Effect of nanosecond pulsed electric field on Escherichia coli in water: inactivation and impact on protein changes.

AIMS: This article shows the effect of nanosecond pulsed electric field (nsPEF) on Escherichia coli, which could imply a durable change in protein expressions and then impacted the phenotype of surviving bacteria that might lead to increase pathogenicity. METHODS AND RESULTS: The effects of nsPEF on E. coli viability and membrane permeabilization were investigated. One log10 reduction in bacterial counts was achieved at field strength of 10(7) V m(-1) with a train of 500 successive pulses of 60 × 10(-9) s. Incubation of germs after treatment with propidium iodide showed that membrane permeabilization was reversible. Possible protein changes of surviving bacteria were checked to assess potential phenotypical changes using two-dimensional electrophoresis. In our study, after 40 generations, only UniProt #P39187 was up-regulated with P ≤ 0·05 compared with the control and corresponded to the uncharacterized protein YtfJ. Antibiograms were used to check whether or not the pattern of cultivable bacteria after nsPEF deliveries changed. CONCLUSIONS: The results tend to show that nsPEFs are able to inactivate bacteria and have probably no serious impact in E. coli protein patterns. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of nsPEF is a safe promising new nonthermal method for bacterial inactivation in the food processing and environmental industry.

Distribution and prognostic value of the fibroblast growth factor-2 low-affinity binding sites in human breast cancer.

We performed a competitive binding study with 125I-labelled FGF (fibroblast growth factor)-2 and unlabelled FGF-2 in an unselected series of two hundred and thirty human primary breast cancers. One hundred and ninety-two breast cancer biopsies possessed FGF-2 low-affinity binding sites (FGF-2 LABS). The median dissociation constant was 2.4 nM (range, 1.03-18) and the median concentration of membrane protein was 6187.5 fmol/mg (range, 831-90,000). FGF-2 LABS concentrations were positively correlated to the progesterone receptor level. Cox univariate analyses showed that the FGF-2 LABS (> or = upper quartile) was associated to a longer overall survival (p = 0.05; RR = 0.042); node involvement, estrogen receptor progesterone receptor and histoprognostic grading were also prognostic. In Cox multivariate analyses, only the progesterone receptor, estrogen receptor, node involvement and FGF-2 LABS were prognostic factors; the FGF-2 LABS were associated with a longer overall survival (p = 0.033; RR = 0.068). The present study showed that FGF-2 LABS have only a limited role as a prognostic factor in breast cancer.

Basic fibroblast growth factor receptors and their prognostic value in human breast cancer.

We performed a saturation binding study with 125I-labeled FGF (fibroblast growth factor)-2 in a nonselected series of 250 human primary breast cancers. Two hundred twenty-five breast cancer biopsies possessed bFGFR (basic FGF receptor). The median dissociation constant was 0.35 nM (range, 0.014-1.9), and the median concentration was 1126 fmol/mg protein (range, 49-7328). FGFR-1 was localized, using a specific monoclonal antibody, in cancerous cells and in epithelial cells in normal breast or in benign tumors. In all of the tissues studied, light stromal cell staining was also observed. Thus, the localization of FGFR-1 in carcinoma cells supports the hypothesis that an important part of FGF-2 binding reflects binding to FGFR-1. bFGFR concentrations were positively correlated to estrogen receptor and progesterone receptor levels. Cox univariate analyses showed that the bFGFR (> or = upper quartile) was associated to longer relapse-free survival [P = 0.004; RR (risk ratio), 0.46] and overall survival (P = 0.001; RR, 0.35); age, estrogen receptor levels, progesterone receptor levels, node involvement, tumor diameter, and histoprognostic grading were prognostic, also. In Cox multivariate analyses, only the bFGFR, age, node involvement, and histoprognostic grading were prognostic factors; the bFGFR was associated with longer relapse-free survival (P = 0.03; RR, 0.4) and overall survival (P = 0.009; RR, 0.3). The present study confirms that FGF could be an important regulator of human breast cancer growth and that patients with a high level of bFGFR had a better prognosis.

Differential growth factor production, secretion, and response by high and low metastatic variants of B16BL6 melanoma.

Low levels of tyrosine and phenylalanine alter the metastatic phenotype of B16BL6 murine melanoma. In this study, we investigated expression and secretion of fibroblast growth factor-like (FGF-like) and transforming growth factor beta-like (TGF beta-like) molecules as well as the biological effect of basic FGF (bFGF) and TGF beta 1 on high (NDP) and low (LTP) metastatic variants of B16BL6 melanoma. Both NDP and LTP cells expressed bFGF-like and TGF beta-like polypeptides as detected by Western blot analysis. An M(r) 29,000 bFGF-like form eluted from heparin-Sepharose by 0.6 M NaCl was found in extracts of both NDP and LTP cells. Elution at 0.6 M NaCl suggested that this M(r) 29,000 form might be more closely related to FGF-5 than to bFGF. In addition, cell extracts of LTP, but not NDP cells, contained an M(r) 47,000 monomeric bFGF-like form that was not retained on heparin-Sepharose. Three major specific immunoreactive forms of M(r) 44,000, 36,000, and 29,000 were present in conditioned medium from NDP cells. The M(r) 29,000 form present in the conditioned medium of NDP cells was retained on heparin-Sepharose. Only the M(r) 44,000 and 36,000 FGF-like molecules were detected in conditioned medium from LTP cells, and they were also not retained on heparin-Sepharose. Anti-TGF beta antibody that recognized both TGF beta 1 and TGF beta 2 detected 3 different TGF beta-like forms (M(r) 25,000, 23,000 and 22,000) in NDP and LTP cell extracts. Conditioned medium from NDP cells contained an M(r) 38,000 form of TGF beta; however, no immunoreactive forms were found in conditioned medium from LTP cells. Thus, the NDP-LTP differences in this melanoma system were primarily in growth factor secretion, not expression. The effect of exogenous bFGF and TGF beta 1 on proliferation of LTP and NDP cells was determined by [methyl-3H]thymidine uptake. bFGF stimulated proliferation of NDP cells; whereas, LTP cells exhibited no increase in proliferation. Both NDP and LTP cells responded to TGF beta 1. Proliferation of NDP cells was inhibited more by this growth factor than was proliferation of LTP cells. When NDP and LTP cells were incubated with 5 ng/ml TGF beta 1 and various amounts of bFGF, the effect of TGF beta 1 was masked. Antibody depletion of bFGF-like molecules from NDP conditioned medium resulted in the decreased proliferation of NDP cells but not LTP cells. Depletion of TGF beta-like molecules resulted in increased proliferation of LTP cells but did not affect NDP cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Basic fibroblast growth factor is a testicular germ cell product which may regulate Sertoli cell function.

