Studies on beta-galactosidase production in transient operation cultures

Summary Beta-galactosidase production in transient operation culture was studied considering cycles of 90 and 180 min.The results obtained after 14 cycles periods proved that significant increases in the especific enzyme activity are attained in transient operation namely 3.83 to 4.81 umg^–1 compared with those obtained in steady state continuous culture which ranged between 1.71 to 2.2 Umg^–1.     

Lysine acetylation can generate highly charged enzymes with increased resistance toward irreversible inactivation

This paper reports that the acetylation of lysine ε-NH₃⁺ groups of α-amylase—one of the most important hydrolytic enzymes used in industry—produces highly negatively charged variants that are enzymatically active, thermostable, and more resistant than the wild-type enzyme to irreversible inactivation on exposure to denaturing conditions (e.g., 1 h at 90°C in solutions containing 100-mM sodium dodecyl sulfate). Acetylation also protected the enzyme against irreversible inactivation by the neutral surfactant TRITON X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)phenyl ether), but not by the cationic surfactant, dodecyltrimethylammonium bromide (DTAB). The increased resistance of acetylated α-amylase toward inactivation is attributed to the increased net negative charge of α-amylase that resulted from the acetylation of lysine ammonium groups (lysine ε-NH₃⁺ → ε-NHCOCH₃). Increases in the net negative charge of proteins can decrease the rate of unfolding by anionic surfactants, and can also decrease the rate of protein aggregation. The acetylation of lysine represents a simple, inexpensive method for stabilizing bacterial α-amylase against irreversible inactivation in the presence of the anionic and neutral surfactants that are commonly used in industrial applications.     

Fitness, Flux and Phantoms in Temporally Variable Environments

The evolutionary problem of selection in temporally variable environments is addressed by investigating a metabolic model describing the approach to steady state of a flux emanating from a simple linear pathway of unsaturated enzymes catalyzing reversible monomolecular reactions. Analysis confirms previous claims that steps having no influence on the steady state flux may influence transient behavior, and that enzymes immune to natural selection at steady state may become subject to selection when fitness is a function of individual transient metabolic events. Indeed, calculations show that the β-galactosidase of Escherichia coli, which exerts a negligible effect on the steady state lactose flux, controls the approach to steady state. However, after 6 sec the lactose flux is within 0.1% of steady state, and so an ever changing environment must be invoked to continually expose β-galactosidase to selection. Analysis of the metabolic model undergoing multiple transient events reveals that fitness differences remain unaffected if enzyme activities remain constant and become minimized if enzyme activities differ among environments. Until suitable data become available, claims that metabolic behavior away from steady state necessarily exposes a far greater proportion of allozymes to natural selection should be treated with great skepticism.     

Intramolecular disorder and its relation to mesophase structure in lipid/water mixtures

NMR spin-half pair dipolar echo measurements are reported for the lamellar (dispersions and multibilayer stacks) and hexagonal phases of potassium palmitate/²H₂O mixtures. In the lamellar L_β and L_γ (gel) phases the alkyl chains are rigid and perfectly ordered, while in the lamellar L_α and hexagonal phases they are flexible and disordered. In particular, the measurements show that in the fluid lamellar L_α phase the chain is “bent” at the C₉–C₁₀ segment; but is “straight” in the hexagonal phase.     

The effects of progesterone, prostaglandin F2α and oxytocin on the calcium-activation of the uterus

