Effects of glucose,glutamine, and malate on the metabolism of spodoptera frugiperda clone 9 (sf9) cells

Optimal design and operation of bioreactors for insect cell culture is facilitated by functional relations providing quantitative information on cellular metabolite consumption kinetics, as well as on the specific cell growth rates (μ_G). Initial specific consumption rates of glucose, malate, and oxygen, and associated changes in μ_G, were measured forSpodoptera frugiperda clone 9 (Sf9) cells grown in batch suspension culture in medium containing 7–35 mM glucose, 0–16 mM malate, and 4–16 mM glutamine. The initial specific glucose consumption rate (q _G ) could be described by a modified Michaelis-Menten equation treating malate as a “competitive” inhibitorK ₁ = 6.5 mM) and glutamine as a “noncompetitive” inhibitorK _I = 14 mM) ofq _G , with aK _m of 7.1 mM for glucose. All three carbon sources were found to increase μ_G in a saturable manner, and a modified Monod equation was employed to describe this relationship (μ_Gmax = 0.047 h^-1). The initial specific oxygen consumption rate (q_O2) in Sf9 cells could be related to μ_G by the maintenance energy model, and it was calculated that, under typical culture conditions, about 15–20% of the cellular energy demand comes from functions not related to growth. Fitted parameters in mathematical expression for μg: K₄, Monod constant for glucose (mM); K₅, modified Monod constant for malate (mM); K₆, Monod constant for glutamine (mM); m_o2, specific consumption rate of oxygen by the cells under zero-growth conditions (nmol/cell/h); q_F, initial specific fumarate production rate (nmol/cell/ h);q _G , initial specific glucose consumption rate (nmol/cell/h); q_Gmax, maximum initial specific glucose consumption rate (nmol/cell/h);q _M , initial specific malate consumption rate (nmol/cell/h); q_o2, initial specific oxygen consumption rate (nmol/cell/h); Y_o2, cell yield on oxygen (cells/nmol); μ, initial specific cell growth rate (h^-1); μ_g, initial specific cell growth rate (h^-1); μG_max, maximum initial specific cell growth rate (h^-1).     

Effect of expression of manganese superoxide dismutase in baculovirus-infected insect cells

It has previously been demonstrated that baculovirus infection of the Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines leads to oxidative stress as measured by protein and membrane lipid oxidation and that this oxidative damage contributes to cell death. As a result of these findings, it was hypothesized that baculovirus infection stimulates superoxide radical (O ₂ ^·— synthesis in the mitochondria and that the resulting O ₂ ^·— accumulation overwhelms the cells’ antioxidant defenses. We investigated the ability of manganese superoxide dismutase (MnSOD) expression (which reduces O ₂ ^·— to H₂O₂) to overcome the oxidative damage caused by baculovirus infection. It was found that MnSOD expression significantly reduced oxidative damage in baculovirus-infected Tn-5B1-4 cells but had no significant effect on oxidative damage in baculovirus-infected Sf-9 cells. The results are consistent with the hypothesis that O ₂ ^·— accumulation in the mitochondria is at least partially responsible for the oxidative damage resulting from the baculovirus infection of insect cells.     

Simulaaneous ethanol and cellobiose inhibition of cellulose hydrolysis studied with integrated equations assuming constant or variable substrate concentration

The integrated forms of the Michaelis-Menten equation assuming variable substrate (depletion) or constant substrate concentration were used to study the effect of the simultaneous presence of two exoglucanase Cel7A inhibitors (cellobiose and ethanol) on the kinetics of cellulose hydrolysis. The kinetic parameters obtained, assuming constant substrate (K _m =21 mM, K _ic =0.035 mM; K _icl =1.5×10¹⁵mM; k _cat=12 h⁻¹) or assuming variable substrate (K _m =16 mM, K _ic =0.037 mM; K _icl =5.8×10¹⁴ mM; k _cat=9 h⁻¹), showed a good similarity between these two alternative methodologies and pointed out that bothethanol and cellobiose are competitive inhibitors. Nevertheless, ethanol is a very weak inhibitor, as shown by the large value estimated for the kinetic constant K _icl . In addition, assuming different concentrations of initial accessible substrate present in the reaction, both inhibition and velocity constants are at the same order of magnitude, which is consistent with the obtained values. The possibility of using this kind of methodology to determine kinetic constants in general kinetic studies is discussed, and several integrated equations of different Michaelis-Menten kinetic models are presented. Also examined is the possibility of determining inhibition constants without knowledge of the true accessible substrate concentration.     

