Effect of DG5128 on epinephrine and glucagon induced glucose output from the isolated perfused rat liver

The effect of a specific alpha 2-adrenergic antagonist 2-[2-(4,5-dihydro-1.H-imidazol-2-yl)-1-phenyl-ethyl] pyridine dihydrochloride sesquihydrate (DG5128), on the glucose output by epinephrine and/or glucagon was studied using the perfused rat liver. The administration of DG5128 alone did not affect the glucose output. However, DG5128 produced a significant inhibition of the increased glucose output when induced by 10(-6) M epinephrine alone or 10(-6) M epinephrine plus 1.4 x 10(-10) M glucagon. There were no significant changes of the glucose output by 1.4 x 10(-10) M or 7.0 x 10(-11) M glucagon alone. On the other hand, addition of 1 mU/ml insulin to the perfusate suppressed the 7.0 x 10(-11) M glucagon-induced glucose output, but failed to decrease the 1.4 x 10(-10) M glucagon effect. DG5128 suppressed further the glucagon (7.0 x 10(-11) M)-induced increase of glucose output in the presence of insulin. These results suggest that DG5128 produces a hypoglycemic effect partly through an inhibition of the increased hepatic glucose output elicited by epinephrine and glucagon.     

Calorimetric versus Growth Microbial Analysis of Cellulase Enzymes Acting on Cellulose

Assay of cellulase enzymology on cellulose was investigated by two methods: (i) plate colony counting to determine microbial growth and (ii) microbial calorimetry. These methods were chosen because they accept raw samples and have the potential to be far more specific than spectrophotometric reducing sugar assays. Microbial calorimetry requires ca. 0.5 to 1 h and 10 to 100 μM concentrations of cellulolytic lower sugars (glucose and cellobiose). Growth assay (liquid culture, plating, colony counting) requires 15 to 20 h and ca. 0.5 mM sugars. Microbial calorimetry requires simply aerobic metabolism, whereas growth assay requires completion of the cell cycle. A stripping technique is described for use in conjunction with the calorimetric method to enable separate analysis of the two sugars. Mixtures of glucose and cellobiose are equilibrated with Escherichia coli and spun out to remove glucose. The supernatant is calorimetrically combusted with Klebsiella sp. to quantitate cellobiose, and the same organism combusting the nonstripped mixture gives heat proportional to the sum of the two sugars. Calorimetry of cellulolysis products from individual exo- and endocellulases, and from their reconstituted mixture, was carried out to develop a microbial calorimetric means for demonstrating enzyme synergism.     

Degradation of pentachlorophenol by a Flavobacterium species grown in continuous culture under various nutrient limitations

A Flavobacterium sp. was grown in continuous culture limited for growth with ammonium, phosphate, sulfate, glucose, glucose + pentachlorophenol (PCP) (0.065 h -1), or PCP. Cells ere harvested, washed, and suspended to 3 x 10(7) cells ml (-1) in shake flasks containing a complete mineral salts medium without added carbon or supplemented with 50 mg of PCP ml(-1) or 50 mg of PCP ml(-1) + 100 mg of glucose ml(-1). The PCP concentration and the viable cell density were determined periodically. Cells that were grown under phosphate, glucose, or glucose + PCP limitation were more sensitive to PCP and took longer to degrade 50 mg of PCP ml(-1) than did cells that very were grown under ammonium, sulfate, or PCP limitation. Glucose stimulated viability and PCP degradation in all cases except when the cells were grown under carbon limitation with glucose and PCP added together as the carbon source. These results indicate that there is a relationship between nutrient limitation, phenotypic variation, and the sensitivity to and degradation of PCP by this organism.     

Degradation of pentachlorophenol by a Flavobacterium species grown in continuous culture under various nutrient limitations.

A Flavobacterium sp. was grown in continuous culture limited for growth with ammonium, phosphate, sulfate, glucose, glucose + pentachlorophenol (PCP) (0.065 h -1), or PCP. Cells ere harvested, washed, and suspended to 3 x 10(7) cells ml (-1) in shake flasks containing a complete mineral salts medium without added carbon or supplemented with 50 mg of PCP ml(-1) or 50 mg of PCP ml(-1) + 100 mg of glucose ml(-1). The PCP concentration and the viable cell density were determined periodically. Cells that were grown under phosphate, glucose, or glucose + PCP limitation were more sensitive to PCP and took longer to degrade 50 mg of PCP ml(-1) than did cells that very were grown under ammonium, sulfate, or PCP limitation. Glucose stimulated viability and PCP degradation in all cases except when the cells were grown under carbon limitation with glucose and PCP added together as the carbon source. These results indicate that there is a relationship between nutrient limitation, phenotypic variation, and the sensitivity to and degradation of PCP by this organism.     

