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Chromobacterium violaceum is a ß-proteobacterium found widely worldwide with important biotechnological properties and is associated to lethal sepsis in immune-depressed individuals. In this work, we report the discover, complete sequence and annotation of a plasmid detected in C. violaceum that has been unnoticed until now. We used DNA single-molecule analysis to confirm that the episome found was a circular molecule and then proceeded with NGS sequencing. After DNA annotation, we found that this extra-chromosomal DNA is probably a defective bacteriophage of approximately 44 kilobases, with 39 ORFs comprising, mostly hypothetical proteins. We also found DNA sequences that ensure proper plasmid replication and partitioning as well as a toxin addiction system. This report sheds light on the biology of this important species, helping us to understand the mechanisms by which C. violaceum endures to several harsh conditions. This discovery could also be a first step in the development of a DNA manipulation tool in this bacterium.

The rapid spread of antibiotic resistance - currently one of the greatest threats to human health according to WHO - is to a large extent enabled by plasmid-mediated horizontal transfer of resistance genes. Rapid identification and characterization of plasmids is thus important both for individual clinical outcomes and for epidemiological monitoring of antibiotic resistance. Toward this aim, we have developed an optical DNA mapping procedure where individual intact plasmids are elongated within nanofluidic channels and visualized through fluorescence microscopy, yielding barcodes that reflect the underlying sequence. The assay rapidly identifies plasmids through statistical comparisons with barcodes based on publicly available sequence repositories and also enables detection of structural variations. Since the assay yields holistic sequence information for individual intact plasmids, it is an ideal complement to next generation sequencing efforts which involve reassembly of sequence reads from fragmented DNA molecules. The assay should be applicable in microbiology labs around the world in applications ranging from fundamental plasmid biology to clinical epidemiology and diagnostics.

Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples.

Studies of circular DNA confined to nanofluidic channels are relevant both from a fundamental polymer-physics perspective and due to the importance of circular DNA molecules in vivo. We here observe the unfolding of DNA from the circular to linear configuration as a light-induced double strand break occurs, characterize the dynamics, and compare the equilibrium conformational statistics of linear and circular configurations. This is important because it allows us to determine to which extent existing statistical theories describe the extension of confined circular DNA. We find that the ratio of the extensions of confined linear and circular DNA configurations increases as the buffer concentration decreases. The experimental results fall between theoretical predictions for the extended de Gennes regime at weaker confinement and the Odijk regime at stronger confinement. We show that it is possible to directly distinguish between circular and linear DNA molecules by measuring the emission intensity from the DNA. Finally, we determine the rate of unfolding and show that this rate is larger for more confined DNA, possibly reflecting the corresponding larger difference in entropy between the circular and linear configurations., 21 pages, 7 figures, 1 table

Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification cif pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.

Rose hips and sea buckthorn berries contain high amounts of beneficial bioactive compounds. In this study the content and stability of natural vitamins and antioxidants were investigated in two fruit beverages made from rose hip powder, sea buckthorn berry puree and white grape juice concentrate, and wheat germ oil in one of the beverages. Beverages were formulated, pasteurised and filled aseptically in Tetra Brik packages and stored up to 35 days at 4 degrees C or 22 degrees C. Samples were analysed for the content of ascorbic acid, carotenoids, major phenolics, tocochromanols, total phenols and total antioxidant capacity as ferric reducing ability. The largest changes occurred during the first 5 days of storage, thereafter mainly ascorbic acid decreased. In the wheat germ oil supplemented beverage, alpha-tocopherol was more rapidly degraded. Negative correlations between the decrease of some carotenoids and tocochromanols, and the increase of some phenolics, suggest relationships of redox reactions specific to the amount of oil supplemented. From the present study we can conclude that additions of oil to beverages should be carefully selected to avoid oxidations of bioactive compounds. Furthermore, packaging of the beverages should be carried out at higher temperatures and thereafter it should be stored at low temperatures (4 degrees C), which decreased oxidation of all bioactive compounds except ascorbic acid. (Less)