The relationship between “activator-calcium” (A-Ca), progesterone (P), prostaglandin F2α (PGF2α) and oxytocin (Oxy) has been examined in 100 uterine strips of 34 pregnant and 100 strips of 34 post partum rabbits. At the 25th day of gestation, uterine P was 13.9±1.3 ng/g, while within 3–12 hours post partum 3.3±0.3 ng/g tissue (P<0.001). Uterine strips, mounted isometrically in Krebs' solution, sustained maximum excitability in a steady state when exposed every 30 seconds for 4 seconds to an electric field of 12 V/5 cm (a.c.). The maximally contracting muscles were then rinsed at intervals of 6 minutes with Ca-free Krebs.In Ca-free Krebs, the post partum uterus lost 31% of its Ca and 96% of its excitability in a short 25 minutes, while the pregnant uterus lost 30% of its Ca and 93% of its excitability in 50 minutes (P<0.001). Since the extracellular space is 30% in the uterus, this 30% Ca, lost by both muscles, most probably was extracellular Ca and the small A-Ca fraction which is presumably “bound” more strongly at the membrane systems of the P-dominated pregnant, than the non-dominated post partum uterus. The significantly faster and more complete recovery from Ca-deficiency and inexcitability of the pregnant than the post partum uterus (P<0.001), at different levels of external Ca, further substantiates this premise. So does the demonstration that exposure to Ca-free Krebs increases ⁴⁵Ca-efflux 400% in the post partum and only 110% in the pregnant uterus (P<0.001). Exposure to 100 ng/ml PGF2α in normal Krebs has a similar effect on the ⁴⁵Ca-efflux of the post partum uterus, while the response of the pregnant uterus is indistinct (P<0.001).These highly significant differences between the post partum and the pregnant uteri in their Ca-efflux explain the higher threshold (P<0.001) and lower “sensitivity” to PGF2α and Oxy (P<0.001) of the pregnant than the post partum uterus. The already very highly significant differences between the two muscles, in threshold and sensitivity to these two most potent oxytocics, were increased still further by rendering the uterine strips Ca-deficient. All together, these findings substantiate the early contention (1–7,18,19) that uterine function at the cellular level is regulated by opposing actions of the suppressor P and the intrinsic stimulant PG or other oxytocic agents on threshold, excitability and the Ca-activation of the contractile process.     

The “interleukin 1 receptor antagonist” is a partial agonist of prostaglandin synthesis by human decidual cells

In many systems the interleukin-1 receptor antagonist opposes the effects of interleukin-1β. We considered that it might block interleukin-1β-stimulated prostaglandin production from human decidual cells. Very high levels of interleukin-1 receptor antagonist (>1000 pg/ml) had limited inhibitory effects on IL-1β-stimulated PGE2 synthesis, and lower levels of antagonist (<1000 pg/ml) increased the effects of IL-1β. Low concentrations of the antagonist alone (1–100 pg/ml) increased basal PGE2 production, whereas higher levels (10–100 ng/ml) had less effect. It seems, therefore, that in human decidua the “antagonist” is more accurately described as a partial agonist. It has been suggested that the IL-1 receptor antagonist could be used to inhibit decidual prostaglandin synthesis and thereby prevent preterm labor, but this report shows that caution should be exercised before using the receptor antagonist.     

Expression of frutalin, an alpha-D-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris

Frutalin is an α-d-galactose-binding lectin expressed in breadfruit seeds. Its isolation from plant is time-consuming and results in a heterogeneous mixture of different lectin isoforms. In order to improve and facilitate the availability of the breadfruit lectin, we cloned an optimised codifying frutalin mature sequence into the pPICZαA expression vector. This expression vector, designed for protein expression in the methylotrophic yeast Pichia pastoris, contains the Saccharomyces α-factor preprosequence to direct recombinant proteins into the secretory pathway. Soluble recombinant frutalin was detected in the culture supernatants and recognised by native frutalin antibody. Approximately 18–20 mg of recombinant lectin per litre medium was obtained from a typical small scale methanol-induced culture purified by size-exclusion chromatography. SDS–PAGE and Edman degradation analysis revealed that frutalin was expressed as a single chain protein since the four amino-acid linker peptide “T-S-S-N”, which connects α and β chains, was not cleaved. In addition, incomplete processing of the signal sequence resulted in recombinant frutalin with one Glu-Ala N-terminal repeat derived from the α-factor prosequence. Endoglycosidase treatment and SDS–PAGE analysis revealed that the recombinant frutalin was partly N-glycosylated. Further characterisation of the recombinant lectin revealed that it specifically binds to the monosaccharide Me-α-galactose presenting, nevertheless, lesser affinity than the native frutalin. Recombinant frutalin eluted from a size-exclusion chromatography column with a molecular mass of about 62–64 kDa, suggesting a tetrameric structure, however it did not agglutinate rabbit erythrocytes as native frutalin does. This work shows that the galactose-binding jacalin-related lectins four amino-acid linker peptide “T-S-S-N” does not undergo any proteolytic cleavage in the yeast P. pastoris and also that linker cleavage might not be essential for lectin sugar specificity.     

