Interleukin 1 beta is localized in the cytoplasmic ground substance but is largely absent from the Golgi apparatus and plasma membranes of stimulated human monocytes.

The subcellular location of IL-1 beta was determined using a postsectioning immunoelectron microscopic method on ultrathin frozen sections of human monocytes stimulated with LPS. This methodology permits access of antibody probes to all sectioned intracellular compartments, and their visualization at high resolution. Staining was performed with a rabbit antibody that specifically recognized amino acids 197-215 in the 33-kD IL-1 beta precursor molecule, followed by affinity-purified goat anti-rabbit IgG conjugated to 10 nm colloidal gold particles. Approximately 90% of the IL-1 beta antigens were localized in the ground substance of the cytoplasm at 4 or 20 h after activation, when both intracellular and extracellular accumulation of IL-1 beta was well underway. No significant IL-1 beta staining was observed on the outer cell membrane, nor within the lumens of the endoplasmic reticulum (ER), the Golgi apparatus, or secretory vesicles. In contrast, lysozyme was localized in the ER and dense secretory granules using these methods. Our results suggest that IL-1 beta is not anchored on the plasma membrane, and that its secretion occurs by a novel mechanism that does not use a secretory leader sequence, nor the classical secretory pathway involving the ER and Golgi apparatus.

5-lipoxygenase and 5-lipoxygenase-activating protein are localized in the nuclear envelope of activated human leukocytes.

The intracellular distribution of the enzyme 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) in resting and ionophore-activated human leukocytes has been determined using immuno-electronmicroscopic labeling of ultrathin frozen sections and subcellular fractionation techniques. 5-LO is a 78-kD protein that catalyzes the conversion of arachidonic acid to leukotrienes. FLAP is an 18-kD membrane bound protein that is essential for leukotriene synthesis in cells. In response to ionophore stimulation, 5-LO translocates from a soluble to a sedimentable fraction of cell homogenates. In activated leukocytes, both FLAP and 5-LO were localized in the lumen of the nuclear envelope. Neither protein could be detected in any other cell compartment or along the plasma membrane. In resting cells, the FLAP distribution was identical to that observed in activated cells. In addition, subcellular fractionation techniques showed > 83% of immunoblot-detectable FLAP protein and approximately 64% of the FLAP ligand binding activity was found in the nuclear membrane fraction. A fractionation control demonstrated that a plasma membrane marker, detected by a monoclonal antibody PMN13F6, was not detectable in the nuclear membrane fraction. In contrast to FLAP, 5-LO in resting cells could not be visualized along the nuclear envelope. Except for weak labeling of the euchromatin region of the nucleus, 5-LO could not be readily detected in any other cellular compartment. These results demonstrate that the nuclear envelope is the intracellular site at which 5-LO and FLAP act to metabolize arachidonic acid, and that ionophore activation of neutrophils and monocytes results in the translocation of 5-LO from a nonsedimentable location to the nuclear envelope.

The interleukin-1 beta-converting enzyme (ICE) is localized on the external cell surface membranes and in the cytoplasmic ground substance of human monocytes by immuno-electron microscopy.

