The influence of contemporary and historic landscape features on the genetic structure of the sand dune endemic, Cirsium pitcheri (Asteraceae).

Narrow endemics are at risk from climate change because of their restricted habitat preferences, lower colonization ability and dispersal distances. Landscape genetics combines new tools and analyses that allow us to test how both past and present landscape features have facilitated or hindered previous range expansion and local migration patterns, and thereby identifying potential limitations to future range shifts. We have compared current and historic habitat corridors in Cirsium pitcheri, an endemic of the linear dune ecosystem of the Great Lakes, to determine the relative contributions of contemporary migration and post-glacial range expansion on genetic structure. We used seven microsatellite loci to characterize the genetic structure for 24 populations of Cirsium pitcheri, spanning the center to periphery of the range. We tested genetic distance against different measures of geographic distance and landscape permeability, based on contemporary and historic landscape features. We found moderate genetic structure (Fst=0.14), and a north-south pattern to the distribution of genetic diversity and inbreeding, with northern populations having the highest diversity and lowest levels of inbreeding. High allelic diversity, small average pairwise distances and mixed genetic clusters identified in Structure suggest that populations in the center of the range represent the point of entry to the Lake Michigan and a refugium of diversity for this species. A strong association between genetic distances and lake-level changes suggests that historic lake fluctuations best explain the broad geographic patterns, and sandy habitat best explains local patterns of movement.

Plasminogen kringle 5 reduces vascular leakage in the retina in rat models of oxygen-induced retinopathy and diabetes.

AIMS/HYPOTHESIS: Retinal vascular leakage is an early pathological feature in diabetic retinopathy and can lead to macular oedema and loss of vision. Previously we have shown that plasminogen kringle 5 (K5), an angiogenic inhibitor, inhibits retinal neovascularisation in the rat model of oxygen-induced retinopathy (OIR). The purpose of this study was to examine the effect of K5 on vascular leakage in the retina. METHODS: Neonatal rats were exposed to hyperoxia to induce OIR. Diabetes was induced in adult rats by injecting streptozotocin. Vascular permeability was measured by Evans blue method. Expression of vascular endothelial growth factor (VEGF) was evaluated using immunohistochemistry and western blot analysis. RESULTS: Rats with OIR and diabetes showed abnormal vascular hyperpermeability in the retina and iris. Intravitreal injection of K5, reduced vascular permeability in both animal models, but did not affect permeability in normal rats. K5 reduced vascular permeability at doses substantially lower than that required for inhibition of retinal neovascularisation. The K5-induced reduction in vascular permeability correlated with its down-regulation of VEGF expression in the retina. Moreover, K5 inhibited IGF-1-induced hyperpermeability, which is known to arise through up-regulation of endogenous VEGF expression. However, K5 had no effect on the hyperpermeability induced by injection of exogenous VEGF. CONCLUSIONS/INTERPRETATION: Very low doses of K5 reduce pathological vascular leakage in the retina. K5 thus has therapeutic potential in the treatment of diabetic macular oedema. This effect can be ascribed, at least in part, to the down-regulation of endogenous VEGF expression.

Kallikrein-binding protein inhibits retinal neovascularization and decreases vascular leakage.

AIMS/HYPOTHESIS: Kallikrein-binding protein (KBP) is a serine proteinase inhibitor (serpin). It specifically binds to tissue kallikrein and inhibits kallikrein activity. Our study was designed to test its effects on retinal neovascularization and vascular permeability. METHODS: Endothelial cell proliferation was determined by [(3)H] thymidine incorporation assay and apoptosis quantified by Annexin V staining and flow cytometry. Effect on retinal neovascularization was determined by fluorescein angiography and count of pre-retinal vascular cells in an oxygen-induced retinopathy (OIR) model. Vascular permeability was assayed by the Evans blue method. Vascular endothelial growth factor (VEGF) was measured by Western blot analysis and ELISA. RESULTS: Kallikrein-binding protein specifically inhibited proliferation and induced apoptosis in retinal capillary endothelial cells. Intravitreal injection of KBP inhibited retinal neovascularization in an OIR model. Moreover, KBP decreased vascular leakage in the retina, iris and choroid in rats with OIR. Blockade of kinin receptors by specific antagonists showed significantly weaker inhibition of endothelial cells, when compared to that of KBP, suggesting that the anti-angiogenic activity of KBP is not through inhibiting kallikrein activity or kinin production. KBP competed with (125)I-VEGF for binding to endothelial cells and down-regulated VEGF production in endothelial cells and in the retina of the OIR rat model. CONCLUSION/INTERPRETATION: Kallikrein-binding protein is a multi-functional serpin, and its vascular activities are independent of its interactions with the kallikrein-kinin system. Inhibition of VEGF binding to its receptors and down-regulation of VEGF expression could represent a mechanism for the vascular activities of KBP.

