Cold atoms in non-Abelian gauge potentials: from the Hofstadter "moth" to lattice gauge theory.

We demonstrate how to create artificial external non-Abelian gauge potentials acting on cold atoms in optical lattices. The method employs atoms with k internal states, and laser assisted state sensitive tunneling, described by unitary k x k matrices. The single-particle dynamics in the case of intense U2 vector potentials lead to a generalized Hofstadter butterfly spectrum which shows a complex mothlike structure. We discuss the possibility to realize non-Abelian interferometry (Aharonov-Bohm effect) and to study many-body dynamics of ultracold matter in external lattice gauge fields.

Deterministic nonlinear characteristics of in vivo blood flow velocity and arteriolar diameter fluctuations.

We have performed a nonlinear analysis of fluctuations in red cell velocity and arteriolar calibre in the mesenteric bed of the anaesthetized rat. Measurements were obtained under control conditions and during local superfusion with NG-nitro-L-arginine (L-NNA, 30 microM) and tetrabutylammonium (TBA, 0.1 mM), which suppress NO synthesis and block Ca2+ activated K+ channels (KCa), respectively. Time series were analysed by calculating correlation dimensions and largest Lyapunov exponents. Both statistics were higher for red cell velocity than diameter fluctuations, thereby potentially differentiating between global and local mechanisms that regulate microvascular flow. Evidence for underlying nonlinear structure was provided by analysis of surrogate time series generated from the experimental data following randomization of Fourier phase. Complexity indices characterizing time series under control conditions were in general higher than those derived from data obtained during superfusion with L-NNA and TBA.

Determination of microvascular flow pattern formation in vivo.

Blood flow in microvessels differs significantly from that of red blood cells (RBC) flowing through long, straight glass tubes in vitro. The in vivo situation is characterized by the presence of plasma favoring aggregation, by the irregular geometry of vessel segments, and by frequent branching points. Here, a method is presented to characterize flow patterns in microvascular blood flow during intravital microscopy based on Fourier analysis of recorded light intensity patterns. The interpretation of the resulting power spectra in terms of pattern size distribution was validated by model experiments employing artificial textures and by reverse transformation of idealized spectra. The determined size of RBC flow patterns in microvessels ranged from approximately 8 microm in capillaries to approximately 14 microm in vessels of >30 microm. With increasing shear rate above approximately 100 s(-1) pattern size increased, possibly reflecting formation of short-lived flow clusters. Below approximately 100 s(-1) an increase of pattern size with decreasing shear rate was found in experiments using local occlusion and treatment with high-molecular-weight dextran, suggesting the formation of aggregates. The dynamic process of generation and destruction of RBC flow patterns could well contribute to flow resistance in vivo in peripheral vascular beds.

Microvascular blood flow resistance: role of endothelial surface layer.

Observations of blood flow in microvascular networks have shown that the resistance to blood flow is about twice that expected from studies using narrow glass tubes. The goal of the present study was to test the hypothesis that a macromolecular layer (glycocalyx) lining the endothelial surface contributes to blood flow resistance. Changes in flow resistance in microvascular networks of the rat mesentery were observed with microinfusion of enzymes targeted at oligosaccharide side chains in the glycocalyx. Infusion of heparinase resulted in a sustained decrease in estimated flow resistance of 14-21%, hydrodynamically equivalent to a uniform increase of vessel diameter by approximately 1 micron. Infusion of neuraminidase led to accumulation of platelets on the endothelium and doubled flow resistance. Additional experiments in untreated vascular networks in which microvascular blood flow was reduced by partial microocclusion of the feeding arteriole showed a substantial increase of flow resistance at low flow rates (average capillary flow velocities < 100 diameters/s). These observations indicate that the glycocalyx has significant hemodynamic relevance that may increase at low flow rates, possibly because of a shear-dependent variation in glycocalyx thickness.

Kinetic analysis of the intestinal iron absorption process in situ. The potential of vascularly autoperfused intestinal loops.