Denitrification in presence of benzene,toluene, and <Emphasis Type="Italic">m</Emphasis>-xylene

Denitrification of the electron donors toluene-C(15–100 mg/L), m-xylene-C (15–70 mg/L), benzene-C (5–25 mg/L), and acetate-C as experimental reference (50–140 mg/L) was carried out in batch culture. An initial concentration of 1.1±0.15 g of volatile suspended solids/L of denitrifying sludge without previous exposure to aromatic compounds was used as inoculum. The results showed toluene and nitrate consumption efficiency (E _T and E _N′ respectively) of 100%. Toluene was completely mineralized (oxidized) to CO₂. In all cases, the N₂ (γ_N2) and HCO ₃ ⁻ yields (γ_HCO3) were 0.97±0.01 and 0.8±0.05, respectively. The consumption efficiency (E _x ) of m-xylene (53±5.7%) was partial. The γ_N2 and γ_HCO3 were 0.96±0.01 and 0.86±0.02, respectively. Benzene was not consumed under denitrifying conditions. The specific consumption rates of toluene (q _T ) and m-xylene (q _X ) were lower than that of acetate (q _A ). The differences in specific consumption rates were probably owing to the negative effect of benzene, toluene, and isomers of xylene on the cell membrane.     

Comparison of electrical double layer models postulating the linear or quadratic dependence of the anion adsorption energy on the electrode charge

Equations for calculating the concentration of a singly charged specifically adsorbed anion, the electrode potential, the differential capacitance, and interface tension are derived under the condition that the anion transition from the outer to the inner Helmholtz plane is described by the Frumkin isotherm and the energy of this transition (βG _A) is a quadratic function of the electrode charge q. By the example of the Hg/[H₂O + mc NH₄NO₃ + (1 − m)c NH₄F] system, it is shown that in contrast to the linear ΔG _A vs. q dependence, the quadratic dependence allows the differential capacitance curves to be adequately described throughout the m range. However, this model gives no way of describing the transition from the quadratic δG _A vs. q dependence for small m to the linear dependence observed experimentally in binary solutions with m = 1.     

d-xylose transport by Candida succiphila and Kluyveromyces marxianus

The kinetics and regulation of d-xylose uptake were investigated in the efficient pentose fermentor Candida succiphila, and in Kluyveromyces marxianus, which assimilate but do not ferment pentose sugars. Active high-affinity (K _m ∼ 3.8 mM; V _max ∼ 15 nmol/[mg·min]) and putative facilitated diffusion low-affinity (K _m ∼ 140 mM; V _max ∼ 130 nmol/[mg·min]) transport activities were found in C. succiphila grown, respectively, on xylose or glucose. K. marxianus showed facilitated diffusion low-affinity (K _m ∼ 103 mM; V _max ∼ 190 nmol/[mg·min]) transport activity when grown on xylose under microaerobic conditions, and both a low-affinity and an active high-affinity (K _m ∼ 0.2 mM; V _max ∼ 10 nmol/[mg·min]) transport activity when grown on xylose under fully aerobic conditions.     

Entrapment of living microbial cells in covalent polymeric networks

The kinetics of oxidative phenol degradation with microbial cellsCandida tropicalis, immobilized in a polyacrylamide and polymethacrylamide matrix, were mathematically simulated assuming zero-order and Michaelis-Menten rate equations. For zero-order kinetics an expanded equation for catalytic effectiveness as a function of the Thiele modulus, Biot number, and partition coefficients was derived and compared with numerical solutions for Michaelis-Menten kinetics. Errors with regard to the zero-order approximation become negligible ifc _o/K _M >2. Experimentally determined catalyst activities as a function of particle size and cell concentration were compared to calculated ones. Additional experiments to determine the diffusion and oxygen consumption ratios have been carried out in an effort to resolve the physical parameters to be used in the above mentioned calculations. Furthermore, experiments on cell growth during reincubation with nutrients and oxygen are reported; an increase in activity up to a factor of ten was observed. These experiments demonstrate that the microbial cells are entrapped in the polymer matrix in the living state.     