The output from whole genome sequencing is a set of contigs, i.e. short non-overlapping DNA sequences (sizes 1-100 kilobasepairs). Piecing the contigs together is an especially difficult task for previously unsequenced DNA, and may not be feasible due to factors such as the lack of sufficient coverage or larger repetitive regions which generate gaps in the final sequence. Here we propose a new method for scaffolding such contigs. The proposed method uses densely labeled optical DNA barcodes from competitive binding experiments as scaffolds. On these scaffolds we position theoretical barcodes which are calculated from the contig sequences. This allows us to construct longer DNA sequences from the contig sequences. This proof-of-principle study extends previous studies which use sparsely labeled DNA barcodes for scaffolding purposes. Our method applies a probabilistic approach that allows us to discard "foreign" contigs from mixed samples with contigs from different types of DNA. We satisfy the contig non-overlap constraint by formulating the contig placement challenge as a combinatorial auction problem. Our exact algorithm for solving this problem reduces computational costs compared to previous methods in the combinatorial auction field. We demonstrate the usefulness of the proposed scaffolding method both for synthetic contigs and for contigs obtained using Illumina sequencing for a mixed sample with plasmid and chromosomal DNA.

Objective: To evaluate the impact of an extruded muesli product based on -glucan-rich oat bran on postprandial glycaemia and insulinaemia. Subject/Design: The study is divided in two series. Blood glucose and serum insulin responses were studied after subjects consuming test meals including a serving of muesli with 3 g (series 1) and 4 g (series 2) of -glucans, respectively. The muesli was a component in a single serving packet with muesli and yoghurt. This was served together with white wheat bread in the morning after an overnight fast. The compositions were standardized to contain 50 g available carbohydrates. As a reference meal a serving packet without -glucans was included. The study was performed at Applied Nutrition and Food Chemistry, Lund University, Sweden. Nineteen and thirteen healthy volunteers with normal body mass index were recruited for series 1 and 2, respectively. Results: Muesli with 3 g of -glucans, included in a mixed bread meal, gave no significant differences in glycaemic response compared to a reference meal without muesli and -glucans. In contrast, muesli with 4 g of -glucans significantly (P

The extension of DNA confined to nanochannels has been studied intensively and in detail. Yet quantitative comparisons between experiments and model calculations are difficult because most theoretical predictions involve undetermined prefactors, and because the model parameters (contour length, Kuhn length, effective width) are difficult to compute reliably, leading to substantial uncertainties. Here we use a recent asymptotically exact theory for the DNA extension in the "extended de Gennes regime" that allows us to compare experimental results with theory. For this purpose we performed new experiments, measuring the mean DNA extension and its standard deviation while varying the channel geometry, dye intercalation ratio, and ionic buffer strength. The experimental results agree very well with theory at high ionic strengths, indicating that the model parameters are reliable. At low ionic strengths the agreement is less good. We discuss possible reasons. Our approach allows, in principle, to measure the Kuhn length and effective width of a single DNA molecule and more generally of semiflexible polymers in solution., Revised version, 6 pages, 2 figures, 1 table, supplementary material

By simultaneous coherent anti-Stokes Raman scattering (CARS) and 2-photon fluorescence microscopy of Thioflavin-S stained Alzheimer´s diseased human brain tissues, we show evidence of lipid deposits co-localizing with fibrillar β-amyloid (Aβ) plaques. Two lipid morphologies can be observed; lamellar structures and coalescing macro-aggregates of sub-micron sizes to ~25 μm. No significant lipid deposits were observed in non-fibrillar, diffuse plaques identified by Aβ immuno-staining. CARS microscopy of unlabeled samples confirms the lamellar and macro-aggregate lipid morphologies. The composition of the plaques was analyzed by CARS microspectroscopy and Raman microscopy; vibrational signatures of lipids with long acyl chains co-localize with the β-sheet vibrations. The lipid fluidity was evaluated from the CARS spectra, illustrating that the lipid composition/organization varies throughout the plaques. Altogether this indicates close amyloid-lipid interplay in fibrillar Aβ plaques, rendering them more dynamic compositions than previously believed and, hence, potential sources of toxic oligomers.

We demonstrate a single DNA molecule optical mapping assay able to resolve a specific Escherichia coli strain from other strains. The assay is based on competitive binding of the fluorescent dye YOYO-1 and the AT-specific antibiotic netropsin. The optical map is visualized by stretching the DNA molecules in nanofluidic channels. We optimize the experimental conditions to obtain reproducible barcodes containing as much information as possible. We implement a multi-ligand transfer matrix method for calculating theoretical barcodes from known DNA sequences. Our method extends previous theoretical approaches for competitive binding of two types of ligands to many types of ligands and introduces a recursive approach that allows long barcodes to be calculated with standard computer floating point formats. The identification of a specific E. coli strain (CCUG 10979) is based on mapping of 50–160 kilobasepair experimental DNA fragments onto the theoretical genome using the developed theory. Our identification protocol introduces two theoretical constructs: a P-value for a best experiment-theory match and an information score threshold. The developed methods provide a novel optical mapping toolbox for identification of bacterial species and strains. The protocol does not require cultivation of bacteria or DNA amplification, which allows for ultra-fast identification of bacterial pathogens.

