Steady-State Effects of 2,5,2′,5′-Tetrachlorobiphenyl on Growth, Photosynthesis, and P Uptake in Selenastrum capricornutum

The steady-state effect of 2,5,2′,5′-tetrachlorobiphenyl (TCBP) on the green alga Selenastrum capricornutum was investigated in a P-limited two-stage chemostat system. The partition coefficient of this polychlorinated biphenyl congener was 5.9 × 10⁴ in steady-state cultures. At a cellular TCBP concentration of 12.2 × 10⁻⁸ ng · cell⁻¹, growth rate was not affected. However, photosynthetic capacity (P_max) was significantly enhanced by TCBP (56 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ versus 34 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ in the control). Photosynthetic efficiency, or the slope of the photosynthesis-irradiance curve, was also significantly higher. There was little difference in the cell chlorophyll a content, and therefore the difference in these photosynthetic characteristics was the same even when they were expressed on a per-chlorophyll a basis. Cell C content was higher in TCBP-containing cells than in TCBP-free cells, but approximately 36% of the C fixed by cells with TCBP was not incorporated as cell C. The maximum P uptake rate was also enhanced by TCBP, but the half-saturation concentration appeared to be unaffected.     

Effect of Dissolved Oxygen, Temperature, Initial Cell Count, and Sugar Concentration on the Viability of Saccharomyces cerevisiae in Rapid Fermentations

By using 7 x 10(8) cells of Saccharomyces cerevisiae per ml with which 25 degrees Brix honey solutions were fermented to 9.5% (wt/vol; 12% vol/vol) ethanol in 2.5 to 3 h at 30 C, i.e., rapid fermentation, the death rate was found to be high, with only 2.1% of the yeast cells surviving at the end of 3 h under anaerobic conditions. As the dissolved oxygen in the medium was increased from 0 to 13 to 20 to 100% in rapid fermentations at 30 C, there was a progressive increase in the percentage of cells surviving. The ethanol production rate and total were not seriously affected by a dissolved oxygen concentration of 13%, but fermentation was retarded by 20% dissolved oxygen and still further decreased as the dissolved oxygen content reached 100%. When the fermentation temperature was decreased to 15 C (at 13% dissolved oxygen), the rate of fermentation decreased, and the fermentation time to 9.5% ethanol (wt/vol) increased to 6 h. It was found that the higher the temperature between 15 and 30 C, the greater the rate of death as initial cell counts were increased from 1.1 x 10(7) to 7.8 x 10(8) cells per ml. At the lowest level of inoculum, 1.1 x 10(7) cells per ml, there was actual multiplication, even at 30 C; however, the fermentation was no longer rapid. The addition of 15% sugar, initially followed after an hour by the remaining 10%, or addition of the sugar in increments of 2.5 or 5% yielded a better survival rate of yeast cells than when the fermentation was initiated with 25% sugar.     

干旱胁迫下尖果沙枣幼苗的根系活力和光合特性

以尖果沙枣1年生实生苗为材料,研究了自然干旱时不同土壤相对含水量对幼苗叶片细胞质膜相对透性、叶片相对含水量、根系活力、光合色素含量和光合参数等指标的影响.结果表明:土壤相对含水量从70%(CK)降到40%时,幼苗根系活力和净光合速率均逐渐上升并达到最大值,分别为1178μg.g-1.h-1和21.9μmol.m-2.s-1;光合色素含量稳步上升;蒸腾速率和水分利用效率均保持稳定;叶片细胞质膜相对透性保持较低水平.土壤相对含水量从40%降到20%时,幼苗叶片相对含水量仍在50%以上,叶片细胞质膜相对透性仍保持较低水平;根系活力和光合色素含量仍较高;但其他光合参数开始缓慢下降.土壤相对含水量从10%降到5%时,幼苗叶片细胞质膜相对透性急剧上升;叶片相对含水量、根系活力、总叶绿素含量、光合参数均极显著下降;而土壤相对含水量为10%时幼苗表现出最高的水分利用效率.尖果沙枣土壤相对含水量最好控制在40%~50%,其1年生实生苗的永久萎蔫系数为4.3%(土壤相对含水量).     

