Decay of messenger ribonucleic acid from the lactose operon of Escherichia coli as a function of growth temperature

The inactivation rates of the first, β-galactosidase, and last, transacetylase, messages of the lactose operon of Escherichia coli were measured at different growth temperatures. The inactivation rate of each message appears to increase exponentially with temperature. The rate constant for this increase is almost twice as high for transacetylase message as it is for β-galactosidase message. The inactivation rate is more a direct function of growth temperature than of growth rate. At 15 °C transacetylase message is inactivated about 2.5 times more slowly than is β-galactosidase message. This difference is not paralleled by a different rate of chemical loss of the β-galactosidase message compared to the distal lac mRNA; all parts of the molecule appear to be lost at the same rate. This same pattern is observed in decay of the total mRNA; loss of capacity to direct peptide synthesis (functional inactivation) occurs at variable rates whereas loss of mRNA mass (chemical degradation) seems to occur at a uniform rate.We conclude that each message has a unique target for inactivation with a specifie temperature coefficient of sensitivity, and the inactivation of a message need not be associated with chemical destruction of the molecule.     

Promoting the Avoidance of High-Calorie Snacks: Priming Autonomy Moderates Message Framing Effects

The beneficial effects of gain-framed vs. loss-framed messages promoting health protective behaviors have been found to be inconsistent, and consideration of potential moderating variables is essential if framed health promotion messages are to be effective. This research aimed to determine the influence of highlighting autonomy (choice and freedom) and heteronomy (coercion) on the avoidance of high-calorie snacks following reading gain-framed or loss-framed health messages. In Study 1 (N = 152) participants completed an autonomy, neutral, or heteronomy priming task, and read a gain-framed or loss-framed health message. In Study 2 (N = 242) participants read a gain-framed or loss-framed health message with embedded autonomy or heteronomy primes. In both studies, snacking intentions and behavior were recorded after seven days. In both studies, when autonomy was highlighted, the gain-framed message (compared to the loss-framed message) resulted in stronger intentions to avoid high-calorie snacks, and lower self-reported snack consumption after seven days. Study 2 demonstrated this effect occurred only for participants to whom the information was most relevant (BMI>25). The results suggest that messages promoting healthy dietary behavior may be more persuasive if the autonomy-supportive vs. coercive nature of the health information is matched to the message frame. Further research is needed to examine potential mediating processes.     

Immediate stoichiometric appearance of β-galactosidase products in the medium of Escherichia coli cells incubated with lactose

Products of β-galactosidase action on lactose by intact E. coli cells appeared in the medium as soon as lactose was added and the amount of product was equal to the lactose used. No detectable levels of β-galactosidase were found in the medium and lactose was not significantly broken down unless lac permease was present. The appearance did not depend upon the presence of any of the commonly known galactose or glucose permease systems. The Km of product appearance from whole cells was equal to the K_t for lactose transport by lac permease. When the cells were broken the Km became the normal β-galactosidase Km.     

Recombinant Protein Production by the Baculovirus-Insect Cell System in Basal Media Without Serum Supplementation

The production of β-galactosidase by Sf9 cells infected with recombinant Autographa californica nucleopolyhedrovirus (AcNPV) was investigated in shake-flask culture using two serum-free basal media: Grace's medium and TNM-FH (Grace's medium supplemented with lactalbumin hydrolysate and yeast extract). At the time of infection, cells grown in serum-supplemented TNM-FH were transferred into fresh basal media without adaptation. The absence of serum depressed the β-galactosidase yield considerably in Grace's medium, but to a much lesser extent in TNM-FH, where it reached around 2/3 of the level obtained in TNM-FH supplemented with 10% fetal bovine serum (FBS). While both lactalbumin hydrolysate and yeast extract promoted β-galactosidase production, their removal by medium replacement on post-infection day 1 gave a β-galactosidase yield nearly equal to that obtained in their continuous presence. Supplementation of basal media with phosphatidic acid (PA) from egg yolk lecithin, which has been shown to enhance cell growth and recombinant protein production in serum-free culture of Chinese hamster ovary (CHO) cells, was also effective in increasing β-galactosidase yield. Elevating the multiplicity of infection (MOI) from 2 to 10 plaque-forming units per cell (pfu/cell) also resulted in an increase in product yield. These results provide information important to the development of cost-effective serum-free culture technology for use in large-scale production of recombinant proteins by the baculovirus-insect cell system.     

