Collagen receptors mediate early events in the attachment of epithelial (MDCK) cells

Summary Madin-Darby canine kidney (MDCK) cells kept in suspension culture for 12–15 hr displayed high-affinity binding sites for¹²⁵I-lathyritic (soluble) collagen (120,000/cell,K _D =30nm) and preferred collagens types I and IV over laminin or fibronectin as substrates during the first hour of attachment. On the other hand, after 4 hr, attachment to all four substrates was equally efficient. Upon challenge with a collagen substrate, the high-affinity sites were rapidly recruited on it (T1/2=6 min). Their occupancy by soluble collagen triggered the exocytosis of a second large population of low-affinity collagen binding sites that included laminin and seems to be involved in a second cell-attachment mechanism. These results are compatible with a twostep model of MDCK cell attachment to the substrate: first, via high-affinity collagen binding sites, and second, via laminin of cellular origin.     

Prophylactic and curative effects of Bacopa monniera in gastric ulcer models.

Bacopa monniera Wettst. (BM, syn. Herpestis monniera L; Scrophulariaceae), is an Ayurvedic drug used as a rasayana. Its fresh juice was earlier reported to have significant antiulcerogenic activity. In continuation, methanolic extract of BM (BME) standardized to bacoside-A content (percentage-38.0 ± 0.9), when given in the dose of 10–50 mg/kg, twice daily for 5 days, showed dose-dependent anti-ulcerogenic on various gastric ulcer models induced by ethanol, aspirin, 2 h cold restraint stress and 4 h pylorus ligation. BME in the dose of 20 mg/kg, given for 10 days, twice daily showed healing effects against 50% acetic acid-induced gastric ulcers. Further work was done to investigate the possible mechanisms of its action by studying its effect on various mucosal offensive acid-pepsin secretion and defensive factors like mucin secretion, mucosal cell shedding, cell proliferation and antioxidant activity in rats. BME 20 mg/kg showed no effect on acid-pepsin secretion, increased mucin secretion, while it decreased cell shedding with no effect on cell proliferation. BME showed significant antioxidant effect per se and in stressed animals. Thus, the gastric prophylactic and curative effects of BME may be due to its predominant effect on mucosal defensive factors.     

Carboxylierungsreaktionen in Agaricus bisporus III. Pyruvat und Phosphoenolpyruvat als CO2-Acceptoren

Zusammenfassung Zellfreie Extrakte aus Agaricus bisporus bilden Malat, Fumarat und Aspartat einerseits aus Pyruvat und CO₂ in Gegenwart von Mn²⁺ und andererseits aus Phosphoenolpyruvat und CO₂ in Gegenwart von Mg²⁺.Die Carboxylierung von Pyruvat wird durch ATP und NADPH₂ deutlich gefördert, ist aber unabhängig von der Anwesenheit von CoA-Estern. Die Reaktion erfährt durch pCMB, Oxalat und Avidin eine Hemmung.Die Carboxylierung von Phosphoenolpyruvat wird durch ADP, nicht aber durch GDP und IDP gefördert.Aus den Ergebnissen wird geschlossen, daß bei der Carboxylierung von Pyruvat sowohl Pyruvatcarboxylase als auch Malatenzym wirksam sind, während für die Oxalacetatsynthese aus Phosphoenolpyruvat PEP-Carboxykinase verantwortlich ist.Die Bedeutung der drei Enzyme im Zusammenhang mit der Ernährung des Kulturchampignons aus dem natürlichen Substrat, mit der Glucogenese und der Steuerung des Citronensäurecyclus wird diskutiert.

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Carboxylation reactions in Agaricus bisporus III. Pyruvate and phosphoenolpyruvate as CO₂-acceptors

Summary Cell-free extracts from Agaricus bisporus catalyze the synthesis of malate, fumarate and aspartate from pyruvate and CO₂ in the presence of Mn²⁺, and from phosphoenolpyruvate and CO₂ with Mg²⁺ (partially replaceable by Mn²⁺).The carboxylation of pyruvate is highly stimulated by ATP and NADPH₂, but is not affected by CoA-esters. The reaction is inhibited by pCMB, oxalate and avidin.The carboxylation of phosphoenolpyruvate is stimulated by ADP, but not by IDP and GDP.From cofactor-requirement and inhibitor studies it is concluded, that there are two enzymes, pyruvatecarboxylase and malic enzyme, which catalyze the carboxylation of pyruvate. Phosphoenolpyruvate carboxykinase is responsible for the CO₂-fixation into oxaloacetate.The significance of these three enzymes is discussed in connection with the nutrition of the fungus from its natural growth substrate and with the regulation of glycogenesis and the citric acid cycle.

     

Hyperosid,das Hauptflavonolglykosid von Davidia involucrata Baill.

