Response of human epithelial cells to culture surfaces with varied roughnesses prepared by immobilizing dendrimers with/without D-glucose display

To investigate the response of human epithelial cells to substrates with nanoscale modifications, dendrimer-immobilized surfaces were prepared with or without D-glucose displayed as a terminal ligand, giving topographic structures with mean roughnesses (R(a)) of 1.8-11.0 nm. With an increase in the R(a) value up to 4.0 nm, the epithelial cells cultured on naked dendrimer surface without D-glucose display were somewhat stretched in their morphology compared with those on a nonmodified plain surface. However, for the R(a) values higher than 4.0 nm, such cell stretching was inhibited, resulting in the predominant existence of round-shaped cells. The change in cell morphology was appreciable on the surfaces with D-glucose-displayed dendrimers. When the R(a) value increased up to 4.5 nm on these surfaces, in particular, the enhancement of cell stretching was recognized, and fluorescence microscopic observation supported the hypothesis that the glucose-transporter-mediated adhesion of cells to the surface encouraged the development of filopodia and stress fibers, thereby improving focal contact with the surface. Our results suggest that the combination of displaying D-glucose and modulating roughness can promote cytoskeletal formation accompanied by marked cell elongation on culture surfaces.     

Lack of effect of epidermal growth factor treatment in late-pregnant ewes on subsequent lactation

Twin-bearing ewes were treated with epidermal growth factor (EGF) to determine its effect on mammogenesis and resultant milk production and composition. The EGF was infused intravenously at a dose rate of 0.5 mg/d in 300 ml saline between days 117 and 139 of gestation; control animals received placebo infusions of saline. All animals then received continuous infusions of 300 ml/d saline on days 139-144. Following parturition 1-5 d later, ewes were milked by hand for 10 d and thereafter were machine-milked until day 16 of lactation. At this level of treatment, EGF was not detected in the circulation during infusion and feed intake was not affected. All ewes gave birth to healthy twin lambs. There were no effects of EGF on birth weights of lambs, live weights of ewes or lengths of gestation. An EGF-immunoreactive material was detected in the mammary secretions of control ewes at a mean concentration of 2 micrograms/l on day 1 of lactation. Two ewes had detectable levels on day 2, but none was found in the milk thereafter. In the EGF-infused group, concentrations of EGF in colostrum were approximately 10 times higher than in the control ewes on day 1 of lactation and EGF was detected in mammary secretions on day 2 but not in subsequent milk samples. A range of 0.3-0.5% of the EGF infused appeared in mammary secretions over the first 2 d of lactation. No other differences were observed for colostrum composition, subsequent milk yield or composition between the two groups of ewes indicating that mammary gland development and function were unaffected. The levels of EGF observed in the mammary secretions of treated and control ewes indicate that the mammary glands accumulate and store EGF in the pre partum period.     

Hepatocyte growth factor exerts multiple biological functions on bovine mammary epithelial cells

The met proto-oncogene product Met is a member of the family of tyrosine kinase growth factor receptors, and hepatocyte growth factor/scatter factor (HGF/SF) has been identified as its only ligand. Bovine Met and HGF/SF have been recently cloned and their expression has been characterized in the mammary gland, but no data regarding the biological effects of this ligand/receptor couple in bovine mammary cells are yet available. We examined the role of HGF/SF and its receptor in a bovine mammary epithelial cell line (BME-UV). Expression of Met at the mRNA level in BME-UV mammary epithelial cells evaluated by real-time PCR was similar to the expression in MDCK cells, a widely used model for Met biology. Met expression in BME-UV at the protein level was confirmed by western blot. The analysis of some signal transductional pathways downstream from the Met receptor revealed that HGF/SF addition to BME-UV cells induced activation of the extracellular signal-regulated kinase ½ proliferative pathway and the Akt antiapoptotic pathway. The BME-UV cells treated with HGF responded with increased proliferation, cell scatter, and motility. Met activation by HGF induced degradation of the extracellular matrix and migration through matrigel coated transwells. Moreover, BME-UV cells included in a 3-dimensional matrix of collagen and treated with HGF developed tubular structures, reminiscent of the mammary gland ducts. These data indicate that HGF and Met might be important regulators of mammary gland growth, morphogenesis, and development in the bovine.     

Effect of sialoadenectomy on the ability of mouse serum to induce deoxyribonucleic acid synthesis in mammary epithelial cells: possible role of epidermal growth factor

Serum was collected from sham-operated or sialoadenectomized mice and added to serum starved cultures of normal mouse mammary epithelial cells. Synthesis of DNA, estimated with a pulse of 1 microCi/ml [3H]thymidine between 17 and 18 h after adding serum, was increased by serum. Sialoadenectomy did not affect the ability of low serum concentrations (less than 4%) to increase DNA synthesis, but DNA synthesis induced by higher serum concentrations was reduced by sialoadenectomy. Physiological concentrations of epidermal growth factor restored the ability of serum from sialoadenectomized mice to increase DNA synthesis. Antiepidermal growth factor reduced DNA synthesis in cultures treated with high concentrations of serum from sham-operated mice, but not in cultures treated with serum from sialoadenectomized mice. Time course studies indicated that the time of maximum DNA synthesis was similar between cultures treated with serum from sham-operated and sialoadenectomized mice. Treatment with serum from sialoadenectomized mice for 4 d resulted in lower cell number than did treatment with serum from sham-operated mice. This effect of sialoadenectomy could be overcome by epidermal growth factor. These data indicate that sialoadenectomy, which has previously been shown to depress mammary gland development, alters the endocrine status of mice, such that serum from sialoadenectomized mice is less effective in inducing DNA synthesis by mammary epithelial cells. These differences in ability of serum to induce DNA synthesis can be explained by differences in epidermal growth factor concentration or the concentration of epidermal growth factor-like constituents.     