A threshold exists for the stimulatory effect of insulin on plasma L-carnitine clearance in humans

Maintaining hyperinsulinemia ( approximately 160 mU/l) during steady-state hypercarnitinemia ( approximately 550 mumol/l) increases skeletal muscle total carnitine (TC) content by approximately 15% within 5 h. The aim of the present study was to further examine the relationship between serum insulin concentration and skeletal muscle carnitine accumulation by attempting to identify the serum insulin concentration at which this stimulatory effect of insulin on carnitine retention becomes apparent. On four randomized experimental visits, eight healthy men (body mass index 23.8 +/- 0.9 kg/m(2)) underwent a 6-h euglycemic insulin clamp of 5, 30, 55, or 105 mU x m(-2) x min(-1) accompanied by a 5-h iv infusion of l-carnitine (15 mg/kg bolus followed by 10 mg x kg(-1) x h(-1)). The clamps produced steady-state serum insulin concentrations of 10.1 +/- 0.5, 48.8 +/- 1.0, 88.9 +/- 2.8, and 173.9 +/- 6.5 mU/l, respectively. During l-carnitine infusion, plasma TC concentration remained above 450 mumol/l during all four visits. However, there was a significant treatment effect of insulin (P < 0.001), such that by the end of infusion the plasma TC concentration in the 55- and 105-mU clamps was lower than that seen in the 5- (P < 0.05 and P < 0.01, respectively) and 30-mU (P < 0.01) clamps. The findings demonstrate that only high circulating serum insulin concentrations (> or =90 mU/l) are capable of stimulating skeletal muscle carnitine accumulation. This is of relevance to athletes, and the treatment of obesity and type 2 diabetes, where increasing skeletal muscle carnitine content may be used as tool to modify skeletal muscle energy metabolism.     

Physiological characterization of Dunaliella sp. (Chlorophyta, Volvocales) from Yucatan, Mexico

Physiological responses of Dunaliella salina and Dunaliella viridis, isolated from solar saltworks on the Yucatan Peninsula, were studied. Optimal growth temperature for D. salina was 22 degrees C (3.06 x 10(6) cells mL(-1)) and 26 degrees C for D. viridis (4.04 x 10(6)cells mL(-1)). Total carotenoid content in D. salina increased with temperature to a maximum of 35.14 pg cell(-1) at 38 degrees C. Dunaliella salina alpha-carotene and beta-carotene content was 0.083+/-0.003 and 0.598+/-0.020 mg 100g dry wt(-1) respectively, whereas lower values were found in D. viridis cultured under same experimental conditions (0.018+/-0.002 and 0.136+/-0.012 mg 100g dry wt(-1) respectively). The highest specific growth rate in D. salina was obtained at 10% NaCl (0.28 d(-1)), while its cell volume increased from 524 to 2066.93 microm(3) when cultured from 10% to 35% NaCl. Maximum photosynthetic rates were attained when increasing from optimal growing temperature to 30 degrees C for D. viridis (108 n mol O(2)microg chl alpha h(-1)) and D. salina (139 n mol O(2)microg chl alpha h(-1)). Photosynthetic responses to temperature variations indicated physiological adjustments in both species, with higher acclimation in D. salina. Evaluation of physiological attributes of these species will be used for to carry out mass cultivation.     