Corticotropin-releasing factor (CRF) is involved in the acute anorexic effect of alpha-melanocyte-stimulating hormone: a study using CRF-deficient mice

Alpha-melanocyte-stimulating hormone (α-MSH) and its receptors are critical and indispensable for maintaining appropriate feeding behavior and energy homeostasis in both mice and humans. Corticotropin-releasing factor (CRF) is a candidate for mediating the anorexic effect of α-MSH. In the present study, we examined whether CRF and its receptors are involved in the anorexic effect of α-MSH, using CRF-deficient (CRFKO) mice and a CRF receptor antagonist. Intracerebroventricular administration of NDP-MSH, a synthetic α-MSH analogue, suppressed food intake in wild-type (WT) mice. This effect was abolished by pretreatment with a non-selective CRF receptor antagonist, astressin, suggesting that the effect of α-MSH-induced anorexia was mediated by a CRF receptor. In CRFKO mice, administration with NDP-MSH did not affect food intake at an early phase (0–4 h). In addition, CRF mRNA levels in the hypothalamus were significantly increased in NDP-MSH-treated mice. Therefore, our findings, using CRFKO, strongly support evidence that CRF is involved in the acute anorexic effect of α-MSH. On the other hand, NDP-MSH administered to CRFKO mice led to suppressed food intake at the late phase (4–12 h), similar to the effect in WT mice. Further, NDP-MSH similarly reduced food intake during the late phase in all types of mice, including WT, CRFKO, and CRFKO with corticosterone replacement. The results would suggest that α-MSH-induced suppression of food intake at late phase was independent of glucocorticoids and CRF.     

DFT and MP2 studies on the C2C2α bond cleavage in thiamin catalysis

Thiamin diphosphate (ThDP), the biologically active derivative of vitamin B₁, is an important cofactor of several enzymes that catalyze the oxidative and non-oxidative conversion of α-keto acids. The final step of non-oxidative decarboxylation of pyruvate by pyruvate decarboxylase – the liberation of acetaldehyde – requires deprotonation of the α-hydroxyl group and cleavage of the C2–C2α bond of the transitory 2-(1-hydroxyethyl)-ThDP intermediate. It has been proposed that the cofactor 4′-amino/imino function is essentially involved in the deprotonation of the α-hydroxyl group. Proton transfer and C2–C2α cleavage may occur in a stepwise manner, or, alternatively in a concerted mechanism. Here, density functional theory (DFT) calculations as well as second order Møller–Plesset perturbation theory (MP2) studies were performed on a simple model for the enzyme using the program package Gaussian 03. Calculations favor a stepwise mechanism with initial formation of the C2α alkoxide, followed by C2–C2α bond cleavage.     