Previously, a proteinacious factor secreted by a mixture of rat testicular spermatocytes and round spermatids was shown to stimulate transferrin mRNA and protein levels in Sertoli cells. To identify the germ cell-secreted proteins which affect Sertoli cell functions, concentrated germ cell-conditioned medium was fractionated by reverse-phase HPLC. The fraction which eluted at 35% acetonitrile increased transferrin secretion in Sertoli cell cultures 2.4-fold above the basal level. Both the active fraction and a protein extract from cultured germ cells were positive for basic fibroblast growth factor (bFGF) as determined by Western blot analysis and immunoprecipitation. The apparent molecular sizes of the immunoreactive proteins were 30, 27, and 24 kilodaltons (kDa). By immunohistochemistry, bFGF was shown to be present in pachytene spermatocytes and Leydig cells. The bFGF receptor was also examined by immunohistochemistry and found to be present in Leydig cells, round and elongated spermatids, and Sertoli cells. The presence of receptors was more pronounced in stages I-VIII. Western blot analysis confirmed that the receptors were expressed in isolated round spermatids, elongated spermatids, and Sertoli cells. Two major receptor species with apparent molecular sizes of 120 and 145 kDa were detected in the rat testis. Germ cells contained both of these receptors, but Sertoli cells possessed only the 120-kDa receptor. From these experiments, it is evident that bFGF is a germ cell product which may regulate Sertoli cell function. The expression of bFGF and its receptor may be an important component of germ cell-Sertoli cell and/or germ cell-germ cell communication during spermatogenesis.

Production of anti-fibroblast growth factor receptor monoclonal antibodies by in vitro immunization.

Monoclonal antibodies against the high affinity tyrosine kinase Fibroblast Growth Factor (FGF) receptor may help define receptor epitopes involved in FGF binding and signal transduction which mediate coronary and tumor angiogenesis (the development of blood vessels). Monoclonal antibodies against the FGF receptor were made by in vitro immunization. Receptor was PAGE-purified and transblotted to nitrocellulose. The nitrocellulose was dissolved with acetonitrile, and the receptor used as antigen in an in vitro immunization system. Screening for FGF receptor positive clones was done using membrane preparations from Coronary Venular Endothelial Cells (CVEC) expressing the FGF receptor, both by Enzyme Linked Immunosorbent Assay (ELISA) and Western blot. Culture supernatants of several clones tested positive for IgM or IgG monoclonal antibodies against the FGF receptor. Antibodies were affinity purified. Ascites fluid was produced in Balb/c mice primed with Incomplete Freund's adjuvant (IFA). The monoclonal antibodies were also found to be suitable for receptor immunoprecipitation. Immunocytochemistry was done on sections from a variety of species. Further characterization of these antibodies, as well as the production of the remainder of a panel of anti-FGF receptor monoclonal antibodies, is underway.

Evidence of high and low affinity binding sites for basic fibroblast growth factor in mouse placenta.

The placenta has been shown to contain bFGF, but the presence of specific binding sites for this growth factor in this tissue remained to be established. In order to study the role of bFGF in the placenta growth, we looked for specific binding sites on mouse placental cell membranes at days 12, 14, 16, and 18 of pregnancy. At day 12, Scatchard analyses indicated that two classes of specific interaction sites for bFGF were detected. One class of high affinity binding sites was characterized by an apparent Kd of 10 pM and a binding capacity of 10 fmoles per mg of membrane protein. A second class of low affinity binding sites was detected with an apparent Kd of 60 nM and a binding capacity of 26 pmoles per mg of membrane protein. At days 14, 16 or 18, Scatchard analyses only showed low affinity binding sites with an apparent Kd of 24 nM and a binding capacity of 230 pmoles per mg of membrane protein. The characterization of these binding sites was performed by cross linking experiments that revealed two forms of specific complexes. This result suggested that the high affinity binding sites correspond to putative receptors with relative molecular masses equal to 65,000 and 85,000. The dramatic decrease of the high affinity receptor number after the 12th day of pregnancy, which is synchronous with the 9-fold increase of the low affinity binding site number, suggests that the biological activity of bFGF could be regulated by a balance between both the numbers of high and low affinity binding sites on placenta cell membranes. Thus, as it was shown for other growth factors, bFGF could only be involved at specific pregnancy stages.