Cell cycle- and growth phase-dependent variations in size distribution, antibody productivity, and oxygen demand in hybridoma cultures

Simultaneous determination of cell size and DNA content of hybridomas (HB-32) revealed a direct correlation between average cell volume and progression through the cell cycle. Pseudocontinuous experiments showed that G(1) cells, as estimated from cell size measurements, secreted monoclonal antibody at rates higher than those of cells in other stages of interphase and mitosis. Similarly, fed-batch and batch experiments suggested that specific oxygen uptake rate (qO(2)) is also a function of cell cycle, being minimum for cells in G(0) and G(1) phase. In batch cultures, HB-32 showed a rapid decrease in oxygen uptake rate (OUR) just prior to reaching maximum cell concentration. The OUR steadily increased from 0.01-0.05 to 0.5-0.7 mmol O(2)/L h as the cells went from the lag to the midexponential phase. The qO(2) increased from 0.3 x 10(-10)-0.9 x 10(-10) mmol O(2)/cell h at inoculation to 3.3 x 10(-10)-3.7 x 10(-10) mmol O(2)/cell h during the early exponential phase where it remained relatively constant. Several hours before maximum cell concentration was reached, OUR and qO(2) rapidly decreased to levels below those observed at inoculation. The time at which the shift in OUR and qO(2) occurred and the onset of decrease in the average cell size corresponded to the time of glutamine depletion. Based on monitoring OUR on-line in batch cultures, glutamine was supplemented, resulting in increased cell concentration, extension of culture viability, and increased MAb concentration.     

Effect of glucose,maltose, soluble starch,and CO<Subscript>2</Subscript> on the growth of the hyperthermophilic archaeon<Emphasis Type="Italic"> Pyrococcus furiosus</Emphasis>

The hyperthermophilic archaeon Pyrococcus furiosus was cultivated in batch and continuous fermentations on different carbon substrates. The cultivation of P furiosus on soluble starch as the only carbon source resulted in cell densities three times higher than in cultivations on maltose, 1.06 x 10(10) cells/ml compared to 3.4 x 10(9) cells/ml. The yield coefficient, Y(x/y) = 0.12 g/g, and the growth rate, mu = 0.33 h(-1), were almost equal on soluble starch and on maltose, but on glucose no growth could be detected. An inhibitory effect of glucose, when added to other carbon substrates, also could not be found. Isobutyric and isovaleric acid were detected as novel metabolites produced by P. furiosus. Inhibitory effects of these acids, as well as of the well-known products acetic acid, propionic acid, and alanine, could be precluded. Concentrations of 10% CO2 in the gas supply respective to the exhaust gas enhanced the growth of P furiosus significantly. The maximum cell number was two orders of magnitude higher than was observed with pure nitrogen. Further increase of the CO2 concentration up to 100% had no significant effect on the growth of P. furiosus.     

Optimization of biomass, total carotenoids and astaxanthin production in Haematococcus pluvialis Flotow strain Steptoe (Nevada, USA) under laboratory conditions

The microalga Haematococcus pluvialis Flotow is one of the natural sources of astaxanthin, a pigment widely used in salmon feed. This study was made to discover optimal conditions for biomass and astaxanthin production in H. pluvialis from Steptoe, Nevada (USA), cultured in batch mode. Growth was carried out under autotrophic (with NaNO3, NH4Cl and urea) and mixotrophic conditions (with 4, 8, 12 mM sodium acetate) under two photon flux densities (PFD) (35 and 85 mumol m-2 s-1). The carotenogenesis was induced by 1) addition of NaCl (0.2 and 0.8%), 2) N-deprivation and 3) high PFD (150 mumol m-2 s-1). Total carotenoids were estimated by spectrophotometry and total astaxanthin by HPLC. Ammonium chloride was the best N-source for growth (k = 0.7 div day-1, 228-258 mg l-1 and 2.0 x 10(5)-2.5 x 10(5) cells ml-1 at both PFD, respectively). With increasing acetate concentration, a slight increment in growth occurred only at 85 mumol m-2 s-1. Light was the best inductive carotenogenic factor, and the highest carotenoid production (4.9 mg l-1, 25.0 pg cell-1) was obtained in cultures pre-grown in nitrate at low light. The NaCl caused an increase in carotenoid content per cell at increasing salt concentrations, but resulted in a high cell mortality and did not produce any increment in carotenoid content per volume compared to cultures grown at 150 mumol m-2 s-1. The highest carotenoid content per cell (22 pg) and astaxanthin content per dry weight (10.3 mg g-1) (1% w/w) were obtained at 85 mumol m-2 s-1 with 0.8% NaCl.     