1 Blood sampling from mesenteric venules during absorption in situ is a useful tool to analyse intestinal absorption kinetics and prehepatic metabolism in different sections of the rat small intestine. By use of a micromanipulator, the method can be applied to the duodenum. This part of the small intestine shows the strongest adaptation of non-haem iron absorption to the demand for iron. 2 Iron absorption kinetics was linear in duodenal and jejunal segments. In iron-deficient animals, intestinal iron absorption capacity was increased in the duodenum, while simultaneously determined galactose absorption showed no change. 3 In situ perfusion and cannulation of mesenteric venules in duodenal segments are described. The use of a micromanipulator permits varying the blood volume collected by changing the vertical angle between the cannula and the mesenteric vessel. 4 Intestinal iron absorption rates remained close to constant when blood flow rates were varied by a factor of about ten. Plasma concentrations of absorbed iron vs mesenteric blood flow rates followed a hyperbolic function, as the plasma concentration of absorbed iron in mesenteric venules increased to the same extent as the blood flow decreased. 5 As the plasma transferrin concentration did not change over the experimental period, the concentration of absorbed iron in the mesenteric plasma exceeded the iron-binding capacity of plasma transferrin at low blood flow rates. This observation shows that enhancement of intestinal iron absorption does not require a corresponding increase in plasma iron-binding capacity in the intestinal tissue. 6 Vascularly perfused gut loops were also used to measure prehepatic metabolism, which may influence organotropism of carcinogenic metabolites. Therefore, this type of preparation is likely to find a variety of toxicological applications.

Pathways in the binding and uptake of ferritin by hepatocytes.

The binding and uptake of rat liver ferritin by primary cultures of rat liver hepatocytes was studied in order to assess the relative importance of saturable, high-affinity pathways and nonspecific processes in the incorporation of the protein by the cells. To minimize artifacts, ferritin not subjected to heat treatment and labeled in vivo with 59Fe was used. Binding to cell membranes was estimated from incubations performed at 4 degrees C. After 2 h, when a steady state in cell-associated ferritin had been achieved, approx. 4-10(4) binding sites per cell were observed, with an affinity constant for ferritin of 1 x 10(9) M-1. At 37 degrees C, the maximal uptake from these sites was 1.3 x 10(5) ferritin molecules/cell per h. For ferritin molecules bearing an average of 2400 iron atoms, this uptake amounts to 5 x 10(6) iron atoms/cell per min. Half-maximal uptake was achieved at a ferritin concentration, or KM1, of 3 x 10(-9) M. Although uptake rates at least a thousand times greater could be achieved by binding to the much larger number of low-affinity sites, the apparent KM2 for such 'nonspecific' uptake was 4 x 10(-7) M. At ferritin concentrations up to 2 nM, at least 90% of ferritin bound and taken up by hepatocytes involves saturable, high-affinity sites, presumably true ferritin receptors.

Subcellular distribution of recently absorbed iron and of transferrin in the mouse duodenal mucosa.

A successful method for the analytical subcellular fractionation of mouse duodenal mucosa organelles was established. 59Fe(III)-nitrilotriacetate (pH 7.2) was injected into tied-off duodenal segments in vivo and, after 2-20 min, mucosal homogenates were subjected to subcellular fractionation. Radioactivity was recovered in the cytosolic fractions and in the gradient at a density of 1.18-1.20 g/ml. Enhanced iron absorption was achieved by placing the animals in a hypobaric chamber for 3 days. These animals had a higher proportion of particulate 59Fe compared to controls. Homogenisation in sucrose medium containing the selective plasma membrane perturbant digitonin shifted the particulate iron fraction to a higher density region of the gradient indicating a localisation of the iron binding site to the plasma membrane region of the mucosal cells. No significant radioactive iron was observed in the brush-border region of the gradient. Transferrin immunoreactivity was found only in the cytosolic region of the gradient and was not associated with any organelle.

Intestinal iron absorption and mucosal transferrin in rats subjected to hypoxia.

Three days hypoxia (0.5 atm) increased the haemoglobin and haematocrit values in rats paralleled by enhanced intestinal iron absorption. The destination of recently-absorbed iron was primarily the erythropoietic system, viz. bone marrow, spleen and red cells. Total plasma transferrin, was increased by 30%, but no significant changes in mucosal transferrin were found. No increase in labelling of mucosal transferrin by absorbed iron was observed. These results suggest that mucosal transferrin does not play a major role in the regulation of intestinal iron absorption in hypoxia.

