An improved method for primary culture of ovarian androgen-producing cells in serum-free medium: Effect of lipoproteins,insulin, and insulinlike growth factor-I

Summary Although luteinizing hormone (LH) alone stimulates ovarian interstitial cells cultured in serum-free medium to synthesize large amounts of androgens, there seem to be additional factors in vivo that modulate the time course and magnitude of the cellular responses to LH. In an attempt to develop a more nearly physiologic cell culture model, lipoproteins, insulin, and insulinlike growth factor-I (IGF-I) were added to the serum-free medium. The effects of these modifications on androgen biosynthesis by dispersed cells from ovaries of hypophysectomized immature rats cultured in 96-well tissue culture plates were examined. A saturating dose of LH stimulated a 25-fold increase in androsterone synthesis at 2 d, which decreased at 4 and 6 d. Addition of human high density (hHDL) or human low density lipoprotein (hLDL) caused a 2.5-fold increase in LH-stimulated androsterone synthesis. Cells were approximately twice as sensitive to hHDL (ED₅₀=5.5±0.5 μg cholesterol/ml) compared to hLDL (ED₅₀=9.1±1.1 μg cholesterol/ml). Surprisingly, rat HDL caused only a 40% increase in LH-stimulated androsterone synthesis. When insulin alone was added to cells cultured with a saturating dose of LH, there was a 2.8-fold increase in androsterone synthesis. Addition of hHDL and insulin together caused a synergistic increase in LH-stimulated androsterone synthesis. In contrast to hHDL, which did not change the time course of LH-stimulated androsterone production, insulin prolonged maximal LH-stimulated androsterone synthesis at 4 and 6 d. Inasmuch as the ED₅₀ for insulin action (1.3±0.1 μg/ml) was supraphysiologic, the effects of IGF-I on LH-stimulated androgen synthesis were examined. IGF-I mimicked all of the effects of insulin, but at a physiologic concentration (ED₅₀=2.5±0.3 ng/ml). Ovarian cells cultured in serum-free medium supplemented with hHDL and insulin or IGF-I exhibit responses that closely approximate the physiologic responses observed in vivo. These results suggest that lipoproteins and IGF-I are important physiologic stimulators of ovarian theca-interstitial cell androgen biosynthesis which, when added to the serum-free medium, make the cellular responses in this in vitro model more nearly approximate the responses in vivo. This research was supported by research center grant HD 12303 from the National Institute of Child Health and Human Development, Bethesda, MD, and USCD Academic Senate grant RM-169M     

Spring migration of Atlantic mackerel,Scomber scombrus,in relation to water temperature through Cabot Strait (Gulf of St. Lawrence)

Synopsis Sea surface temperatures across Cabot Strait (Gulf of St. Lawrence) ranged from 6 to 9°C on June 3, 1989, but only from 3 to 5°C on June 2, 1990. Periods of peak commercial landings of mackerel in eastern Cape Breton Island extended from May 22 to June 3 in 1989, and from May 28 to June 2 in 1990. In late May 1990, Atlantic mackerel were captured with a purse-seiner during exploratory fishing in Cabot Strait; commercial quantities were caught in water as cold as 2.8°C. The presence of mackerel in water a full 4°C colder than its reported lower tolerance limit indicates that the development of the 7°C isotherm is not a requirement for the vernal appearing of mackerel. The overlap of the periods of peak commercial landings between 1989 and 1990, despite: marked differences in warming chronology, suggests that the movements of mackerel are not as closely linked to water temperature as previously reported. The fish's thermal preferences could be subordinate to their reproductive requirements at this stage of their spawning migration.     