Effect of cationic and non-ionic surfactants on the hydrolysis of N-glutaryl- -phenylalanine catalysed by chymotrypsin iso-enzymes

The hydrolysis of N-glutaryl- -phenylalanine p-nitroanilide catalysed by various chymotrypsin (CT) iso-enzymes (α-CT, β-CT, δ-CT, and γ-CT) has been studied in the presence of cationic and non-ionic surfactants at concentration higher than the critical micellar concentration. The enzyme activity was tested in the presence of the following surfactants: cetyltrimethylammonium bromide (CTABr), cetyldimethylethylammonium bromide (CDMEABr), cetyltripropylammonium bromide (CTPABr), Triton X100 (TX100) and polyoxyethylene 9 lauryl ether (PO9). The activity of the iso-enzymes depends on the surfactant concentration and it varies with the surfactant head group dimensions (CTPABr>CDMEABr>CTABr). For all the iso-enzymes, superactivity has been detected only in the presence of CTPABr and CDMEABr. The extent of superactivity depends on the enzyme used (δ-CT>β-CT>γ-CT>α-CT). The observed reaction rate has been compared with the prediction of a theoretical model for enzymatic activity in the presence of surfactant aggregates in aqueous media developed in a previous paper. The results can be explained by introducing an equilibrium relation between the enzyme confined in the free bulk water and in the bound water pseudo-phase, and by allowing for different catalytic behaviours of the two forms of enzyme.The theoretical model enables the initial reaction rate to be related to the substrate concentration with an overall Michaelis–Menten equation. Good agreement has been found between experimental and model predicted values of the kinetic parameters.     

The efficacy of the extraovular “Prostaglandin-Impact” in provoking first trimester abortion

In good agreement with earlier findings (1–8) legal abortion had been induced successfully with Csapo's method of “Prostaglandin Impact” (PGI) in 44 out of 50 sedated patients. They were 25±1.1 years of age, 11.3±0.2 weeks pregnant, para 1.1±0.2. Only a PGI (10 mg PG F2α) was delivered into the extraovular space. In 44 women, this single PGI provoked 40% progesterone (P)-withdrawal in 3 hours (P < 0.001) and 64% P-withdrawal (P < 0.001) in 15.3±0.9 hours, when the patients aborted. The remaining 6 women, whose P-withdrawal was only 14% at 3 hours without continuation during 24 hours, failed to abort. Thus in PG-induced abortions the regulatory significance of rapid and continued P-withdrawal (1–8) had been verified.The side effects were mild, transient and acceptable. The “Abortion Score” was 86. There were no serious complaints or complications during the study and followup. Since even in the 6 cases of failure the cervix dilated sufficiently to allow curettage (without surgical dilatation), the therapeutic benefits of the single PGI technique should be further examined in those services, where constant medical supervision (for determining the necessity and timing of repeated PGI) is not available.     

MT1-MMP Initiates Activation of pro-MMP-2 and Integrin αvβ3 Promotes Maturation of MMP-2 in Breast Carcinoma Cells

We evaluated cellular mechanisms involved in the activation pathway of matrix prometalloproteinase-2 (pro-MMP-2), an enzyme implicated in the malignant progression of many tumor types. Membrane type-1 matrix metalloproteinase (MT1-MMP) cleaves the N-terminal prodomain of pro-MMP-2 thus generating the activation intermediate that then matures into the fully active enzyme of MMP-2. Our results provide evidence on how a collaboration between MT1-MMP and integrin αvβ3 promotes more efficient activation and specific, transient docking of the activation intermediate and, further, the mature, active enzyme of MMP-2 at discrete regions of cells. We show that coexpression of MT1-MMP and integrin αvβ3 in MCF7 breast carcinoma cells specifically enhances in trans autocatalytic maturation of MMP-2. The association of MMP-2′s C-terminal hemopexin-like domain with those molecules of integrin αvβ3 which are proximal to MT1-MMP facilitates MMP-2 maturation. Vitronectin, a specific ligand of integrin αvβ3, competitively blocked the integrin-dependent maturation of MMP-2. Immunofluorescence and immunoprecipitation studies supported clustering of MT1-MMP and integrin αvβ3 at discrete regions of the cell surface. Evidently, the identified mechanisms appear to be instrumental to clustering active MMP-2 directly at the invadopodia and invasive front of αvβ3-expressing cells or in their close vicinity, thereby accelerating tumor cell locomotion.     

