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PubMed1990

Automated enzymatic assays for the determination of intestinal permeability probes in urine. 1. Lactulose and lactose.

Lactulose is becoming the disaccharide of choice in the dual sugar assessment of passive permeability of the small intestinal mucosa. However its more widespread use is hampered by current analytical methods which are tedious and time consuming. An automated spectrophotometric technique for the assay of this sugar in urine is presented in which lactulose is linked by a series of enzyme reactions to the equimolar production of NADPH. In addition to lactulose, the procedure also gives accurate values for lactose, glucose and fructose in the urine sample. The assay has been shown to be highly specific for lactulose and lactose and was not affected by high concentrations of other sugars or other urinary constituents. Within assay and between assay precision were similar with the coefficient of variation for both sugars in the range 0.4-1.6%. The technique represents a significant improvement in time, simplicity and precision on existing methods of analysis.

Disaccharides↗
PubMed1986

Absence of a unique relationship between active transport of lactose and protonmotive force in E. coli.

The relationship between active transport of lactose via the lactose permease and the protonmotive force has been determined in E. coli cells using either the respiratory chain inhibitor cyanide or protonophores to decrease the protonmotive force progressively. In contradiction with the prediction of the delocalized chemiosmotic theory, two different relationships were obtained depending on the method used.

Biological Transport, Active↗
PubMed1994

The human HIP gene, overexpressed in primary liver cancer encodes for a C-type carbohydrate binding protein with lactose binding activity.

HIP was originally identified as a gene expression in primary liver cancers, and in normal tissues such as pancreas and small intestine. Based on gene data base homologies, the HIP protein should consist of a signal peptide linked to a single carbohydrate recognition domain. To test this hypothesis HIP and the putative carbohydrate recognition domain encoded by the last 138 C-terminal amino acids, were expressed as glutathione-S-transferase proteins (GST-HIP and GST-HIP-142, respectively). Both recombinant proteins were purified by a single affinity purification step from bacterial lysates and their ability to bind saccharides coupled to trisacryl GF 2000M were tested. Our results show that HIP and HIP-142 proteins bind to lactose, moreover the binding requires divalent cations. Thus the HIP protein is a lactose-binding lectin with the characteristics of a C-type carbohydrate recognition domain of 138 amino acids in the C-terminal region.

Antigens, Neoplasm↗
PubMed1984

Dietary lactose and the metabolic activity of the caecal microfloras of weanling and adult rats.

Weanling or adult (9 wk old) rats were fed diets containing 0, 250 or 500 g lactose/kg for 10 days, after which the activities of six caecal microbial enzymes (azoreductase, beta-glucosidase, beta-glucuronidase, nitrate reductase, nitroreductase and urease) were determined. Adult controls had larger caeca than weanlings, but the numbers of bacteria were not significantly different. Expressed in relation to body weight, caecal microbial enzyme activities were significantly lower in adult controls, with the exceptions of beta-glucuronidase and urease. Lactose caused caecal enlargement; this was greatest in weanling animals, which also showed a decreased concentration of bacteria. Lactose increased total nitrate reductase and urease activities in both age groups, but decreased total azoreductase and nitroreductase activities in weanlings. Enzyme activities per 10(9) bacteria were decreased for azoreductase, beta-glucosidase, beta-glucuronidase and nitroreductase in both age groups, while urease activity increased. Azoreductase and nitroreductase activities were highly correlated but nitrate reductase and urease did not correlate significantly with any other enzyme activity.

Aging↗
PubMed1988

Lactose-hydrolyzing beta-glycosidases of barley meal.

A beta-glucosidase and a beta-galactosidase were purified to homogeneity from barley meal. The beta-glucosidase is a single basic polypeptide (pI greater than 8.5) with an Mr of 53,000 acting optimally at pH 4.5-5.0. The beta-galactosidase is composed of two subunits with an Mr of 42,000 and 33,000, respectively, and is acidic in nature (pI less than 5.7). Both enzymes are able to hydrolyze lactose with Michaelis constants lower than the concentration of this sugar in milk whey. Consequently, barley seems to be an inexpensive source of lactose-splitting enzymes.

