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At least 1,045 records · Page 58Linked to original sources
PubMed1986

Energetic studies of lactose active transport in Escherichia coli membrane vesicles.

The energetics of D-lactate-driven active transport of lactose in right-side-out Escherichia coli membrane vesicles has been investigated with a microcalorimetric method. Changes of enthalpy (delta Hox), free energy (delta Gox), and entropy (delta Sox) during the D-lactate oxidation reaction in the presence of membrane vesicles are -39.9 kcal, -46.4 kcal, and 22 cal/deg per mole of D-lactate, respectively. The free energy released by this reaction is utilized to form a proton electrochemical potential (delta-microH+) across the membrane. The higher observed heat in the D-lactate oxidation reaction in the presence of carbonylcyanide m-chlorophenylhydrazone (a proton ionophore) supports the postulate that delta-microH+ is formed across the membrane vesicles. Thermodynamic quantities for the formation of delta-microH+ are delta Hm = 14.1 kcal, delta Gm = 0.6 kcal, and delta Sm = 45 cal/deg per mole of D-lactate. The efficiency in the free energy transfer from the oxidation reaction to the formation of delta-microH+ (defined by delta Gm/delta Gox) was 2%, as compared to that in the heat transfer (defined by delta Hm/delta Hox) of 35%. The energetics of the movement of lactose in symport with proton across the membrane as a consequence of the formation of delta-microH+ are delta H1 = -19 kcal, delta G1 = -0.5 kcal, and delta S1 = -62 cal/deg per mole of lactose. No heat of reaction is contributed by lactose movement across the membrane without symport with H+.

Biological Transport, Active↗
PubMed1981

Inhibition of growth of Escherichia coli by lactose and other galactosides.

A study has been made of the inhibition of growth caused by the addition of lactose or other galactosides to lac constitutive Escherichia coli growing in glycerol minimal medium. The effect was greater at pH 5.9 and pH 7.9 than at pH 7.0. Inhibition of growth by lactose was observed also in the case of a beta-galactosidase negative mutant. However, a lacY mutant, which has a defect in the entry of protons normally coupled with galactoside transport, showed only slight inhibition of growth on the addition of galactosides. In the case of the parental strain the addition of lactose resulted in a sharp fall in delta pH across the cell membrane and a reduction in intracellular ATP, and the recovery was slow. Under the same conditions the lacY mutant showed a smaller and only transient effect. It is postulated that the sudden entry of protons associated with lactose uptake lowers the protonmotive force, reducing the ATP levels and inhibiting growth of the cells. This hypothesis would account also for the selection of lacY mutants found when E. coli is grown in the presence of isopropyl-beta-D-thiogalactoside.

Escherichia coli↗
PubMed1986

Use of a hydrolysable probe, [14C]lactose, to distinguish between pre-lysosomal and lysosomal steps in the autophagic pathway.

[14C]Lactose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, is sequestered autophagically in the same way as the established sequestration probe, [14C]sucrose. However, unlike the inert sucrose molecule, lactose is rapidly hydrolysed in the lysosomes, and can therefore be used to probe the last step of the autophagic pathway (i.e. fusion with the lysosome). During autophagy lactose is present only at a low, steady-state level in pre-lysosomal vacuoles (probably autophagosomes), serving as a useful marker for these organelles. If autophagosome-lysosome fusion is blocked with vinblastine (Kovács et al., Exp cell res 137 (1982) 191), [14C]lactose will accumulate continuously as a function of the sequestration rate, and reach a high level in the pre-lysosomal vacuoles. Density gradient analysis, using chloroquine (CLQ) to alter lysosomal density, suggests that these organelles have a broad density distribution (1.08-1.13 g/ml), thus differing significantly from the distribution of lysosomes.

Adenine↗
PubMed1985

Implication of proteases in the respiration dependent inactivation of the lactose permease of E. coli.

