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At least 919 records · Page 51Linked to original sources
PubMed1986

Site-directed mutagenesis of the gag-myc gene of avian myelocytomatosis virus 29: biological activity and intracellular localization of structurally altered proteins.

Transfection of chicken embryo cells with pMC29, a plasmid vector containing the sequences for the acute transforming virus MC29, and a cloned transformation-defective helper virus, p delta Mst, resulted in morphological transformation, the synthesis of P110gag-myc (the product of the gag-myc oncogene), and the production of infectious virus. MC29 mutants bearing site-directed deletions within the gag-specific sequences or within the middle portion of the myc sequences efficiently induced transformation of chicken embryo cells in culture. However, variants containing deletions of sequences in the amino-terminal half or carboxy-terminal portion of the myc gene were defective for transformation. The gag-myc proteins encoded by these variants efficiently localized to the cell nucleus. Premature termination mutants were isolated which encoded gag-myc proteins lacking the carboxy-terminal 185 residues; these truncated proteins localized to both the nucleus and the cytoplasm. Deletion of as few as 11 residues within the middle of the myc-specific sequences (residues Ile-239 to Glu-249) significantly reduced the efficiency of chicken hematopoietic cell transformation.

Animals↗
PubMed1994

Structure and localization of the IGFBP-1 gene and its expression during liver regeneration.

Insulin-like growth factor-binding protein-1s are important modulators of the insulin-like growth factors that may have both positive and negative effects on the ability of insulin-like growth factors to stimulate cell growth. The IGFBP-1 gene is one of the most highly induced immediate-early genes after partial hepatectomy. The IGFBP-1 gene is also expressed at a high level during fetal liver development and in response to nutritional changes and diabetes. Therefore it may have important roles in liver growth and metabolism. To begin to examine the regulation of this gene, we cloned and sequenced the entire mouse IGFBP-1 gene. Its structure is highly similar to that of the human gene, and, in addition to the exonic regions, the two genes are highly conserved in specific regions in the promoter and first intron. Analysis of this conservation allows us to predict important regulatory sites that define the tissue specific and insulin-mediated regulation of the gene and identify potential sites that might be important for the transcriptional induction during liver regeneration. The mouse gene is located on mouse chromosome 11; it is found at the boundary between regions in the mouse genome homologous to human chromosomes 22 and 7. We found IGFBP-1 mRNA in both parenchymal and nonparenchymal RNA after partial hepatectomy. Using in situ hybridization of IGFBP-1 mRNA in regenerating rat liver tissue, we demonstrated IGFBP-1 transcripts in several cell types. We found that IGFBP-1 gene induction after partial hepatectomy is paralleled by protein expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2001

XAS studies of Ni and Zn sorbed to hydrous manganese oxide.

Adsorption mechanisms of Zn2+ and Ni2+ on hydrous manganese oxide (HMO) were investigated using XAS. Analyses reveal that both metal ions retain their primary hydration shell when sorbed to HMO, which is consistent with physical sorption. These local structures are invariant of pH (3.0-7.0) and adsorbate loading (10(-4) - 10(-2) mol (g of HMO)-1), suggesting one average type of adsorption mechanism. In addition to the first shell, a second shell was observed with 6-8 O atoms at 3.34 A for Ni and 8-10 O atoms at 3.49 A for Zn. The lack of Ni or Zn contributions in the second shell eliminates the possibility of surface precipitation or polymerization. Likewise, the absence of Mn atoms in the second shell suggests outer-sphere adsorption. Interestingly, the local structure of Ni and Zn sorbed to HMO did not change with reaction time when as much as 90% of the sorbed contribution was due to the slow sorption process, thus supporting intraparticle diffusion as the rate-limiting mechanism. This result demonstrates that adsorption sites located along the micropore walls of HMO are similar to ones located on the external surface. Overall, metals from the same group in the Periodic Table appear to form similar adsorption complexes with HMO.

Absorptiometry, Photon↗
PubMed2006

NMR measurements of scalar-coupling distributions in disordered solids.