Glutathione replenishment capacity is lower in isolated perivenous than in periportal hepatocytes.

The zonal distribution of GSH metabolism was investigated by comparing hepatocytes obtained from the periportal (zone 1) or perivenous (zone 3) region by digitonin/collagenase perfusion. Freshly isolated periportal and perivenous cells had similar viability (dye exclusion, lactate dehydrogenase leakage and ATP content) and GSH content (2.4 and 2.7 mumol/g respectively). During incubation, periportal cells slowly accumulated GSH (0.35 mumol/h per g), whereas in perivenous cells a decrease occurred (-0.14 mumol/h per g). Also, in the presence of either L-methionine or L-cysteine (0.5 mM) periportal hepatocytes accumulated GSH much faster (3.5 mumol/h per g) than did perivenous cells (1.9 mumol/h per g). These periportal-perivenous differences were also found in cells from fasted rats. Efflux of GSH was faster from perivenous cells than from periportal cells, but this difference only explained 10-20% of the periportal-perivenous difference in accumulation. Furthermore, periportal cells accumulated GSH to a plateau 26-40% higher than in perivenous cells. There was no significant difference in gamma-glutamylcysteine synthetase or glutathione synthetase activity between the periportal and perivenous cell preparations. The periportal-perivenous difference in GSH accumulation was unaffected by inhibition of gamma-glutamyl transpeptidase or by 5 mM-glutamate or -glutamine, but was slightly diminished by 2 mM-L-methionine. This suggests differences between periportal and perivenous cells in their metabolism and/or transport of (sulphur) amino acids. Our results suggest that a lower GSH replenishment capacity of the hepatocytes from the perivenous region may contribute to the greater vulnerability of this region to xenobiotic damage.     

Photosynthetic acclimation of the filamentous cyanobacterium, Plectonema boryanum UTEX 485, to temperature and light

Photosynthetic acclimation to temperature and irradiance was studied in the filamentous, non-heterocystous cyanobacterium Plectonema boryanum UTEX 485. Growth rates of this cyanobacterium measured at ambient CO2 were primarily influenced by temperature with minimal effects of irradiance. Both growth temperature and irradiance affected linolenic (18:3) and linoleic acid (18:2) levels in the four major lipid classes in an independent but additive manner. In contrast, photosynthetic acclimation was not due to either growth temperature or irradiance per se, but rather, due to the interaction of these environmental factors. P. boryanum grown at low temperature and moderate irradiance mimicked cells grown at high light. Compared to cells grown at either 29 degrees C/150 micromol m(-2) s(-1) (29/150) or 15/10, P. boryanum grown at either 15/150 or 29/750 exhibited: (1) reduced cellular levels of Chl a and phycobilisomes (PBS), and concomitantly higher content of an orange-red carotenoid, myxoxanthophyll; (2) higher light saturated rates (Pmax) when expressed on a Chl a basis but lower apparent quantum yields of oxygen evolution and (3) enhanced resistance to high light stress. P. boryanum grown at 15/150 regained normal blue-green pigmentation within 16 h after a temperature shift to 29 degrees C at a constant irradiance of 150 micromol m(-2) s(-1). DBMIB and KCN but not DCMU and atrazine partially inhibited the change in myxoxanthophyll/Chl a ratio following the shift from 15 to 29 degrees C. We conclude that P. boryanum responds to either varying growth temperature or varying growth irradiance by adjusting the ability to absorb light through decreasing the cellular contents of Chl a and light-harvesting pigments and screening of excessive light by myxoxanthophyll predominantly localized in the cell wall/cell membrane to protect PSII from over-excitation. The possible role of redox sensing/signalling for photosynthetic acclimation of cyanobacteria to either temperature or irradiance is discussed.     