Driving forces and current-voltage characteristics of amino acid transport in Riccia fluitans

The complete steady-state I–V relationship of α-aminoisobutyric acid transport across the plasmalemma of rhizoid cells from Riccia fluitans has been measured and analysed with special emphasis on α-aminoisobutyric acid equilibrium and saturation conditions. (A) The electrical data show that: (1) the amino acid-induced electrical current saturates after the addition of the amino acid, regardless of the concentration; (2) a steady state is reached 1–2 h after incubation in α-aminoisobutyric acid, but after less that 5 min in the presence of 1 mM CN⁻; (3) the steady-state I–V characteristic of α-aminoisobutyric acid transport is a sigmoid curve and fairly symmetric in current with respect to the voltage axis; and (4) the equilibrium potential is clearly a function of the amino acid accumulation ratio. It is suggested that the sigmoid curve represents the characteristic of carrier-mediated α-aminoisobutyric acid transport with a voltage-insensitive step, possibly the translocation of the unloaded carrier, rate-limiting. Since under normal conditions the voltage-sensitive rate constant k_oi is much greater than k_io, it is further suggested that the energy to drive this system is put into the transfer of positive charge from outside to the cytoplasm. (B) Accumulation ratios have been determined by inspection of current-voltage data, and additionally by compartmental analysis on green thalli from Riccia fluitans. Both methods give ratios far too low compared with the thermodynamically possible accumulation of about 10⁴. It is suggested that substantial leakages via different non-electrical pathways prevent equilibrium at steady state, and it is concluded that in such leaky systems the thermodynamic equilibrium condition is not suitable for estimating stoichiometries.     

Effects of succinylacetone on dimethylsulfoxide-mediated induction of heme pathway enzymes in mouse Friend virus-transformed erythroleukemia cells

Heme has been reported to exert a control over its own biosynthesis and to affect the erythroid differentiation process at different sites. In this study, succinylacetone, a powerful inhibitor of δ-aminolevulinic acid dehydrase was used to block heme synthesis and to study the effects of heme depletion on the dimethylsulfoxide (DMSO)-mediated induction of the heme pathway enzymes in Friend virus-transformed erythroleukemia cells. The presence of succinylacetone in the medium during the DMSO treatment (1) potentiates the induction of δ-aminolevulinic acid synthetase (the first enzyme of the pathway) and this effect is reversed by the addition of exogenous hemin; (2) does not affect the induction of δ-aminolevulinic acid dehydrase (the second enzyme); (3) prevents the induction of porphobilinogen deaminase (the third enzyme), since no increase could be detected in either the enzyme activity or the immunoreactive protein and this effect could not be reversed by the addition of exogenous hemin; (4) does not affect the induction of ferrochelatase. The possible role of heme or of intermediate metabolites of the pathway on the induction of these enzymes during the erythroid differentiation process is discussed.     

The contribution of β1 integrins to neuronal migration and differentiation depends on extracellular matrix molecules

The interaction of β1 integrin receptors and different extracellular matrix molecules during neuronal development was investigated by comparing both migration and morphological differentiation of D3 wild-type embryonic stem (ES) cell line-derived neural precursor cells with those of the β1 integrin knockout ES cell line G201. Analysing neurosphere explants on laminin and fibronectin as major β1 integrin ligands, the maximal spreading of outward migrating neuronal cells was determined. Compared with gelatine as a standard substrate, migration was found to be significantly increased for D3-derived neurospheres on fibronectin and laminin-1. These matrix effects were found to be even enhanced for G201 preparations. In addition, also the differentiation of wild-type and β1 integrin −/− neurones – as determined by MAP-2- and HNK-1-immunoreactive processes – was found to be increased on fibronectin and laminin when compared to gelatine standards. In the respective knockout preparations on these matrices, again perturbation effects were less pronounced than on gelatine. Our observations indicate that laminin and fibronectin are involved both in β1 integrin-dependent and -independent signalling mechanisms during neurogenesis. Upregulation of compensatory mechanisms such as β1 integrin-independent receptors for laminin and fibronectin might be responsible for the much less pronounced perturbations of G201 neural precursor migration and differentiation on these two substrates than on gelatine.     