Scanning assay of β-galactosidase activity

β-galactosidase, encoded by the lacZ gene in E. coli, can cleave lactose and structurally related compounds to galactose and glucose or structurally related products. Its activity can be measured using an artificial substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG). Miller firstly described the standard quantitative assay of β-galactosidase activity in the cells of bacterial cultures by disrupting the cell membrane with the permeabilization solution instead of preparing cell extracts. Therefore, β-galactosidase became one of the most widely used reporters of gene expression in molecular biology to reflect intracellular gene expression difference. But the Miller assay procedure could not monitor the β-galactosidase reaction in real time and its results were greatly influenced by some operations in the Miller procedure, such as permeabilization time, reaction time and concentration of the cell suspension. A scanning method based on the Miller method to determine the intracellular β-galactosidase activity in E. coli Tuner (DE3) expressing β-galactosidase in real time was developed and the permeabilization time of cells was optimized for that. The comparison of 3 assays of β-galactosidase activity (Miller, colorimetric and scanning) was made. The results proved that scanning method for the determination of enzyme activity with using ONPG as substrate is simple, fast and reproducible.     

Efficient cellular delivery of β-galactosidase mediated by NrTPs, a new family of cell-penetrating peptides

Nucleolar targeting peptides (NrTPs), a recently developed family of cell-penetrating peptides, have been shown to be very efficient in entering cells and accumulating in their nucleoli. In this work, we have used conjugates of NrTP6 (YKQSHKKGGKKGSG) covalently linked to β-galactosidase in order to demonstrate the capacity of NrTP for intracellular delivery of large molecules. NrTP6/β-galactosidase conjugates, prepared by maleimide-based chemistry, were stable and enzymatically active on the standard 4-methylumbelliferyl β-d-galactopyranoside substrate. Their translocation into HeLa cells, monitored by β-galactosidase activity as a readout of the uptake, showed efficient cellular entry and thus demonstrated the potential of NrTPs for intracellular delivery of large-size cargos with preservation of biological activity.     

Activity of digestive proteinases and carbohydrases in the alimentary tract of the fig leaf roller,Choreutis nemorana Hübner (Lepidoptera: Choreutidae)

.The fig leaf roller or Fig-tree Skeletoniser, Choreutis nemorana (Lep.: Choreutidae), is a destructive pest of fig trees found in some fig-growing areas of Iran. The larvae feed on the upper level of leaves, near the main vein. In this study, digestive carbohydrases including α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase and proteinases including trypsin, chymotrypsin and elastase were investigated. The results showed that the carbohydrases were present in the alimentary tracts of the pest. Optimum pH for α-glucosidase and β-glucosidase activity was at pH 6.0 and 7.0, respectively. Maximum activity of α-galactosidase and β-galactosidase occurred at pH 6.0. Total proteolitic activity against the substrate azocasein was optimally occurred at pH 10.0. The greatest activity of trypsin, chymotrypsin and elastase was determined at pH 10.0, 11.0 and 11.0, respectively. Zymogram analyses using nitrocellulose membrane revealed two trypsin isoforms in which one of them was completely inhibited by Soybean Kunitz inhibitor and the other was notably inhibited.     

β-Galactosidases of Lilium pollen

Lily (Lilium auratum) pollen contains very high levels of β-galactosidase. There are three forms: β-galactosidase I and II differ in M_r, while β-galactosidase III is firmly bound in the pollen wall. The two cytoplasmic forms were separated and partially purified using a combination of chromatography on DEAE-cellulose, Sephadex G-200 and Sepharose 6B. Forms I and II appear to be glycoprotein in nature as shown by binding to Con A-Sepharose. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. The wall-bound enzyme, β-galactosidase III effectively hydrolysed nitrophenyl β-galactosidase but not lactose, and could not be released from the wall polysaccharide matrix by high salt concentrations or detergents. The total β-galactosidase activity of lily pollen remained constant during in vitro germination. A possible role for this enzyme may be in degradation of stylar arabinogalactans providing a carbon source for pollen tube nutrition.     