THE QUENCHING OF TYROSINE AND TRYPTOPHAN FLUORESCENCE BY H2O AND D2O*

Abstract— The quantum yields and lifetimes of the fluorescence of tyrosine and tryptophan were determined in D₂O-H₂O and glycerol-H₂O solvent mixtures of varying composition from 10 vol.% to 100% H₂O at 15°C. Forboth amino acids the ratio of the quantum yields in D₂O and H₂O (i.e., q_D/q_H) was smaller than the ratio of the corresponding lifetimes (_D/_H). For tyrosine the ratio of the quantum yields in glycerol and H₂O (q_G/q_H) was also smaller than the corresponding _G/_H ratio, but for tryptophan q_G/g_HG/_H. The proximity of the q vs. plots for tyrosine in the two solvent mixtures indicates that at 15°C neither D₂O nor glycerol, in the pure state or when diluted with H₂O, quench tyrosine significantly. However, H₂O quenches tyrosine by a dynamic process, which increases both the radiative and the nonradiative rate constant. The quenching action is attributed to a tyrosine-H₂O exciplex, whose formation is independent of bulk viscosity and dielectric constant. Unlike tyrosine, tryptophan is quenched weakly by D₂O by a static process at 15°C (i.e., involving no change in), but H₂O quenches tryptophan much more efficiently by a dynamic process, which involves the nonradiative rate constant, but not the radiative constant. These results are explained on the basis of electrostatic complexation of the ammonium group to the carbon atom adjacent to the ring nitrogen with a lifetime which is longer thanin D₂O but shorter thanin H₂O, with solvent reorientation possibly also being an important factor in the quenching. This explanation is consistent with the fact that concentrated (8 M) urea increases q andof aqueous tryptophan ? 15–20%, while guanidine hydrochloride (6.4 M) has the opposite effect, i.e., it decreases q and t of tryptophan ? 15–20%, and with the fact that neither 8 M urea nor 6.4 M guanidine hydrochloride affects any fluorescence parameter of tyrosine at all.     

Intracellular release of recombinant green fluorescent protein (gfp uv ) from Escherichia coli

The recombinant green fluorescent protein (gfp _uv ) was expressed by Escherichia coli DH5-α cells transformed with the plasmid pGFPuv. The gfp _uv was selectively permeabilized from the cells in buffer solution (25 mM Tris-HCl, pH 8.0), after freezing (−70°C for 15 h), by four freeze (−20°C)/thaw cycles interlaid by sonication. The average content of released gfp _uv (experiment 2) was 7.76, 34.58, 39.38, 12.90, and 5.38%, for the initial freezing (−70°C) and the first, second, third and fourth freeze/thaw cycles, respectively. Superfusion on freezing was observed between −11°C and −14°C, after which it reached −20°C at 0.83°C/min.     

Statistical Determination of Optimal Baculovirus Infection Condition for Recombinant Protein Production in <Emphasis Type="Italic">Drosophila</Emphasis> S2 Cells

Insect Drosophila melanogaster S2 cell was developed as plasmid-based and, therefore, a nonlytic expression system for functional foreign proteins. To achieve multiple protein expressions, it was suggested that baculovirus be used on S2 cell system because baculovirus can infect S2 cells but cannot replicate inside the cells. Therefore, establishment of baculovirus infection conditions is the first important step and this should be properly optimized for production yield. We used statistical methodology to optimize the baculovirus infection conditions using green fluorescent protein (GFP) as a reporter protein. Consequently, we arrived at optimal infection conditions through a statistical regression method. The secreted GFP yield from vMT-GFP baculovirus-infected wild-type S2 cells under optimal infection conditions was >15-fold higher than that under nonoptimal conditions and comparable to that from stably transfected recombinant S2 cells.     

<Emphasis Type="Italic">k</Emphasis>-resonance in Toroidal Polyhexes*

This paper considers the k -resonance of a toroidal polyhex (or toroidal graphitoid) with a string (p, q, t) of three integers (p ≥ 2, q ≥ 2, 0 ≤ t ≤ p − 1). A toroidal polyhex G is said to be k-resonant if, for 1≤ i ≤ k, any i disjoint hexagons are mutually resonant, that is, G has a Kekulé structure (perfect matching) M such that these hexagons are M-alternating (in and off M). Characterizations for 1, 2 and 3-resonant toroidal polyhexes are given respectively in this paper. *This work is supported by FRG, Hong Kong Baptist University; NSFC and TRAPOYT.     

Purification and characterization of an exoinulinase from Aspergillus fumigatus

An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg⁺⁺, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower K _m (0.25 mM) and higher V _max (333.3 IU/mg) values for inulin.     

