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PubMed1999

Conjugation of plasmid DNAs with lactose via diazocoupling enhances resistance to restriction enzymes and acquires binding affinity to galactose-specific lectin.

Oligosaccharide-plasmid DNA conjugates were synthesized simply and effectively via the diazocoupling method. Plasmids (pUC19, pTRI-beta-actin, and pEGFP-C1) were treated with an N-beta-lactoside-substituted diazonium salt to yield diazocoupling products with degree of substitutions of 2.5-3.1 mol% of overall nucleobases. The lactose-pUC19 conjugate was found to resist restriction enzymes more strongly than the nonconjugated plasmid DNA and to acquire a strong binding affinity to galactose-specific lectin RCA(120). The diazocoupling modification of pTRI-beta-actin plasmid DNA little influenced in vitro transcription with T7 RNA polymerase. When lactose-pEGFP-C1 conjugate was transfected to baby hamster kidney (BHK) cells by means of cationic lipids, transduced gene was expressed in BHK cells similarly with the nonconjugated pEGFP-C1. The modification of plasmid DNA with carbohydrate enhanced the resistance to restriction enzymes and developed a strong binding affinity to galactose-specific lectin.

Actins↗
PubMed1996

Conformation, protein-carbohydrate interactions and a novel subunit association in the refined structure of peanut lectin-lactose complex.

The structure of the complex of the tetrameric peanut lectin with lactose has been refined to an R-value of 16.4% using 2.25 angstroms resolution X-ray diffraction data. The subunit conformation in the structure is similar to that in other legume lectins except in the loops. It has been shown that in the tertiary structure of legume lectins, the short five-stranded sheet plays a major role in connecting the larger flat six-stranded and curved seven-stranded sheets. Furthermore, the loops that connect the strands at the two ends of the seven-stranded sheet curve toward and interact with each other to produce a second hydrophobic core in addition to the one between the two large sheets. The protein-lactose interactions involve the invariant features observed in other legume lectins in addition to those characteristic of peanut lectin. The "open" quaternary association in peanut lectin is stabilised by hydrophobic, hydrogen-bonded and water-mediated interactions. Contrary to the earlier belief, the structure of peanut lectin demonstrates that the variability in quaternary association in legume lectins, despite all of them having nearly the same tertiary structure, is not necessarily caused by covalently bound carbohydrate. An attempt has been made to provide a structural rationale for this variability, on the basis of buried surface areas during dimerisation. A total of 45 water molecules remain invariant when the hydration shells of the four subunits are compared. A majority of them appear to be involved in stabilising loops.

Carbohydrate Metabolism↗
PubMed1993

Stability analysis of the Lactococcus lactis DRC1 lactose plasmid using pulsed-field gel electrophoresis.

Pulsed-field agarose gel electrophoresis of SmaI digests of genomic DNA was used to examine lactose plasmid copy number and stability in Lactococcus lactis. In L. lactis strain DRC1, the plasmid was found to exist as a single-copy plasmid. Transconjugants of strain HID113 carrying this plasmid were unstable. Variants were isolated with improved phenotypic stability resulting from improved maintenance of the lactose plasmid or from integration of part of the plasmid into the lactococcal chromosome.

Conjugation, Genetic↗
PubMed1996

Vitamin D3, lactose, and xylitol stimulate chromaffin cell proliferation in the rat adrenal medulla.