Slow inactivation and reactivation of the K+ channel in squid axons. A tail current analysis.

Potassium current inactivation and reactivation in squid axons were measured from tail current amplitudes after voltage clamp prepulses to the potassium equilibrium potential, EK, in seawater containing elevated levels of potassium ion concentration, Ko. Little or no inactivation resulted with prepulses lasting less than 100 ms. Longer pulses caused the current to inactivate in two phases, one between 0.1 and 1 s, and a second phase between 5 and 100 s. Inactivation was incomplete. The time constant of the tail current after a prepulse to EK was independent of pulse duration (0.1-120 s). Inactivation was independent of Ko (10 less than or equal to Ko less than or equal to 300 mM), and it was independent of membrane potential, V, for -40 less than or equal to V less than or equal to 0 mV. Reactivation was measured with a three-pulse protocol. The reactivation time course was sigmoidal with a delay of approximately 100 ms before significant reactivation occurred. These results were described by a model consisting of three inactivated states arranged in a linear sequence. The rate constants of the model are of the form (A + B exp (CV), or 1/(A + B exp (CV], which are required to describe the non-inactivating conductance component.     

Proteolytic degradation of fused protein A-β-galactosidase in Escherichia coli

The stability of the fusion protein staphylococcal protein A-E. coli β-galactosidase (SpA-βgal) produced in E. coli has been studied both in cell disintegrate and in purified preparations. SpA-βgal was degraded by a proteolytic cleavage between the two functional parts of the molecule, resulting in one β-galactosidase tetramer and four protein A molecules. Intermediates were detected, namely β-galactosidase containing three, two and one protein A. The β-galactosidase was stable with respect to enzyme activity and molecular weight, while protein A was further degraded. In cell disintegrate the half-life of SpA-βgal was found to be 6 h at 20°C and 1.5 h at 37°C. The protease responsible for initial proteolytic cleavage of SpA-βgal was shown to be cell debris associated.     

LacI(Ts)-regulated expression as an in situ intracellular biomolecular thermometer

In response to needs for in situ thermometry, a temperature-sensitive vector was adapted to report changes in the intracellular heat content of Escherichia coli in near-real time. This model system utilized vectors expressing increasing quantities of β-galactosidase in response to stepwise temperature increases through a biologically relevant range (22 to 45°C). As judged by calibrated fluorometric and colorimetric reporters, both whole E. coli cells and lysates expressed significant repeatable changes in β-galactosidase activity that were sensitive to temperature changes of less than 1°C (35 to 45°C). This model system suggests that changes in cellular heat content can be detected independently of the medium in which cells are maintained, a feature of particular importance where the medium is heterogeneous or nonaqueous, or otherwise has a low heat transfer capacity. We report here that the intracellular temperature can be reliably obtained in near-real time using reliable fluorescent reporting systems from cellular scales, with a 20°C range of detection and at least 0.7°C sensitivity between 35 and 45°C.     

I-Cell disease: Activities of lysosomal enzymes toward natural and synthetic substrates

Cultured skin fibroblasts from a patient with I-Cell disease (mucolipidosis II) were assayed for a number of lysosomal enzymes using both natural and synthetic substrates. The cells from this patient were found to have very low activity for galactosylceramide β-galactosidase, lactosylceramide β-galactosidases (using two assay methods that measure different enzymes), G_M1 ganglioside β-galactosidase and sphingomyelinase. Glucosylceramide β-glucosidase activity was found to be normal. Acid hydrolase activities toward many synthetic substrate were measured and all except β-glucosidase and acid phosphatase were found to be extremely low (as has been reported by others). Acid phosphatase and β-glucosidase were in the low normal range. These studies expand on previously published reports on I-Cell disease that only present data from synthetic substrates, and also report the fibroblast culture deficiencies of galactosyl-ceramide β-galactosidase (the Krabbe disease enzyme) and sphingomyelinase (the Niemann-Pick disease enzyme) activities for the first time. Those two enzymes do not have a readily available synthetic analog to assay. Acid β-galactosidase activity measured with both the 4-methylumbelliferyl derivative and G_M1 ganglioside was partially deficient in leukocytes prepared from this patient. New methods for measuring 4-methylumbelliferyl-β-D-glucoside and glucosylceramide β-glucosidase activities are also presented.     