The measurement of scalar (J) couplings by solid-state NMR is a field of great interest, since this interaction is a rich source of local structural information, complementary to dipolar and chemical shift interactions. Here, we first demonstrate that J-coupling distributions exist and can be observed in disordered solids, as illustrated with the observation of a pair-specific distribution of (2)J((31)P-N-(31)P) couplings in a bis-phosphino amine, and we investigate the potential effects of such distributions on the measurement of average J-coupling constants. Second, we show that the measurement of two-dimensional (2D) distributions of J-couplings provides a much richer probe of local structural disorder than one-dimensional distributions, and we introduce new methods that provide different (selective or non-selective) ways of measuring 2D J distributions in a wide range of disordered systems. These methods are finally applied to a slightly disordered polymorphic sample of fully (13)C-enriched cellulose, and then to the bis-phosphino amine sample, from which 2D (2)J(PP)-coupling distributions are clearly identified and interpreted.

Journal Article↗
PubMed1999

Sorption of Metal Ions on Clay Minerals.

The mechanism of Co uptake from aqueous solution onto hectorite (a magnesian smectite) and its impact on the stability of this clay mineral were investigated as a function of Co concentration (TotCo = 20 to 200 µM, 0.3 M NaNO(3)) and ionic strength (0.3 and 0.01 M NaNO(3), TotCo = 100 µM) by combining kinetics measurements and Co K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy. The morphology of the sorbent phase was characterized by atomic force microscopy (AFM) and consists of lath-type particles bounded by large basal planes and layer edges. At low ionic strength (0.01 M NaNO(3)), important Co uptake occurred within the first 5 min of reaction, consistent with Co adsorption on exchange sites of hectorite basal planes. Thereafter, the sorption rate dramatically decreased. In contrast, at high ionic strength (0.3 M NaNO(3)), Co uptake rate was much slower within the first 5 min and afterward higher than at 0.01 M NaNO(3), consistent with Co adsorption on specific surface sites located on the edges of hectorite. Time-dependent isotherms for Co uptake at high ionic strength indicated the existence of several sorption mechanisms having distinct equilibration times. The dissolution of hectorite was monitored before and after Co addition. A congruent dissolution regime was observed prior to Co addition. Just after Co addition, an excess release of Mg relatively to congruent dissolution rates occurred at both high and low ionic strengths. At high ionic strength, this excess release nearly equaled the amount of sorbed Co. The dissolution rate of hectorite then decreased at longer Co sorption times. EXAFS spectra of hectorite reacted with Co at high and low ionic strengths and for reaction times longer than 6 h, exhibited similar features, suggesting that the local structural environments of Co atoms are similar. Spectral simulations revealed the occurrence of approximately 2 Mg and approximately 2 Si neighboring cations at interatomic distances characteristic of edge-sharing linkages between Co and Mg octahedra and corner-sharing linkages between Co octahedra and Si tetrahedra, respectively. This local structure is characteristic of inner sphere mononuclear surface complexes at layer edges of hectorite platelets. The occurrence of these complexes even at low ionic strength apparently conflicts with kinetics results, as exchangeable divalent cations are known to form outer sphere surface complexes. To clarify this issue, the amount of Co adsorbed on exchange sites was calculated from the solute Co concentration, assuming that cation exchange was always at equilibrium. These calculations showed that sorbed Co was transferred within 48 h from exchange sites to edge sorption sites. Copyright 1999 Academic Press.

Journal Article↗
PubMed1987

Structure and localization of synaptic complexes in the cardiac ganglion of a portunid crab.

The cardiac ganglion of Portunus sanguinolentus exhibits spontaneous rhythmic activity when isolated. The ganglion contains five large and four small intrinsic neurons and is innervated by three pairs of fibres originating in the thoracic ganglia. We have identified the processes of the large neurons in electron micrographs by injecting these cells with two electron-dense markers, horseradish peroxidase (HRP) and Procion Rubine (PR). In addition we have studied the processes of the four smaller neurons by light microscopy serial reconstructions and by electron microscopy of selected regions. Both markers were found only in neuronal processes and not in glial cells nor in the extracellular space, except close to the soma of the injected cell. We found contacts between the small secondary (collateral) processes of the large cells but not between their somata or their primary processes (axons and dendrites). Two specialized structures present at the contacts between the collateral processes were small membrane close appositions, possibly the site of electrotonic junctions, and chemical synapses. Contacts between processes marked by HRP and those marked by PR were common, as were contacts between processes marked by either HRP or PR and those of the other intrinsic neurons. Adjacent processes stained by PR could contain PR deposits of different densities, but it is unclear whether this finding was due to intercellular diffusion of the dye or to its diffusing at different rates into branches of the same process. Identified processes of all the intrinsic neurons contained the same type of vesicles, which were different from those found in processes of the extrinsic fibres. Chemical synapses were present at contacts between processes of the extrinsic and intrinsic neurons, as well as at contacts between processes of the intrinsic neurons. The axons of three small cells made a series of contacts at which extensive arrays of membrane close appositions, but not chemical synapses, were found. These three axons also formed contacts, either directly or through their collateral branches, with processes of the large cells, at which both membrane close appositions and chemical synapses were present. The axon of the fourth small cell could not be followed in our series.