Methanol biosynthesis by covalently immobilized cells of Methylosinus trichosporium: batch and continuous studies

The DEAE-cellulose linked cells of Methylosinus trichosporium displaying high specific methane mono-oxygenase activity (66 mumol methane oxidized/h mg cells) were used for methanol biosynthesis from biogas derived methane in a batch and a continuous cell reactor. The optimum cell-to-carrier ratio was determined to be 0.5 g cells/g dry weight cellulose. Batch experiments indicated that 100 mM phosphate ion concentration was necessary to inhibit further oxidation of methanol; excess oxygen supply favored methanol accumulation with an increase in methane conversion efficiency to 27%. A pulse of 40 mM sodium formate at the end of 6 h resulted in restoration of methanol accumulation by regenerating NADH(2) required for the sustained activity of methane mono-oxygenase. Maximum methanol level of 50 mumol/mg cells was obtained in the batch reactor. In a standard 50-mL ultrafiltration continuous reactor, the covalently linked cells produced methanol at a continuous rate of 100 mumol/h for the first 10 h, after which the methanol accumulation rate fell low due to the depletion of NADH(2). The methanol accumulation could be stimulated by supplying sodium formate (40 mM) in either 20 or 100 mM phosphate buffer. Maximum methanol accumulation rate of 267 mumol/h was obtained when 20 mM formate was supplied in the feed stream containing 100 mM phosphate ions, and this level of biosynthesis was maintained for over 72 h. The stoichiometric balance made at various points of formate addition indicated that the molar amount of methanol generated at steady state is dependent on the equimolar addition of sodium formate to the feed. The half-life t(1/2) and thermal denaturation rate constant K(d) were computed to be 108 h and 6.42 x 10(-3) h(-1), respectively.     

Effects of cell density and glucose and glutamine levels on the respiration rates of hybridoma cells

The effects of cell density as well as the concentration levels of glucose and glutamine on the specific respiration rate of a hybridoma cell line were investigated. The experimental oxygen consumption rate was found to be constant over a wide range of dissolved oxygen levels if the suspension medium contained glutamine. In glutamine-free medium, however, the rate of oxygen consumption decreased slowly with time.In a stationary flask batch culture, the specific respiration rate decreased from about 7 to 2.9 mumol/min per 10(9) cells as the cell density increased exponentially from 1 x 10(5) to 1.2 x 10(6)/mL. To isolate the effect of cell density, cells were re suspended in fresh culture medium so that nutrient concentrations were the same for all experiments. The specific respiration rate decreased with increasing cell density in the same manner as in the stationary flask culture, falling from 8 to 4 mumol/min per 10(9) cells as the cell density increased from 10(5) to 10(6) cells/mL, then declining to 2 mumol/min per 10(9) cells when the cell density reached 10(7) cells/mL.Cells suspended in Hanks balanced sale solution (HBSS) were used to elucidate the effect of glucose and glutamine levels on respiration. The addition of glucose in concentrations of 0.25, 0.50, and 0.75 g/L had no observable effect on the specific oxygen uptake rate; however, a glucose concentration of 1 g/L reduced the uptake rate by 22%. Glutamine in a concentration of 0.30 g/L increased the specific respiration rate in HBSS containing 0 and 1 g/L glucose by approximately 13%.     

ATP Sulfurylase from Penicillium chrysogenum: Is the Internal Level of the Enzyme Sufficient to Account for the Rate of Sulfate Utilization?