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE) is a novel cysteine protease that cleaves the 31-kD inactive cytoplasmic IL-1 beta precursor into active extracellular 17-kD IL-1 beta. The ICE gene product is a 45-kD proenzyme that requires proteolytic processing to activate ICE. Active ICE is a heterodimer consisting of equal amounts of p20 and p10 subunits. Generation of active ICE is affected by the removal of an 11-kD NH2-terminal precursor domain (p11) and an internal 19-amino acid sequence that separates the 20- and 10-kD subunits. Immuno-electron microscopy was performed on human monocytes with immunoglobulins recognizing the active (p20) or precursor (p11) domains of ICE. Elutriated monocytes were stimulated with 50 pM lipopolysaccharide followed by heat-killed Staphylococcus aureus under conditions that induce maximal rates of IL-1 beta secretion. Ultrathin cryosections were cut from fixed frozen pellets of these monocytes and were immunogold labeled with either antibody. Active and precursor domain ICE epitopes were localized in the cytoplasmic ground substance, but they were not detected within the endoplasmic reticulum, the Golgi apparatus, and secretory granules of activated or inactive monocytes. Importantly, numerous ICE p20 epitopes were also observed on the extracellular surfaces of the cell membrane, and were concentrated on the microvilli. Very similar patterns of ICE localization were obtained with unstimulated blood monocytes. In contrast, ICE p11 epitopes were not detected on the surfaces of these monocytes. Likewise, labeling of fixed ultrathin cryosections of monocytes with a biotinylated irreversible ICE inhibitor [Ac-Tyr-Val-Lys(biotin)-Asp-(acyloxy)-methyl-ketone] showed that the compound localized on the outer cell surface as well, and to a lesser extent, within the cytoplasmic ground substance. Furthermore, antipeptide antibodies specific for either the mature or precursor domains of IL-1 beta were both localized upon the cell membrane after stimulation of IL-1 beta secretion. Lipopolysaccaride-primed monocytes that synthesized, but did not secrete IL-1 beta, exhibited only cytoplasmic staining. The data suggests that mature IL-1 beta is generated via cleavage of the 31-kD inactive cytoplasmic IL-1 beta precursor by ICE after association with the plasma membrane during secretion.

Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts.

We have recently observed a transmembrane association between extracellular fibronectin (FN) fibers and elongated focal patches or fibers of vinculin (VN) in G1-arrested stationary Nil 8 hamster fibroblasts, with double-label immunofluorescence microscopy (Singer and Paradiso, 1981, Cell. 24:481-492). We hypothesized that these FN-VN complexes might correspond to focal contacts, the membrane sites that are probably mainly responsible for attaching cells to their substrata, because vinculin is often localized in focal contacts. However, because fibronectin-vinculin associations may not be restricted to the substrate adhesive surface of the cell, it became necessary to determine whether some or all of the various kinds of FN-VN complexes which we described are in proximity to the substrate. Using interference reflection optics and double-label immunofluorescence microscopy for fibronectin and vinculin, many elongated (up to 38 micrometer) FN-VN associations were found to be strikingly coincident with focal contacts in the perinuclear area of extremely flattened arrested Nil 8 fibroblasts in 0.3% fetal bovine serum (FBS). In addition, the long FN-VN adhesion complexes were precisely aligned with the major phase-dense stress fibers observed at the ventral surfaces of these stationary cells with phase contrast microscopy. Fibronectin was neither associated with vinculin-containing focal contacts of Nil 8 cells cultured in medium with 5% FBS nor with vinculin-negative focal contacts located at the extreme edges of stationary cells arrested in 0.3 FBS. Our time-course experiments suggest that early FN-VN lacking-focal contacts, which form at the cellular margins, develop into mature substrate adhesion complexes containing both fibronectin and vinculin, localized in the major stress fibers at the centers of sessile fibroblasts.

Adhesomes: specific granules containing receptors for laminin, C3bi/fibrinogen, fibronectin, and vitronectin in human polymorphonuclear leukocytes and monocytes.

We have localized several major extracellular matrix protein receptors in the specific granules of human polymorphonuclear (PMN) and monocytic leukocytes using double label immunoelectron microscopy (IEM) with ultrathin frozen sections and colloidal-gold conjugates. Rabbit antibodies to 67-kD human laminin receptor (LNR) were located on the inner surface of the specific granule membrane and within its internal matrix. LNR antigens co-distributed with lactoferrin, a marker of specific granules, but did not co-localize with elastase in azurophilic granules of PMNs. Further, CD11b/CD18 (leukocyte receptor for C3bi, fibrinogen, endothelial cells, and endotoxin), mammalian fibronectin receptor (FNR), and vitronectin receptor (VNR) antigens were also co-localized with LNR in PMN specific granules. A similar type of granule was found in monocytes which stained for LNR, FNR, VNR, CD18, and lysozyme. Activation of PMNs with either PMA, f-met-leu-phe (fMLP), tumor necrosis factor (TNF), or monocytic leukocytes with lipopolysaccharide (LPS), induced fusion of specific granules with the cell membrane and expression of both LNR and CD18 antigens on the outer cell surface. Further, stimulation led to augmented PMN adhesion on LN substrata, and six- to eightfold increases in specific binding of soluble LN that was inhibited by LNR antibody. These results indicate that four types of extracellular matrix receptors are located in leukocyte specific granules, and suggest that up-regulation of these receptors during inflammation may mediate leukocyte adhesion and extravasation. We have thus termed leukocyte specific granules adhesomes.