The use of glucagon in nifedipine poisoning complicated by clonidine ingestion.

Glucagon has been used to treat the hypotension associated with calcium channel antagonist poisoning in adult patients. We describe the successful use of glucagon in a pediatric patient poisoned with nifedipine and clonidine whose hypotension was unresponsive to fluid resuscitation, calcium chloride, and dopamine.

Observation of unique cross-linked lattices between multiantennary carbohydrates and soybean lectin. Presence of pseudo-2-fold axes of symmetry in complex type carbohydrates.

The tetrameric lectin from Glycine max (soybean) (SBA) has been shown to cross-link and precipitate with N-linked multiantennary complex type oligosaccharides containing nonreducing terminal Gal residues (Bhattacharyya, L., Haraldsson, M., & Brewer, C. F. (1988) Biochemistry 27, 1034-1041). In the present study, negative stain electron micrographs of the precipitates of SBA with a series of naturally occurring and synthetic multiantennary carbohydrates with terminal Gal or GalNAc residues show the presence of highly ordered cross-linked lattices for many of the complexes. The precipitates of SBA with a "bisected" and "nonbisected" N-linked biantennary complex type oligosaccharide containing Gal residues at the nonreducing termini show similar two-dimensional patterns. However, the pattern observed for the precipitates of a tetraantennary complex type oligosaccharide with SBA is distinct from those of the two biantennary carbohydrates. Furthermore, the precipitates formed between the lectin and a synthetic O-linked biantennary ("cluster") glycoside with terminal GalNAc residues show a pattern that is different from those above. Four biantennary pentasaccharide analogs of the blood group I antigen containing beta-LacNAc moieties at the 2,3-, 2,4-, 2,6-, and 3,6-positions of the core Gal also showed ordered patterns in their precipitates with SBA. X-ray crystallographic data and mixed quantitative precipitation profiles of binary mixtures of the four analogs demonstrate that each analog possesses a unique cross-linked lattice with the protein. A common structural feature of the naturally occurring and synthetic carbohydrates that show highly organized cross-linked lattices with SBA is the presence of a pseudo-2-fold axis of symmetry in each oligosaccharide relating the terminal binding epitopes on each arm. This suggests that the symmetry features of certain naturally occurring branch chain oligosaccharides facilitate formation of highly ordered, homogeneous cross-linked complexes with specific lectins.

Observation of unique cross-linked lattices between multiantennary carbohydrates and soybean lectin. Presence of pseudo-2-fold axes of symmetry in complex type carbohydrates.

The tetrameric lectin from Glycine max (soybean) (SBA) has been shown to cross-link and precipitate with N-linked multiantennary complex type oligosaccharides containing nonreducing terminal Gal residues (Bhattacharyya, L., Haraldsson, M., & Brewer, C. F. (1988) Biochemistry 27, 1034-1041). In the present study, negative stain electron micrographs of the precipitates of SBA with a series of naturally occurring and synthetic multiantennary carbohydrates with terminal Gal or GalNAc residues show the presence of highly ordered cross-linked lattices for many of the complexes. The precipitates of SBA with a "bisected" and "nonbisected" N-linked biantennary complex type oligosaccharide containing Gal residues at the nonreducing termini show similar two-dimensional patterns. However, the pattern observed for the precipitates of a tetraantennary complex type oligosaccharide with SBA is distinct from those of the two biantennary carbohydrates. Furthermore, the precipitates formed between the lectin and a synthetic O-linked biantennary ("cluster") glycoside with terminal GalNAc residues show a pattern that is different from those above. Four biantennary pentasaccharide analogs of the blood group I antigen containing beta-LacNAc moieties at the 2.3-, 2.4-, 2.6-, and 3.6-positions of the core Gal also showed ordered patterns in their precipitates with SBA. X-ray crystallographic data and mixed quantitative precipitation profiles of binary mixtures of the four analogs demonstrate that each analog possesses a unique cross-linked lattice with the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Brefeldin A inhibits degradation as well as production and secretion of collagen in human lung fibroblasts.