Alzheimera's disease (AD) is a progressive neurodegenerative disorder currently without cure, characterized by the presence of extracellular plaques surrounded by dystrophic neurites. In an effort to understand the underlying mechanisms, biochemical analysis (protein immunoblot) of plaque extracts reveals that they consist of amyloid-beta (Aβ) peptides assembled as oligomers, protofibrils and aggregates. Their spatial distribution has been confirmed by Thioflavin-S or immuno-staining with fluorescence microscopy. However, it is increasingly understood that the protein aggregation is only one of several mechanism that causes neuronal dysfunction and death. This raises the need for a more complete biochemical analysis. In this study, we have complemented 2-photon fluorescence microscopy of Thioflavin-S and Aβ immuno-stained human AD plaques with CARS microscopy. We show that the chemical build-up of AD plaques is more complex and that Aβ staining does not provide the complete picture of the spatial distribution or the molecular composition of AD plaques. CARS images provide important complementary information to that obtained by fluorescence microscopy, motivating a broader introduction of CARS microscopy in the AD research field.

Several studies have shown that there is a strong physical interaction between cholesterol and sphingomyelin (SM). The critical factor is thought to be the high degree of saturation in the very long acyl chains of SM. In this study we examined the effects of SM on cholesterol absorption in the rat and compared them with those of phosphatidylcholine (PC). Cholesterol absorption was studied by use of the dual-isotope plasma ratio method. We also studied the effect of sterols on the fecal excretion of undigested SM and its metabolites after a single oral meal of (3)H-dihydrosphingosine-labeled SM. When cholesterol was given dissolved in soybean oil, without addition of SM or other phospholipids, absorption was 68 +/- 12% in the rat intestine. As a general feature the absorption was less efficient from the cholesterol/phospholipid dispersions. In dispersions with cholesterol and SM, the lowest cholesterol absorption (9 +/- 2%) was seen with a cholesterol:SM molar ratio of 1:1. With dispersions of cholesterol and different PC substrates the absorption of cholesterol was lower with saturated PC (16 +/- 8%) than with soybean-PC (22 +/- 4%) or dioleoyl PC (23 +/- 8%). Uptake of SM in the rat intestine was reduced by sterols. For example, percentage recovery of (3)H radioactivity in fecal lipids was 38 +/- 8% when SM was given with cholesterol and 16 +/- 3% without any sterol. One third of the radioactivity in feces was present as ceramide. Sitostanol had the same effect on uptake of SM as cholesterol. This study shows that when rats are fed mixtures of SM and cholesterol the intestinal uptake of both lipids is decreased. By feeding mixtures of SM and sterols the exposure of the colon to ceramide can be increased.

BACKGROUNDThere is a renewed interest in metabolism of sphingolipids because of their role in signal transduction. Sphingomyelin is the dominating phospholipid in human milk but its metabolism and possible function in the gastrointestinal tract of breast fed infants is unknown. We explored whether bile salt-stimulated milk lipase has a role in sphingolipid metabolism.METHODSIn vitro assays of sphingomyelinase and ceramidase activities, using radiolabeled substrates, human milk samples and purified native and recombinant variants of bile salt-stimulated milk lipase with or without known activators or inhibitors.RESULTSHuman whey and purified lipase catalysed hydrolysis of palmitoyl-labeled ceramide with the highest rate around pH 8.5-9.0. 1 mg of lipase hydrolysed 0.7 micromol ceramide in one hour at pH 8.5 in presence of 4 mM bile salt. The activity of whey was inhibited by antibodies towards human bile salt-stimulated milk lipase, indicating that this lipase accounted for virtually all ceramidase activity in the milk. In contrast, bile salt-stimulated milk lipase showed no activity against sphingomyelin. However we give evidence of a separate, hitherto unknown, acid sphingomyelinase in human milk. Under the used in vitro conditions this sphingomyelinase could account for hydrolysis of half of milk sphingomyelin in one hour.CONCLUSIONSHuman milk bile salt-stimulated milk lipase hydrolyses ceramide and may thus have a role in sphingomyelin digestion, but only after initial hydrolysis to ceramide and phosphorylcholine. Part of the latter could be carried out in the stomach by the acid milk sphingomyelinase now described. We speculate that these two milk enzymes may be of importance for optimal use of human milk sphingolipids. (Less)