In vivo co-distribution of fibronectin and actin fibers in granulation tissue: immunofluorescence and electron microscope studies of the fibronexus at the myofibroblast surface.

The fibronexus ( FNX ), a very close transmembrane association of individual extracellular fibronectin fibers and actin microfilaments, was found previously at the substrate-binding surface of fibroblasts in tissue culture (Singer, 1. 1., 1979, Cell, 16:675-685). To determine whether the fibronexus might be involved in fibroblast adhesion during wound healing in vivo, we looked for co-localization of actin and fibronectin in granulation tissue formed within full-thickness guinea pig skin wounds. At 7-9 d, most of the actin fibers were observed to be coincident with congruent fibronectin fibers using double-label immunofluorescence microscopy. These fibronectin and actin fibers were co-localized at the myofibroblast surface surrounding the nucleus, and along attenuated myofibroblast processes which extended deeply into the extracellular matrix. This conspicuous co-distribution of fibronectin and actin fibers prompted us to look for fibronexuses at the myofibroblast surface with electron microscopy. We observed three kinds of FNXs : (a) tandem associations between the termini of individual extracellular fibronectin fibers and actin microfilament bundles at the tips of elongate myofibroblast processes, (b) plaque-like and, (c) track-like FNXs , in which parallel fibronectin and actin fibers were connected by perpendicular transmembranous fibrils. Goniometric studies on the external and internal components of these cross-linking fibrils showed that their membrane-associated ends are probably co-axial. Using immunoelectron microscopy on ultrathin cryosections, we confirmed that the densely staining external portion of these various FNXs does indeed contain fibronectin. The finding that these FNXs appear to connect collagen fibers to intracellular bundles of actin microfilaments is particularly significant. Our studies strongly suggest that the fibronexus is an important in vivo cell surface adhesion site functioning in wound repair, and perhaps within fibronectin-rich tissues during embryogenesis, tumor growth, and inflammation.

The fibronectin cell attachment sequence Arg-Gly-Asp-Ser promotes focal contact formation during early fibroblast attachment and spreading.

Cultured fibroblasts form focal contacts (FCs) associated with actin microfilament bundles (MFBs) during attachment and spreading on serum- or fibronectin (FN)-coated substrates. To determine if the minimum cellular adhesion receptor recognition signal Arg-Gly-Asp-Ser (RGDS) is sufficient to promote FC and MFB formation, rat (NRK), hamster (Nil 8), and mouse (Balb/c 3T3) fibroblasts in serum-free media were plated on substrates derivatized with small synthetic peptides containing RGDS. These cultures were studied with interference reflection microscopy to detect FCs, Normarski optics to identify MFBs, and immunofluorescence microscopy to observe endogenous FN fiber formation. By 1 h, 72-78% of the NRK and Nil 8 cells plated on RGDS-containing peptide had focal contacts without accompanying FN fibers, while these fibroblasts lacked FCs on control peptide. This early FC formation was followed by the appearance of coincident MFBs and colinear FN fibers forming fibronexuses at 4 h. NRK and Nil 8 cultures on substrates coated with native FN or 75,000-D FN-cell binding fragment showed similar kinetics of FC and MFB formation. In contrast, the Balb/c 3T3 mouse fibroblasts plated on Gly-Arg-Gly-Asp-Ser peptide-derivatized substrates, or on coverslips coated with 75,000-D FN cell-binding fragment, were defective in FC formation. These results demonstrate that the apparent binding of substrate-linked RGDS sequences to cell surface adhesion receptors is sufficient to promote early focal contact formation followed by the appearance of fibronexuses in some, but not all, fibroblast lines.

Cell surface distribution of fibronectin and vitronectin receptors depends on substrate composition and extracellular matrix accumulation.