Animals↗
PubMed2002

Planarian cytochrome b(561): conservation of a six transmembrane structure and localization along the central and peripheral nervous system.

Cytochrome b(561) is a major transmembrane protein of catecholamine and neuropeptide secretory vesicles in the central and peripheral nervous systems of higher animals. We succeeded in cloning a full-length cDNA encoding planarian cytochrome b(561). The deduced amino acid sequence shows a very similar six transmembrane topology to those of cytochromes b(561) of higher vertebrates and contains both putative ascorbate- and monodehydro ascorbate-binding sites. Among the six totally-conserved His residues of cytochrome b(561) in higher vertebrates, one is substituted with an Asn residue, indicating that His88 and His161 of bovine cytochrome b(561) play roles as heme b ligands at the extravesicular side. Northern- and Western-blot analyses confirmed the expression of the mRNA and protein with the expected sizes in planarians. The distributions of the mRNA and apoprotein were analyzed by in situ hybridization and immunohistochemical staining, respectively, showing two morphologically distinct structures, a pair of ventral nerve cords and the cephalic ganglion cluster in the head region. The present results suggest that the usage of ascorbate to supply electron equivalents to neuroendocrine-specific copper-containing monooxygenases is likely to have originated in organisms with a very simple nervous system.

Amino Acid Sequence↗
PubMed2000

Molecular cloning, genomic structure, chromosomal localization, and alternative splice forms of the platelet collagen receptor glycoprotein VI.

Glycoprotein VI (GPVI) is the major collagen receptor underlying platelet activation. We cloned the full-length cDNA for GPVI (GPVI-1) and its two isoforms (GPVI-2 and -3) from phorbol-ester-stimulated CMK cells. The GPVI-1 cDNA was identical in the coding region with the cDNA that has recently been reported to belong to the immunoglobulin superfamily. The GPVI gene consisted of 8 exons spanning over 23 kbp and was mapped on the chromosome 19q13. 4. The promoter of GPVI gene lacked TATA and CAAT boxes and had multiple transcription start sites like other megakaryocytic genes. When COS-7 cells were cotransfected with the GPVI isoforms and Fc receptor gamma chain, Fc receptor gamma chain was associated with GPVI-1 and -2 but did not affect the GPVI expression levels. GPVI-1 and -2 could bind the collagen-related peptide, which exhibits triple-helical and polymeric structure of collagen to activate platelets via GPVI.

Alternative Splicing↗
PubMed1999

A multifrequency electron spin resonance study of T4 lysozyme dynamics.

Electron spin resonance (ESR) spectroscopy at 250 GHz and 9 GHz is utilized to study the dynamics and local structural ordering of a nitroxide-labeled enzyme, T4 lysozyme (EC 3.2.1.17), in aqueous solution from 10 degrees C to 35 degrees C. Two separate derivatives, labeled at sites 44 and 69, were analyzed. The 250-GHz ESR spectra are well described by a microscopic ordering with macroscopic disordering (MOMD) model, which includes the influence of the tether connecting the probe to the protein. In the faster "time scale" of the 250-GHz ESR experiment, the overall rotational diffusion rate of the enzyme is too slow to significantly affect the spectrum, whereas for the 9-GHz ESR spectra, the overall rotational diffusion must be accounted for in the analysis. This is accomplished by using a slowly relaxing local structure model (SRLS) for the dynamics, wherein the tether motion and the overall motion are both included. In this way a simultaneous fit is successfully obtained for both the 250-GHz and 9-GHz ESR spectra. Two distinct motional/ordering modes of the probe are found for both lysozyme derivatives, indicating that the tether exists in two distinct conformations on the ESR time scale. The probe diffuses more rapidly about an axis perpendicular to its tether, which may result from fluctuations of the peptide backbone at the point of attachment of the spin probe.

Bacteriophage T4↗
PubMed2006

Structure and bonding of alkanethiols on Cu(111) and Cu(100).