Dietary sphingomyelin (SM) undergoes sequential cleavage to ceramide and sphingosine in the intestine. A distinctive intestinal sphingomyelinase (SMase) with alkaline pH-optimum was earlier identified by us. The activity was highest in middle and lower small intestine, but its role in SM digestion has not been clarified. In this study we examined the extension and capacity of SM digestion in vivo. After feeding rats 0.2, 6.6, or 32 μmol SM containing 2 μCi 3H-sphingosine-labeled milk SM (3H-SM), radioactivity was analyzed in intestinal contents and tissues 2, 4, and 8 hr later. The proportion of radioactivity in the contents of small intestine increased with the dose of SM; 9% of given dose with 0.2 μmol, 34% with 6.6 μmol, and 71% with 32 μmol, respectively after 2 hr. Lowest tissue radioactivity was found in duodenum and proximal jejunum and highest in distal jejunum and proximal ileum. Three to twenty one percent of radioactivity in the intestinal tissue was in ceramide, the proportion varying with the dose given, region of the intestine, and time after administration. After administration of 6.6 or 32 μmol SM, significant amounts of intact SM and ceramide was found in intestinal contents, colon, and excreted faeces. Colon was exposed to ceramide derived from exogenous SM in amounts that were rather proportional to the dose of SM fed. SM digestion is thus a process extending over the whole intestine and occurring mainly in the middle and lower parts of the small intestine. The site of digestion coincides with the distribution of the alkaline SMase, indicating that this enzyme catalyzes the first step in the digestion. The extension and limited capacity of the SM digestion leads to an exposure of the lower small intestine and colon to SM and sphingolipid metabolites.

There is convincing evidence that consumption of fish and fish oil rich in long-chain (LC)n-3 PUFA (n-3 LCPUFA), EPA (20 : 5n-3) and DHA (22 : 6n-3) reduce the risk of CHD. The aim of the present study was to investigate whethern-3 LCPUFA-enriched food products provide similar beneficial effects as fish oil with regard to incorporation into plasma lipids and effects on cardiovascular risk markers. A parallel 7-week intervention trial was performed where 159 healthy men and women were randomised to consume either 34 g fish pâté (n44), 500 ml fruit juice (n38) or three capsules of concentrated fish oil (n40), all contributing to a daily intake of approximately 1 g EPA and DHA. A fourth group did not receive any supplementation or food product and served as controls (n37). Plasma fatty acid composition, serum lipids, and markers of inflammation and oxidative stress were measured. Compared with the control group, plasman-3 LCPUFA and EPA:arachidonic acid ratio increased equally in all intervention groups. However, no significant changes in blood lipids and markers of inflammation and oxidative stress were observed. In conclusion, enriched fish pâté and fruit juice represent suitable delivery systems forn-3 LCPUFA. However, although the dose given is known to reduce the risk of CVD, no significant changes were observed on cardiovascular risk markers in this healthy population.

The properties of sphingomyelin obtained from bovine milk were investigated. In particular, the properties of liposomes and emulsions prepared from the spingomyelin, as well as the liquid crystalline behavior, were investigated and compared to those of related phosphatidylcholine systems. Like sphingomyelins from other sources, sphingomyelin from milk contains a large fraction of long and saturated acyl groups, which results in a high gel-to-liquid crystal transition temperature (Tc 35–82°C, depending on the lipid concentration). At high sphingomyelin concentrations, a lamellar phase forms above Tc, while a swollen gel phase is obtained below Tc. The gel phase swells to about 20 wt% water, whereas the swelling continues to about 40 wt% water above Tc. The limiting areas per molecule are 51 and 68 A2 below and above Tc, respectively. Sphingomyelin from milk forms liposomes readily in the presence of cholesterol. The liposomes formed have a diameter of about 100 nm and are stable, even at 0.1 M NaCl or HCl. Materials entrapped in the liposomes are released rather slowly (typically 40% over 5 h). A comparison shows that the sphingomyelin liposomes behave similarly to those formed by phosphatidylcholine systems. Furthermore, sphingomyelin from milk forms stable oil-in-water emulsions with soybean oil. The size of the emulsion droplets obtained was about 200 nm. Both the size of the emulsion droplets and its dependence on electrolyte addition correlate closely with those of emulsions formed by the corresponding phosphatidylcholine system. Therefore, it is possible to use sphingomyelin as an alternative for saturated phosphatidylcholines, which may be advantageous for oral and dermal pharmaceutical applications, as well as in cosmetics.