We used antibodies against the alpha subunits of the human fibronectin receptor (FNR) and vitronectin receptor (VNR) to localize simultaneously FNR and VNR at major substrate adhesion sites of fibroblasts and melanoma cells with double-label immunofluorescence microscopy. In early (2-6-h) serum-containing cultures, both FNR and VNR coaccumulated in focal contacts detected by interference reflection microscopy. Under higher resolution immunoscanning electron microscopy, FNR and VNR were also observed to be distributed randomly on the dorsal cell surface. As fibronectin-containing extracellular matrix fibers accumulated beneath the cells at 24 h, FNR became concentrated at contacts with these fibers and was no longer detected at focal contacts. VNR was not observed at matrix contacts but remained strikingly localized in focal contacts of the 24-h cells. Since focal contacts represent the sites of strongest cell-to-substrate adhesion, these results suggest that FNR and VNR together play critical roles in the maintenance of stable contacts between the cell and its substrate. In addition, the accumulation of FNR at extracellular matrix contacts implies that this receptor might also function in the process of cellular migration along fibronectin-containing matrix cables. To define the factors governing accumulation of FNR and VNR at focal contacts, fibroblasts in serum-free media were plated on substrates coated with purified ligands. Fibronectin-coated surfaces fostered accumulation of FNR but not VNR at focal contacts. On vitronectin-coated surfaces, or substrata derivatized with a tridecapeptide containing the cell attachment sequence Arg-Gly-Asp, both FNR and VNR became concentrated at focal contacts. These observations suggest that the availability of ligand is critical to the accumulation of FNR and VNR at focal contacts, and that FNR might also recognize substrate-bound vitronectin.

Aldosterone-induced protein in toad urinary bladder.

Simultaneous electrophysiological and biochemical experiments demonstrated a specific aldosterone-induced protein in paired urinary hemibladders isolated from the toad Bufo marinus. Whenever aldosterone stinlmulated short-circuit current, aldosterone specifically increased [(35)S] methionine incorporation into a low-molecular-weight protein (about 12,000). Comparative studies with dexamethasone and insulin and inhibitory studies with spironolactone and actinomycin D suggest mineralocorticoid specificity.

Effects of tetrodotoxin on excitability of squid giant axons in sodium-free media.

The effect of tetrodotoxin on excitability of internally perfused squid giant axons immersed in various sodium-free media was examined. Action potentials were found to be suppressed by this substance, with or without sodium ion in the external medium. Tetrodotoxin showed a strong suppressive effect upon action potentials produced in media containing salts of only divalent cations (CaCl(2), CaBr(2), SrCl(2), or BaCl(2)). Our findings concerning the action of tetrodotoxin do not support the separate-channel hypothesis for excitable membranes.

Agent-based model for friendship in social networks.

A model is proposed to understand the structuring of social networks in a fixed setting such as, for example, inside a university. The friendship formation is based on the frequency of encounters and mutual interest. The model shows distinctive single-scale behavior and reproduces accurately the measurable experimental quantities such as clustering coefficients, degree distribution, degree correlation, and friendship distribution. The model produces self-organized community structures and can be described as a network of densely interconnected networks. For the friendships, we find that the mutual interest is the dominant factor, which optimizes the network and that the number of encounters determines the statistically relevant distributions.

Lithium-induced nephrogenic diabetes insipidus: in vivo and in vitro studies.

The physiological basis for the polyuria and polydipsia occurring in some manic-depressive patients treated with lithium salts was studied in vivo and in vitro. Three lithium-treated polyuric patients, in whom other causes of a concentrating defect were excluded, had abnormal urinary concentrating abilities after a standard water depreviation test. Two of these patients failed to respond to exogenous vasopressin (ADH) and one had a subnormal response. The abilities of these patients to excrete solute-free water (C(H2O)) was comparable to normal subjects during steady-state water diuresis, suggesting no gross abnormalities in sodium transport. However, each of these patients demonstrated abnormally low capacities to reabsorb solute-free water (T(C) (H2O)) under hydropenic conditions after administration of hypertonic saline and vasopressin. These in vivo findings demonstrate at least a nephrogenic basis for the diabetes insipidus syndrome manifested by these three patients. The defect in water transport was further characterized in toad urinary bladders in vitro. Short-circuit current (I) and water flow (W) were studied under basal, ADH-stimulated, and cyclic adenosine 3',5'-monophosphate (c-AMP)-stimulated conditions. Increasing mucosal [Li(+)] progressively inhibited basal I, and both I and W induced by ADH. Significant inhibition of basal and ADH-induced I was observed at mucosal [Li(+)] < 1.1 mEq/liter, and of ADH-induced W at mucosal [Li(+)] = 11 mEq/liter. On the other hand, at these lithium concentrations, neither c-AMP-stimulated W nor I was inhibited. Increasing serosal [Li(+)] produced significant inhibition of basal I only at [Li(+)] at least 50-fold greater than at the mucosal (urinary) surface. These in vitro studies confirm that mucosal lithium inhibits the action of ADH, but not c-AMP. Hence, lithium appears to be a significant inhibitor of ADH-stimulated water flow, probably acts from the urinary surface, and appears to exert its effect at a site biochemically proximal to c-AMP action.