Quantifying the Responses of Mixed Rumen Microbes to Excess Carbohydrate

The aim of this study was to determine if a mixed microbial community from the bovine rumen would respond to excess carbohydrate by accumulating reserve carbohydrate, energy spilling (dissipating excess ATP energy as heat), or both. Mixed microbes from the rumen were washed with N-free buffer and dosed with glucose. Total heat production was measured by calorimetry. Energy spilling was calculated as heat production not accounted by (i) endogenous metabolism (heat production before dosing glucose) and (ii) synthesis of reserve carbohydrate (heat from synthesis itself and reactions yielding ATP for it). For cells dosed with 5 mM glucose, synthesis of reserve carbohydrate and endogenous metabolism accounted for nearly all heat production (93.7%); no spilling was detected (P = 0.226). For cells dosed with 20 mM glucose, energy spilling was not detected immediately after dosing, but it became significant (P < 0.05) by approximately 30 min after dosing with glucose. Energy spilling accounted for as much as 38.7% of heat production in one incubation. Nearly all energy (97.9%) and carbon (99.9%) in glucose were recovered in reserve carbohydrate, fermentation acids, CO₂, CH₄, and heat. This full recovery indicates that products were measured completely and that spilling was not a methodological artifact. These results should aid future research aiming to mechanistically account for variation in energetic efficiency of mixed microbial communities.     

Culture of insect cells in helical ribbon impeller bioreactor

An 11-L helical ribbon impeller (HRI) bioreactor was tested for the culture of Spodoptera frugiperda (Sf-9) cells. This impeller and surface baffling ensured homogeneous mixing and high oxygen transfer through surface aeration and surface-induced babble generation. Serum-supplemented and serum-free cultures, using TNMFH and IPL/41 media, respectively, grew a similar specific growth rates(0.031 and 0.028 h(-1)) to maximum cell densities of 5.5 x 10(6)-6.0 x 10(6) cells. mL(-1) with viability exceeding 98% during exponential growth phase. Growth limitation coincided with glucose and glutamine depletion and production of significant amounts of alanine. The bioreactor was further tested under more stringent conditions by infecting a serum-free medium culture with a recombinant baculovirus. Heterologous protein production of approximately 35 mug per 10(6) cells was comparable to yields obtained in serum-free cultures grown in spinner flasks and petri dishes. Average specific oxygen up-take and carbon dioxide production rates of the serum-free culture prior to infection as measured by on-line mass spectroscopy were 0.20 mumol O(2)mu.(10(6) cells)(-1) h(-1) and 0.22 mumol CO(2) . (10(6) cells)(-1)h(-1) and increased by 30-40% during infection. Therefore, the mixing and oxygenation conditions of this bioreactor were suitable for insect cell culture and recombinant protein production, with limitation being mainly attributed to nutrient depletion and toxic by-product generation.     

High-frequency phage-mediated gene transfer among Escherichia coli cells, determined at the single-cell level

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3x10(-8) to 2x10(-6), 1x10(-8) to 4x10(-8), and <4x10(-9) to 4x10(-8) per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7x10(-4) to 1x10(-3), 9x10(-4) to 3x10(-3), and 5x10(-4) to 4x10(-3) for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.     

Propionate supplementation did not increase whole body glucose turnover in growing lambs fed rye grass

The objective of the present study was to investigate the effects of propionate supplementation on whole body glucose turnover in growing lambs fed frozen rye-grass at 1.5 x maintenance using [1-13C]-glucose. Intraruminal infusion of propionate (0.55 and 0.91 mol x d(-1)) increased the ruminal molar proportions of propionate from 25% with the control to 40% with the highest propionate treatment. It did not however modify glucose turnover (26 mmol x d(-1) x kg(-1)), nor the conversion of its carbon into L-lactate (21%) and alanine (21%), nor glucose recycling (9%). All of the results suggest that in the present conditions glucose turnover and metabolism were not influenced by the supply of propionate.     

On-line determination of animal cell concentration in two industrial high-density culture processes by dielectric spectroscopy.