Galactosidases↗
PubMed1992

Novobiocin, brilliant green, glycerol, lactose agar: a new medium for the isolation of Salmonella strains.

We describe a medium, novobiocin, brilliant green, glycerol, lactose (NBGL) agar, for the routine isolation of Salmonella strains from stool samples. The NBGL agar principle involved the use of the antisaprophytic effect of brilliant green and novobiocin. Glycerol and lactose were added in order to distinguish between Citrobacter and Salmonella. NBGL was used in parallel with salmonella-shigella (SS) and Hektoen (H) agar for culturing 2,853 stool samples, of which 184 were confirmed to be salmonellae. NBGL showed a high sensitivity: 94% in direct plating compared to 74% (p < 10(-3)) and 65% (p < 10(-5)) for H and SS, respectively, and 96% in enrichment broth plating vs. 83% (p < 10(-4)) and 86% (p < 10(-3)), respectively, for H and SS. In direct plating using NBGL, 95% of black-centred colonies were confirmed to be salmonellae (vs. 31% and 36%, H and SS). In enrichment plating using NBGL, this figure was 82% (vs. 26% and 28%). The results suggest that NBGL agar is advantageous for the isolation of non-Typhi H2S+ salmonellae.

Anti-Infective Agents, Local↗
PubMed1987

Effect of dietary calcium supplementation with lactose on bone in vitamin D-deficient rats.

The relationship between plasma calcium and bone length, chemical and histomorphometric bone parameters was studied in vitamin D-deficient rats in order to determine whether the effects of vitamin D on bone could be attributed to the effect of vitamin D on serum calcium. Plasma calcium was varied over a wide range of dietary manipulation. Four groups of vitamin D-deficient rats were given for 6 weeks: a vitamin D-deficient diet (D-, n = 6), the D--diet with calcium supplementation (D-Ca+, n = 6), the D-Ca+-diet with lactose substituted for dextrose (D-Ca+lac, n = 6) or a normal diet (D+, n = 8). After 6 weeks the mean plasma calcium concentrations were 6.1; 7.0; 9.8; and 10.4 mg/dl, respectively. In the vitamin D-deficient rats (groups D-, D-Ca+, D-Ca+lac) plasma calcium was correlated with bone length, bone ash, volumetric density of osteoid in the metaphysis of the tibia, and volumetric density of trabecular bone in the same bone section. In the D-Ca+lac group these bone parameters approximated the values of the D+ group, but were still significantly lower. It is concluded that in vitamin D-deficient rats longitudinal bone growth, bone mineral content and bone histomorphometry can be brought close to normal by supplying additional dietary calcium with lactose, without vitamin D repletion. The study does not exclude the possibility that residual amounts of vitamin D are required to obtain this effect.

Animals↗
PubMed2005

The aggregation of pig articular chondrocyte and synthesis of extracellular matrix by a lactose-modified chitosan.

A reductive amination reaction (N-alkylation) obtained exploiting the aldheyde group of lactose and the amino group of the glucosamine residues of chitosan (d.a. 89%) afforded a highly soluble engineered polysaccharide (chitlac) for a potential application in the repair of the articular cartilage. Chitosan derivatives with 9% and 64% of side chain groups introduced have been prepared and characterized by means of potentiometric titration, (1)H-NMR and intrinsic viscosity. Both polymers, with respect to the unmodified chitosan, induce cell aggregation when in contact with a primary culture of pig chondrocytes, leading to the formation of nodules of considerable dimensions (up to 0.5-1 mm in diameter). The nodules obtained from chondrocytes treated with chitlac with the higher degree of substitution have been studied by means of optical and electron microscopy (SEM, TEM) and the production of glycosaminoglycans (GAGs) and collagen has been measured by means of colorimetric assays. The chondro-specificity of GAG and collagen was determined by RT-PCR. The results show that the lactose-modified chitosan is non-toxic and stimulates the production of aggrecan and type II collagen.