5-Lipoxygenase is located in the euchromatin of the nucleus in resting human alveolar macrophages and translocates to the nuclear envelope upon cell activation.

5-Lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) are two key proteins involved in the synthesis of leukotrienes (LT) from arachidonic acid. Although both alveolar macrophages (AM) and peripheral blood leukocytes (PBL) produce large amounts of LT after activation, 5-LO translocates from a soluble pool to a particulate fraction upon activation of PBL, but is contained in the particulate fraction in AM irrespective of activation. We have therefore examined the subcellular localization of 5-LO in autologous human AM and PBL collected from normal donors. While immunogold electron microscopy demonstrated little 5-LO in resting PBL, resting AM exhibited abundant 5-LO epitopes in the euchromatin region of the nucleus. The presence of substantial quantities of 5-LO in the nucleus of resting AM was verified by cell fractionation and immunoblot analysis and by indirect immunofluorescence microscopy. In both AM and PBL activated by A23187, all of the observable 5-LO immunogold labeling was found associated with the nuclear envelope. In resting cells of both types, FLAP was predominantly associated with the nuclear envelope, and its localization was not affected by activation with A23187. The effects of MK-886, which binds to FLAP, were examined in ionophore-stimulated AM and PBL. Although MK-886 inhibited LT synthesis in both cell types, it failed to prevent the translocation of 5-LO to the nuclear envelope. These results indicate that the nuclear envelope is the site at which 5-LO interacts with FLAP and arachidonic acid to catalyze LT synthesis in activated AM as well as PBL, and that in resting AM the euchromatin region of the nucleus is the predominant source of the translocated enzyme. In addition, LT synthesis is a two-step process consisting of FLAP-independent translocation of 5-LO to the nuclear envelope followed by the FLAP-dependent activation of the enzyme.

Enhanced attenuation of meningeal inflammation and brain edema by concomitant administration of anti-CD18 monoclonal antibodies and dexamethasone in experimental Haemophilus meningitis.

Antiinflammatory therapy has been shown to reduce the adverse pathophysiological consequences that occur in bacterial meningitis and to improve outcome from disease. In the present study, modulation of two principal steps of the meningeal inflammatory cascade was accomplished by concomitant administration of dexamethasone to diminish overproduction of cytokines in response to a bacterial stimulus and of a monoclonal antibody directed against adhesion-promoting receptors on leukocytes to inhibit recruitment of white blood cells into the subarachnoid space. Dexamethasone and antibody therapy produced a marked attenuation of all indices of meningeal inflammation and reduction of brain water accumulation after H. influenzae-induced meningitis in rabbits compared with results of each agent given alone and of untreated animals. In addition, the enhanced host's meningeal inflammatory reaction that follows antibiotic-induced bacterial lysis was profoundly ameliorated when dual therapy was administered without affecting clearance rates of bacteria from cerebrospinal fluid and vascular compartments. The combination of both therapeutic approaches may offer a promising mode of treatment to improve further the outcome from bacterial meningitis.

Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints.