Self-bonding in an amorphous polymer below the glass transition: A T-peel test investigation

Films of amorphous polystyrene (PS) with a weight-average molecular weight (M_w) of 225 × 10³ g/mol were bonded in a T-peel test geometry, and the fracture energy (G) of a PS/PS interface was measured at the ambient temperature as a function of the healing time (t_h) and healing temperature (T_h). G was found to develop with (t_h)^1/2 at T_h = T_g-bulk − 33 °C (where T_g-bulk is the glass-transition temperature of the bulk sample), and log G was found to develop with 1/T_h at T_g-bulk − 43 °C ≤ T_h ≤ T_g-bulk − 23 °C. The smallest measured value of G = 1.4 J/m² was at least one order of magnitude larger than the work of adhesion required to reversibly separate the PS surfaces. These three observations indicated that the development of G at the PS/PS interface in the temperature range investigated (<T_g-bulk) was controlled by the diffusion of chain segments feasible above the glass-transition temperature of the interfacial layer, in agreement with our previous findings for fracture stress development at several polymer/polymer interfaces well below T_g-bulk. Close values of G = 8–9 J/m² were measured for the symmetric interfaces of polydisperse PS [M_w = 225 × 10³, weight-average molecular weight/number-average molecular weight (M_w/M_n) = 3] and monodisperse PS (M_w = 200 × 10³, M_w/M_n = 1.04) after healing at T_h = T_g-bulk − 33 °C for 24 h. This implies that the self-bonding of high-molecular-weight PS at such relatively low temperatures is not governed by polydispersity. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 1861–1867, 2004     

Enzyme kinetics and glycan structural characterization of secreted alkaline phosphatase prepared using the baculovirus expression vector system

Secreted human alkaline phosphatase (SEAP, a model protein containing a single N-glycan chain) was expressed in Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines infected with recombinant Autographa californica multiple nuclear polyhedrovirus expressing SEAP under control of the polyhedrin promoter. SDS-PAGE showed that both systems expressed fairly pure rSEAP products. The rSEAP expression level was 7.0 U/mL in Tn-5B1-4, higher than the 4.1 U/mL produced by Sf-9. Kinetic analysis showed that V _max and K _m of human placental SEAP were approx 10-fold higher than that of rSEAP, whereas the V _max and K _m of rSEAP prepared using both insect cell lines were comparable. To characterize the recombinant SEAP (rSEAP) glycosylation, the purified rSEAP was digested with PNGase F to release the N-glycan chains. Glycan analysis showed the presence of oligomannose-type N-linked glycans (i.e., Man_2–8 GlcNAc₂ and FucMan_{3 or 4}GlcNAc₂) in rSEAP from Sf9 and Tn-5B1-4 cell lines. The proportions of these oligosaccharide structures were different in the two cell lines. Man₄GlcNAc₂ and FucMan₄GlcNAc₂ were the major rSEAP N-glycans produced in Sf-9 cells, while Man₂GlcNAc₂ was the major rSEAP N-glycan produced in Tn-5B1-4 cells.     

Comparative energetics of glucose and xylose metabolism in recombinant Zymomonas mobilis

Recombinant Zymomonas mobilis CP4:pZB5 was grown with pH control in batch and continuous modes with either glucose or xylose as the sole carbon and energy source. In batch cultures in which the ratio of the final cell mass concentration to the amount of sugar in the medium was constant (i.e., under conditions that promote “coupled growth”), maximum specific rates of glucose and xylose consumption were 8.5 and 2.1 g/(g of cell…h), respectively; maximum specific rates of ethanol production for glucose and xylose were 4.1 and 1.0 g/(g of cell…h), respectively; and average growth yields from glucose and xylose were 0.055 and 0.034 g of dry cell mass (DCM)/g of sugar respectively. The corresponding value of Y_ATP for glucose and xylose was 9.9 and 5.1 g of DCM/mol of ATP, respectively. Y_ATP for the wild-type culture CP4 with glucose was 10.4g of DCM/mol of ATP. For single substratechem ostat cultures in which the growth rate was varied as the dilution rate (D), the maximum or “true” growth yield (max Y_a/s) was calculated from Pirt plots as the inverse of the slope of the best-fit linear regression for the specific sugar utilization rate as a function of D, and the “maintenance coefficient” (m) was determined as the y-axis intercept. For xylose, values of max Y _s/s and m were 0.0417g of DCM/g of xylose (Y_ATP=6.25) and 0.04g of, xylose/(g of cell…h), respectively. However, with glucose there was an observed deviation from linearity, and the data in the Pirt plot was best fit with a second-order polynomial in D. At D>0.1/h, Y_ATP=8.71 and m=2.05g of glu/(g of cell…h) whereas at D<0.1/h, Y_ATP=4.9g of DCM/mol of ATP and m=0.04g of glu/(g of cell…h). This observation provides evidence to question the validity of the unstructured growth model and the assumption that Pirt's maintenance coefficient is a constant that is in dependent of the growth rate. Collectively, these observations with individual sugars and the values assign ed to various growth and fermentation parameters will be useful in the development of models to predict the behavior of rec Zm in mixed substrate fermentations of the type associated with biomass-to-ethanol processes.     