Animals↗
PubMed2005

The synthesis and biological evaluation of lactose-based sialylmimetics as inhibitors of rotaviral infection.

Rotaviruses are the most significant cause of gastroenteritis in young children and are responsible for over 600,000 infant deaths annually. The rotaviral haemagglutinin protein (VP8*) of some strains has been implicated in early recognition and binding events of host cell-surface sialoglycoconjugates, and is therefore an attractive target for potential therapeutic intervention. Since N-acetylneuraminic acid alpha(2,3)-linked to galactose is believed to be the minimum binding epitope of rotavirus to host cells, we report here our development of an efficient and flexible synthetic route to a range of lactose-based sialylmimetics of alpha(2,3)-linked thiosialosides. These compounds were biologically evaluated as inhibitors of rotaviral infection using an in vitro neutralisation assay. The results suggest that these lactose-based sialylmimetics are not inhibitors of the rhesus rotavirus strain; however, they do exhibit modest inhibition of the human (Wa) strain, presumably through inhibition of the rotaviral adhesion process.

Animals↗
PubMed2006

Molecular mobility and dynamic site heterogeneity in amorphous lactose and lactitol from erythrosin B phosphorescence.

We have used phosphorescence from erythrosin B to characterize the molecular mobility and dynamic heterogeneity in dry films of amorphous lactose and lactitol from -25 to 120 degrees C. The phosphorescence emission spectra red-shifted and broadened with temperature in both sugars, indicating that both the rate of dipolar relaxation and the extent of inhomogeneous broadening increased dramatically at higher temperature. Phosphorescence intensity decays were well fit using a stretched exponential decay model; the rate constant for non-radiative quenching due to collisions with the matrix was calculated from the lifetimes. Arrhenius plots of this rate were non-linear, increasing very gradually at low and dramatically at high temperatures in both sugars. The rate of quenching was significantly lower in a 1:1 (wt/wt) mixture of lactose/lactitol in both the glass and the melt, providing strong evidence that specific interactions within the mixture lowered the matrix mobility. The lifetimes varied systematically with emission wavelength in both matrixes; analysis of the temperature dependence indicated that the activation energy for non-radiative quenching of the triplet state varied somewhat with emission wavelength. Time-resolved emission spectra collected as a function of delay time following pulsed excitation exhibited significant shifts to higher energy as a function of time. These data support a photophysical model in which erythrosin B molecules are distributed among matrix sites that vary such that blue-emitting sites with slower rates of matrix dipolar relaxation also have slower rates of molecular collisions. The amorphous matrixes of lactose and lactitol in both the glass and the melt state are thus characterized by dynamic site heterogeneity in which different sites vary in terms of their overall molecular mobility.

Erythrosine↗
PubMed2004

Development of a digital video-microscopy technique to study lactose crystallisation kinetics in situ.

Polarised light microscopy was employed non-invasively to monitor lactose crystallisation from non-seeded supersaturated solutions in real time. Images were continuously recorded, processed and characterised by image analysis, and the results were compared with those obtained by refractometry. Three crystallisation temperatures (10, 20 and 30 degrees C) and three different levels of initial relative supersaturation (C/C(s)=1.95; 2.34; 3.15) were investigated. Induction times using the imaging technique proved to be substantially lower than those determined using refractive index. Lactose crystals were isolated digitally to determine geometrical parameters of interest, such as perimeter, diameter, area, roundness and Feret mean, and to derive crystal growth rates. Mean growth rates obtained for single crystals were fitted to a combined mass transfer model (R(2)=0.9766). The model allowed the effects of temperature and supersaturation on crystallisation rate to be clearly identified. It also suggested that, in this set of experiments, surface integration seemed to be the rate controlling step. It is believed that a similar experimental set-up could be implemented in a real food system to characterise a particular process where crystallisation control is of interest and where traditional techniques are difficult to implement.