Sugars fermented byBifidobacterium infantis ATCC 27920 in relation to growth and α-galactosidase activity

Summary The ability of Bifidobacterium infantis ATCC 27 920 to ferment glucose, galactose, lactose, melibiose and raffinose was investigated with respect to -galactosidase (-d-galactoside galactohydrolase, E.C. 3.2.1.22). The sugars were tested at three concentrations: 0.5, 1.0 and 2.0%. The growth of B. infantis was slower on glucose compared with the other sugars. The highest specific growth rate was observed on melibiose followed by lactose. High cell numbers could be rapidly obtained on galactose-containing sugars. For each carbohydrate, enzyme activity was maximal at the end of the exponential phase and the highest specific -galactosidase activities were recorded on the two -1,6 galactosaccharides (melibiose and raffinose: 3.0 and 4.5 nkat · 10⁹ colony-forming units, respectively).Contribution no. 186 from the Food Research and Development Centre Offprint requests to: D. Roy     

EMG power spectra of elbow extensors during ramp and step isometric contractions

The goal of the present study was to compare electromyogram (EMG) power spectra obtained from step (constant force level) and ramp (progressive increase in the force level) isometric contractions. Data windows of different durations were also analysed for the step contractions, in order to evaluate the stability of EMG power spectrum statistics. Fourteen normal subjects performed (1) five ramp elbow extensions ranging from 0 to 100% of the maximum voluntary contraction (MVC) and (2) three stepwise elbow extensions maintained at five different levels of MVC. Spectral analysis of surface EMG signals obtained from triceps brachii and anconeus was performed. The mean power frequency (MPF) and the median frequency (MF) of each power spectrum were obtained from 256-ms windows taken at 10, 20, 40, 60 and 80% MVC for each type of contraction and in addition on 512-, 1024- and 2048-ms windows for the step contractions. No significant differences (P greater than 0.05) were found in the values of both spectral statistics between the different window lengths. Even though no significant differences (P greater than 0.05) were found between the ramp and the step contractions, significant interactions (P less than 0.05) between these two types of contraction and the force level were found for both the MPF and the MF data. These interactions point out the existence of different behaviours for both the MPF and the MF across force levels between the two types of contraction.     

Ca2+ regulation of thyroid NADPH-dependent H2O2 generation

A thyroid particulate fraction contains an NADPH-dependent H2O2-generating enzyme which requires Ca2+ for activity. A Chaps solubilized extract of the thyroid particulate fraction partially purified by DEAE chromatography did not show a dependence on Ca2+ for activity. Preincubation of the particulate fraction with Ca2+ yielded a preparation insensitive to Ca2+. The non-particulate fraction obtained after incubation of the particles in the presence of Ca2+ was able to inhibit, in the presence of EGTA, the Ca2+-desensitized particulate fraction and the enzyme isolated on DEAE. It is concluded that the reversible Ca2+ activation of the NADPH-dependent H2O2 generation was modulated in porcine thyroid tissue by (a) calcium-releasable inhibitor protein(s).     

L-649,923, sodium (beta S*, gamma R*)-4-(3-(4-acetyl-3-hydroxy-2-propylphenoxy)-propylthio)- gamma-hydroxy-beta-methylbenzenebutanoate, a selective, orally active leukotriene receptor antagonist

L-649,923, Sodium (beta S*, gamma R*)-4-(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propylthio)- gamma- hydroxy-beta-methylbenzenebutanoate is a selective and competitive inhibitor of [3H]leukotriene D4 (Ki value of 400 nM) and to a lesser extent [3H]leukotriene C4 (Ki value of 8.6 microM) binding in guinea-pig lung homogenates. Functionally, it selectively antagonized contractions of guinea pig trachea induced by leukotriene C4, D4, E4, and F4 but not those induced by acetylcholine, histamine, serotonin, prostaglandin F2 alpha, or U-44069 (stable endoperoxide analogue). Schild plot analysis indicated a competitive inhibition of contractions of guinea-pig ileum induced by leukotriene D4 (pA2 8.1) and contractions of guinea-pig trachea induced by leukotrienes E4 and F4 (pA2 7.1 and 6.9, respectively). In contrast, contractions of guinea-pig trachea induced by leukotrienes C4 (pA2 7.2; slope 0.6) and D4 (pA2 7.2; slope 0.7) were inhibited in a noncompetitive fashion. In vivo, intravenously administered L-649,923 selectively blocked bronchoconstriction induced in anesthetized guinea pigs by leukotriene C4 and D4 (ED50 values i.v. 0.38 and 0.26 mg/kg, respectively) but not that induced by histamine, arachidonic acid, serotonin, U-44069, or acetylcholine. Following intraduodenal administration, L-649,923, blocked leukotriene D4 induced bronchoconstriction (5 and 10 mg/kg). The present findings indicate that selective antagonists, such as L-649,923, may be useful for defining the role of leukotrienes in diseases such as bronchial asthma.     