Summary Hyperosid (quercetin-3-galactoside) was isolated from the bracts of the dove tree and identified by paper chromatography, melting point determinations, UV- and IR-spectroscopy. The yield of crystalline hyperosid was approximately 1% of the dry weight. It is the main glycoside of the bracts and also appears in the green leaves, but is absent in the bark, wood and fruit. Quercitrin (quercetin-3-rhamnoside) is also present, in minor amount. No other flavonol glycoside could be detected. The analytical results are discussed in relation to their possible taxonomic and physiological implications.     

Expression of a maize proteinase inhibitor gene is induced in response to wounding and fungal infection: systemic wound-response of a monocot gene

The isolation and characterization of cDNA and genomic clones encoding a proteinase inhibitor protein (MPI) in maize is reported. Accumulation of the MPI mRNA is induced in response to fungal infection in germinating maize embryos. The expression pattern of the MPI gene, in healthy and fungal infected maize tissues, was examined and compared with the expression pattern of a gene that codes for a pathogenesis-related protein (the PRms protein) from maize. These two genes are induced by fungal infection, however different signals trigger their activation. Accumulation of the proteinase inhibitor mRNA is more a consequence of the wound produced by the penetration and colonization of the host tissues by the pathogen, than the result of a direct molecular recognition of the pathogen by the plant, as is the case for the induction of the PRms gene. Wounding, or treatment with abscisic acid or methyl jasmonate, stimulate MPI mRNA accumulation, but not PRms mRNA accumulation. Local and systemic induction of the MPI gene expression in response to wounding occurs in maize plants. To the authors' knowledge, this is the first example of a gene from a monocotyledonous species that clearly shows a systemic wound response. The possible functional implications for the existence of different signal transduction pathways that simultaneously activate a battery of defense mechanisms against potential pathogens are discussed.     

Spatial and temporal coding in single neurons

Convergence between cells which differ in both spatial and temporal properties create higher order neurons with response properties that are distinctly different from those of the input neurons. The spatial properties of target neurons are not necessarily cosinetuned. In addition, unlike the independence between spatial and temporal properties in cosine-tuned afferent neurons, higher-order target cells generally exhibit a dependence of temporal dynamics on spatial properties. The response properties of target neurons receiving spatio-temporal convergence (STC) from tonic and phasic-tonic or phasic afferents is investigated here by considering a general case where the dynamic input is represented by a fractional, leaky, derivative transfer function. It is shown that, at frequencies below the corner frequency of the dynamic input, the temporal properties of target neurons can be described by leaky differentiators having time constants that are a function of spatial direction. Thus, STC target neurons exhibit tonic temporal response properties during stimulation along some spatial directions (having small time constants) and phasic properties along other directions (having large time constants). Specifically, target neurons encode the complete derivative of the stimulus along certain spatial directions. Thus, STC acts as a directionally specific high-pass filter and produces complete derivatives from fractional, leaky derivative afferent signals. In addition, spatio-temporal transformations can generate novel temporal dynamics in the central nervous system. These observations suggest that spatio-temporal computations might constitute an alternative to parallel, independent spatial and temporal channels.     

Plant regeneration from stem and petiole-derived callus ofHumulus lupulus L. (hop) clone Bragança and var. Brewer’s Gold

Summary Plant regeneration was achieved from both a spontaneous clone (Bragan?a) and Brewer's Gold variety ofHumulus lupulus. The results obtained for these two different genotypes were compared. The organogenic ability of petiole and stem segments was tested on three different basal media supplemented with 0.025 mg (0.14 μM) indole-3-acetic acid/L and 2 mg (8.87 μM) 6-benzylaminopurine (N⁶-benzyladenine)/L. These conditions induced rather heterogeneous responses, which depended mainly on the explant source and the genotype. Because of the high organogenic competence revealed by the spontaneous clone on modified Murashige and Skoog medium, several hormones in different combinations were tested to optimize conditions for adventitious shoot regeneration in this clone. The best relation between the average shoot number/callus and the regeneration rate was achieved with 0.025 mg (0.14 μM) indole-3-acetic acid/L and 2 mg (8.87 μM) 6-benzylaminopurine/L or with 0.02 mg (0.11 μM) indole-3-acetic acid/L and 1.5 mg (6.97 μM) kinetin/L, which enabled 72 and 59% of regeneration, respectively. The regenerated plantlets could be acclimatized with 90% success.     