Dielectric spectroscopy was applied to two industrial high cell density culture processes and used to determine on-line the concentration of CHO cells immobilized on macroporous microcarriers in a stirred bioreactor and in a packed-bed of disk carriers. The cell concentration predicted from the spectroscopic data was in excellent agreement with off-line cell counting data for both processes. Deviations between the two counting methods only occurred in the case of a significant decrease of the cell viability, from 93% to 64%, which induced a change of the average cell size in the culture. Results for the packed-bed process were further confirmed by the application of indirect yield models based on the measurement of glucose, lactate, and the protein of interest. Moreover, dielectric spectroscopy was used as a tool to characterize the packed-bed process. It was possible to determine both the maximum cell concentration that could be reached in the culture system, 2.0 x 10(11) cell per kg of disk carrier, and to quantify the increase of specific protein productivity induced by the production phase, from 5.14 x 10(-8) microg x cell(-1) x h(-1) to 4.24 x 10(-7) microg x cell(-1) x h(-1).     

Laboratory-scale continuous reactor for soluble selenium removal using selenate-reducing bacterium,Bacillus sp. SF-1

A model continuous flow bioreactor (volume 0.5 L) was constructed for removing toxic soluble selenium (selenate/selenite) of high concentrations using a selenate-reducing bacterium, Bacillus sp. SF-1, which transforms selenate into elemental selenium via selenite for anaerobic respiration. Model wastewater contained 41.8 mg-Se/L selenate and excess lactate as the carbon and energy source; the bioreactor was operated as an anoxic, completely mixed chemostat with cell retention time between 2.2-95.2 h. At short cell retention times selenate was removed by the bioreactor, but accumulation of selenite was observed. At long cell retention times soluble selenium, both selenate and selenite, was successfully reduced into nontoxic elemental selenium. A simple mathematical model is proposed to evaluate Se reduction ability of strain SF-1. First-order kinetic constants for selenate and selenite reduction were estimated to be 2.9 x 10(-11) L/cells/h and 5.5 x 10(-13) L/cells/h, respectively. The yield of the bacterial cells by selenate reduction was estimated to be 2.2 x 10(9) cells/mg-Se.     

Duplicated dockerin subdomains of Clostridium thermocellum endoglucanase CelD bind to a cohesin domain of the scaffolding protein CipA with distinct thermodynamic parameters and a negative cooperativity

Mutagenized dockerin domains of endoglucanase CelD (type I) and of the cellulosome-integrating protein CipA (type II) were constructed by swapping residues 10 and 11 of the first or the second duplicated segment between the two polypeptides. These residues have been proposed to determine the specificity of cohesin-dockerin interactions. The dockerin domain of CelD still bound to the seventh cohesin domain of CipA (CohCip7), provided that mutagenesis occurred in one segment only. Binding was no longer detected by nondenaturing gel electrophoresis when both segments were mutagenized. The dockerin domain of CipA bound to the cohesin domain of SdbA as long as the second segment was intact. None of the mutated dockerins displayed detectable binding to the noncognate cohesin domain. Isothermal titration calorimetry showed that binding of the CelD dockerin to CohCip7 occurred with a high affinity [K(a) = (2.6 +/- 0.5) x 10(9) M(-1)] and a 1:1 stoichiometry. The reaction was weakly exothermic (DeltaHdegrees = -2.22 +/- 0.2 kcal x mol(-1)) and largely entropy driven (TDeltaSdegrees = 10.70 +/- 0.5 kcal x mol(-1)). The heat capacity change on complexation was negative (DeltaC(p) = -305 +/- 15 cal x mol(-1) x K(-1)). These values show that cohesin-dockerin binding is mainly hydrophobic. Mutations in the first or the second dockerin segment reduced or enhanced, respectively, the hydrophobic character of the interaction. Due to partial enthalpy-entropy compensation, these mutations induced only small changes in binding affinity. However, the binding affinity was strongly decreased when both segments were mutated, indicating strong negative cooperativity between the two mutated sites.     

Characterization of energy conversion based on metabolic flux analysis in mixotrophic liverwort cells, Marchantia polymorpha

In order to characterize the contributions of respiratory and photosynthetic actions to energy conversions, the mixotrophic cells of Marchantia polymorpha were cultivated in the medium containing 10kg/m(3) glucose as an organic carbon source. The cultures were conducted with the supply of ordinary air (0.03% CO(2)) at constant incident light intensities of 50 and 180W/m(2). From the results of metabolic analysis, it was found that the cell yield based on ATP synthesis was estimated to be 6.3x10(-3)kg-dry cells/mol-ATP in these cultures. Under the examined conditions, energy conversion efficiency through respiration was larger than that through photosynthesis, and efficiency of overall energy conversion to ATP was maximized when the sum of energies from glucose and light captured by the cells was approximately 7.2x10(5)J/(hkg-dry cells). Taking into account the efficiency of overall energy conversion, a batch culture of M. polymorpha in a bioreactor was carried out by regulating incident light intensity ranging from 9 to 58W/m(2). In the culture with light regulation, the cell yield of 6.2x10(-9)kg-dry cells/J was achieved on the basis of energy provided to the system throughout the culture, and this value was 2.3 and 9.3 times as large as those obtained in the cultures under constant incident light intensities of 50 and 180W/m(2), respectively.     