Crystallization↗
PubMed2006

Synthesis of multivalent lactose derivatives by 1,3-dipolar cycloadditions: selective galectin-1 inhibition.

Acetylene derivatives of phenylalanine, phenethylamine and the multifunctional unnatural amino acids, phenyl-bis-alanine and phenyl-tris-alanine, were synthesized and functionalized with 2-azidoethyl beta-D-galactopyranosyl-(1-->4)-beta-D-glucopyranoside via regioselective copper(I)-mediated 1,3-dipolar cycloaddition to give a panel of mono-, di- and trivalent lactoside derivatives. Evaluation of the compounds as inhibitors against the tumour- and inflammation-related galectin-1, -3, -4N, -4C, -4, -7, -8N and -9N revealed a divalent compound with a Kd value as low as 3.2 microM for galectin-1, which corresponded to a relative potency of 30 per lactose unit as compared to the natural disaccharide ligand lactose. This divalent compound had at least one order of magnitude higher affinity for galectin-1 than for any of the other galectins investigated.

Acetylene↗
PubMed2006

Modelling, property verification and behavioural equivalence of lactose operon regulation.

Understanding biochemical pathways is one of the biggest challenges in the field of molecular biology nowadays. Computer science can contribute in this area by providing formalisms and tools to simulate and analyse pathways. One formalism that is suited for modelling concurrent systems is Milner's Calculus of Communicating Systems (CCS). This paper shows the viability of using CCS to model and reason about biochemical networks. As a case study, we describe the regulation of lactose operon. After describing this operon formally using CCS, we validate our model by automatically checking some known properties for lactose regulation. Moreover, since biological systems tend to be very complex, we propose to use multiple descriptions of the same system at different levels of abstraction. The compatibility of these multiple views can be assured via mathematical proofs of observational equivalence.

Lactose↗
PubMed2005

The use of inverse gas chromatography and gravimetric vapour sorption to study transitions in amorphous lactose.

The aim of this study was to measure the glass transition of amorphous lactose under well-controlled temperature and humidity, using inverse gas chromatography (IGC) and to relate these data to gravimetric vapour sorption experiments. Amorphous lactose (spray-dried) was exposed to a stepwise increment in the relative humidity (%RH) under isothermal conditions in an IGC. At the end of each conditioning step a decane injection was made, and the retention volumes were calculated using the maximum peak height (V(max)) method. The pressure drop across the column was recorded using the pressure transducers. These measurements were performed at various temperatures from 25 to 40 degrees C. The extent of water sorption at identical humidity (%RH) and temperature conditions was determined gravimetrically using dynamic vapour sorption (DVS). At each T, it was possible to determine: (1) a transition at low RH relating to the onset of mobility; (2) changes in retention volume relating to the point, where T(g) = T; (3) changes in pressure drop, which were related to the sample collapse. The rate and extent of water sorption was seen to alter at T(g) and also at a collapse point. Combinations of temperature and critical %RH (%cRH required to lower the dry glass transition temperature to the experimental temperature) obtained from IGC were comparable to those obtained from DVS. It was shown that at each T, the sample spontaneously crystallised, when T(g) was 32 degrees C below T. Inverse gas chromatograph can be used in this novel way to reveal the series of transitions that occur in amorphous materials.

Absorption↗
PubMed2006

Lactose: a definitive guide to polymorph determination.

Lactose is a well-known molecule capable of forming a number of different polymorphs with varied chemical and physical properties. To date, no definitive guide for distinguishing between polymorphs using simple analytical techniques has been available. The information presented in this article aims to provide a conclusive guide for identifying the polymorphs of lactose and to successfully unravel years of contradictory research. Data have been collected on single phase polymorphs, prepared from an identical source, adopting the use of in situ and ex situ powder X-ray diffraction, CCD-Raman, FT-IR and (13)C-(1)H cross-polarisation magic angle spinning NMR (CP-MASNMR) spectroscopy, in order to provide simple methods to discriminate between the polymorphs.