Immobilization of a recombinant cutinase by entrapment and by covalent binding

Fusarium solani pisi recombinant cutinase, immobilized by entrapment in calcium alginate and by covalent binding on porous silica, was used to catalyze the hydrolysis of tricaprylin. The influence of relevant parameters on the catalytic activity such as pH, temperature, and the substrate concentration were studied. Cutinase immobilized by entrapment presented a Michaelis-Menten kinetics for tricaprylin concentrations up to 200 mM. At higher concentrations of substrate, inhibition was observed. For covalent binding immobilization, diffusional limitations were observed at low substrate concentrations and substrate inhibition occurred for concentrations higher than 150 mM. The stability of immobilized cutinase was also evaluated. The enzyme immobilized by entrapment showed a high stability, in contrast to the immobilization on porous silica.     

Planar Hypercoordinate Carbons in Alkali Metal Decorated CE32− and CE22− Dianions

After exploring the potential energy surfaces of M_mCE₂^p (E=S−Te, M=Li−Cs, m=2, 3 and p=m-2) and M_nCE₃^q (E=S−Te, M=Li−Cs, n=1, 2, q=n-2) combinations, we introduce 38 new global minima containing a planar hypercoordinate carbon atom (24 with a planar tetracoordinate carbon and 14 with a planar pentacoordinate carbon). These exotic clusters result from the decoration of V-shaped CE₂²⁻ and Y-shaped CE₃²⁻ dianions, respectively, with alkali counterions. All these 38 systems fulfill the geometrical and electronic criteria to be considered as true planar hypercoordinate carbon systems. Chemical bonding analyses indicate that carbon is covalently bonded to chalcogens and ionically connected to alkali metals.     

Extended molecular symmetry groups

The group theoretical analysis of Longuet-Higgins molecular symmetry groupsG(m, n) andGB(m) and their torsional extensionsG ^q (m, n) andGB ²(m) of molecules consisting of two coaxially rotated parts is presented. The structure of the groups is described in terms of direct and semi-direct products. The groupsG ^q (m, n) andGB ²(m) are shown to be group extensions of the groupsG(m,n) andGB(m), respectively. All irreducible representations of the groupsG(m,n),GB(m), andGB ²(m) are derived using standard techniques of the extension or induction. A restriction method is proposed for derivation of irreducible representations of the groupG ^q (m,n). The class structure of the groups is determined and the character tables are given in the most general case.     

A representation of the polarization term in the interaction energy between a molecule and a point-like charge

The interaction energy of a molecule M with a point-like charge q can be partitioned into simpler contributions, two of which can be expressed in terms of the charge distribution _M of the sole M. The first term, qV(r), represents the interaction of q with the undistorted charge _M ⁰ of M while the second q ² P(r) gives the additional contributions due to the polarization of _M ⁰ under the influence of the charge q placed at the point r. In this paper we investigate the possibility of getting an inexpensive and sufficiently accurate analytical representation of P(r) over the whole space outside the van der Waals volume of M.     

Enzymatic kinetic of cellulose hydrolysis

The ethanol effect on the Trichoderma reesei cellulases was studied to quantify and clarify this inhibition type. To determine inhibition parameters of crude cellulase and purified exoglucanase Cel7A, integrated Michaelis-Menten equations were used assuming the presence of two inhibitors: cellobiose as the reaction product and ethanol as a possible bioproduct of cellulose fermentation. It was found that hydrolysis of cellulose by crude enzyme follows a model that considers noncompetitive inhibition by ethanol, whereas Cel7A is very slightly competitively inhibited. Crude cellulase is much more inhibited (K _iul=K _icl=151.9 mM) than exoglucanase Cel7A (K _icl=1.6 × 10¹⁵ mM). Also, calculated inhibition constants showed that cellobiose inhibition is more potent than ethanol inhibition both for the crude enzyme as well as exoglucanase Cel7A.