Cooperation between two ClpB isoforms enhances the recovery of the recombinant β-galactosidase from inclusion bodies

Bacterial ClpB is a molecular chaperone that solubilizes and reactivates aggregated proteins in cooperation with the DnaK chaperone system. The mechanism of protein disaggregation mediated by ClpB is linked to translocation of substrates through the central channel within the ring-hexameric structure of ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and the truncated ClpB80 (ClpBΔN), which does not contain the N-terminal domain. The functional specificity of the two ClpB isoforms and the biological role of the N-terminal domain are still not fully understood. Recently, it has been demonstrated that ClpB may achieve its full potential as an aggregate-reactivating chaperone through the functional interaction and synergistic cooperation of its two isoforms. It has been found that the most efficient resolubilization and reactivation of stress-aggregated proteins occurred in the presence of both ClpB95 and ClpB80. In this work, we asked if the two ClpB isoforms functionally cooperate in the solubilization and reactivation of proteins from insoluble inclusion bodies (IBs) in Escherichia coli cells. Using the model β-galactosidase fusion protein (VP1LAC), we found that solubilization and reactivation of enzymes entrapped in IBs occurred more efficiently in the presence of ClpB95 with ClpB80 than with either ClpB95 or ClpB80 alone. The two isoforms of ClpB chaperone acting together enhanced the solubility and enzymatic activity of β-galactosidase sequestered into IBs. Both ClpB isoforms were associated with IBs of β-galactosidase, what demonstrates their affinity to this type of aggregates. These results demonstrate a synergistic cooperation between the two isoforms of ClpB chaperone. In addition, no significant recovery of the β-galactosidase from IBs in ΔclpB mutant cells suggests that ClpB is a key chaperone in IB protein release.     

Determination of the transgalactosylation activity of Aspergillus oryzae β-galactosidase: effect of pH, temperature, and galactose and glucose concentrations

The catalytic potential of β-galactosidase is usually determined by its hydrolytic activity over natural or synthetic substrates. However, this method poorly predicts enzyme behavior when transglycosylation instead of hydrolysis is being performed. A system for determining the transgalactosylation activity of β-galactosidase from Aspergillus oryzae was developed, and its activity was determined under conditions for the synthesis of galacto-oligosaccharides and lactulose. Transgalactosylation activity increased with temperature up to 55 °C while the effect of pH was mild in the range from pH 2.5 to 5.5, decreasing at higher values. The effect of glucose and galactose on transgalactosylation activity was also assessed both in the reactions for the synthesis of galacto-oligosaccharides and lactulose and also in the reaction of hydrolysis of o-nitrophenyl β-d-galactopiranoside. Galactose was a competitive inhibitor and its effect was stronger in the reactions of transgalactosylation than in the reaction of hydrolysis. Glucose was a mild activator of β-galactosidase in the reaction of hydrolysis, but its mechanism of action was more complex in the reactions of transgalactosylation, having this positive effect only at low concentrations while acting as an inhibitor at high concentrations. This information is relevant to properly assess the effect of monosaccharides during the reactions of the synthesis of lactose-derived oligosaccharides, such as galacto-oligosaccharides and lactulose.     