Crystallization↗
PubMed2006

Systematic lactose-functionalization of amphiphilic octaamine macrocycle as a gene carrier. Optimization of the charge, size, toxicity, and receptor factors for hepatocyte targeting.

We investigated the transfection properties of singly and triply lactose-functionalized derivatives, Lac(1) and Lac(3), and non-glycosylated one, Lac(0), of calix[4]resorcarene-based amphiphilic octaamine 1 in light of those previously reported for more extensively glycosylated compounds Lac(5) and Lac(8). They all strongly bind to the luciferase-encoding plasmid DNA pCMVluc (7040 base-pairs) with a saturation stoichiometry of Lac(n)/P approximately =0.5 (n=0, 1, or 3) or 0.7 (n=5 or 8), where P stands for a phosphate moiety of the plasmid DNA. The resulting carrier-DNA conjugates are positively charged and monomeric (monomolecular with respect to DNA) as such when n=0 or 1, neutral and monomeric when n=3, or neutral and aggregated when n=5 or 8. Transfection of HeLa (uterine) and HepG2 (hepatic) cell lines shows a general trend of decreasing luciferase expression efficiencies (E) in lively cells as well as cytotoxicities with increasing n's. The cell selectivities for HepG2 over HeLa sharply increase with increasing n's; E(HepG2)/E(HeLa)=0.3, 0.6, 7, 14, and 120 for Lac(0), Lac(1), Lac(3), Lac(5), and Lac(8), respectively, as a result of specific receptor pathway involving the asialoglycoprotein receptors on the hepatic (HepG2) cell surfaces and clustering lactose moieties of the carrier-DNA conjugates. The toxicity-corrected, overall efficiency of gene delivery to hepatocytes is optimized at Lac(3), which forms compactly packed (approximately 40 nm), charge-masked (xi approximately = 0 mV), and less toxic virus-like particles capable of receptor-mediated hepatocyte targeting.

Asialoglycoprotein Receptor↗
PubMed2005

Quantitative analysis of amorphous content of lactose using CCD-Raman spectroscopy.

A Raman spectroscopy method was developed for the quantification of the amorphous content of lactose. Both physical mixtures and spray-dried samples were used and the results were compared with the IMC determinations. Sample inhomogeneities were averaged out by collecting multiple spectra from each sample, and the total measurement time remained below 10 min due to the high sensitivity of the CCD-Raman spectrometer used in the measurements. The obtained calibration error (SEC) for the physical mixtures was 1.3% (w/w) in the 0-100% amorphous content range and was reduced to 0.2% (w/w) in the 0-10% range of more practical interest. The crystallization heat values of the spray-dried samples showed a linear correlation with the Raman quantifications in the amorphous content range of 0-80%, but saturated over the 80% concentration. This finding suggests a reference value of ca. 60 J/g for the spray-dried samples, instead of the crystallization heat of amorphous lactose (ca. 50 J/g) valid in the IMC determinations of physical mixtures.

Lactose↗
PubMed2004

Dietary additions of lactose, casein and soy protein exerted only moderate effects on calcium homeostasis in calcitriol deficient piglets.

It has been reported from rats and mice that blood and bone calcium can be normalised in the absence of Vitamin D hormone or its receptor by dietary means. It was the aim of this study to test, whether a similar result can be obtained with pigs. Piglets with inherited calcitriol deficiency were fed with high calcium and Pi diets and supplemented with soy protein or casein and lactose or corn starch, which have been shown to normalise plasma and bone calcium in Vitamin D deficient rats and in mice. In the calcitriol deficient piglets none of the diets was capable to prevent the development of hypocalcemia. However, additions of lactose and soy protein improved somewhat plasma calcium (P < 0.001). Feeding of soy protein also had a significant positive effect on plasma phosphate concentration (P < 0.001). The study shows that in contrast to rats, calcitriol is essential for maintaining a normal plasma and bone calcium status. Responses of this type, when obtained with rats or mice can probably not directly be transferred to pigs and perhaps also not to humans.

Animals↗