Epidermal growth factor and hepatocyte growth factor receptors collaborate to induce multiple biological responses in bovine mammary epithelial cells

The aim of this work was to explore whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) could increase the biological responses of a mammary epithelial cell line of bovine origin when added simultaneously. We also investigated a possible molecular mechanism underlying this cooperation. The development of mammary gland requires several circulating and locally produced hormones. Hepatocyte growth factor and its tyrosine kinase receptor, mesenchymal-epithelial transition factor (MET), are expressed and temporally regulated during mammary development and differentiation. Epidermal growth factor receptor and its ligands have also been implicated in the growth and morphogenesis of the mammary epithelium. Both EGF and HGF seem to exert a morphogenic program in this tissue; therefore, we hypothesized that these cytokines could act cooperatively in bovine mammary epithelial cells. We have already shown that the bovine BME-UV cell line, a nontumorigenic mammary epithelial line, expresses both MET and EGF receptor. Simultaneous treatment with HGF and EGF elicited an increase in proliferation, dispersion, degradation of extracellular matrix, and motility. Following EGF treatment, BME-UV mammary cells exhibited an increase in MET expression at both the mRNA and protein levels. Long-term treatment of BME-UV cells with HGF and EGF together increased the level of activation of the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathways when compared with HGF or EGF alone. These data outline a possible cooperative role of the EGF and HGF pathways and indicate that cross-talk between their respective receptors may modulate mammary gland development in the cow.     

Substratum modulation of epidermal growth factor receptor expression by normal mouse mammary cells

Normal mouse mammary cells synthesize the basement membrane scaffold on which the cells rest in vivo. This extracellular matrix material serves important functions in the growth and differentiation of the mammary epithelium. Components of the basement membrane negatively regulate basement membrane collagen biosynthesis in response to epidermal growth factor stimulation. This effect is shown to correlate with changes in growth factor receptor regeneration following ligand-induced receptor down-regulation. The results suggest that while control of basement membrane synthesis may be manifest in part by the availability of growth factors, the environment in which the mammary cell finds itself is also very important.     

Effects of epidermal growth factor,interleukin 1 and nitric oxide on prostaglandin production by guinea-pig uterus

Initial experiments in the present study investigated the effects of epidermal growth factor (EGF), interleukin 1beta (IL-1beta) and sodium nitroprusside (a nitric oxide donor) on the output of prostaglandins from guinea-pig uterus on day 7 of the oestrous cycle. Superfusion of day 7 guinea-pig uterus in vitro with either EGF or sodium nitroprusside increased the output of PGF(2alpha) and 6-keto-PGF(1alpha), but not of PGE(2). IL-1beta had no effect on the output of these three prostaglandins. EGF still increased the output of PGF(2alpha), but did not increase the output of 6-keto-PGF(1alpha) in a calcium-depleted superfusate. Subsequent experiments investigated the effect of sodium nitroprusside on contractile activity of day 7 guinea-pig uterus. Basal spontaneous activity of both the intact uterus and isolated myometrium superfused in vitro was low. Sodium nitroprusside increased the contractile activity of these tissues two- to fourfold. EGF did not affect the contractile activity of the uterus, indicating that sodium nitroprusside-induced contractions are not due to increased prostaglandin production. Overall, the findings indicate that EGF and nitric oxide may act as mediators in the mechanism by which oestradiol acting on a progesterone-primed uterus stimulates the increase in PGF(2alpha) production by the guinea-pig uterus necessary for luteolysis. Nitric oxide may increase the spontaneous activity of the uterus when this activity is low.     

Effect of Bacillus cereus exocellular factors on human intestinal epithelial cells

To gain insight on the biological effects of the exocellular factors produced by Bacillus cereus, culture filtrate supernatants of different strains were coincubated with differentiated Caco-2 cells. Exocellular factors were able to detach enterocyte-like cells from the substratum after 1 h of incubation. In addition, microvilli effacing and dramatic changes on the cellular surface of enterocytes were found after incubation periods as short as 20 min. Since cell detachment was not inhibited by fetal calf serum, thiol activated cholesterol-binding cytolysin, cereolysin O, does not seem to be involved. Also, translocation of phosphatidylserine from the inner to the outer leaflets of the plasma membrane was demonstrated by using fluorescein isothiocyanate (FITC)-Annexin V. In contrast to the high capability of detaching Caco-2 cells shown by all the strains under study, the mitochondrial dehydrogenase activity was lowered by culture filtrate supernatants in a strain-dependent manner. For strain M2, the decrease in dehydrogenase activity was already evident after 30 min of incubation. Production of biologically active factors depends on the growth phase, and maximal activity was found in late exponential-early stationary phases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of concentrated exocellular factors showed a very complex scenery supporting the multifactorial character of the biological activity of B. cereus.     