The lactose permease of E. coli becomes irreversibly inactivated during lactose transport under conditions of high respiratory activity. This inactivation is characterized by a decrease in the steady state of lactose accumulation, a decrease in the influx rate of lactose, and a decrease in the transmembrane electrical potential. We report here that inhibitors of serine proteases (phenylmethylsulfonyl fluoride and N-alpha-P-tosyl-L-lysine chloromethyl ketone) prevent this inactivation, thus implicating proteases in this process.

Escherichia coli↗
PubMed1985

Basis for lactose aversion in the weanling rat.

As reported previously, weanling rats will choose to consume more of a liquid diet composed of 12% lactose (low-L) than of an isocaloric liquid diet composed of 47% lactose (high-L) despite the greater sweetness of the latter diet. To investigate the basis for this, saccharin was added to the high-L diet to overwhelm any unpalatable qualities. The results of several choice tests indicate that pups initially prefer the saccharin-sweetened diet. However, if the low-L diet is the alternate choice, the preference shifts to the low-L diet after several days of exposure. This initial preference for the sweetened high-L diet suggests that the high lactose aversion is due to post-ingestive consequences rather than to low palatability of the high-L diet. Furthermore, it appears that the aversion to a high concentration of lactose takes precedence over a preference for the increased sweetness of the high-L diet.

Animals↗
PubMed1987

Characterization of self-transmissible plasmids determining lactose fermentation and multiple antibiotic resistance in clinical strains of Klebsiella pneumoniae.

The lactose fermentation (Lac+) and antibiotic resistance (R+) phenotypes were conjugally transferred from Klebsiella pneumoniae strains (K166, K182, K186, K218, and K220) to Salmonella typhi, S. typhimurium, Shigella flexneri, and Vibrio cholerae. The genes for lactose fermentation and antibiotic resistance were located on the plasmids. Further analysis of plasmid DNA from these isolates indicated the presence of multiple plasmids (Mr ranged less than 2.7 to 70 X 10(6)). The Lac+R+ plasmids p166 and p182 were members of the FII incompatibility group. The fertility inhibition property of plasmids, p182, p218, and p220 was fi+ type. Furthermore, phage typing experiments showed that plasmids p166 and p218 (Lac+R+) conferred the ability to inhibit the multiplication of bacteriophages 12 and 13 in S. typhimurium. However, the plasmids p182, p186, and p220 (Lac+R+) could inhibit the visible lysis of all the 30 phages in S. typhimurium. This study describes the characterization of Lac+R+ plasmids and the medical significance of an intergeneric transfer of lactose fermentation to non-lactose-fermenting pathogens.

Escherichia coli↗
PubMed1986

Chronic (89-week) feeding study with hydroxypropyl distarch phosphate, starch acetate, lactose and sodium alginate in mice.

A chronic feeding study was carried out in mice with two chemically modified potato starches, hydroxypropyl distarch phosphate (HP-starch) and starch acetate (AC-starch), and with lactose and sodium alginate. Each of the materials was fed to a group of 75 male and 75 female mice for 89 wk. The dietary level of the test products was gradually increased until the diets contained (by weight) 55% HP-starch, 55% AC-starch, 55% lactose or 25% alginate. The control diet contained 55% pregelatinized potato starch. Each of the four test materials caused increased water consumption, distinct caecal and colonic enlargement, a slightly increased incidence of intratubular nephrosis and, with the exception of AC-starch, also slightly lower body weights. An increased incidence of gastric trichobezoars was observed in mice fed either the modified starches or lactose. The occurrence of concrements in the renal pelvis along with slight urinary changes, such as increased amounts of amorphous material in the urine and increased urinary Ca content, in mice fed HP-starch, AC-starch or lactose was regarded as an effect of little, if any, toxicological significance. Alginate fed at 25% (w/w) of the diet was nephrotoxic to mice, as shown by extremely high water consumption, high urine production, urinary incontinence, high pH and low specific gravity of the urine, increased level of blood urea nitrogen, increased kidney weights, distension of the renal calyx and the high incidence of dilated distal tubules. Caecal and colonic enlargement and changes in urinalysis were found to be reversible and had completely or largely disappeared within 2-5 wk of the cessation of the treatment in wk 87. The incidence of intratubular calcinosis or of concrements in the pelvic space was not reduced during the recovery period. The study did not provide any evidence of carcinogenicity of the products tested.

