ca2+ medium containing 0.1 mM colchicine had a reduced proportion of elongated cells; b) treatment of the cells with the calcium ionophore A23187 in low calcium medium resulted in an increase in the number of elongated cells which had more polymerized tubulin; and d) treatment of the cells with cyclic-AMP in low Ca2+ medium had no observable effect on cell morphology. These results indicate that high levels of Ca2+ either favor tubulin polymerization or stabilize the polymerized state.

Breast↗

PubMed1989

Altered queuine modification of transfer RNA involved in the in vitro transformation of Chinese hamster embryo cells.

Altered queuine modification of tRNA has been correlated to neoplastic transformation, but no direct cause and effect relationship has been defined. In the present study, a potential role for this alteration has been assigned. The tRNA in normal Chinese hamster embryo cells is significantly more queuine modified than the tRNA in their transformed Chinese hamster embryo counterparts, even though the specific activity of the queuine modification enzyme is much lower in Chinese hamster embryo cells than in transformed Chinese hamster embryo cells. Substrate availability appears to be responsible for the queuine hypomodification of tRNA in the transformed cells, since addition of excess exogenous queuine to the culture medium results in incorporation of queuine into the anti-codon of the undermodified tRNAs. Most importantly, the excess queuine inhibits anchorage-independent growth of transformed Chinese hamster embryo cells, thereby implicating queuine hypomodification of tRNA in the expression of this transformed phenotype.

Animals↗

PubMed1987

Inhibition of phorbol ester-mediated phenotypic changes in cultured cells by hypoxanthine.

Hypoxanthine induces the differentiation of certain transformed cells in vitro, so analyses were undertaken to determine whether this purine metabolite might influence the expression of transformed phenotypes induced in normal cells by chemical agents. Chinese hamster embryo cells and human skin fibroblasts in culture were treated with the promoting agent phorbol-12,13-didecanoate (PDD) with or without prior treatment with 3-methylcholanthrene (MCA), and various phenotypic effects were monitored. Hypoxanthine was found to inhibit significantly the formation of type III foci and the increase in saturation density observed for Chinese hamster cells treated with MCA plus the phorbol ester. Inosine and the hypoxanthine analogue allopurinol could also mediate the effect on saturation density, while xanthosine could not. An increase in the saturation density of human skin fibroblasts, which can be induced by the phorbol ester alone, was also inhibited by hypoxanthine. There was no significant effect on the growth rate or the intracellular nucleotide pools with hypoxanthine-treated cells. The results suggest that a normal purine metabolite, hypoxanthine, can modulate the expression of transformed phenotypes induced in vitro by the known tumor promotor PDD. These observations could help in elucidating the cellular basis for promotion of carcinogenesis.

Animals↗