Eight crossbred, ovariectomized gilts (Swedish Landrace x Swedish Yorkshire x Hampshire), with a mean age of 7.5 mo, were studied during anesthesia and surgery, control-sampling, relocation, and grouping during a period of 3 wk. Acute treatment with a hydrocortisone injection (5 mg, i.v.) was also given. Blood samples were taken frequently (every 10 min) during the intensive part of the experimental days. The blood samples were analyzed for catecholamines (CA), adrenaline (A) and noradrenaline (NA), cortisol, and corticosteroid-binding globulin (CBG). The surgical period with anesthesia and surgery showed significant effects on CA, cortisol, and CBG. The A level increased immediately after the surgery. A biphasic increase in the cortisol level was observed on the day of surgery; the greatest increase was seen when the animals had regained consciousness after surgery but still were drowsy and staggering. The CBG level decreased on the day after the surgery. The study showed that the levels of the analyzed substances were back to normal 2 d after surgery. During relocation and grouping there was a short, significant increase in CA and cortisol levels, whereas the CBG level was not affected. Treatment with hydrocortisone significantly increased the cortisol level, but no change was seen in plasma CA or CBG levels. In conclusion, anesthesia and surgery induced significant effects on the levels of CA, cortisol, and CBG in gilts and the animals needed 2 d to recover before regaining their normal condition. During relocation and a limited grouping period, the significant increases in CA and cortisol levels were of short duration and CBG did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND: Sphingomyelin occurs in modest amounts in the diet, in sloughed mucosal cells, and in bile. It is digested by the mucosal enzymes alkaline sphingomyelinase and ceramidase. In humans, alkaline sphingomyelinase is also secreted in bile. The digestion of sphingomyelin is slow and incomplete, which has been linked to the inhibition of cholesterol absorption and colonic carcinogenesis. OBJECTIVE: We evaluated whether the supply of moderate amounts of milk sphingomyelin increases the exposure of the colon to sphingomyelin and its metabolites. DESIGN: Two experimental series were performed. In experiment A, we measured the content of sphingomyelin and ceramide in human ileostomy content by HPLC during 8 h after consumption of a test meal containing 250 mg milk sphingomyelin. In experiment B, we measured the molecular species of sphingomyelin and ceramide by HPLC-tandem mass spectrometry after doses of 50, 100, or 200 mg sphingomyelin. RESULTS: In experiment A, the average increase in ileostomy content of ceramide plus sphingomyelin amounted to 19% of the fed dose of sphingomyelin. In experiment B, the output of C-22:0-sphingomyelin, C-23:0-sphingomyelin, C-24:0-sphingomyelin, and sphingosine increased significantly and palmitoyl-sphingomyelin increased proportionally less. Outputs and concentrations of palmitoyl-ceramide and sphingosine showed great individual variation, and stearoyl-sphingomyelin and stearoyl-ceramide did not increase after the meals. Although the output of long-chain sphingomyelin species increased significantly, the data indicated that >81% of all measured sphingomyelin species had been digested. CONCLUSIONS: Humans digest and absorb most of the sphingomyelin in normal diets. The level of sphingolipid metabolites to which the colon is exposed can, however, be influenced by realistic amounts of dietary sphingomyelin. (Less)

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder without cure, characterized by the presence of extracellular plaques surrounded by dystrophic neuritis . Fundamental understanding of the molecular processes involved is urgently needed in order to develop strategies to inhibit steps in the AD pathogenesis. Current understanding is that the senile plaques are composed of peptide fragments (Amyloid- β peptides) formed after the cleavage of the transmembrane protein amyloid protein precursor (APP) . This knowledge relies primarily on biochemical analysis of the plaques in cell extracts together with fluorescence microscopy of tissue samples exposed to harsh preparation procedures and labeling. Thus, it is disputed to which extent the observations reported reflect the true biochemistry and morphology of AD brain tissue. By the powerful combination of Raman microspectroscopy, Coherent Anti-Stokes Raman Scattering (CARS) and 2-photon fluorescence microscopy, we show that the molecular composition of AD plaques is more complex and consequently the mechanisms behind their formation.