Alginates↗
PubMed1975

Inhibition by cyclic AMP of lactose production in lactating guinea pig mammary gland slices.

Addition of the cyclic AMP phosphodiesterase inhibitors theophylline (10- minus 2 M) or papaverine (10- minus 4 M) leads to a complete inhibition of lactose synthesis in incubated guinea pig mammary gland slices. Addition of 10- minus 5 M cyclic AMP or dibutyryl cyclic AMP results in 1 30-40% inhibition of the synthesis, which effect is not increased by applying higher concentrations of these compounds. A 30-40% inhibition can also be obtained with epinephrine (5 - 10- minus 5 M), or isoproterenol (10- minus 4 M), but the polypeptide hormones glucagon (10- minus 7 M), insulin (1 munit/ml) and relaxin (10 mug/ml) do not significantly affect lactose synthesis. Cytochalasin B (5 mug/ml) inhibits lactose production by 58and colchicine (10- minus 5 M) by 25%. These experiments suggest that an increase in the intracellular level of cyclic AMP either through its addition, through hormonal stimulation of its synthesis, or through inhibition of its intracellular breakdown, leads to an inhibition of lactose production in lactating mammary gland. This effect of cyclic AMP is similar to that of progesterone, which is known to inhibit lactation in vivo and the withdrawal of which at parturition has been postulated to initiate lactogenesis.

Animals↗
PubMed1977

Cloning of chemically synthesized lactose operators.

Recombinant DNA molecules, constructed from the ColE1-Mk5 hybrid plasmid PMB9 and a chemically synthesized wild-type lactose operator segment, have been used to transform Escherichia coli. Up to 10% of the transformants (selected for the tetracycline-resistance property of PMB9) are partially constitutive for the lactose operon enzyme beta-galactosidase. In vitro studies demonstrate that these partially constitutive transformants contain plasmid DNA molecules which carry one or more lactose operators, and which will bind purified lactose repressor. Preliminary results with some modified operator sequences are also presented.

Coliphages↗
PubMed2004

Lactose biosensor based on Langmuir-Blodgett films of poly(3-hexyl thiophene).

An amperometric lactose biosensor was developed by immobilizing lactase (EC 3.2.1.23) and galactose oxidase (GaO) (EC 1.1.3.9) in Langmuir-Blodgett (LB) films of poly(3-hexyl thiophene) (P3HT)/stearic acid (SA) for estimation of lactose in milk and its products to prevent "lactose intolerance". The enzyme immobilized LB film was used as working electrode and platinum as reference electrode. The enzyme electrodes show a linearity 1-6 g/dL of lactose and have a shelf life more than 120 days. The reusability of electrode was found ten times with 3% loss in current response. The enzyme electrode was characterized by Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and kinetic parameters such as pH, temperature and stability. The working electrode may be used for the estimation of lactose/galactose in food and biological fluids.

Animals↗
PubMed2006

Lactones of disialyl lactose: characterisation by NMR and mass spectra.

The lactonisation of alpha-Neup5Ac-(2-->8)-alpha-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-D-Glc (disialyl lactose) was investigated. (1)H and (13)C NMR chemical shifts of disialyl lactose and alpha-Neup5Ac-(2-->8, 1-->9)-alpha-Neup5Ac-(2-->3, 1-->2)-beta-D-Galp-(1-->4)-D-Glc (disialyl lactose-dilactone) were assigned based on 1D and 2D NMR results, including edited HSQC, HSQC-TOSCY and HMBC. The time course of lactonisation was followed by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) with electrospray ionisation (ESI) mass spectrometry (MS) detection. The rate of lactonisation between alpha-(8)Neu5Ac and alpha-(3)Neu5Ac residues (lactonisation at the alpha-(2-->8) linkage) was faster than that of lactonisation between alpha-(3)Neu5Ac and Gal residues (lactonisation at the alpha-(2-->3) linkage). The mass spectra of disialyl lactose, its lactones, alpha-Neup5Ac-(2-->8)-alpha-Neup5Ac (alpha-(2-->8) disialic acid) and alpha-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-D-Glc-lactone (3'-sialyllactose-lactone) showed that the alpha-(2-->8) linkage between Neu5Ac residues is difficult to cleave in the ESI-MS, compared with the alpha-(2-->3) linkage between Neu5Ac and Gal residues.