Chronic consumption by rats of diets rich in sugars or sugar alcohols leads to an increased incidence of pheochromocytomas. This relationship is hypothesized to be based on altered Ca2+ homeostasis due to increased intestinal Ca2+ absorption. Other agents associated with pheochromocytomas in rats in long-term toxicity studies have been shown to increase chromaffin cell proliferation, leading to the suggestion that the tumors occur secondarily to increased chromaffin cell turnover. We have demonstrated marked stimulation of chromaffin cell proliferation by vitamin D3, a potent stimulus to Ca2+ absorption not previously associated with adrenal medullary toxicity. This effect is detectable during the first week of dietary supplementation and persists throughout a 4-week time course. Lactose and xylitol, representative of sugars and sugar alcohols associated with pheochromocytomas, are also mitogenic but to a lesser extent, with their effects first detectable during Week 4 of dietary supplementation. Vitamin D3, its active metabolite calcitriol, lactose, and xylitol all fail to stimulate proliferation of rat chromaffin cells in vitro. The mitogenic effects of these agents may be mediated presynaptically in vivo. The data suggest that altered Ca2+ homeostasis may increase chromaffin cell proliferation and support the hypothesis that diets containing high concentrations of sugars and sugar alcohols cause pheochromocytomas in rats secondarily by this mechanism.

Adrenal Medulla↗
PubMed1996

Interregional foodborne salmonellosis outbreak due to powdered infant formula contaminated with lactose-fermenting Salmonella virchow.

Spain's Salmonella surveillance system backed by regionally-based epidemiologists around the country made it possible to detect and halt the spread of a foodborne salmonellosis outbreak due to powdered infant formula contaminated with a lactose-fermenting strain of Salmonella virchow. Forty-eight cases involving children, mostly under 7 months old, were detected in 14 out of Spain's 17 Regions. The outbreak started in January and ended in June 1994. All cases were microbiologically confirmed. The implicated strain had a 3.6 kb plasmid, was susceptible to all antimicrobials tested except nitrofurantoin and was phagetype 4a. Isolates from 8 of 24 Brand "A" milk samples tested had the same characteristics as case isolates. All affected/suspect batches of Brand "A" milk were destroyed and the product withdrawn from sale, which led to the end of the outbreak. This incident underscores the importance of maintaining surveillance systems able to detect and prevent foodborne outbreaks and alert to the possibility of isolating unusual lactose-fermenting Salmonella serotypes in especially sensitive food products.

Animals↗
PubMed1990

Characterization of a plasmid involved with cointegrate formation and lactose metabolism in Lactococcus lactis subsp. lactis OZS1.

A 55 kilobase (kb) plasmid (pOZS550) in the non-clumping Lactococcus lactis subsp. lactis strain OZS1 carrying genes for lactose metabolism was characterised. A mobilizable cointegrate plasmid which is formed between pOZS550 and pOZS448 carries the necessary information for conjugation and transfer. Cointegrate formation was found to involve an insertional element located on pOZS550. The insertion sequence was found to be identical to ISS1 located on pSK08 in the clumping L. lactis subsp. lactis strain ML3. Restriction maps of pOZS550 and pSK08 were similar suggesting a close ancestral relationship, although pSK08, in addition to the lactose metabolism genes, expressed genes for proteinase activity and cell clumping, which were not expressed by pOZS550, and carried two copies of ISS1 compared to one on pOZS550. Furthermore, hybridization of the 18 base pair inverted repeat, of the insertion sequence, with various L. lactis subsp. lactis strains and two L. lactis subsp. cremoris strains showed moderate to strong hybridization to one plasmid in each organism.

Base Sequence↗
PubMed1979

Multiple integration sites for the lactose transposon Tn 951 on plasmid RP 1 and establishment of a coordinate system for Tn 951.

Various molecules generated by transposition of the lactose transposon Tn 951 from plasmid pGC1 to plasmid RP1 were examined by DNA heteroduplex and restriction endonuclease analysis. Tn 951 was found to transpose to at least eight different sites on RP 1 in both possible orientations. A coordinate system for the lactose transposon Tn 951 is constructed.

DNA Restriction Enzymes↗
PubMed1976

Persistence of high intestinal lactase activity (lactose tolerance) in Afghanistan.

