Rotavirus Infection Reduces Sucrase-Isomaltase Expression in Human Intestinal Epithelial Cells by Perturbing Protein Targeting and Organization of Microvillar Cytoskeleton

Rotavirus infection is the most common cause of severe infantile gastroenteritis worldwide. These viruses infect mature enterocytes of the small intestine and cause structural and functional damage, including a reduction in disaccharidase activity. It was previously hypothesized that reduced disaccharidase activity resulted from the destruction of rotavirus-infected enterocytes at the villus tips. However, this pathophysiological model cannot explain situations in which low disaccharidase activity is observed when rotavirus-infected intestine exhibits few, if any, histopathologic changes. In a previous study, we demonstrated that the simian rotavirus strain RRV replicated in and was released from human enterocyte-like Caco-2 cells without cell destruction (N. Jourdan, M. Maurice, D. Delautier, A. M. Quero, A. L. Servin, and G. Trugnan, J. Virol. 71:8268–8278, 1997). In the present study, to reinvestigate disaccharidase expression during rotavirus infection, we studied sucrase-isomaltase (SI) in RRV-infected Caco-2 cells. We showed that SI activity and apical expression were specifically and selectively decreased by RRV infection without apparent cell destruction. Using pulse-chase experiments and cell surface biotinylation, we demonstrated that RRV infection did not affect SI biosynthesis, maturation, or stability but induced the blockade of SI transport to the brush border. Using confocal laser scanning microscopy, we showed that RRV infection induces important alterations of the cytoskeleton that correlate with decreased SI apical surface expression. These results lead us to propose an alternate model to explain the pathophysiology associated with rotavirus infection.     

Basic chitinases are correlated with the morphogenic response of flax cells

Flax ( Linum usitatissimum ) hypocotyl protoplasts immobilized in a calcium‐alginate matrix give rise to embryo‐like structures. A direct correlation was established between the presence of a set of ionically‐bound cell wall proteins, which includes the basic polypeptides P184 and P183 with an apparent molecular mass of 25 kDa, and this morphogenic response. Microsequencing of tryptic fragments from P184 and P183 indicated homologies with the chitinase family. These homologies were confirmed by demonstrating that, after renaturation, such proteins express a potential chitinase activity in SDS‐PAGE gel containing glycol chitin as synthetic substrate. Using degenerate primers from P184 internal sequences, we isolated one partial genomic sequence of a chitinase of 626 bp from which a putative 74‐amino acid sequence, disrupted by one intron, was deduced. High degrees of homology with several plant chitinases, including those expressed during somatic embryogenesis or in seeds, were observed. P184 microsequences match the corresponding sequence deduced from the chitinase PCR‐fragment perfectly.     

Dedicated Roles of Plastid Transketolases during the Early Onset of Isoprenoid Biogenesis in Pepper Fruits

Isopentenyl diphosphate (IPP), which is produced from mevalonic acid or other nonmevalonic substrates, is the universal precursor of isoprenoids in nature. Despite the presence of several isoprenoid compounds in plastids, enzymes of the mevalonate pathway leading to IPP formation have never been isolated or identified to our knowledge. We now describe the characterization of two pepper (Capsicum annuum L.) cDNAs, CapTKT1 and CapTKT2, that encode transketolases having distinct and dedicated specificities. CapTKT1 is primarily involved in plastidial pentose phosphate and glycolytic cycle integration, whereas CapTKT2 initiates the synthesis of isoprenoids in plastids via the nonmevalonic acid pathway. From pyruvate and glyceraldehyde-3-phosphate, CapTKT2 catalyzes the formation of 1-deoxy-xylulose-5-phosphate, the IPP precursor. CapTKT1 is almost constitutively expressed during the chloroplast-to-chromoplast transition, whereas CapTKT2 is overexpressed during this period, probably to furnish the IPP necessary for increased carotenoid biosynthesis. Because deoxy-xylulose phosphate is shared by the plastid pathways of isoprenoid, thiamine (vitamin B₁), and pyridoxine (vitamin B₆) biosynthesis, our results may explain why albino phenotypes usually occur in thiamine-deficient plants.     

Xyloglucan Octasaccharide XXLGol Derived from the Seeds of Hymenaea courbaril Acts as a Signaling Molecule

Treatment of the xyloglucan isolated from the seeds of Hymenaea courbaril with Humicola insolens endo-1,4-β-d-glucanase I produced xyloglucan oligosaccharides, which were then isolated and characterized. The two most abundant compounds were the heptasaccharide (XXXG) and the octasaccharide (XXLG), which were examined by reference to the biological activity of other structurally related xyloglucan compounds. The reduced oligomer (XXLGol) was shown to promote growth of wheat (Triticum aestivum) coleoptiles independently of the presence of 2,4-dichlorophenoxyacetic acid (2,4-D). In the presence of 2,4-D, XXLGol at nanomolar concentrations increased the auxin-induced response. It was found that XXLGol is a signaling molecule, since it has the ability to induce, at nanomolar concentrations, a rapid increase in an α-l-fucosidase response in suspended cells or protoplasts of Rubus fruticosus L. and to modulate 2,4-D or gibberellic acid-induced α-l-fucosidase.     