Synergistic effect of nisin and heat treatment on the growth of Escherichia coli O157:H7

A combination of nisin and heat treatment was found to inhibit Escherichia coli O157:H7 effectively. After organisms were heated at 50, 52.5, and 55 degrees C for 5, 10, and 15 min, respectively, nisin was incorporated into the plates of E. coli O157:H7 at 0, 25, 50, and 100 IU/ml. The concentration of 100 IU/ml nisin significantly inhibited the growth of E. coli O157:H7 heated at 50 and 52.5 degrees C for 15 min. Nisin treatment at 100 IU/ml for 6 h resulted in the elimination of E. coli O157:H7 heated at 55 degrees C for 10 and 15 min.     

Resveratrol metabolites have an antiproliferative effect on intestinal epithelial cancer cells

Trans-resveratrol (RV) is an active polyphenol with numerous physiological properties including antitumour activity, especially in colon cancer. RV is metabolized in the intestine and then in the liver to sulphated and glucuronidated forms that are exported to target organs. After exerting their effects, they are eliminated in the urine and stools. There are few and contradictory findings on the biological effects of RV metabolites. On the basis of RV metabolism, we selected three metabolites RV 3-O-sulphate, RV 3-O-glucuronide and RV 4′-O-glucuronide, and studied their effects on cell growth inhibition, the cell cycle and apoptosis using human adenocarcinoma cell line (Caco-2 cell) cultures.     

The protective effect of chlorogenic acid on bovine mammary epithelial cells and neutrophil function

Chlorogenic acid (CGA) is the ester of caffeic acid and quinic acid and plays an important role in antibacterial activity and anti-inflammatory properties. The objective of this study was to examine the effects of CGA on the growth of Staphylococcus aureus and the mRNA levels of the genes encoding the inflammatory response cytokines, κ-casein, and neutrophil function in bovine mammary epithelial cells (BMEC) exposed to S. aureus. Chlorogenic acid has important antibacterial, antioxidant, and anti-inflammatory functions; however, the effect of CGA on BMEC and neutrophils exposed to S. aureus has not been investigated previously. Our results demonstrated that 10, 20, and 30 μg/mL CGA had no cytotoxic effects on BMEC in culture, and that 20 μg/mL CGA enhanced the viability of BMEC exposed to S. aureus, whereas 30 μg/mL CGA reduced S. aureus growth after 9 h compared with controls. The rate of S. aureus invasion into BMEC was also attenuated by 30 μg/mL CGA compared with controls, whereas this treatment led to reduced abundance of IL6, IL8, and TLR2 mRNA in S. aureus-exposed BMEC. Migration of bovine polymorphonuclear leukocytes was significantly decreased in S. aureus-exposed BMEC with 10 and 20 μg/mL CGA treatment when compared with S. aureus treatment alone. In addition, incubation with 20 or 30 μg/mL CGA enhanced the phagocytic ability of polymorphonuclear leukocytes compared with the control group. Importantly, levels of κ-casein were enhanced by treatment of S. aureus-exposed BMEC with CGA. Our results suggest that the use of CGA may be a potent therapeutic tool against bovine mastitis caused by S. aureus.     

Synergistic effect of sous-vide and fruit-extracted enzymes on pork tenderization

Food Science and Biotechnology - A combination of sous-vide (SV) and enzymatic treatment (one commercial, Neutrase (NE), and two fruit-extracted enzymes obtained from kiwifruit and pineapple, KE...     

Synergistic effect of active oxygen species and alginate on chitinase production by Wasabia japonica cells and its application

The specific chitinase productivity of a Wasabia japonica cell suspension culture under pure oxygen aeration was 3.8 times higher than that of a suspension culture aerated with ordinary air. During aeration with pure oxygen, both oxygen consumption by the cells and the H2O2 concentration in the medium increased. Addition of H2O2 to the cultivation medium also promoted the specific chitinase productivity. H2O2 could pass freely through the cell membrane. It was assumed that the excess oxygen was converted into active oxygen species such as H2O2, and that the promotion of chitinase production was probably due to the generated active oxygen species. Addition of alginate oligomer (AO, an endogenous elicitor-like substance) to cultures aerated with pure oxygen or supplemented with H2O2 resulted in synergistic increases in chitinase production. Based on these results, the development of a simple and efficient chitinase production system was investigated. Cells were immobilized in alginate gel (instead of adding AO to the medium) and cultivated in a medium containing H2O2. The specific chitinase productivity increased to the levels observed in the suspension culture system. During repeated batch cultivation of immobilized cells, the chitinase production remained stable for three repeated batches. When immobilized protoplasts were cultivated in a medium containing H2O2, there was 7-fold increase in chitinase production compared with that of immobilized cells.