The local structure of the sulfur atom of methanethiolate and ethanethiolate on the Cu(111) and Cu(100) surfaces was investigated from first principles employing the periodic supercell approach in the framework of density functional theory. On the 111 surface, we investigated the (square root 3 x square root 3)R30 degrees and (2 x 2) structures, whereas on the 100 surface, we investigated the p(2 x 2) and c(2 x 2) structures. The landscape of the potential energy surface on each metal surface presents distinctive features that explain the local adsorption structure of thiolates found experimentally. On the Cu(111) surface, the energy difference between the hollow and bridge sites is only 3 kcal/mol, and consequently, adsorption sites ranging from the hollow to the bridge site were observed for increasing surface coverages. On the Cu(100) surface, there is a large energy difference of 12 kcal/mol between the hollow and bridge sites, and therefore, only the 4-fold coordination was observed. The high stabilization of thiolates on the hollow site of Cu(100) may be the driving force for the pseudosquare reconstruction observed experimentally on Cu(111). Density of states analysis and density difference plots were employed to characterize the bonding on different surface sites. Upon interaction with the metal d bands, the pi* orbital of methanethiolate splits into several peaks. The two most prominent peaks are located on either edge of the metal d band. They correspond to bonding and antibonding S-Cu interactions. In the case of ethanethiolate, all the back-bonds are affected by the surface bonding, leading to alternating regions of depletion and accumulation of charge in the successive bonds.

Journal Article↗
PubMed1987

Superhelical DNA with local substructures. A generalization of the topological constraint in terms of the intersection number and the ladder-like correspondence surface.

The presence of certain local structural elements in superhelical DNA, such as cruciforms and denatured loops, complicates the topological and geometric analysis of these molecules. In particular, the duplex axis is often difficult to define. In consequence, the usual conservation condition, Lk = Tw + Wr, is often inapplicable as formulated in terms of the winding of either strand of the DNA about the duplex axis. We present here a more general formulation of the topological conservation condition in terms of a model in which the two strands of DNA are regarded as twisting about one another, and in which one of the two strands is considered to writhe. We define a ladder-like correspondence surface, which connects the two strands nd is independent of whether or not a unique duplex axis is locally available. These considerations lead to the definition of a new topological property of superhelical DNA, the intersection number, In. This quantity describes the complexity of a local structural element; in the case of a cruciform, for example, the intersection number is a measure of the number of duplex turns removed from the major segment of the DNA by the cruciform formation. In more general terms, the topological constraint applicable to closed circular DNA is given by Lk(W,C) + In(S,C) = Tw(W,C) + Wr (C), where W and C represent the two strands of the DNA and S is the ladder-like correspondence surface that connects the two strands.

DNA, Superhelical↗
PubMed1977

Human kidney gamma-glutamyl transpeptidase. Catalytic properties, subunit structure, and localization of the gamma-glutamyl binding site on the light subunit.

Human kidney gamma-glutamyl transpeptidase has been purified by a procedure involving Lubrol extraction, acetone precipitation, treatment with bromelain, and column chromatography on DEAE-cellulose and Sephadex G-150. The final preparation is a glycoprotein (molecular weight of approximately 84,000) composed of two nonidentical glycopeptides (molecular weights of 62,000 and 22,000). The isozymic forms, separable by isoelectric focusing, have different contents of sialic acid. The utilization of L-glutamine (which is both a gamma-glutamyl donor and acceptor) is stimulated about 3-fold by maleate in contrast to 10-fold stimulation of glutamine utilization by the rat kidney enzyme. The gamma-glutamyl analogs, 6-diazo-5-oxo-L-norleucine (DON) and L-azaserine inactivate the human kidney enzyme with respect to its transpeptidase and hydrolase activities. Inactivation is prevented by gamma-glutamyl substrates (but not by acceptor substrates) and is accelerated by maleate. [14C]DON reacts covalently and stoichiometrically at the gamma-glutamyl site, which was localized to the light subunit of the enzyme. The light subunit of human transpeptidase closely resembles that of rat kidney enzyme in having the gamma-glutamyl binding site, and similar molecular weight and amino acid composition. The heavy subunits of the two enzymes are markedly different in both molecular weight and amino acid content; this may account for differences observed in acceptor amino acid specificity and in the magnitude of the maleate effect.

Amino Acids↗
PubMed2005

Molecular-dynamics study of anomalous volumetric behavior of water-benzene mixtures in the vicinity of the critical region.