Comparison of Binding Properties and Early Biological Effects of Elicitins in Tobacco Cells

Elicitins are a family of small proteins secreted by Phytophthora species that have a high degree of homology and elicit defense reactions in tobacco (Nicotiana tabacum). They display acidic or basic characteristics, the acidic elicitins being less efficient in inducing plant necrosis. In this study we compared the binding properties of four elicitins (two basic and two acidic) and early-induced signal transduction events (Ca²⁺ influx, extracellular medium alkalinization, and active oxygen species production). The affinity for tobacco plasma membrane-binding sites and the number of binding sites were similar for all four elicitins. Furthermore, elicitins compete with one another for binding sites, suggesting that they interact with the same receptor. The four elicitins induced Ca²⁺ influx, extracellular medium alkalinization, and the production of active oxygen species in tobacco cell suspensions, but the intensity and kinetics of these effects were different from one elicitin to another. As a general observation the concentrations that induce similar levels of biological activities were lower for basic elicitins (with the exception of cinnamomin-induced Ca²⁺ uptake). The qualitative similarity of early events induced by elicitins indicates a common transduction scheme, whereas fine signal transduction tuning is different in each elicitin.     

Immunolocalization of β-(1→4) and β-(1→6)-D-galactan epitopes in the cell wall and Golgi stacks of developing flax root tissues

Summary Most cell wall components are carbohydrate including the major matrix polysaccharides, pectins and hemicelluloses, and the arabinogalactan-protein proteoglycans. Both types of molecules are assembled in the Golgi apparatus and transported in secretory vesicles to the cell surface. We have employed antibodies specific to -(16) and -(14)-D-galactans, present in plant cell wall polysaccharides, in conjunction with immunofluorescence and electron microscopy to determine the location of the galactan-containing components in the cell wall and Golgi stacks of flax root tip tissues. Immunofluorescence data show that -(14)-D-galactan epitopes are restricted to peripheral cells of the root cap. These epitopes are not expressed in meristematic and columella cells. In contrast, -(16)-D-galactan epitopes are found in all cell types of flax roots. Immunogold labeling experiments show that both epitopes are specifically located within the wall immediately adjacent to the plasma membrane. They are also detected in Golgi cisternae and secretory vesicles, which indicates the involvement of the Golgi apparatus in their synthesis and transport. These findings demonstrate that the synthesis and localization of -(14)-D-galactan epitopes are highly regulated in developing flax roots and that different -linked D-galactans associated with cell wall polysaccharides are expressed in a cell type-specific manner.     

The Ambiguity of the Word "Complexity" a Proposal for Clarification

There are two different ways of defining complexity.1) Traditionally, the word "complexity" is considered synonymous to "organization". The transformation of species is an expression of victory against random indifferencism.2) The means of measuring complexity that was conceived by Kolmogorov has the advantage of having an external reference. Therefore, its logical reliability is considerable. But Kolmogorov's complexity will be at its best in cases of pure randomness.These mutually incompatible definitions explicitly demand a classification system.The first definition of complexity is contrary to the second one. This must be explained more precisely in order not to disturb the logic of Kolmogorov's conception and to enable to add to this conception, as closely as possible, along the logical rules derived from Gödel's incompleteness.The author proposes a beginning of complexity typology, founded on the necessity of laws that rule a random substract aiming at organization. The generality of laws to be selected will have a direct effect on the logical strength of the "biologists' definition" of complexity.Two fundamental laws are expressed, one derived from mathematics and the other from physics, two fields alien to biology. This fact improves the logical accordance to the argument.     

Acyl Transfer Activity of an Amidase from Rhodococcus sp. Strain R312: Formation of a Wide Range of Hydroxamic Acids