Animals↗
PubMed2006

Enzymatically oxidized lactose and derivatives thereof as potential protein cross-linkers.

The enzyme galactose oxidase [EC 1.1.3.9] was applied to convert lactose, lactylamine and lactobionic acid into their corresponding 6'-aldehyde compounds. The potential protein cross-linking ability of these oxidized lactose and derivatives thereof was investigated using n-butylamine as the model compound. First, oxidized lactose gave double Maillard reaction products that were stable under mild alkaline conditions. Second, reductive amination of lactose followed by enzymatic oxidation gave cross-links that were stable under both neutral and alkaline conditions. Third, stable cross-links were obtained through enzymatic oxidation and amidation of lactobionic acid.

Aldehydes↗
PubMed2005

Separation of lactose from human milk oligosaccharides with simulated moving bed chromatography.

The successful separation of the disaccharide lactose from a complex mixture of human milk oligosaccharides (HMOS) with the continuous chromatography of simulated moving bed (SMB) technique is described. Since lactose is the main carbohydrate in human milk with well-known functions for the infant, it is necessary to separate it from the rest of the oligosaccharides to divide them into less complex fractions and analyse their partial unknown functions. For separation of lactose from HMOS two different stationary phases (size-exclusion gel as well as ion-exchange gel) were used. As the main result, it is shown that a size-exclusion gel with the particle size of 50-100 microm and porosity of 50 A was the preferred stationary phase for our separation process with almost complete lactose separation and stable conditions.

Chromatography, Gel↗
PubMed2005

Calorimetric determination of amorphous content in lactose: a note on the preparation of calibration curves.

Calorimetric methods (isothermal or solution calorimetry) offer the ability to detect amorphous contents to 0.5% (w/w) or better in processed pharmaceuticals and calorimetric data are becoming more widely accepted in regulatory submissions. However, both methods require the construction of calibration curves, prepared using quantitative physical mixtures of entirely amorphous and entirely crystalline material. If the sample under investigation exists in two or more isomers or polymorphs, and the enthalpy of solution (solution calorimetry) or the enthalpy of crystallisation (isothermal calorimetry) are different for the isomers or polymorphs, then it must be ensured that the batch of material used to prepare the calibration samples has the same isomeric or polymorphic composition as the (processed) material to be tested. Here, we demonstrate the problems that may arise using lactose as a pharmaceutically important model substance. Calibration curves were prepared from solution calorimetry and isothermal gas perfusion calorimetry data using two batches of lactose (one predominantly anhydrous alpha-lactose and one predominantly beta-lactose). The calibration curves are shown to be significantly different for the two batches, and it is shown that quantification of the amorphous content of a processed sample of unknown isomeric composition is impossible, unless the calibration curve is prepared from the same batch of material as the processed sample. In addition, some of the other problems inherent in using isothermal gas perfusion calorimetry for amorphous content determination, such as wetting issues and the preparation of calibration standards that mimic processes samples, are discussed.

Calibration↗
PubMed2004

Engineered autolytic yeast strains secreting Kluyveromyces lactis beta-galactosidase for production of heterologous proteins in lactose media.

