Variable electric fields for high throughput electroporation protocol design in curvilinear coordinates

The mathematical solution to the electric field equation in cylindrical coordinates, has suggested to us a new experimental methodology and device for reducing experimental effort in designing electroporation protocols. Using a new cylindrical electroporation system, we show, with an Escherichia coli cell model, how key electroporation parameters emerge precisely from single experiments rather than through interpolation from numerous experiments in the conventional Cartesian electroporation system.     

Lipophilic phosphonium-lanthanide compounds with magnetic, luminescent, and tumor targeting properties

Multifunctional phosphonium-lanthanide compounds that simultaneously possess paramagnetism, luminescence, and tumor mitochondrial targeting properties were prepared by use of a facile method. These compounds were fully characterized by use of ¹H, ¹³C, ³¹P NMR, FT-IR, and elemental analyses. The thermal properties of these compounds including melting points and decomposition temperatures were investigated using DSC and TGA analyses. In addition, the paramagnetism, luminescence, and tumor targeting properties of these multifunctional compounds were confirmed by respective use of SQUID, fluorescence, and cell cytotoxicity studies. All compounds exhibited paramagnetism at room temperature, which could provide target delivery of these compounds to parts of the body containing tumor cells using a strong external magnetic field. In addition, these compounds display two major characteristic emissions originating from Dy^3 +, which can be utilized for imaging tumor cells. The IC₅₀ values of these compounds measured against normal breast cell line (Hs578Bst) are significantly greater than those measured against the corresponding carcinoma breast cell line (Hs578T), clearly indicating the selective tumor targeting properties of these compounds. Confocal fluorescence microscopy studies were used to confirm the yellowish-green fluorescence corresponding to the emission of dysprosium thiocyanate anion within cancer cells upon exposure of cancer cell lines such as human pancreatic carcinoma cell line (MIAPaCa-2) and human breast carcinoma (MDA-MB-231) to a solution of these phosphonium-dysprosium compounds.     

A simple and reliable EDDA method for mycobactin production in mycobacteria: Optimal conditions and use in mycobacterial speciation

Ethylenediamino-di-(0-hydroxyphenylacetic acid) (EDDA) was found to be a suitable iron chelator for investigating mycobactin production in mycobacteria. The optimal conditions of EDDA concentration and time of exposure were established and were used to develop a twostep method for mycobactin production. Applied to representative strains of selected species, the method was found to yield reliable results useful for identification and speciation of mycobacteria.     

Characteristics of human types 1, 2 and 3 17β-hydroxysteroid dehydrogenase activities: Oxidation/reduction and inhibition

Following transfection of types 1, 2 and 3 17β-hydroxysteroid dehydrogenase (17β-HSD) cDNAs into transformed embryonal kidney (293) cells, we have characterized the selective directional and inhibitory characteristics of these activities. While homogenates of transfected cells could catalyze interconversion of the substrate and product, in agreement with the general belief on the activity of these enzymes, the same activities measured in intact cells, in order to better reflect the physiological conditions, showed an unidirectional reaction. Types 1 and 3 17β-HSD catalyzed the reduction of estrone to estradiol and 4-androstenedione to testosterone, respectively, while type 2 17β-HSD catalyzed the oxidative transformation of both testosterone and 17β-estradiol to 4-androstenedione and estrone, respectively. In addition, types 1, 2 and 3 17β-HSD activities showed different pH optima. While types 1 and 3 showed pH optimum values centered at around 5 and 6, respectively, type 2 17β-HSD activity, which preferentially catalyzes the oxidation reaction, has higher activity at an alkaline pH (8–10). Differences in the optimum incubation temperatures were also observed: type 1 17β-HSD shows a relatively high temperature tolerance (55°C). In contrast, type 2 and 3 functioned best at 37°C. Types 1, 2 and 3 17β-HSD activities could be also differentiated by their sensitivity toward various specific inhibitors: type 1 was potently inhibited by an estradiol derivative containing a bromo/or iodopropyl group at position 16. On the other hand a derivative of estrone containing a spiro-γ-lactone at position 17 showed a potent inhibitory effect on type 2 17β-HSD, whereas type 3 was strongly inhibited by 1,4-androstadiene-1,6,17-trione.     

Linkage analysis of 26 Canadian breast and breast-ovarian cancer families

We have examined 26 Canadian families with hereditary breast or ovarian cancer for linkage to markers flanking the BRCA1 gene on chromosome 17q12–q21. Of the 15 families that contain cases of ovarian cancer, 94% were estimated to be linked to BRCA1. In contrast, there was no overall evidence of linkage in the group of 10 families with breast cancer without ovarian cancer. A genetic recombinant in a breast-ovarian cancer family indicates a placement of BRCA1 telomeric to D17S776, and helps to define the region of assignment of the cancer susceptibility gene. Other cancers of interest that appeared in the BRCA1-linked families included primary peritoneal cancer, cancer of the fallopian tube, and malignant melanoma.     