The enantioselective amidase from Rhodococcus sp. strain R312 was produced in Escherichia coli and was purified in one chromatographic step. This enzyme was shown to catalyze the acyl transfer reaction to hydroxylamine from a wide range of amides. The optimum working pH values were 7 with neutral amides and 8 with α-aminoamides. The reaction occurred according to a Ping Pong Bi Bi mechanism. The kinetic constants demonstrated that the presence of a hydrophobic moiety in the carbon side chain considerably decreased the K_mamide values (e.g., K_mamide = 0.1 mM for butyramide, isobutyramide, valeramide, pivalamide, hexanoamide, and benzamide). Moreover, very high turnover numbers (k_cat) were obtained with linear aliphatic amides (e.g., k_cat = 333 s⁻¹ with hexanoamide), whereas branched-side-chain-, aromatic cycle- or heterocycle-containing amides were sterically hindered. Carboxylic acids, α-amino acids, and methyl esters were not acyl donors or were very bad acyl donors. Only amides and hydroxamic acids, both of which contained amide bonds, were determined to be efficient acyl donors. On the other hand, the highest affinities of the acyl-enzyme complexes for hydroxylamine were obtained with short, polar or unsaturated amides as acyl donors (e.g., K_mNH2OH = 20, 25, and 5 mM for acetyl-, alanyl-, and acryloyl-enzyme complexes, respectively). No acyl acceptors except water and hydroxylamine were found. Finally, the purified amidase was shown to be l-enantioselective towards α-hydroxy- and α-aminoamides.Many bacterial amidases (EC 3.5.1.4) have been described previously because of their amide hydrolysis activities. Wide-spectrum amidases from Rhodococcus sp. strain R312 (26) and Pseudomonas aeruginosa (1), which are very similar, hydrolyze only short-chain amides. These enzymes are made up of four and six identical subunits having molecular weights of about 45,000 and 35,000, respectively. Based on the results of experiments performed with inhibitors, they have been classified as belonging to a branch of sulfhydryl enzymes (1, 26). The other amidases, the enantioselective amidases from Pseudomonas chlororaphis B23 (5), Rhodococcus erythropolis MP50 (12, 27), Rhodococcus sp. strain R312 (20), Rhodococcus sp. strain N-774 (10), Rhodococcus sp. (21), and Rhodococcus rhodochrous J1 (14), belong to a group of amidases containing a GGSS signature in the amino acid sequence (4) and are made up of two (or eight) identical subunits. The corresponding genes are located in clusters containing genes encoding the two subunits of a nitrile hydratase (EC 4.2.1.84). These amidases were also previously classified as sulfhydryl enzymes (5, 15), but no active amino acid residue was identified in any of them. Recently, Kobayashi et al. (15) showed that the real active site residues of the amidase from R. rhodochrous J1 were Asp-191 and Ser-195 rather than the generally accepted Cys-203 residue. These authors showed that aspartic acid and serine residues of this enzyme were also present in the active site sequences of aspartic proteinases and suggested that there is an evolutionary relationship between amidases and aspartic proteinases.All of the different amidases also exhibit an acyl transfer activity in the presence of hydroxylamine: RCONH₂ + NH₂OH ↔ RCONHOH + NH₃. This kind of reaction was previously described for the wide-spectrum amidase from Rhodococcus sp. strain R312 (6), but there has been no detailed study examining the acyl transfer reaction of amidases belonging to the GGSS signature-containing group. The final reaction products (hydroxamic acids) are known to possess high chelating properties. Some of them (particularly α-aminohydroxamic acid derivatives) are potent inhibitors of matrix metalloproteases, a family of zinc endopeptidases involved in tissue remodelling (3). Some other hydroxamic acids (α-aminohydroxamic acids, synthetic siderophores, acetohydroxamic acid, etc.) have also been investigated as anti-human immunodeficiency virus agents or antimalarial agents or have been recommended for treatment of ureaplasma infections and anemia (2, 8, 13, 28). Moreover, some fatty hydroxamic acids have been studied as inhibitors of cylooxygenase and 5-lipooxygenase with potent antiinflammatory activity (9).Apart from these medical applications, some hydroxamic acids (particularly polymerizable unsaturated hydroxamic acids and mid-chain or long-chain hydroxamic acids) have also been extensively investigated in wastewater treatment and nuclear technology studies as a way to eliminate contaminating metal ions (11, 16, 18).In this paper we describe the formation of a wide range of hydroxamic acids with the enantioselective amidase (a 120,000-dalton homodimer) from Rhodococcus sp. strain R312, and we provide some additional information which enhanced our comprehension of the reaction mechanism of this amidase.     

Activation of MAPK homologues by elicitors in tobacco cells

Elicitors of plant defence reactions (such as cryptogein, an elicitin produced by Phytophthora cryptogea , or oligogalacturonides (OGs)), induced in tobacco cell suspensions ( Nicotiana tabacum var Xanthi) a rapid and transient activation of two protein kinases (PKs) with apparent molecular masses of 50 and 46 kDa, respectively. These PKs activated and phosphorylated at tyrosine residues, phosphorylated myelin basic protein (MBP) at serine/threonine residues. Both are recognized by anti-MAPK antibodies. The two MBP kinases possessed the same kinetics of activation, and their activation depended, to the same extent, on different exogenously applied compounds (staurosporine, lanthanum, EGTA). We demonstrate here that the activation of the MBP kinases is calcium dependent and sensitive to staurosporine, a protein kinase inhibitor which annihilates all known responses of tobacco cells to cryptogein. The activation of MBP kinases appeared to be independent of the production of active oxygen species (AOS) and insensitive to calyculin A, a protein phosphatase type 1 and 2A inhibitor. The activation of MAPKs is discussed in relation to the early responses induced by cryptogein.