Some Characteristics of Practical Relevance of the β -Galactosidase from Potential Probiotic Strains ofPropionibacterium acidipropionici

In the present study, factors influencing the synthesis and activity of β-galactosidase of two strains of Propionibacterium acidipropionici with some probiotic properties are described for the first time. The enzyme 6-phospho-β-D-galactosidase of the PEP-PTS system was not detected, suggesting that P. acidipropionici metabolize lactose only by using β-galactosidase. The highest enzymatic activities were obtained from cultures developed in a basal broth medium containing 1.0% sodium lactate or 0.25% lactose. Maximum β-galactosidase activity from cell-free extracts of the strains was obtained at pH 7.0 and 50°C, but a high activity was even detected at 37°C. The enzyme was competitively inhibited by lactose and activated by glucose and sodium lactate. The remaining activities after heating cell-free extracts up to 20 min at 60°C were 70% and 25% of untreated control activities for P. acidipropionici Q4 and CRL 1198, respectively. Cations like Mg²⁺, Mn²⁺, Li⁺, Na⁺, and K⁺ acted as stimulators of the β-galactosidase activity whereas Ca²⁺, Co²⁺, Ni²⁺, Hg²⁺ and Cu²⁺ showed inhibitory effect in different extent. These results suggest that the environmental conditions commonly present in the human's intestine may be adequate for the synthesis and activity of β-galactosidase from these strains of Propionibacterium. The enzyme resist the cooking temperature of Swiss-type cheeses in different extent depending on the strain tested and most of the cations present in milk stimulate the enzymatic activity. Our results suggest that a cheese would be an appropriate vehicle for delivery of β-galactosidase from propionibacteria to the host and efforts to develop a Swiss-type probiotic cheese for lactose intolerant persons should be done.     

The expression of β-galactosidase by Escherichia coli during continuous culture

We have used the technique of continuous culture to study the expression of β-galactosidase in Escherichia coli. In these experiments the cultures were grown on carbon-limited media in which half of the available carbon was supplied as glycerol, glucose, or glucose 6-phosphate, and the other half as lactose. Lactose itself provided the sole source of inducer for the lac operon. The steady-state specific activity of the enzyme passed through a maximal value as a function of dilution rate. Moreover, the rate at which activity was maximal (0.40 h^?1) and the observed specific activity of the enzyme at a given growth rate were found to be identical in each of the three media tested. This result was unexpected, since the steady-state specific activity can be shown to be equal to the differential rate of enzyme synthesis, and since it is known that glycerol, glucose, and glucose-6-P-cause different degrees of catabolite repression in batch culture. The differential rate of β-galactosidase synthesis was an apparently linear function of the rate of lactose utilization per milligram protein regardless of the composition of the input medium. That is, it is independent of the rate of metabolism of substrates other than lactose which are concurrently being utilized and the enzyme level appears to be matched to the metabolic requirement for it. If this relationship is taken to indicate the existence of a fundamental control mechanism, it may represent a form of attenuation of the rate of β-galactosidase synthesis which is independent of cyclic AMP levels.     

Models for decay of Escherichia coli lac messenger RNA and evidence for inactivating cleavages between its messages.

The size distributions of decaying polycistronic Escherichia coli lac messenger RNA have been followed on polyacrylamide gels. At the same time, equations have been derived that generate the theoretical size distributions of decaying macromolecules for different mechanisms of degradation. Using observed values of lac mRNA metabolism, it was possible to reproduce the in vivo patterns with a model in which cleavage occurs at the start of each of the three messages² and is followed by a net 5′ to 3′ wave of mass loss. Other models of degradation could not generate the observed in vivo patterns. These alternative mechanisms include: (1) the same number of primary cleavage sites (three) but at different positions on the full-length molecule; (2) an exclusive 5′ to 3′ directional degradation from the start of the lac mRNA (no cleavages); or (3) the presence of many internal targets. Further support for primary cleavage at the start of messages came from the observed accumulation of the intact z mRNA released by cleavage at the $\text{z}\text{y}$ boundary and from the predictable effects of specific deletions on the resultant size distributions.The significance of these cleavages has been assessed; they could be necessary “processing” events or, conversely, inactivate the message for translation. Full-length molecules as well as cleavage fragments have been fractionated by successive sucrose gradient centrifugation and tested for their capacity to form translation initiation complexes in vitro. Full-length lac RNA could form such complexes at one or more of its three ribosome-loading sites, whereas the y or a message fragments were inactive. These results suggest that a cleavage at or near the start of a message inactivates it.