Two hundred and seventy apparently healthy adult subjects from Afghanistan, mainly from the central and eastern parts of the country, were subjected to a lactose tolerance test. The change of blood glucose from the fasting concentration at 20 min after the administration of lactose showed a bimodal distribution. Forty-seven subjects had a rise of blood glucose concentration of more than 1.1 mmol/l and were classified as persistence of high intestinal lactase activity (PHILA), a term which lays emphasis on the fact that high lactase activity in the adult is an unusual state whose prevalence in some populations requires explanation. In the Afghan sample there were no significant differences of the frequency of PHILA in different ethnic groups.

Adult↗
PubMed1993

Monoclonal antibody against a lactose epitope of glycosphingolipids binds to melanoma tumour cells.

Mice were immunized with a neoglycoprotein consisting of a chemically modified carbohydrate moiety (reductively aminated 3'-sialyllactose) linked to human serum albumin. By this procedure an antibody response to the normally non-immunogenic carbohydrate structure was obtained. Hybridomas were established, and monoclonal antibodies were selected in ELISA based on their binding to the saccharide hapten, or to a lactosylceramide-mimicking neoglycolipid, lactose-bis-sulfone. One of the selected antibodies, 2H4, was of particular interest, since it also bound to glycolipids present on melanoma cells. FACS analysis of a panel of 14 melanoma cell lines showed that the 2H4 antibody bound to the majority of these. In frozen, non-fixed sections or paraffin sections of biopsies the monoclonal antibody 2H4 stained melanoma cells, but not tumour infiltrating lymphocytes or normal skin. Detailed immunochemical analysis of 2H4, using thin layer chromatography revealed that it recognized an internal lactose epitope in several glycosphingolipids.

Animals↗
PubMed1981

A family study of lactose tolerance.

Survey data from 38 families consisting of both parents and at least one biological offspring provided no support for the belief that lactose tolerance resulted from having at least one dominant gene on an autosomal gene pair and did not support the belief that Caucasians are more frequently tolerant of lactose than are persons of Oriental or of mixed Caucasian-Oriental ancestry.

Adult↗
PubMed1993

Variations of neutral oligosaccharides and lactose in human milk during the feeding.

There exist only few data concerning the variation of oligosaccharides in human milk. In this study the variations of neutral oligosaccharides and of lactose in human milk during the feeding were determined from five women at day 8 and at day 57 post partum. The milk of the investigated feedings was divided in four parts of equal volumes during sampling; the concentrations of neutral oligosaccharide fractions were determined by gel permeation chromatography on Fractogel TSK HW 40 (S) columns. No significant differences in the concentrations of the neutral oligosaccharide groups monofucosyllactoses, difucosyllactose, lacto-N-tetraoses, monofucosyllacto-N-tetraoses and difucosyllacto-N-tetraoses and of lactose were found in the four milk parts. The results of this study favor the use of so-called mid-feed samples, a simple and convenient sampling method for analytical studies of human milk. Mid-feed samples are representative of the whole feeding as concerned for neutral oligosaccharides.

Adult↗
PubMed2004

Molecularly defined lactose malabsorption, peak bone mass and bone turnover rate in young finnish men.