Brefeldin A (BFA) inhibits protein secretion, collapses the Golgi complex into the endoplasmic reticulum (ER), causes redistribution of processing enzymes normally resident in the Golgi to the ER, and uncouples the proximal and distal regions of the secretory pathway. We used BFA to determine where intracellular degradation of newly synthesized collagen degradation occurs. In normal human fetal lung fibroblasts, BFA (50 ng/ml) completely blocked collagen secretion and reduced collagen production by two-thirds. In cells synthesizing collagen under normal conditions, intracellular degradation was about 16%; BFA (50 ng/ml) reduced degradation to less than 5%. In cells induced to synthesize structurally abnormal collagen (by incubation with the proline analog cis-hydroxyproline), degradation was approximately 33%; BFA reduced this level to less than 10%. When the y axes were scaled appropriately, the dose-response curves for collagen degradation +/- cis-hydroxyproline versus BFA concentration coincided. A pulse-chase experiment demonstrated that BFA did not inhibit hydroxylation of prolyl residues, a major posttranslational modification of collagen that occurs in the ER, and that inhibition of degradation was independent of inhibition of collagen synthesis. Immunofluorescence examination revealed that BFA redistributed Golgi glycoproteins to the ER. At the ultrastructural level, Golgi complex could not be found in fibroblasts exposed to BFA for 1 h; however, clusters of small vesicles were observed. A different structure, comprising one or two lamellae and resembling a partial Golgi complex, was observed in cells incubated with BFA for 6 h. This structure was adjacent to ER but far from the nucleus. In addition, the ER was devoid of ribosomes. The inhibition of intracellular collagen degradation by BFA indicates that collagen degradation does not occur in the ER. Rather, it suggests that collagen degradation occurs beyond the BFA block, perhaps in the trans-Golgi network.

Properties of heart fibroblasts of adult rats in culture.

In the heart of the adult rat, fibroblasts are mainly responsible for the synthesis and deposition of the collagenous matrix. Because these cells in vitro may serve as an important model system for studies of collagen metabolism in heart tissue, we have cultured and characterized rat-heart fibroblasts from young adult and old animals. Conditions included use of media of different compositions with and without addition of ascorbate. Cells used were either cultured directly from fresh tissues or thawed previously frozen cells. Cultured cells were studied with respect to growth properties, morphology and ultrastructure and patterns of collagen. Heart fibroblasts generally resembled fibroblasts cultured from other tissues, but were more like skeletal muscle fibroblasts in that they deposited, in addition to type I collagen, type IV collagen and laminin. The fibroblasts showed a typical appearance in phase-contrast microscopy and electron microscopy. In the case of cells grown with added ascorbate, aligned collagen fibrils in the extracellular matrix showed a periodicity typical of type I collagen. The deposition of type I collagen occurred only in medium supplemented with ascorbate, and in that circumstance increased as a function of time past confluence; this was independent of the age of the animal from which the cells were obtained or of other changes of medium composition studied. Immunofluorescence studies with specific antibodies revealed that the cells deposited types I and IV collagens, laminin and fibronectin. In contrast to the case of type I collagen, the deposition of type IV collagen occurred in cells grown either with or without ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding and precipitating activities of Lotus tetragonolobus isolectins with L-fucosyl oligosaccharides. Formation of unique homogeneous cross-linked lattices observed by electron microscopy.