The in vivo rate of sulfate activation in Penicillium chrysogenum (wild-type strain ATCC 24791) was determined to be 0.19 +/- 0.09 mumol g(-1) (dry weight) min(-1) by the following methods. (i) The maximum growth of the organism in synthetic medium was a linear function of the initial Na(2)SO(4) concentration between 0 and 8 x 10(-4) Na(2)SO(4). The growth yield was 1.64 x 10(-2) g (dry weight) of mycelium per mumol of added sulfate, corresponding to a minimum sulfur requirement of 61 mumol/g (dry weight). Under these conditions (limiting sulfate) the minimum doubling time of P. chrysogenum in submerged culture was about 3.8 h, corresponding to a maximum exponential growth rate constant of 3.0 x 10(-3) min(-1). If all the sulfur in this mycelium passed through adenosine-5'-phosphosulfate, the rate of sulfate activation in vivo must have been 0.183 mumol min(-1) g(-1) (dry weight). (ii) In the presence of excess (35)SO(4) (2-), the total organic (35)S produced varied with the mycelial growth rate. However, until the culture approached maximum density, the product of [(growth rate constant) x (organic (35)S content)] was nearly constant at 0.24 to 0.28 mumol min(-1) g(-1) (dry weight). (iii) A sulfur-starved mycelium pulsed with 10(-4) M (35)SO(4) (2-) produced organic (35)S at a rate of about 0.10 mumol min(-1) g(-1) (dry weight) under conditions where the internal concentrations of ATP and sulfate would permit ATP sulfurylase to operate at about 70% of its V(max). Cell-free extracts of P. chrysogenum growing rapidly on excess sulfate contained 0.22 U of ATP sulfurylase per g (dry weight). Thus, in spite of the relatively low specific activity of homogeneous ATP sulfurylase (0.13 U/mg of protein, corresponding to an active site turnover of 7.15 min(-1)), the mycelial content of the enzyme was sufficient to account for the observed growth rate of the organism on inorganic sulfate as the sole sulfur source.     

Arbuscular Mycorrhizal Fungi Alter Fractal Dimension Characteristics of Robinia pseudoacacia L. Seedlings Through Regulating Plant Growth,Leaf Water Status,Photosynthesis, and Nutrient Concentration Under Drought Stress

The influence of arbuscular mycorrhizal fungi (AMF), Funneliformis mosseae and Rhizophagus intraradices, on plant growth, leaf water status, chlorophyll concentration, photosynthesis, nutrient concentration, and fractal dimension (FD) characteristics of black locust (Robinia pseudoacacia L.) seedlings was studied in pot culture under well-watered, moderate drought stress, and severe drought stress treatments. Mycorrhizal seedlings had higher dry biomass, leaf relative water content (RWC), and water use efficiency (WUE) compared with non-mycorrhizal seedlings. Under all treatments, AMF colonization notably enhanced net photosynthetic rate, stomatal conductance, and transpiration rate, but decreased intercellular CO₂ concentration. Leaf chlorophyll a and total chlorophyll concentrations were higher in AM seedlings than those in non-AM seedlings although there was no significant difference between AMF species. AMF colonization improved leaf C, N, and P concentrations, but decreased C:N, C:P, and N:P ratios. Mycorrhizal seedlings had a larger FD value than non-mycorrhizal seedlings. The FD value was positively and significantly correlated to the plant growth parameters, photosynthesis, RWC, WUE, and nutrient concentration but negatively correlated to leaf/stem ratio, C:N and C:P ratios, and intercellular CO₂ concentration. We conclude that AMF lead to an improvement of growth performance of black locust seedlings under all growth conditions, including drought stress via improving leaf water status, chlorophyll concentration, photosynthesis, and nutrient uptake. Moreover, FD technology proved to be a powerful non-destructive method to characterize the effect of AMF on the physiology of host plants during drought stress.     

Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart

The bioenergetic basis by which the Krebs cycle substrate pyruvate increased cardiac contractile function over that observed with the Embden-Meyerhof substrate glucose was investigated in the isovolumic guinea pig heart. Alterations in the content of the high energy phosphate metabolites and the rate of high energy phosphate turnover were measured by 31P NMR. These were correlated to the changes in contractile function and rates of myocardial oxygen consumption. Maximum left ventricular developed pressure (LVDP) and high energy phosphates were observed with 16 mM glucose or 10 mM pyruvate. In hearts perfused with 16 mM glucose, the intracellular phosphocreatine (PCr) concentration was 15.2 +/- 0.6 mM with a PCr/Pi ratio of 10.3 +/- 0.9. The O2 consumption was 5.35 mumol/g wet weight/min, and these hearts exhibited a LVDP of 97 +/- 3.7 mm Hg at a constant paced rate of 200 beats/min. In contrast, when hearts were switched to 10 mM pyruvate, the PCr concentration was 18.3 +/- 0.4 mM, the PCr/Pi ratio was 30.4 +/- 2.2, the O2 consumption was 6.67 mumol/g wet weight/min, and the LDVP increased to 125 +/- 3.3 mm Hg. From NMR saturation transfer experiments, the steady-state flux of ATP synthesis from PCr was 4.9 mumol/s/g of cell water during glucose perfusion and 6.67 mumol/s/g of cell water during pyruvate perfusion. The flux of ATP synthesis from ADP was measured to be 0.99 mumol/s/g of cell water with glucose and calculated to be 1.33 mumol/s/g of cell water with pyruvate. These results suggest that pyruvate quite favorably alters myocardial metabolism in concert with the increased contractile performance. Thus, as a mechanism to augment myocardial performance, pyruvate appears to be unique.     

Effects of in vitro fertilization conditions on preimplantation development and quality of pig embryos

The present study was to investigate the effects of in vitro fertilization conditions on in vitro development and structural integrity of pig embryos. Porcine oocytes matured in vitro were co-incubated with four different spermatozoa concentrations (0.6 x 10(5), 1.2 x 10(5), 2.5 x 10(5) and 5 x 10(5) cells/ml) for 6 h, and at a spermatozoa concentration (1.2 x 10(5) cells/ml) for 2, 4 and 6 h, respectively. Spermatozoa penetration and blastocyst formation were observed at 10 and 144 h post insemination, respectively. The allocation of a blastocyst to inner cell mass (ICM) and trophectoderm (TE) cells was determined by using a differential staining method. Polyspermy frequency increased with increasing spermatozoa concentrations. The spermatozoa-oocyte co-incubation period of 2 h provided for decreased in vitro development rate than 4 and 6 h groups (P < 0.05), although no difference was detected in polyspermy frequency between spermatozoa-oocyte co-incubation periods. Interestingly, blastocysts derived from the groups with greater spermatozoa concentrations (2.5 x 10(5) and 5 x 10(5) cells/ml) had significantly fewer ICM cell nuclei as compared with those groups with lesser spermatozoa concentrations (0.6 x 10(5) and 1.2 x 10(5) cells/ml). There was no difference in the structural integrity of blastocysts among the co-incubation periods. Blastocysts derived from respective experiments were individually classified into three groups (I: <20%; II: 20-40% and III: >40%) based on the ratio of ICM to total cells. Proportion of blastocysts in Group II, with a presumptive normal range of structural integrity, was slightly decreased in the groups with greater spermatozoa concentrations (2.5 x 10(5) and 5 x 10(5) cells/ml). The results indicate that the spermatozoa concentration during in vitro fertilization may be important for developmental competence and quality of pig embryos.     