Lactose malabsorption (LM; adult-type hypolactasia), an autosomal recessive condition, results from the down-regulation of the activity of lactase enzyme in the intestinal wall. In previous studies the effect of LM on bone mass, bone turnover rate, development of osteoporosis and osteoporotic fractures has remained controversial. We have recently identified a single nucleotide polymorphism (SNP), a C to T change residing 13910 base pairs upstream of the lactase (LCT) gene at chromosome 2q21-22, which shows complete association with lactase persistence, with the C/C-13910 genotype defining LM and the genotypes C/T-13910 and T/T-13910 lactase persistence. The present study was undertaken to examine the relationship of the C/T-13910 polymorphism to peak bone mass, bone turnover rate, and stress fractures among young Finnish men. The study population comprised 234 young men, aged 18.3 to 20.6 years, 184 men were recruits of the Finnish Army, and 50 were men of similar age who had postponed their military service for reasons not related to health. Bone mineral content (BMC), density (BMD), and scan area were measured in the lumbar spine and upper femur by dual-energy X-ray absorptiometry (DXA). Blood was sampled for genotyping of the C/T-13910 polymorphism and determination of serum 25-hydroxyvitamin D (25OHD), intact parathyroid hormone (iPTH), type I procollagen aminoterminal propeptide (PINP), and tartrate-resistant acid phosphatase 5b (TRACP5b). Second-void urine samples were collected for the determination of type I collagen aminoterminal telopeptide (NTX). The prevalence of the C/C-13910-genotype of these young adults did not differ significantly from the corresponding population prevalence of C/C-13910 (17.1% vs 18.1%) among Finnish blood donors. Fifteen recruits of the army experienced a stress fracture; 3 of them (20%) had the C/C-13910-genotype. Calcium intake was similar for the three genotypes as were the unadjusted BMCs, scan areas, and BMDs at different measurement sites. The adjustments for age, height, weight, smoking, alcohol consumption, and physical exercise in the multiple regression analysis did not reveal any significant relationships between the lactase genotypes and BMDs at lumbar (P = 0.16), femoral neck (P = 0.99) or total hip (P = 0.96) sites. Serum 25OHD, iPTH, and bone marker levels were similar for the C/C-13910 C/T-13910 and T/T-13910 genotypes. In summary, in young Finnish men, molecularly defined lactose malabsorption does not alter bone turnover rate and impair the acquisition of peak bone mass. Moreover, the C/C-13910 genotype does not seem to be a risk factor for stress fractures in army recruits.

Absorptiometry, Photon↗
PubMed2006

A novel polymorphism associated with lactose tolerance in Africa: multiple causes for lactase persistence?

Persistence or non-persistence of lactase expression into adult life is a polymorphic trait that has been attributed to a single nucleotide polymorphism (C-13910T) in an enhancer element 13.9 kb upstream of the lactase gene (LCT). The -13910*T allele occurs at very high frequency in northern Europeans as part of a very long haplotype (known as A), and promotes binding of the transcription factor Oct-1. However, -13910*T is at very low frequency in many African milk drinking pastoralist groups where lactase persistence phenotype has been reported at high frequency. We report here for the first time, a cohort study of lactose digester and non-digester Sudanese volunteers and show there is no association of -13910*T or the A haplotype with lactase persistence. We support this finding with new genotype/phenotype frequency comparisons in pastoralist groups of eastern African and Middle Eastern origin. Resequencing revealed three new SNPs in close proximity to -13910*T, two of which are within the Oct-1 binding site. The most frequent of these (-13915*G) is associated with lactose tolerance in the cohort study, providing evidence for a cis-acting effect. Despite its location, -13915*G abolishes, rather than enhances Oct-1 binding, indicating that this particular interaction is unlikely to be involved in lactase persistence. This study reveals the complexity of this phenotypic polymorphism and highlights the limitations of C-13910T as a diagnostic test for lactase persistence status, at least for people with non-European ancestry.

Adult↗
PubMed1991

Flow injection analysis of lactose using covalently immobilized beta-galactosidase, mutarotase, and glucose oxidase/peroxidase on a 2-fluoro-1-methylpyridinium salt-activated Fractogel support.

Milk samples were analyzed for their lactose content using flow injection analysis and incorporating immobilized beta-galactosidase or beta-galactosidase/mutarotase and glucose oxidase/peroxidase bioreactors. These enzymes were immobilized, under mild conditions, on to a 2-fluoro-1-methylpyridinium salt-activated Fractogel support. The use of a phosphate buffer (0.15 M) was found to facilitate the rapid mutarotation of alpha-D-glucose and hence could obviate the need for the more expensive mutarotase. The chromogenic agents of choice for monitoring the reaction were 3-methyl-2-benzothiazolinone hydrazone and 3-dimethylaminobenzoic acid. Linearity was observed over the concentration range 16-160 micrograms/ml using lactose standards (r = 0.996). Between 30 and 40 milk samples/h can be analyzed. Comparisons are made with existing HPLC and alkaline methylamine methods for a range of milk matrices. The FIA method consistently gives the lowest standard deviations and coefficient of variation for the various milk matrices analyzed.