We have recently observed that certain asparagine-linked oligosaccharides are multivalent and capable of binding and precipitating with the D-mannose-specific lectin concanavalin A [cf. Bhattacharyya, L., & Brewer, C. F. (1989) Eur. J. Biochem. 178, 721-726] and with a variety of D-galactose-specific lectins [Bhattacharyya, L., Haraldsson, M., & Brewer, C. F. (1988) Biochemistry 27, 1034-1041]. In the present study, we have examined the binding and precipitating activities of a variety of mono- and biantennary L-fucosyl oligosaccharides with three L-fucose-specific isolectins from Lotus tetragonolobus, LTL-A, LTL-B, and LTL-C. The results show that certain difucosyl biantennary oligosaccharides are capable of cross-linking and precipitating with tetrameric isolectins, LTL-A and LTL-C, but not with dimeric isolectin, LTL-B. Quantitative precipitation analyses show that biantennary oligosaccharides containing the Lewis(x) antigen (or type 2 chain of Lewis(a)), Gal beta (1-4)[Fuc alpha (1-3)]GlcNAc, at the nonreducing terminus of each arm are bivalent ligands. However, a biantennary oligosaccharide containing a Lewis(x) determinant on one arm and a type 2 chain of blood group H(O) determinant, Fuc alpha (1-2)Gal beta (1-4)GlcNAc, on the other arm and a monoantennary oligosaccharide containing two fucose residues (analogue of the Lewis(y) antigen) bind but do not precipitate with the isolectins, indicating that the positions and linkage of fucose residues are critical for cross-linking.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of highly ordered cross-linked lattices between asparagine-linked oligosaccharides and lectins observed by electron microscopy.

The interaction of asparagine-linked carbohydrates (N-linked) with carbohydrate binding proteins called lectins has been demonstrated to be involved in a variety of cellular recognition processes. Certain N-linked carbohydrates have been shown to be multivalent and capable of binding, cross-linking, and precipitating lectins (Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., and Brewer, C. F. (1987) J. Biol. Chem. 262, 1288-1293; Bhattacharyya, L., Haraldsson, M., and Brewer, C. F. (1987) J. Biol. Chem. 262, 1294-1299; Bhattacharyya, L., Haraldsson, M., and Brewer, C. F. (1988) Biochemistry 27, 1034-1041). Recent data have further suggested that certain oligomannose and bisected hybrid-type N-linked glycopeptides form homogeneous cross-linked lattices with concanavalin A (Bhattacharyya, L., Khan, M. I., and Brewer, C. F. (1988) Biochemistry 27, 8762-8767). In the present study, evidence has been obtained from electron microscopy for the formation of highly ordered and distinct lattices for two bivalent complex type oligosaccharides cross-linked with soybean lectin (Glycine max) and isolectin A from Lotus tetragonolobus, respectively. The results indicate a new source of specificity for interactions of N-linked carbohydrates with lectins, namely their ability to form highly ordered homogeneous aggregates.

Characterization of a novel 66 kd subunit of mammalian neurofilaments.

A 66 kd protein, pl 5.4, was purified from the Triton-insoluble fraction of rat spinal cord. This protein formed 10 nm filaments in vitro. The 66 kd protein was unique, although it shared homology with the 70 kd neurofilament protein (NF-L) and vimentin. An antiserum (anti-66) specific to the 66 kd protein did not cross-react with any of the neurofilament triplet proteins. In the spinal cord, anti-66 intensely stained the axons of the anterior and lateral columns. However, afferents from dorsal root ganglia and the efferents from the motoneurons were negative. In the cerebellum, anti-66 intensely stained most axons. The 66 kd protein was readily detectable in homogenates of forebrain, cerebellum, brainstem, and spinal cord, but was found only in trace amounts in adult sciatic nerves and was not found in extraneural tissues. The 66 kd protein constituted 0.5% of total protein in the spinal cord, whereas NF-L constituted about 1.5%.

Staphylococcus aureus--human endothelial cell interactions.

Staphylococcus aureus is an invasive pathogen capable of causing life-threatening disease. A major component of this pathogen's virulence is its ability to invade normal endovascular tissue. We examined the interaction of S. aureus with cultured human endothelial cells and with human and rabbit endovascular tissue. Our ultrastructural study demonstrated a sequence of steps which occurred with staphylococcal invasion of human endothelial cells; adhesion, endocytosis, and intracellular replication. Ultimately, this resulted in cell disruption and death. Cytochemical staining of lysosomes demonstrated lysosomal fusion with both viable and killed intracellular bacteria without evidence of staphylococcal degradation. Quantitative studies using an in vitro infection assay demonstrated comparable rates of adhesion by viable and ultraviolet-killed bacteria, phagocytosis at a slower rate, and intracellular replication. The present study demonstrates an active role for the endothelial cell in the development and spread of endovascular staphylococcal infections. It also supports the use of this in vitro tissue culture system as a model for the study of bacterial invasion of the endothelium.