Small intestinal regulation of iron absorption in the rat.

Ultrastructural, biochemical, and immunologic studies of the small intestinal mucosa of rats were undertaken to investigate factors associated with the regulation of iron absorption. The quantity of iron within mucosal cells was proportionate to the degree of iron repletion. Although the quantity of iron-binding substances was similar in iron-deficient and iron-loaded animals, the unsaturated iron-binding capacity of mucosal cells varied inversely with the state of iron repletion of animals in ultrastructural, biochemical, and immunologic observations. Although only certain mucosal cells contained iron-binding substances, their number was not increased in iron-deficient animals. Greater quantities of iron and iron-binding substances were observed in duodenal mucosa than in ileal mucosa. These findings are consistent with the hypothesis that the quantity of unsaturated iron-binding substances within intestinal mucosal cells regulates iron absorption. These findings are only partially explained by changes observed in the concentration of ferritin and transferrin within intestinal mucosa and suggest that other iron-binding substances may also participate in the regulation of iron absorption.

Computational analysis of density gradient distribution profiles.

A method is presented for the construction of distribution profiles following subcellular fractionation by density gradient centrifugation. The method allows reconstruction of the frequency-density distributions over user-defined density limits, enabling averaging of results from multiple experiments. An alternative format for data presentation, that of relative concentration versus density, is also introduced. This overcomes some practical problems associated with frequency-density distribution profiles. The technique utilizes a direct noniterative approach to curve fitting and can easily be implemented on microcomputers.

Independence of in vitro iron absorption from mucosal transferrin content in rat jejunal and ileal segments.

Isolated non blood-perfused intestinal segments from normal and iron-deficient rats were used in vitro. A modification of the luminal perfusion method according to Fisher and Parsons allowed the comparison of iron and transferrin quantities in the serosal fluid at 15 min intervals. Iron transfer in jejunal and ileal segments was directly proportional to the luminal iron concentration within a dose range of 1 to 100 mumol/l, did not show saturation characteristics and was linear over time. Jejunal segments from iron-deficient rats transferred about twice as much iron as the jejunal controls. In ileal segments there was no difference in iron transfer between iron-deficient and control rats; in both cases transfer amounted to approx. 10% of jejunal controls. An exponential correlation was found, when the decreasing transferrin content of the tissue was plotted against the cumulative water transport. Transferrin and albumin release from jejunal and ileal segments into the absorbate cumulated asymptotically, which is typical for wash-out phenomena. As iron transfer cumulated linearly while transferrin release cumulated in an asymptotic manner, the capacity of transferrin to bind iron ions is exceeded roughly 100 times by molar equivalents of iron in the last absorbate fractions. Independence of iron transfer from mucosal transferrin quantities is concluded. As the molar transferrin/albumin ratios do not show significant differences between plasma and the sequence of absorbate samples, a wash-out from the gut's interstitial space is assumed, which makes plasma the most likely origin of transferrin in the mucosa.

Studies on the role of transferrin and endocytosis in the uptake of Fe3+ from Fe-nitrilotriacetate by mouse duodenum.

Addition of iron-binding proteins (human serum transferrin, mouse serum transferrin, human lactoferrin) to the luminal fluid in tied-off segments of mouse intestine in vivo led to reduced 59Fe3+ absorption from 59Fe3+-nitrilotriacetate when compared to 59Fe3+-nitrilotriacetate alone. Assay of transferrin in luminal fluid from tied segments revealed only trace amounts of immunoreactivity. The levels of luminal transferrin are unaltered in chronic hypoxia where iron absorption is significantly enhanced. Studies in vitro revealed that NH4Cl, dansylcadavarine, para-chloromercuribenzoate and trinitrobenzenesulphonate have no effect on initial 59Fe3+ uptake rates from 59Fe3+-nitrilotriacetate, while N-ethylmaleimide (1 mM) caused a 40% inhibition. In vivo 59Fe3+ uptake was unaffected by preincubation of tied-off segments with colchicine (5 mM) for up to 2 h. These results suggest that receptor-mediated endocytosis of transferrin is not a significant mechanism in the uptake of luminal Fe3+ by mouse duodenum.