L-648,051, sodium 4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)- propylsulfonyl]-gamma-oxo-benzenebutanoate: a leukotriene D4 receptor antagonist

L-648,051, sodium 4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy) propylsulfonyl]-gamma-oxo-benzenebutanoate is a selective and competitive inhibitor of [3H]leukotriene D4 (KB value of 4.0 microM) and to a lesser extent [3H]leukotriene C4 (Ki value of 36.7 microM) binding in guinea pig lung homogenates. Functionally, it selectively antagonized contractions of guinea pig trachea induced by leukotrienes C4, D4, E4, and F4 in concentrations that did not antagonize contractions induced by acetylcholine, histamine, serotonin, prostaglandin F2 alpha, or U-44069 (endoperoxide analogue). Schild plot analysis indicated that L-648,051 competitively antagonized contractions of guinea pig ileum induced by leukotriene D4 (pA2 7.7) and contractions of trachea induced by leukotrienes D4, E4, and F4 (pA2 7.3, 7.4, and 7.5, respectively). Contractions of guinea pig trachea induced by leukotriene C4 were inhibited in a noncompetitive fashion (Schild plot slope, 0.45). Developed contractions of trachea induced by the leukotrienes were rapidly reversed by L-648,051 greater than FPL-55712 greater than L-649,923. Intravenous L-648,051 selectively blocked bronchoconstriction induced in anaesthetized guinea pigs by intravenous leukotrienes C4, D4, and E4 but not that induced by arachidonic acid, serotonin, U-44069, or acetylcholine. The compound displayed poor activity following intraduodenal administration. The profile of activity for L-648,051 indicates that it may be a useful topical agent for studying the role of leukotrienes in diseases such as bronchial asthma.     

Mitogenic effect of PMA + IL 2 on subpopulations of corticoresistant thymocytes

The proliferative response of subpopulations of corticoresistant thymocytes (CRT) to phorbol-12-myristate-13-acetate (PMA) + interleukin 2 (IL 2) was investigated. Thymocyte subpopulations were selected by the indirect "panning" technique, and their purity was checked by cytofluorometry. Microcultures were set up with an optimal concentration of PMA, EL4 supernatant, or pure IL 2 obtained by recombinant DNA technology (r-IL 2) in the presence or in the absence of accessory splenic adherent cells (SAC). Under these conditions, only the Lyt-2+ CRT proliferated, and this response was IL 2-dose-dependent and was increased by accessory cells. When the calcium ionophore A23187 was added to the cultures, the proliferation of L3T4+ CRT was greatly increased. These results were confirmed by cultures at limiting dilution of positively selected Lyt-2+ and L3T4+ subpopulations of CRT at optimal concentrations of PMA, r-IL 2, A23187, and accessory cells. These results are consistent with the idea that two signals are necessary to activate L3T4+ CRT, whereas only IL 2 is necessary for PMA-induced proliferation of Lyt-2+ CRT. Finally, unlike the case of lectin-induced proliferation of Lyt-2+ and L3T4+ CRT, the presence of accessory cells or cell-cell contact is important for optimal response to PMA + IL 2.