To examine the activity of matrix metalloproteinases (MMPs) and aggrecanase in control and diseased human articular cartilage, metabolic fragments of aggrecan were detected with monospecific antipeptide antibodies. The distribution and quantity of MMP-generated aggrecan G1 fragments terminating in VDIPEN341 were compared with the distribution of aggrecanase-generated G1 fragments terminating in NITEGE373. Both types of G1 fragments were isolated from osteoarthritic cartilage. The sizes were consistent with a single enzymatic cleavage in the interglobular domain region, with no further proteolytic processing of these fragments. Both neoepitopes were also detected by immunohistochemistry in articular cartilage from patients undergoing joint replacement for osteoarthritis (OA), rheumatoid arthritis (RA), and in cartilage from adults with no known joint disease. In control specimens, the staining intensity for both G1 fragments increased with age, with little staining in cartilage from 22-wk-old fetal samples. There was also an increase with age in the extracted amount of MMP-generated neoepitope in relation to both aggrecan and collagen content, confirming the immunohistochemical results. After the age of 20-30 yr this relationship remained at a steady state. The staining for the MMP-generated epitope was most marked in control cartilage exhibiting histological signs of damage, whereas intense staining for the aggrecanase-generated fragment was often noted in adult cartilage lacking overt histological damage. Intense staining for both neoepitopes appeared in the more severely fibrillated, superficial region of the tissue. Intense immunostaining for both VDIPEN- and NITEGE- neoepitopes was also detected in joint cartilage from patients with OA or RA. Cartilage in these specimens was significantly more degraded and high levels of staining for both epitopes was always seen in areas with extensive cartilage damage. The levels of extracted VDIPEN neoepitope relative to collagen or aggrecan in both OA and RA samples were similar to those seen in age-matched control specimens. Immunostaining for both types of aggrecan fragments was seen surrounding the cells but also further removed in the interterritorial matrix. In some regions of the tissue, both neoepitopes were found while in others only one was detected. Thus, generation and/or turnover of these specific catabolic aggrecan fragments is not necessarily coordinated. Our results are consistent with the presence in both normal and arthritic joint cartilage of proteolytic activity against aggrecan based on both classical MMPs and "aggrecanase."

VDIPEN, a metalloproteinase-generated neoepitope, is induced and immunolocalized in articular cartilage during inflammatory arthritis.

The destruction of articular cartilage in immune inflammatory arthritic disease involves the proteolytic degradation of its extracellular matrix. The role of activated matrix metalloproteinases (MMPs) in the chondrodestructive process was studied by identifying a selective cleavage product of aggrecan in murine arthritis models initiated by immunization with either type II collagen or proteoglycan. We conducted semiquantitative immunocytochemical studies of VDIPEN341 using a monospecific polyclonal antibody requiring the free COOH group of the COOH-terminal Asn for epitope detection. This antibody recognizes the aggrecan G1 domain fragment generated by MMP [i.e., stromelysin (SLN) or gelatinase A] cleavage of aggrecan between Asn341-Phe342 but does not recognize intact aggrecan. VDIPEN was undetectable in normal mouse cartilage but was observed in the articular cartilage (AC) of mice with collagen-induced arthritis 10 d after immunization, without histological damage and clinical symptoms. This aggrecan neoepitope was colocalized with high levels of glycosaminoglycans (GAGs) in pericellular matrices of AC chondrocytes but was not seen at the articular surface at this early time. Digestion of normal (VDIPEN negative) mouse paw cryosections with SLN also produced heavy pericellular VDIPEN labeling. Computer-based image analysis showed that the amount of VDIPEN expression increased dramatically by 20 d (70% of the SLN maximum) and was correlated with GAG depletion. Both infiltration of inflammatory cells into the synovial cavity and early AC erosion were also very prominent at this time. Analysis of adjacent sections showed that both induction of VDIPEN and GAG depletion were strikingly codistributed within sites of articular cartilage damage. Similar results occurred in proteoglycan-induced arthritis, a more progressive and chronic model of inflammatory arthritis. These studies demonstrate for the first time the MMP-dependent catabolism of aggrecan at sites of chondrodestruction during inflammatory arthritis.

Analysis and visualization of multiply oriented lattice structures by a two-dimensional continuous wavelet transform.