Regulation of sex steroid formation by interleukin-4 and interleukin-6 in breast cancer cells

Sex steroids play a predominant role in the development and differentiation of normal mammary gland as well as in the regulation of hormone-sensitive breast cancer growth. There is evidence suggesting that local intracrine formation of sex steroids from inactive precursors secreted by the adrenals namely, dehydroepiandrosterone (DHEA) and 4-androstenedione (4-dione) play an important role in the regulation of growth and function of peripheral target tissues, including the breast. Moreover, human breast carcinomas are often infiltrated by stromal/immune cells secreting a wide spectra of cytokines. These might in turn regulate the activity of both immune and neoplastic cells. The present study was designed to examine the action of cytokines on 17β-hydroxysteroid dehydrogenase (17β-HSD) and 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) activities in human breast cancer cells. The various types of human 17β-HSD (five types) and 3β-HSD (two types), because of their tissue- and cell-specific expression and substrate specificity, provide each cell with necessary mechanisms to control the level of intracellular active androgens and estrogens. We first investigated the effect of exposure to IL-4 and IL-6 on reductive and oxidative 17β-HSD activities in both intact ZR-75-1 and T-47D human breast cancer cells. In ZR-75-1 cells, a 6 d exposure to IL-4 and IL-6 decreased E₂-induced cell proliferation, the half maximal inhibitory effect being exerted at 88 and 26 pM, respectively. In parallel, incubation with IL-4 and IL-6 increased oxidative 17β-HSD activity by 4.4- and 1.9-fold, respectively, this potent activity being observed at ₅₀ values of 22.8 and 11.3 pM, respectively. Simultaneously, reductive 17β-HSD activity leading to E₂ formation was decreased by 70 and 40% by IL-4 and IL-6, respectively. Moreover, IL-4 and IL-6 exerted the same regulatory effects on 17β-HSD activities when testosterone and 4-dione were used as substrates, thus strongly suggesting the expression of the type 2 17β-HSD ZR-75-1 cells. In contrast, in T-47D cells, IL-4 increased the formation of E₂, whereas IL-6 exerts no effect on this parameter. However, we found that T-47D cells failed to convert testosterone efficiently into 4-DIONE, thus suggesting that there is little or no expression of type 2 17β-HSD in this cell line. The present findings demonstrate that the potent regulatory effects of IL-4 and IL-6 on 17β-HSD activities depend on the cell-specific gene expression of various types of 17β-HSD enzymes. We have also studied the effect of cytokines on the regulation of the 3β-HSD expression in both ZR-75-1 and T-47D human breast cancer cells. Under basal culture conditions, there is no 3β-HSD activity detectable in these cells. However, exposure to IL-4 caused a rapid and potent induction of 3β-HSD activity, whereas IL-6 failed to induce 3β-HSD expression. Our data thus demonstrate that cytokines may play a crucial role in sex steroid biosynthesis from inactive adrenal precursors in human breast cancer cells.     

The simultaneous use of 14CO2 and 15N2 labelling techniques to study the carbon and nitrogen economy of legumes grown under natural conditions

A method based on simultaneous short-term exposure to ¹⁴CO₂ and ¹⁵N₂ is described for studying nitrogen fixation and distribution in legumes relative to carbon assimilation and use. Equipment designed to accomodate experiments under natural conditions with very little disturbance of the N₂ fixing association is used. It permits continuous measurement and regulation of variables such as air temperature, humidity and CO₂ concentration as well as soil aeration. Measurements of distribution and use of assimilates, respiration of nodulated roots, quantitative N₂ fixation and the distribution and fate of fixed N as a function of time lead to a precise estimation of C and N budgets for each labelling period. When experiments are done at several phenological stages they give a new insight into the complex C and N interrelations in legume symbiosis.
A series of trials throughout the growth period of Glycine max (L.) Merr. cv. Hodgson demonstrated the sensitivity of the method. The development of the plants from vegetative to reproductive stages was accompanied by a complete change in the distribution patterns of current assimilates and products of nitrogen fixation. Maximum sink strength moved from the leaves to the pods and seeds which ended up receiving 70% of the incoming C and 35% of the fixed N. The fact that up to 85% of fixed N in the plants was in the reproductive organs at maturity can be accounted for by remobilisation from vegetative parts.
The respiration of nodulated roots utilized 33% of carbon translocated to below-ground plant parts before nitrogen fixation started, but as much as 50% during the period of optimal fixation. The advantages and limitations of the isotopic method described are critically discussed as a prelude to future investigations.     

Increased testicular 5α-androstane-3α, 17β-diol formation induced by treatment with [D-Ser(TBU)6, des-Gly-NH210]LHRH ethylamide in the rat

While the intact male adult rats respond to LH with a predominant increase of testicular and plasma testosterone levels, the response to LH stimulation in animals treated with the LHRH agoriist, [D-Ser(TBU)⁶, des-Gly-NH₂10]LHRH ethylamide is characterized by a major production of 5α-androstane-3α, 17β-diol. The marked increase of 5α-androstane-3α, 17β-diol levels in the presence of a 90% decrease of testosterone concentration strongly suggests that 5α-reductase and 3α-hydroxysteroid oxidoreductase activities are increased during testicular desensitization induced by treatment with the LHRH agonist.