Light-enhanced dark respiration in leaves, isolated cells and protoplasts of various types of C4 plants

The rate of respiratory CO2 evolution from the leaves of Zea mays, Panicum miliaceum, and Panicum maximum, representing NADP-ME, NAD-ME, and PEP-CK types of C4 plants, respectively, was increased by approximately two to four times after a period of photosynthesis. This light-enhanced dark respiration (LEDR) was a function of net photosynthetic rate specific to plant species, and was depressed by 1% O2. When malate, aspartate, oxaloacetate or glycine solution at 50 mM concentration was introduced into the leaves instead of water, the rate of LEDR was enhanced, far less in Z. mays (by 10-25%) than in P. miliaceum (by 25-35%) or P. maximum (by 40-75%). The enhancement of LEDR under glycine was relatively stable over a period of 1 h, whereas the remaining metabolites caused its decrease following a transient increase. The metabolites reduced the net photosynthesis rate in the two Panicum species, but not in Z. mays, where this process was stimulated by glycine. The bundle sheath cells from P. miliaceum exhibited a higher rate of LEDR than those of Z. mays and P. maximum. Glycine had no effect on the respiration rate of the cells, but malate increased in cells of Z. mays and P. miliaceum by about 50% and 30%, respectively. With the exception of aspartate, which stimulated both the O2 evolution and O2 uptake in P. maximum, the remaining metabolites reduced photosynthetic O2 evolution from bundle sheath cells in Panicun species. The net O2 exchange in illuminated cells of Z. mays did not respond to CO2 or metabolites. Leaf mesophyll protoplasts of Z. mays and P. miliaceum, and bundle sheath protoplasts of Z. mays, which are unable to fix CO2 photosynthetically, also produced LEDR, but the mesophyll protoplasts, compared with bundle sheath protoplasts, required twice the time of illumination to obtain the maximal rate. The results suggest that the substrates for LEDR in C4 plants are generated during a period of illumination not only via the Calvin cycle reactions, but also by the conversion of endogenous compounds present in leaf cells. The stimulation of LEDR under glycine is discussed in relation to its direct or indirect effect on mitochondrial respiration.     

Superoxide anion and hydrogen peroxide metabolism in soybean embryonic axes during germination.

The total rate of mitochondrial O2- production in the presence of NADH as substrate increased from 200 to 1340 pmol/min per axis between 2 and 30 h of imbibition. The activities of the enzymes involved in hydroperoxide metabolism, e.g., superoxide dismutase, catalase, peroxidase and glutathione and ascorbate peroxidases, markedly changed during the germination of soybean embryonic axes. Superoxide dismutase was the enzymatic activity affected the most during the initial stages of germination. Intracellular O2- steady-state concentration, calculated from the rate of O2- production and superoxide dismutase activity, showed a 2-fold increase from 2 x 10(-8) M to 4 x 10(-8) M in germination phase I, declined in phase II to 2 x 10(-8) M and remained constant over the rest of the incubation period. The reaction of H2O2 and luminol catalyzed by Co2+ was utilized to measure H2O2 diffused out of the soybean axes after 5 to 10 min of incubation. The catalase-sensitive luminol emission of diffusates prepared from axes previously imbibed from 2 to 30 h corresponded to a H2O2 intracellular steady-state concentration in the range of 0.3 to 0.9 microM. The activity of metal-containing antioxidant enzymes was determined in the extracellular fluid. Cell wall peroxidase activity increased from 10 to 300 mumol/min per mg protein and appears as a potentially important pathway for H2O2 utilization. Hydrogen peroxide metabolism in soybean embryonic axes during early inhibition appears to have the following main features: (a) mitochondrial membranes are the most important source of cytosolic O2- and H2O2; (b) H2O2 is regulated at a steady-state concentration of 0.3-0.9 microM; (c) catalase is the main enzyme in terms of H2O2 utilization; (d) H2O2 exo-diffusion is quantitatively important destiny of intracellular H2O2; and (e) extracellular peroxidase located at the cell wall affords an enzymatic system able to use diffused H2O2.     

Isolation of phenol-degrading Bacillus stearothermophilus and partial characterization of the phenol hydroxylase

Bacillus stearothermophilus BR219, isolated from river sediment, degraded phenol at levels to 15 mM at a rate of 0.85 mumol/h (4 x 10(6) cells). The solubilized phenol hydroxylase was NADH dependent, exhibited a 55 degrees C temperature optimum for activity, and was not inhibited by 0.5 mM phenol.