Secretion of the heterologous Kluyveromyces lactis beta-galactosidase into culture medium by several Saccharomyces cerevisiae osmotic-remedial thermosensitive-autolytic mutants was assayed and proved that new metabolic abilities were conferred since the constructed strains were able to grow in lactose-containing media. Cell growth became independent of a lactose-uptake mechanism. Higher levels of extra-cellular and intra-cellular beta-galactosidase production, lactose consumption and growth were obtained with the LHDP1 strain, showing a thermosensitive-autolytic phenotype as well as being peptidase-defective. The recombinant strain LHDP1 presented the highest beta-galactosidase yields from biomass and the lowest ethanol levels from lactose. This strain is effective for the heterologous production and release of K. lactis beta-galactosidase into the extra-cellular medium after osmotic shock.

Cheese↗
PubMed2005

Three forms of thermostable lactose-hydrolase from Thermus sp. IB-21: cloning, expression, and enzyme characterization.

Three thermostable lactose-hydrolases, namely, two beta-glycosidases (bglA and bglB) and one beta-galactosidase (bgaA) genes were cloned from the genomic library of Thermus sp. IB-21. The bglA, bglB, and bgaA consisted of 1311 bp (436 amino acid residues), 1296 bp (431 aa), and 1938 bp (645 aa) of nucleotides with predicted molecular masses of 49,066, 48,679, and 72,714 Da, respectively. These enzymes were overexpressed in Escherichia coli BL21(DE3) using pET21b(+) vector system. The recombinant enzymes were purified to homogeneity by a heat precipitation (70 degrees C, 40 min) and a Ni2+-affinity chromatography. The molecular masses of the purified enzymes estimated by SDS-PAGE agreed with their predicted values. All the purified enzymes showed their optimal pH at around 5.0-6.0. In contrast, the temperature profiles for activity and thermostability patterns were different for each enzyme. BglB beta-glycosidase displayed the best lactose hydrolysis activity of the three enzymes without substrate inhibition up to 200 mM lactose at 70 degrees C and pH 7.0. The specific activities (U/mg) of BglA, BglB, and BgaA on 138 mM lactose at 70 degrees C and pH 7.0 were 36.8, 160.3, and 8.5, respectively.

Amino Acid Sequence↗
PubMed2003

Feedback regulation in the lactose operon: a mathematical modeling study and comparison with experimental data.

A mathematical model for the regulation of induction in the lac operon in Escherichia coli is presented. This model takes into account the dynamics of the permease facilitating the internalization of external lactose; internal lactose; beta-galactosidase, which is involved in the conversion of lactose to allolactose, glucose and galactose; the allolactose interactions with the lac repressor; and mRNA. The final model consists of five nonlinear differential delay equations with delays due to the transcription and translation process. We have paid particular attention to the estimation of the parameters in the model. We have tested our model against two sets of beta-galactosidase activity versus time data, as well as a set of data on beta-galactosidase activity during periodic phosphate feeding. In all three cases we find excellent agreement between the data and the model predictions. Analytical and numerical studies also indicate that for physiologically realistic values of the external lactose and the bacterial growth rate, a regime exists where there may be bistable steady-state behavior, and that this corresponds to a cusp bifurcation in the model dynamics.

Computer Simulation↗
PubMed2000

Identification and synthesis of a trisaccharide produced from lactose by transgalactosylation.

Enzymatic transgalactosylation of lactose by means of Streptococcus thermophilus, subspecies DN-001065, led to a mixture of D-galactose (approximately 4%), D-glucose (approximately 15%), lactose (approximately 51%), minor disaccharides (6%), trisaccharides (approximately 20%) and tetrasaccharides (3%). The major trisaccharide (approximately 16%) was identified by NMR spectroscopy and chemical synthesis as being the known beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->4)-D-glucos e (3'-beta-D-galactopyranosyl-lactose). It was purified from a mixture of peracetylated oligosaccharides by column chromatography followed by deacetylation. For the first time, 3'-beta-D-galactopyranosyl-lactose has been obtained on the 1 g scale, by resorting to simple techniques and equipment. NMR spectra have been unambiguously assigned.

Carbohydrate Sequence↗