PEMC sensor's mass change sensitivity is 20 pg/Hz under liquid immersion

To enhance the mass change sensitivity of the resonating piezoelectric-excited millimeter-sized cantilever (PEMC) sensors, we reduced its length and eliminated one layer of its composite structure. As a result the mass sensitivity of the second flexural mode increased by two orders of magnitude (from 10(-9) to 10(-11)g/Hz) and the resonant frequency increased by more than 5 kHz. We demonstrate the effects of modification by detecting a model pathogen Group A Streptococcus (GAS) at 700 cells/mL. The resonant frequency change of the second mode at concentrations of 700, 7 x 10(3), 7 x 10(5), 7 x 10(6), 7 x 10(7), and 7 x 10(9)cells/mL resulted in, respectively, 3.1+/-0.5, 11.6+/-1, 15.7+/-1, 25.7+/-0.15, 28.5+/-2, and 40.5+/-3 ng (n=3 for all) of pathogen attachment. A kinetic model for the binding is proposed and verified. The observed binding rate constant was found to be in the range of 0.051-0.166 min(-1). The significance of the results we report is that the modified PEMC sensors have high mass sensitivity that pathogens can be detected at very low concentration under liquid immersion conditions.     

添加TCA循环中间产物加速光滑球拟酵母积累丙酮酸

在维生素限制的条件下,研究了添加TCA循环中间产物对光滑球拟酵母多重维生素营养缺陷型菌株CCTCC M202019生长和积累丙酮酸的影响。该菌株能以TCA循环中间产物为唯一碳源进行生长,且在以葡萄糖、乙酸和TCA循环中间产物为复合碳源的平板上菌落数高于分别以葡萄糖和乙酸或TCA循环中间产物为唯一碳源时的菌落数。与其它TCA循环中间产物相比,草酰乙酸更能促进细胞的生长、提高丙酮酸产量和对葡萄糖的得率。草酰乙酸能够促进细胞生长,是因为T. glabrata CCTCC M202019菌株能够利用乙酸作为乙酰辅酶A供体。在含有100 g/L葡萄糖和6 g/L乙酸钠的培养基中再添加10 g/L草酰乙酸进行分批发酵实验,可使菌体浓度从11.8 g/L提高到 13.6 g/L,增长幅度为15%;丙酮酸对葡萄糖的得率(0.66 g/g)以及生产强度(1.19 g·L^{-1<、sup>·h-1<、sup>)分别高出6%和24%,使发酵结束时间提前8～12h。}     

Use of potato extract broth for culturing root-nodule bacteria

Liquid media containing potato extract and 1% of glucose or sucrose were used to culture root-nodule bacteria (rhizobia) in shaken Erlenmeyer flasks. For comparison, these bacteria were also cultured in yeast extract-mannitol broth (YEMB) as a standard medium. Proliferation of rhizobia was monitored by measuring optical densities (OD550) of the cultures and by plate counting of the viable cells (c.f.u) of the bacteria. In general, multiplication of the rhizobia in potato extract-glucose broth (PEGB) and potato extract-sucrose broth (PESB) was markedly faster, as indicated by higher values of OD550, than in YEMB. The numbers of R. leguminosarum by. vicae GGL and S. meliloti 330 in PEGB and PEGB were high and ranged from 1.2 x 10(10) to 4.9 x 10(10) mL(-1) after 48 h of incubation at 28 degrees C. B. japonicum B3S culture in PEGB contained 6.4 x 10(9) c.f.u. ml(-1) after 72 h of incubation. PEGB and YEMB cultures of the rhizobia were similar with respect to their beneficial effects on nodulation of the host-plants of these bacteria.     

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.     