Taxol induces the formation of unusual arrays of cellular microtubules in colchicine-pretreated J774.2 cells.

Taxol is an antimitotic agent with the unique ability to induce the formation of parallel arrays of microtubules in cells. We have studied the effects of taxol on microtubule organization in the cultured macrophage-like cell line, J774.2, and shown that this novel reorganization of cellular microtubules is both a concentration-dependent and time-dependent phenomenon. In this paper, we have examined in detail the unusual microtubule arrays induced by taxol in colchicine-pretreated cells. Interphase cells which are pretreated with the irreversible inhibitor, colchicine, and then treated with taxol form a single microtubule aster associated with the nucleus and numerous discrete sites of apparent microtubule nucleation scattered throughout the cytoplasm. One interesting possibility is that these structures represent nucleation sites for taxol-induced bundles, a result supporting the notion that taxol-induced microtubule arrays are organized assemblies at what are perhaps secondary organizing sites.

A scanning electron microscopic study of the cytoplasmic surface of the toad bladder luminal membrane.

Antidiuretic hormone (ADH) increases water flow across receptor cells in the kidney and amphibian bladder by stimulating the insertion of particles into the luminal (urinary) cell membrane. The particles originate from tubular structures in the cytoplasm which fuse with the luminal membrane. Many of the steps involved in fusion and particle insertion are still unknown. We have been able to separate the luminal cell membrane of ADH-treated toad bladder from the rest of the cell and attach the membranes to glass coverslips with polylysine, with their cytoplasmic surfaces facing up. Inspection of the membranes by scanning electron microscopy reveals subluminal granules and what appear to be fusing tubules. The present communication describes our technique for membrane preparation and adhesion, as well as our initial observations of membrane-associated organelles.

The characterization of human uterine smooth muscle cells in culture.

Primary cultures initiated from normal human uterine endometrium after total enzymatic dissociation contained epithelioid cells and smooth muscle cells. The smooth muscle cells were subsequently isolated by differential trypsinization and grown in culture for 36 +/- 4 generations. Ultrastructural examination of log and post-confluent cultures of cells at low and high population doubling levels revealed characteristics similar to those of published reports on other smooth muscle cells studied in vivo and in vitro. Among the common features present were: (a) abundant bundles of 60--70 A myofilaments; (b) branched mitochondria; (c) stacks of cisternae of rough endoplasmic reticulum; (d) caveolae intracellulares; (e) nexuses. Other features included ovoid nuclei, a well developed Golgi apparatus and abundant free ribosomes. The subcultured cells exhibited features of dedifferentiation in the log phase of growth and at post-confluency. However, the post-confluent cells showed characteristics indicating redifferentiation back towards their in vivo morphology. Smooth muscle cells isolated from endometrial curettings may provide a useful model for biochemical and pharmacological studies of a cell type derived from a hormonal target tissue as the cells "age" in culture.

Intracellular iron-binding macromolecules in HeLa cells.

The concentration of the iron-chelating agent, desferrioxamine (Desferal), that just inhibits iron entry into HeLa cells is also the concentration that inhibits DNA synthesis. As a first step in clarification of the mechanism whereby iron may partake in DNA synthesis, we have partially characterized several of the intracellular iron-binding sites. Most cytoplasmic iron appears to be bound to a polysaccharide containing glucose that sediments at about 32 S. Nucleolar iron is bound to a single protein, the mobility of which is independent of the concentration of sodium dodecyl sulfate in an acrylamide gel. In contrast the pattern and mobility of nuclear iron, other than nucleolar, is heterogeneous and markedly affected by the concentration of sodium dodecyl sulfate. The evidence suggests that nuclear iron is bound to protein through one or more intermediate(s).