Animals↗
PubMed1978

Immobilized bovine lactose synthase. A method of topographical analysis of the active site.

Bovine galactosyltransferase (UDPgalactose: D-glucose 4beta-galactosyltransferase, EC 2.4.1.22) was covalently coupled to Sepharose 4B by reaction at pH 5.0 with the activated mixed disulfide Sepharose-glutathione-2(5-nitropyridyl)-disulfide. The Sepharose-protein conjugate was presumably coupled via the unique highly reactive cysteine of those thiols on the bovine enzyme. The gel-bound N-acetyllactosamine and lactose synthase activity of about 0.4% was consistent with the affects of diffusion and the 90% activity reduction noted upon thiol modification of the dissolved enzyme. The residual lactose biosynthetic activity of the bound enzyme appeared possible only if the reactive thiol were physically distinct from the active site since the bulky Sepharose-glutathione group must not obscure the alpha-lactalbumin binding region.

Animals↗
PubMed1978

Binding of galactose and lactose to ricin. Equilibrium studies.

The interaction of ricin, one of the two lectins of Ricinus sanguineus, with its specific ligands galactose and lactose (4-O-beta-D-galactopyranosyl-D-glucopyranose) has been studied by means of equilibrium dialysis, analytical ultracentrifugation and fluorescence polarization. In the studied concentration range, only one molecule of galactose is bound per molecule of ricin with an association constant, Ka = 6900 m-1 at 4 degrees C. Scatchard plots of equilibrium dialysis data show that two molecules of lactose bind to one molecule of ricin, without modification of molecular weight of the lectin. Together with results of microcalorimetric experiments and agglutination of erythrocytes by ricin, equilibrium dialysis data indicate that the lectin contains two distinct saccharide binding sites. Regardless of the existence of extended sites, it is not possible to select between the two models: (a) two independent sites (Ka1 = 35 000 M-1, Ka2 = 2800 M-1 at 4 degrees C) or (b) two identical sites with negative cooperativity.

Dialysis↗
PubMed1983

The pairing of light and heavy chains of isomeric anti-lactose antibodies.

Sets of polyclonal isomeric anti-carbohydrate antibodies are present in sera of rabbits immunized with vaccines of non-viable streptococcal cells containing antigenic glycans. Anti-lactose antibodies have been purified by affinity chromatography and have been subjected to dissociation, electrophoretic and agar diffusion analyses. The anti-lactose antibody preparation was found to be composed of 16 isomeric proteins which yield 16 heavy chains and 16 light chains.

Animals↗
PubMed1990

The effect of bismuth on the selective oxidation of lactose on supported palladium catalysts.

The selective oxidation of lactose by molecular oxygen has been studied in a batch reactor containing an aqueous slurry of 0.5 kmol m-1 reactant and 1.0 kg m-3 catalyst. The in situ Bi promotion of a commercial Pd-C catalyst resulted in 100% selectivity to sodium lactobionate up to conversions of 95% in the pH range 7-10 and at temperatures up to 333 K. Performing the reaction under such conditions that the oxygen transfer to the liquid phase was rate-controlling allowed the production of sodium lactobionate in high yields in approximately 1 h. A maximum initial reaction-rate of 0.47 mol kg-1 s-1 was found at a molar Bi to Pd ratio of 0.50-0.67. Fifteen batches of lactose were oxidized with the same charge of catalyst without significant loss in initial activity or selectivity. Such other aldoses as maltose, glucose, and galactose could be oxidized analogously with similar selectivities.

Bismuth↗