The Apical Submembrane Cytoskeleton Participates in the Organization of the Apical Pole in Epithelial Cells

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145–3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40–70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical–basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37°C or in n-octyl-β-d-glycoside at 4°C (representative of GPIanchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na⁺– K⁺ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.Cell polarity (asymmetry) is a broadly distributed and highly conserved feature of many different cell types, from prokaryotes to higher eukaryotes (Nelson, 1992). In multicellular organisms it is more conspicuous in, but not restricted to, neurons and epithelial cells. In the latter, the plasma membrane is organized in two different domains, apical and basolateral. This characteristic enables epithelia to accomplish their most specialized roles including absorption and secretion and, in general, to perform the functions of organs with an epithelial parenchyma such as the kidney, liver, intestine, stomach, exocrine glands, etc. (Simons and Fuller, 1985; Rodriguez-Boulan and Nelson, 1989).The acquisition and maintenance of epithelial polarity is based on multiple interrelated mechanisms that may work in parallel. Although the origin of polarization depends on the sorting of apical and basolateral membrane proteins at the trans-Golgi network (Simons and Wandinger-Ness, 1990), the mechanisms involved in the transport of apical or basolateral carrier vesicles, the specific fusion of such vesicles to the appropriate domain, and the retention of membrane proteins in their correct positions are also important (Wollner and Nelson, 1992). Various components of the cytoskeleton seem to be especially involved in these mechanisms (Mays et al., 1994). Among them, the microtubules, characteristically oriented in the apical–basal axis with their minus ends facing toward the apical domain, appear in a strategic position to transport carrier vesicles (Bacallao et al., 1989). This orientation is largely expected because of the apical distribution of centrioles and microtubule organizing centers in epithelial cells (Buendia et al., 1990). The molecular interactions responsible for that localization, however, are unknown.Actin is a widespread component of the membrane skeleton found under apical, lateral, and basal membranes in a nonpolarized fashion (Drenckhahn and Dermietzel, 1988; Vega-Salas et al., 1988). Actin bundling into microvillus cores in the presence of villin/fimbrin, on the other hand, is highly polarized to the apical domain (Ezzell et al., 1989; Louvard et al., 1992). In fact, different isoforms of plastins determine microvillus shape in a tissue-specific manner (Arpin et al., 1994b ). Why this arrangement is not found in other actin-rich regions of the cell is unclear (Louvard et al., 1992; Fath and Burgess, 1995).Fodrin, the nonerythroid form of spectrin, underlies the basolateral domain (Nelson and Veshnock, 1987a ,b) and is known to participate in the anchoring/retention of basolateral proteins (Drenckhahn et al., 1985; Nelson and Hammerton, 1989). Although different groups have found specific cytoskeletal anchoring of apical membrane proteins at the “correct” domain (Ojakian and Schwimmer, 1988; Salas et al., 1988; Parry et al., 1990), no specific apical counterpart of the basolateral fodrin cytoskeleton is known. This is especially puzzling since we showed that MDCK cells can maintain apical polarity in the absence of tight junctions, an indication that intradomain retention mechanisms are operational for apical membrane proteins (Vega-Salas et al., 1987a ).It is known that a network of intermediate filament (IF)¹, the major component of the terminal web, bridges the desmosomes under the apical membrane in brush border cells (Franke et al., 1979; Hull and Staehelin, 1979; Mooseker, 1985), although no specific protein has been identified with this structure. The observation of a remarkable resistance to extractions of apical proteins anchored to cytoskeletal preparations (Salas et al., 1988) comparable to that of intermediate filaments, led us to the study of cytokeratins in polarized cells. We developed an antibody against a 53-kD intermediate filament protein in MDCK cells. This protein was found to be distributed exclusively to the apical domain and to form large (2,900 S) multi-protein complexes with apical plasma membrane proteins. Internal microsequencing of the 53-kD protein showed very high (95– 100%) homology with two polypeptides in the rod domain of cytokeratin 19 (CK19; Moll et al., 1982) a highly conserved and peculiar intermediate filament protein (Bader et al., 1986). A complete identification however, could not be achieved (Rodriguez et al., 1994). The present study was undertaken to establish that identity and to determine the possible functions of this apical membrane skeleton. Because cytokeratins have been poorly characterized in canine cells, and no cytokeratin sequences are available in this species, we decided to switch from MDCK cells to two human epithelial cell lines, CACO-2, an extensively studied model of epithelial polarization that differentiates in culture to form brush border containing cells (Pinto et al., 1983), and MCF-10A (Tait et al., 1990), a nontumorigenic cell line derived from normal mammary epithelia, as a model of nonbrush border cells.To assess possible functions of cytokeratin 19, we chose to selectively reduce its synthesis using anti-sense phosphorothioate oligodeoxy nucleotides, an extensively used approach in recent years (e.g., Ferreira et al., 1992 ; Hubber et al., 1993; Takeuchi et al., 1994). Although we could not achieve a complete knock out, the steady-state levels of cytokeratin 19 were decreased to an extent that enabled us to detect significant changes in the phenotype of CACO-2 and MCF-10A cells.