The mechanism of pH-dependent hydrogen peroxide cytotoxicity in vitro

The present paper is concerned with the influence of hydrogen ion concentration and composition of the medium on clonogenic survival of epithelial cells exposed to hydrogen peroxide in vitro. The survival of cells incubated with H2O2 in phosphate-buffered saline at pH 6.5 was 1 x 10(-2) and increased abruptly to 9 x 10(-2) at pH 7.0. The pH dependence of the cytocidal effect was particularly conspicuous when Eagle's minimum essential medium (SFMEM) was used for cell exposure to H2O2: the survival was characterized by exponential pH dependence and varied from 1 x 10(-1) to 9 x 10(-1) for pH 6.5 and 7.5, respectively, with a superimposed sharp peak value of 9 x 10(-1) at pH 7.0. The enhanced pH dependence of the H2O2 cytotoxicity in SFMEM was found to result from the additive action of glucose and histidine present in this medium. Glucose alone protected the cells with the efficiency decreasing with increasing hydrogen ion concentration. Histidine was responsible for the intermediate maximum in the pH-dependent survival spectrum. In addition, the changes in cell survival were accompanied by pH-dependent release of GSSG from the exposed cells. The GSSG efflux was inhibited by glucose in the medium. The influence of glucose on both the pattern of cell survival and the associated GSSG release indicate that the glutathione peroxidase activity supported by the pentose phosphate pathway is crucial in cell protection against extracellular H2O2 toxicity.     

Calorimetric techniques for metabolic studies of cells and organisms under normal conditions and stress

When cells are subjected to stress, an early result is a shift in type and rate of metabolism to reflect their new conditions. The availability of metabolites, their endogenous vs exogenous origins, and the rates at which they can be used, besides availability of oxygen, dictate cell and tissue response. Measurement of heat output in such a response is a means for monitoring cells and tissues. Differential heat conduction calorimeters are reviewed to provide a listing of instrument parameters important in optimum practical use. Data obtained with one cell system, mammalian sperm, are presented to provide an example of how the combination of calorimetry and carbon balance, plus calculation from thermodynamic constants, permit an assessment of the importance of endogenous metabolism to total cellular metabolism.     

Purification,amino acid sequence,and cDNA cloning of trypsin inhibitors from onion (Allium cepa L.) bulbs

Three protease inhibitors (OTI-1-3) have been purified from onion (Allium cepa L.) bulbs. Molecular masses of these inhibitors were found to be 7,370.2, 7,472.2, and 7,642.6 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. Based on amino acid composition and N-terminal sequence, OTI-1 and -2 are the N-terminal truncated proteins of OTI-3. All the inhibitors are stable to heat and extreme pH. OTI-3 inhibited trypsin, chymotrypsin, and plasmin with dissociation constants of 1.3 x 10(-9) M, 2.3 x 10(-7) M, and 3.1 x 10(-7) M, respectively. The complete amino acid sequence of OTI-3 showed a significant homology to Bowman-Birk family inhibitors, and the first reactive site (P1) was found to be Arg17 by limited proteolysis by trypsin. The second reactive site (P1) was estimated to be Leu46, that may inhibit chymotrypsin. OTI-3 lacks an S-S bond near the second reactive site, resulting in a low affinity for the enzyme. The sequence of OTI-3 was also ascertained by the nucleotide sequence of a cDNA clone encoding a 101-residue precursor of the onion inhibitor.     

Early effect of glucose and oxygen deprivation on the spontaneous acetylcholine release from the myenteric plexus of the guinea pig ileum

The early effects of glucose and oxygen deprivation on the spontaneous acetylcholine output from the myenteric plexus - longitudinal muscle preparation of the guinea pig ileum were studied using an incubation chamber that permitted rapid sample collection in 2-min intervals. Glucose deprivation or hypoxia resulted in a gradual decline in rate of spontaneous acetylcholine collection in 2-min intervals. Glucose deprivation or hypoxia resulted in a gradual decline in rate of spontaneous acetylcholine output. However, glucose deprivation plus hypoxia caused an acceleration in acetylcholine output within 10-15 min, which attained a rate seven times greater than observed under normal conditions. Recovery of low resting rates was obtained by reintroduction of oxygen and glucose into the bath medium. Neither morphine (2.7 x 10(-5) M) nor tetrodotoxin (1.6 x 10(-6) M) prevented the increase in acetylcholine output induced by energy deprivation. The substitution of Ca2+ by Mg2+, in the presence of EGTA, greatly reduced the acetylcholine output induced by energy deprivation. However, a small transitory output of acetylcholine was observed under these conditions which was resistant to tetrodotoxin and ot affected by depolarizing amounts of K+. The transitory output was repeatable by reintroduction of glucose and oxygen to the Ca2+-free medium with subsequent return to conditions of hypoxia and glucose deprivation. These results suggest that energy deprivation initially stimulates normal acetylcholine secretion by (a) increasing Ca2+ influx across the plasma membrane and (b) mobilizing an intracellular Ca2+ poll. This implies that processes involved in maintenance of normal low transmitter release are more sensitive to energy lack than the neurosecretion process itself.