The interactions of the bile salt sodium taurocholate (TC) in 50 mM Trizma-HCl buffer and 150 mM NaCl (pH 9) at 37 degrees C with membranes composed of sphingomyelin (SM) were studied by dynamic light scattering, cryogenic transmission electron microscopy (cryo-TEM) and turbidity measurements. Small unilamellar SM vesicles were prepared by extrusion. Below the CMC of TC, taurocholate addition leads to vesicle growth due to incorporation of the taurocholate molecules into the vesicle bilayer. At around half the CMC of the bile salt, the SM vesicles are transformed into SM/TC mixed worm-like micelles, which are visualized by cryo-TEM for the first time. Further increase in the taurocholate concentration leads to the rupture of these structures into small spherical micelles. Interestingly, large non-spherical micelles were also identified for pure taurocholate solutions. Similar threadlike structures have been reported earlier for the bile salt sodium taurodeoxycholate [Rich, A., Blow, D., 1958. Nature 182, 1777; Blow, D.M., Rich, A., 1960. J. Am. Chem. Soc. 82, 3566-3571; Galantini, L., Giglio, E., La Mesa, C., Viorel-Pavel, N., Punzo, F., 2002. Langmuir 18, 2812] and for mixtures of taurocholate and phosphatidylcholate [Ulmius, J., Lindblom, G., Wennerstrom, H., Johansson, L.B.-A., Fontel, K., Soderman, O., Ardvisson, G., 1982. Biochemistry 21, 1553; Hjelm, R.P., Thiyagarajan, P., Alkan-Onyuksel, H., 1992. J. Phys. Chem. 96, 8653] as determined by various scattering methods.

Rapid characterization of unknown biological samples is under the focus of many current studies. Here we report a method for screening of biological samples by optical mapping of their DNA. We use a novel, one-step chemo-enzymatic reaction to covalently bind fluorophores to DNA at the four-base recognition sites of a DNA methyltransferase. Due to the diffraction limit of light, the dense distribution of labels results in a continuous fluorescent signal along the DNA. The amplitude modulations (AM) of the fluorescence intensity along the stretched DNA molecules exhibit a unique molecular fingerprint that can be used for identification. We show that this labelling scheme is highly informative, allowing accurate genotyping. We demonstrate the method by labelling the genomes of λ and T7 bacteriophages, resulting in a consistent, unique AM profile for each genome. These profiles are also successfully used for identification of the phages from a background phage library. Our method may provide a facile route for screening and typing of various organisms and has potential applications in metagenomics studies of various ecosystems.

The hydrolysis of sphingomyelin has been found to generate important signals regulating cell proliferation, differentiation and apoptosis. However, the enzymes responsible for digestion of dietary sphingomyelin have not been well documented. This study demonstrates the occurrence of a sphingomyelinase (SMase) in both human hepatic bile and gallbladder bile. The enzyme was equally found in both bacteria negative and positive bile samples and in samples obtained from patients with or without gallbladder diseases. A bacteria-free gallbladder bile was used for characterization. It was found that bile SMase hydrolyzed sphingomyelin to phosphorylcholine and ceramide with negligible activity against either phosphatidylcholine or p-nitrophenyl phosphate. The enzyme preferred an alkaline condition and the optimal pH was 9. The activity of this alkaline SMase was bile salt dependent and was fully activated by 4–6 mM bile salts. Triton X-100, the non-ionic detergent did not activate bile SMase. Ca2+ and Mg2+ ions had no significant effect at optimal bile salt concentration. The molecular mass of this enzyme was about 85 kDa as measured by Sephadex G200 gel chromatography. In conclusion, we demonstrated a SMase in bile which differs markedly from the known acid and neutral SMase. Its potential important roles in sphingomyelin digestion and gallbladder diseases require further investigation.