Rapid preparation of highly purified human transferrin.

A rapid, two-step purification for human transferrin using DEAE-Sephacel chromatography followed by phenyl-boronate affinity chromatography is described. The method gives a 70% yield and isolates transferrin of over 97% purity which is hemopexin-free. The remaining protein is due to a single contaminant which may be the glycosylated form of albumin.

Transferrin in isolated cells from rat duodenum and jejunum.

Mucosal transferrin was determined as transferrin-like immunoreactivity (TLIR) by means of a 2-site immunoradiometric assay (IRMA). Scraped-off mucosal tissue as well as isolated mucosal cells from the duodenum and jejunum of normal and iron-deficient rats before and after a washing procedure were examined. In iron-deficient rats there was about twice as much TLIR in scraped-off mucosal tissue as in the untreated animals. In the duodenum and jejunum of normal and iron-deficient rats, TLIR contents of the isolated cells in the magnitude of 320-510 ng/mg dry weight were found. Washing isolated cells three times in ice-cold Hank's solution resulted in a nearly tenfold decrease of TLIR content in all groups. In contrast the cells' RNA content remained unchanged.

Determination of transferrin-like immunoreactivity in the mucosal homogenate of the duodenum, jejunum, and ileum of normal and iron deficient rats.

1. In intestinal mucosal tissue a transferrin-like immunoreactivity (TLIR) was determined after loading with 59Fe-(FeCl3) in vivo. The scraped-off mucosal tissue was homogenized and centrifuged at 100 000 X g. The supernatant was fractionated chromatographically. The TLIR was determined by the Mancini-test. 2. Extrapolated to infinite dilutions of the homogenate the following contents of the TLIR were calculated per cm of the intestinal segment: 28, 20, and 13, microgram/cm in the duodenum, jejunum, and ileum of iron-deficient animals and 15, 14, and 12 microgram/cm in the corresponding intestinal segments of normal rats, respectively. 3. The content of TLIR was compared with the iron uptake of the mucosal tissue from the intestinal lumen during absorption. An "iron turnover number" of 1.6 +/- 0.2 iron atoms per min during the absorption in duodenal and jejunal segments and of 0.5 +/- 0.1 in ileal segments of either iron-deficient and normal animals was calculated for the mucosal transferrin represented by the TLIR.

Influence of fucoidan on the intestinal absorption of iron, cobalt, manganese and zinc in rats.

Fucoidan was extracted from the seaweed Ascophyllum nodosum with boiling water and purified by repeated precipitation steps. Increasing doses of fucoidan (1.2--200 mg) were injected together with 360 nmol 59Fe-(FeCl3) or 60Co-(CoCl2) into tied-off jejunal segments of two groups of rats fed either a normal (160 mg Fe/kg) or a low iron diet (5 mg/kg). Fucoidan together with 360 nmol 54Mn-(MnCl2) or 65Zn-(ZnCl2) was administered in the same manner in rats fed a normal diet only. Fucoidan administered in doses above 30 mg decreased the absorption of iron, cobalt, manganese and zinc in normal rats, and the absorption of iron and cobalt in iron-deficient rats. This inhibitory effect of fucoidan on the absorption of heavy metals is apparently the consequence of the formation of metal complexes which are poorly absorbed from the intestinal lumen.

Capacity of the mucosal transfer system and absorption of iron after oral administration in rats.

1. The dose dependence of the iron absorption shows a saturation characteristic if iron (0.25--5 mumoles Fe/kg body weight) is administered in tied-off intestinal segments of normal and iron-deficient rats. 2. If the iron doses (2.1--570 mumoles Fe/kg body weight) are administered by stomach tube only in normal rats a similar dose dependence curve has been obtained as after administration in tied-off intestinal segments. In iron-deficient rats the shape of the dose dependence curve is changed and shows no clear-cut saturation characteristic. 3. The differences of these dose dependence curves are discussed with respect to the differences of the methodological conditions. A saturation type kinetic for absorption cannot be expected under all experimental conditions despite of the existence of a transfer system with limited capacity for the transport of iron.