Biomedical subjects

L M Varela

Publications and source records attributed to L M Varela.

5 recordsLinked to original sources

PubMed2001

Adsorption of an amphiphilic penicillin onto human serum albumin: characterisation of the complex.

The complex formed by the interaction of the amphiphilic penicillin drug nafcillin and human serum albumin (HSA) in water at 25 degrees C has been characterised using a range of physicochemical techniques. Measurements of the solution conductivity and the electrophoretic mobility of the complexes have shown an ionic adsorption of the drug on the protein surface leading to a surface saturation at a nafcillin concentration of 0.012 mmol kg(-1) and subsequent formation of drug micelles in solutions of higher nafcillin concentration. Measurements of the size of the complex and the thickness of the adsorbed layer by static and dynamic light scattering have shown a gradual change in hydrodynamic radius of the complex with increasing drug concentration typical of a saturation rather than a denaturation process, the magnitude of the change being insufficient to account for any appreciable extension or unfolding of the HSA molecule. The interaction potential between the HSA/nafcillin complexes, and the stability of the complexes were determined from the dependence of diffusion coefficients on protein concentration by application of the DLVO colloidal stability theory. The results indicate decreasing stability of the colloidal dispersion of the drug/protein complexes with an increase in the concentration of added drug.

Adsorption↗

PubMed2001

TNFalpha induces NFkappaB/p50 in association with the growth and morphogenesis of normal and transformed rat mammary epithelial cells.

In contrast to the cytotoxic or cytostatic effect of TNFalpha on many breast cancer cell lines, TNFalpha stimulates growth and morphogenesis of normal rat mammary epithelial cells (MEC). The present studies were carried out to determine whether there are intrinsic differences between normal and malignant MEC which may explain the differing responsiveness to TNFalpha. Freshly isolated rat MEC organoids from normal mammary gland or 1-methyl-1-nitrosourea-induced mammary tumors were treated with TNFalpha for 21 days. Unexpectedly, TNFalpha stimulated growth and morphogenesis of both normal and transformed MEC in primary culture, although in transformed cells its effects were delayed and the majority of the colonies were histologically abnormal, with multiple cell layers and no lumen. Since NFkappaB is a key mediator of TNFalpha action and has been implicated in carcinogenesis, the expression of the p50, p52, p65, and c-rel NFkappaB proteins in normal and transformed MEC was determined. Expression of p52 was significantly reduced in tumor cells, and p50 was absent, although its putative precursor, p105 was abundant. There were no changes in the levels of p65 or c-rel. TNFalpha induced a pronounced and sustained increase of a p50 homodimeric NFkappaB/DNA complex in both normal and transformed MEC. However, in transformed MEC, NFkappaB binding was initially undetectable but then increased in response to TNFalpha. Thus, NFkappaB expression and DNA binding activity are altered during mammary carcinogenesis. In addition, the significant increase in NFkappaB/p50 DNA-binding was temporally coincident with TNFalpha-induced growth and morphogenesis, suggesting that it may play a significant role in both normal development and carcinogenesis.

Adenocarcinoma↗

PubMed1999

Selective changes in EGF receptor expression and function during the proliferation, differentiation and apoptosis of mammary epithelial cells.

Epidermal growth factor (EGF) is a multifunctional regulator of mammary epithelial cells (MEC) that transduces its signals through the EGF receptor (EGFR). To clarify the role of the EGFR in the mammary gland, EGFR expression, localization and function were examined during different developmental stages in rats. Immunoblot analysis demonstrated high levels of EGFR during puberty, pregnancy and involution as well as at sexual maturity, and low levels throughout lactation. An immunohistochemical assay was used to show that EGFR was distinctly expressed in a variety of cell types throughout mammary glands from virgin rats and rats during pregnancy and involution, and was down-regulated in all cell types throughout lactation. To examine the relationship between EGFR expression and function, primary MEC were cultured under conditions that induced physiologically relevant growth, morphogenesis and lactogenesis. Cultured MEC expressed an in vivo-like profile of EGFR. EGFR was high in immature MEC, down-regulated in functionally differentiated MEC, and then up-regulated in terminally differentiated and apoptotic MEC. An inhibitor of the tyrosine kinase domain of EGFR was used to demonstrate that EGFR signaling was required for growth and differentiation of immature MEC, and for survival of terminally differentiated MEC, but not for maintaining functional differentiation.

Adenocarcinoma↗

PubMed1997

The epidermal growth factor receptor is not required for tumor necrosis factor-alpha action in normal mammary epithelial cells.

Our laboratory has shown that tumor necrosis factor-alpha (TNF alpha) can regulate normal mammary epithelial cell (MEC) growth, morphogenesis, and, under certain circumstances, functional differentiation in a manner similar to epidermal growth factor (EGF). As TNF alpha has been shown to up-regulate EGF receptor (EGFR) expression and function in other systems, the present studies were undertaken to determine whether TNF alpha action in MEC was indirect through stimulation of the EGFR. An inhibitor of EGFR tyrosine kinase activity, PD158780, failed to block proliferation induced by 40 ng/ml TNF alpha and only partially inhibited growth in response to 2 ng/ml TNF alpha. PD158780 was also unable to suppress the extensive morphological development induced by either TNF alpha concentration. In contrast, the effects of TNF alpha and PD158780 on functional differentiation (i.e. casein accumulation) were time dependent. When measured on day 7 after 48 h of treatment, casein accumulation was unaffected by either concentration of TNF alpha or by PD158780. When assessed on day 21 after 16 days of treatment, however, casein levels were decreased by 40 ng/ml TNF alpha and increased by PD158780. Significantly, this PD158780-induced increase in casein was not observed in MEC that had been treated with both PD158780 and TNF alpha. These results thus suggest that EGFR tyrosine kinase activity is not necessary for TNF alpha action in normal MEC.

Animals↗

PubMed1996

Tumor necrosis factor-alpha: a multifunctional regulator of mammary gland development.

To determine whether tumor necrosis factor-alpha (TNF alpha) plays a physiological role in normal mammary gland development, TNF alpha and TNF receptor expression were measured in epithelial cells (MEC) isolated from mammary glands of virgin, pregnant, lactating, and postlactational (day 7 of involution) rats. TNF alpha messenger RNA (mRNA) increased significantly during pregnancy, then decreased during lactation and involution. The 26-kDa transmembrane form of TNF alpha protein, undetectable in MEC from virgin rats, increased throughout pregnancy and lactation, and disappeared during involution. In contrast, p55 TNF receptor (TNFR) mRNA levels peaked in early lactation and declined thereafter, whereas p75 TNFR mRNA levels rose steadily through lactation. Using agonistic antibodies specific to either TNFR, the individual roles of each TNFR were investigated in MEC in primary culture. The p55 TNFR was found to be the sole mediator of TNF alpha-induced proliferation. Intriguingly, the two receptors were found to have opposing effects on functional differentiation (casein accumulation), with inhibition occurring through the p55 receptor and stimulation through the p75 receptor. Taken together, these results suggest that TNF alpha plays a role in the growth and development of the mammary gland. In addition, both TNF receptors are important for TNF alpha function and may mediate different effects.

Animals↗