Previous studies indicated that there was an alkaline sphingomyelinase (SMase) activity in small intestine, but its properties have not been studied in detail. In the present work, we studied the distribution of this enzyme activity in rat gastrointestinal tract and characterized it in intestinal mucosal homogenates. Little alkaline SMase activity was detected in the stomach and the duodenum. The activity in both mucosa and intestinal content increased in the small intestine and reached the maximum at the distal jejunum, then declined in the ileum and slightly increased again in the colon. The activity distribution pattern differed markedly from those of acid SMase and alkaline phosphatase. Little alkaline SMase activity could be found in bile, liver and pancreas before or after treatment with trypsin. The optimum pH of the alkaline SMase was 9. It specifically hydrolyzed sphingomyelin (SM), not phosphatidylcholine, to ceramide and phosphocholine. The alkaline SMase was bile salt dependent and was optionally activated by 3 mM bile salts. Triton X-100 could not mimic the effect of bile salt, rather dose-dependently inhibited the enzyme activity. Ca2+, Mg2+ did not change the alkaline SMase activity in the presence of bile salts, and reduced the activity in the absence of bile salt. Trypsin inactivated acid SMase in pancreas, liver and duodenum but had no influence on intestinal alkaline SMase activity. In conclusion, the intestinal alkaline SMase has a specific distribution pattern and the characters of it differ in several respects from the known acid and neutral SMases.

Various markers for stress were evaluated in the plasma of pigs. Treadmill exercise was chosen as a well defined and quantifiable stressor. One group of non-stress-susceptible pigs had to carry out two intense exercise bouts at a one-week interval, and another group remained in their pens and served as controls. Large interindividual differences in the performance and in the hormonal and metabolic responses to exercise were observed. The exercise stress resulted in an immediate increase in the plasma concentrations of cortisol, lactate and hypoxanthine, with a return to the pre-exercise levels within one hour. No direct effect of physical stress on CBG binding capacity or on the concentrations of xanthine and uric acid was observed. In two very nervous pigs, even mild stress in connection with the first blood sampling resulted in an elevated concentration of lactate, but the cortisol and hypoxanthine values remained at the basal level. It is concluded that plasma concentrations of cortisol, lactate and hypoxanthine are valuable markers for physical stress.

Corticosteroid binding globulin (CGB) from term-pregnant mouse serum was isolated and characterized by peptide analysis after treatment with CNBr and Lys-specific protease, respectively. Amino acid sequence analysis of six segments, covering 189 of 383 positions in different regions of the protein, showed unexpectedly low overall homology (60%) to the indirectly deduced human amino acid sequence previously reported. However, some segments displayed a greater resemblance to their human counterparts. Differences were observed in at least two of six potential glycosylation sites. The nature of electrophoretic CBG variants and their immunological properties are described.

A fast and reproducible two-step method with high resolution was developed for purification of murine corticosteroid-binding globulin (CBG). The first step was liquid chromatography on a Sephacryl-S-200 column, and the CBG-containing residual was subsequently chromatographed by fast protein liquid chromatography (FPLC). This enabled us to quickly obtain a highly purified protein and the apparently isolated CBG was tested for its purity by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) and sedimentation equilibrium centrifugation. The CBG concentration in pregnant mouse serum was estimated to 0.78 g/l (1.5% of the total protein). The monomeric organization of the protein was demonstrated by mercaptoethanol treatment. No NH 2 -terminal amino acid could be detected, probably owing to a blocked amino acid. The mol. wt (M r ) of murine CBG was determined to be 52,000 and the sedimentation constant S 20°,w to 3.9 S by analytical ultracentrifugation. The protein showed 5 bands when subjected to isoelectric focusing: 3 bands with apparent isoelectric points (pI) between pH 3.15–3.25 and two between pH 3.40–3.50.

Treatment of mice with ACTH not only increased plasma corticosterone levels, but also reduced the ligand-binding capacity of the glucocorticoid receptor in skeletal muscle cytosol. Non-linear Scatchard plots were obtained for the glucocorticoid receptor following ACTH treatment. This upward concave curvilinearity could not be reproduced by simply adding to the cytosol an amount of corticosterone corresponding to the increase, even though this treatment resulted in reduced binding capacity. The addition of corticosterone resulted in a slower formation of ligand-receptor complexes (lower association rate constant), which offered a partial explanation for the low binding. In addition, ACTH treatment was also found to reduce the binding capacity in adrenalectomized mice that did not respond with corticosterone elevation. However, in this case Scatchard plots were linear.