The phase-field-crystal model [K. R. Elder and M. Grant, Phys. Rev. E 70, 051605 (2004)] produces multigrain structures on atomistic length scale but on diffusive time scales. Since individual atoms are resolved but are treated identically it is difficult to distinguish the exact position of grain boundaries and defects within grains. In order to analyze and visualize the whole grains a two-dimensional wavelet transform has been developed, which is capable of extracting grain boundaries and the lattice orientation of a grain relative to a laboratory frame of reference. This transformation makes it possible not only to easily visualize the multigrain structure, but also to perform exact measurements on low- and high-angle boundaries, grain size distributions and boundary-angle distributions, which can then be compared to experimental data. The presented wavelet transform can also be applied to results of other atomistic simulations, e.g., molecular dynamics or granular materials.

B-vitamin status and concentrations of homocysteine in Austrian omnivores, vegetarians and vegans.

BACKGROUND: A vegetarian diet is considered to promote health and longevity and reduce the risk of cardiovascular diseases and cancer. However, a vegetarian diet may be deficient in some nutrients. Exclusion of animal products in vegetarian diets may affect the status of certain B-vitamins, and further cause the rise of plasma homocysteine concentration. OBJECTIVE: The nutritional status of various B-vitamins (B(1), B(2), B(6), B(12), folic acid) and the concentration of homocysteine in blood plasma of omnivores (n = 40), vegetarians (n = 36) and vegans (n = 42) in Austria was evaluated. METHODS: The evaluation was done using the functional parameters erythrocyte transketolase (ETK), glutathione reductase (EGR) and glutamic oxaloacetic transaminase (EGOT) activation coefficients. Enzyme activity was measured photometrically. The quantity of vitamins B(1), B(2) and B(6) in urine and the concentrations of vitamin B(6) and homocysteine in plasma were determined by HPLC methods with fluorescence detection. Plasma concentration of vitamin B(12) and folic acid were measured with radioimmunoassay. RESULTS: Most of the subjects showed a satisfying vitamin B(1) status. Vegans presented a significantly lower mean plasma vitamin B(12) concentration than omnivores and vegetarians and deficiency in 2.4% of the volunteers but the highest mean value of plasma folate among the investigated groups. A deficient status of folate was found in 18% of omnivores and in approximately 10% of vegans and vegetarians. The status of riboflavin is considered to be deficient in about 10% of omnivores and vegetarians and in over 30% of vegans. According to the activation coefficient of GOT, approximately one third of all subjects showed vitamin B(6) deficiency. Elevated homocysteine concentration in plasma was observed in 66% of the vegans and about 45-50% of the omnivores and vegetarians. Vegan subjects had significantly higher mean plasma homocysteine levels than omnivores. CONCLUSION: Thiamin and folate need not be a problem in a well-planned vegan diet. Vitamins B(12) and B(2) may need attention in the strict vegan diet, especially regarding elevated homocysteine levels in plasma. Pyridoxine status appeared to be independent of the diet.

Subcellular localization of aldosterone-induced proteins in toad urinary bladders.

Paired toad urinary hemibladders were incubated with [35S]methionine in the presence (experimental) or absence (control) of aldosterone. Short-circuit current was used to monitor aldosterone-induced Na+ transport. Protein synthesis in epithelial cell subcellular fractions (cytosolic, microsomal, mitochondrial) was evaluated by gradient polyacrylamide gel electrophoresis and autoradiography. Aldosterone-induced proteins were identified in the cytosolic and microsomal fractions (70 000 and 15 000 daltons, respectively). These results represent the first demonstration of aldosterone-induced proteins in subcellular fractions of epithelial cells derived from single toad urinary hemibladders.

Effects of tetracyclines on aldosterone- and insulin-mediated Na+ transport in the toad urinary bladder.

The effect of oxytetracycline and demethylchlortetracycline on aldosterone- and insulin-mediated Na+ transport (short-circuit current) were examined in toad urinary bladders mounted in modified Ussing chambers. Oxytetracycline had little or no effect on either basal or aldosterone-mediated Na+ transport. In contrast, demethylchlortetracycline markedly inhibited both basal and aldosterone-mediated Na+ transport. Furthermore, demethylchlortetracycline inhibited the aldosterone response significantly out of proportion to its effects on basal Na+ transport. Neither of the drugs had an effect on insulin-mediated Na+ transport. Consequently, the natriuresis observed in certain patients treated with demethylchlortetracyline may be related to drug-induced renal resistance to the effects of aldosterone.