Environmentally-modulated changes in fluorescence distribution in cells with oscillatory genetic network dynamics

We investigated the distribution of green fluorescent protein (GFP) expression levels in a population of E. coli cells expressing an artificial genetic regulatory network, known as the “repressilator”. This network originally constructed by Elowitz and Leibler in 2000 consists of three cyclically-inhibiting promoter–repressor pairs. It is because of this architecture that the network has been known to oscillate at the single-cell level under certain conditions. A series of shake flask experiments were performed and analyzed using flow cytometry to test how cell populations carrying this system could be controlled extracellularly using the inducers anhydrotetracycline (aTc) and isopropyl-β-d-thiogalactopyranoside (IPTG). With variation of [aTc], it exhibits a novel bi-threshold behavior, such that the entire culture reaches one of three steady states at a quasi-time-invariant “reference state.” Also, there is significant hysteresis. Transiently, the middle state shows damping oscillations, while the low and high states show a stable steady state. The addition of IPTG serves to fine-tune the characteristics of the aTc-only expression, lowering the average and coefficient of variation (CV) of the distributions, and possibly perturbing the network to a different state. However, in modeling this system, the multiplicity and bi-threshold behavior are not theoretically possible according to the designed interactions. In order to explain this discrepancy, we hypothesize that one or more of the repressors have a significant nonspecific interaction with a promoter that does not contain its operator site. The new modeling results incorporating these extra interactions qualitatively match our experimental findings. After constructing plasmids to test these hypotheses, we discover that at least four of these interactions exist, which can create the low and high states and multiplicity seen experimentally. This genetic architecture has flexibility in its behavior that has not been demonstrated before, and the combination of experiment and modeling enlightened our understanding of the molecular interactions driving the network's behavior, leading us to discover the significance of nonspecific interactions.     

Glucuronic acid conjugates

The methods of assay in body fluids of 1-β-alkyl, 1-β-phenyl and 1-β-acyl glucuronic acids (“glucuronide conjugates”) have been reviewed. Most of the 78 references cited (from the literature of the period 1990–1997) concern the glucuronide conjugates of drug metabolites, and these have been considered, for reasons of accessibility, within sections of individual drug classes such as analgesics, anti-cancer agents and opioids. Other glucuronide conjugates are considered under “miscellaneous compounds”. A few gas chromatography and capillary electrophoresis methods are described, but the major technique of assay (62 citations) is reversed-phase high-performance liquid chromatography.     

Why the Lysogenic State of Phage λ Is So Stable: A Mathematical Modeling Approach

We develop a mathematical model of the phage λ lysis/lysogeny switch, taking into account recent experimental evidence demonstrating enhanced cooperativity between the left and right operator regions. Model parameters are estimated from available experimental data. The model is shown to have a single stable steady state for these estimated parameter values, and this steady state corresponds to the lysogenic state. When the CI degradation rate (γ_cI) is slightly increased from its normal value (γ_cI 0.0 min⁻¹), two additional steady states appear (through a saddle-node bifurcation) in addition to the lysogenic state. One of these new steady states is stable and corresponds to the lytic state. The other steady state is an (unstable) saddle node. The coexistence these two globally stable steady states (the lytic and lysogenic states) is maintained with further increases of γ_cI until γ_cI 0.35 min⁻¹, when the lysogenic steady state and the saddle node collide and vanish (through a reverse saddle node bifurcation) leaving only the lytic state surviving. These results allow us to understand the high degree of stability of the lysogenic state because, normally, it is the only steady state. Further implications of these results for the stability of the phage λ switch are discussed, as well as possible experimental tests of the model.