Effects of stress on concentrations of cortisol and corticosteroid-binding globulin (CBG) in blood plasma and on glucocorticoid receptor concentrations in muscle cytosol were studied in pigs representing three Halothane (Hal) genotypes (NN, Nn, nn). At 12 wk of age, animals were divided into four groups: pigs subjected to transport (5 h in a truck), pigs treated with amperozide prior to transport, pigs not transported but treated with amperozide and pigs neither transported nor given amperozide. Animals were slaughtered the week they reached 100 kg live weight (3 mo later). The Hal gene showed no major influence on the variables studied except for cortisol concentrations (P = .06) measured directly after transport at 12 wk of age (NN = 66.8 nM, Nn = 61.4 nM, nn = 69.4 nM). However, the response in each Hal genotype differed, depending on whether or not the pigs had been exposed to transport. Two weeks after transport, NN pigs developed higher cortisol concentrations than untransported animals, whereas the response was reversed in nn animals; Nn pigs showed no difference in this regard. At slaughter, the effect of transport (12 wk of age) on cortisol and CBG was still apparent. In NN pigs cortisol and CBG concentrations were elevated (P less than or equal to .05, P = .08, respectively), whereas concentrations tended to be lower in nn pigs (P = .17, P = .07, respectively) when compared with untransported pigs. Transported pigs had lower receptor concentrations at slaughter (P less than or equal to .01) than untransported pigs. However, pigs given amperozide in connection with transport had a receptor concentration comparable to that in untransported pigs. Our study shows conclusively that transport stress had long-term effects on cortisol, CBG and glucocorticoid receptor concentrations. In addition, amperozide had long-term effects on cortisol and receptor concentrations.

Nanofluidic channels have become a versatile tool to manipulate single DNA molecules. They allow investigation of confined single DNA molecules from a fundamental polymer physics perspective as wel ...

Nanofluidic channels have become a versatile tool to manipulate single DNA molecules. They allow investigation of confined single DNA molecules from a fundamental polymer physics perspective as well as for example in DNA barcoding techniques.Circular DNA is of interest since it is found in many biologically relevant contexts, such as bacterial plasmids, viruses and eukaryotic mitochondrial DNA. Furthermore, the circular topology forces two strands in close proximity to each other in nanochannel, which changes the polymer physics compared to linear DNA. Circular DNA is difficult to study with traditional single molecule techniques because such techniques generally require the attachment of handles to the, but is readily accessed using nanofluidics.Circular DNA has less entropy and higher conformational free energy than in its unfolded configuration. Therefore, as a double-strand break occurs and circular DNA opens up, it unfolds to its linear configuration inside the nanochannel. This study compares the static properties of confined linear and circular DNA as well as investigates the dynamics of the transition from circular to linear DNA as a double-strand break occurs.We observe that the difference in extension between the circular and linear configurations depends on the degree of confinement, which we confirm with theoretical predictions. Our data for unfolding of the circular DNA to the linear configuration suggests that hydrodynamic friction between the DNA and the solvent is the main rate-determining factor but that DNA-DNA contacts are also important. Finally, by staining the DNA inhomogeneously, we can follow the local dynamics of the DNA as the folding occurs and conclude, for example, how the two different strands move relative to each other during the unfolding process. We are thus able to study the dynamics of confined DNA with unprecedented resolution and obtain completely new information about confined polymers.

It has long been known that the commonly used fluorescent dye YOYO-1 (YOYO) stains DNA unevenly at sub-stoichiometric concentrations, and that equilibration is slow. We use this uneven staining to increase the resolution in measurements on how YOYO affects the properties of DNA confined in nanofluidic channels. The emission intensity from each DNA-YOYO complex correlates directly with the amount of YOYO bound. We can thus, in one single experiment, study DNA-YOYO molecules ranging from fully saturated to molecules with a low degree of YOYO bound. Interestingly we find that the extension of the DNA does not increase linearly with increasing amount of YOYO bound. Our data instead suggests that there are two different phases with different behavior. We explain this as being due to a transition from a region where intramolecular electrostatics dominates to where also the charges on the channel walls become important.Our observations are of importance not only for YOYO but are general for cationic ligands that bind to DNA by intercalation and the nanochannels are excellently suited for probing such subtle changes in the physics of the DNA-ligand complex.Finally we show how a mix of YOYO and netropsin, a DNA-binding molecule with extreme selectivity for AT-DNA, can be used to map DNA sequences. When adding the two molecules to DNA simultaneously and stretching it in the nanochannel we obtain an emission intensity variation along the DNA that corresponds to the underlying AT/GC ratio. The intensity variation correlates well with the known sequence and we believe that such maps can be useful for example for fast identification of bacterial infections from extremely small sample volumes.