Molecular-dynamics simulations of water-benzene mixtures at 573 K and pressures in the 85-140 bars range have been performed to examine local structure and dynamics of the mixtures, which exhibit anomalously large volume expansion on mixing as recently found by in situ near-infrared measurements. Fractional charges for a simple-point-charge-type potential of water were adjusted so as to reproduce liquid densities and the gas-to-liquid transition pressure of neat water at 573 K. A Lennard-Jones-type potential for benzene was used and the Lorentz-Berthelot combination rule was applied to the water-benzene interaction. Simulations with a N-P-T ensemble of 800-molecule system have been performed and the results reproduce well the anomalous volumetric behavior of the mixtures with the mole fraction of benzene in the 0.3-0.8 range. Pair distribution functions, coordination numbers, and self-diffusion coefficients for the mixtures are calculated, and it is suggested that the local structure around water molecules undergoes drastic change by dissolution of benzene in the vicinity of the critical region, but that around benzene molecules seems to be understood as that of ordinary liquid mixtures.

Journal Article↗
PubMed1996

Biochemical analysis, gene structure and localization of the 24 kDa glutathione S-transferase from Onchocerca volvulus.

Survival of Onchocerca volvulus, a pathogenic human filarial parasite, is likely to depend upon the detoxification activities of the glutathione S-transferases (GSTs). The 24 kDa O. volvulus GST, OvGST2, was expressed in a bacterial system and the recombinant protein was purified to homogeneity by affinity chromatography. Specific activities of the recombinant OvGST2 (rOvGST2) with a variety of substrates, and in the presence of inhibitors, were determined. With the universal substrate 1-chloro-2,4-dinitrobenzene, the specific activity of rOvGST2 was 2130 nmol min-1 mg-1. The rOvGST2 showed relatively limited selenium-independent glutathione peroxidase activity, but secondary products of lipid peroxidation, namely members of the trans,trans-alka-2,4-dienal,trans-alk-2-enal and 4-hydroxyalk-2-enal series, were conjugated to glutathione via OvGST2 dependent activity. The gene encoding the OvGST2 was isolated and the nucleotide sequence determined. The ovgst2 gene was found to possess seven exons with six intervening sequences, with all except one having consensus splice-site junctions. This intron/exon organisation of the ovgst2 gene is almost identical with those described for the mammalian Pi class GST genes, consistent with the protein structural evidence that the OvGST2 is related to the Pi class GSTs. Southern blot analysis with total parasite genomic DNA indicated a single copy gene, with a restriction pattern consistent with that of the isolated gene. The tissue distribution of the OvGST2 was examined in O. volvulus by immunohistochemistry and was shown to be distinct from that of the OvGST1. The OvGST2 was located throughout the syncytial hypodermis of male and female adult worms, as well as in the uterine epithelium. Microfilariae, and infective third stage larvae of O. volvulus, isolated from Simulium neavei, were immunopositive for OvGST2.

Amino Acid Sequence↗
PubMed1993

Iontophoretic transdermal delivery of salicylic acid and lidocaine to local subcutaneous structures.

The depth of penetration of solutes into underlying tissues after transdermal iontophoresis has been evaluated with salicylic acid and lidocaine as model compounds. Concentrations of salicylic acid and lidocaine were measured in plasma and tissues below the donor electrode after iontophoresis in rats. The concentrations obtained were then compared with those obtained after passive delivery (without iontophoresis) of the drugs applied either to intact epidermis or to the exposed dermis (epidermis removed) of rats. Iontophoresis yielded high concentrations of lidocaine in each underlying tissue when compared with passive application to rat epidermis or dermis. Negligible concentrations of lidocaine in plasma were found for each mode of delivery. Similar concentrations of salicylic acid were found in each of the underlying tissues after delivery of salicylic acid either by iontophoresis through intact epidermis or after passive application to the exposed dermis. Negligible concentrations of salicylic acid in underlying tissues were obtained after passive application to intact epidermis. The plasma salicylic acid concentrations observed after both iontophoretic epidermal and passive dermal (epidermis removed) treatments were approximately the same as the tissue salicylic acid concentrations observed at approximately 3-4 mm below the application site. It is concluded that transdermal iontophoresis allows salicylic acid and lidocaine to be effectively delivered across the stratum corneum. Local direct deep tissue penetration of lidocaine is facilitated by iontophoresis. The concentrations of salicylic acid in deeper underlying tissues (> 3-4 mm) tend to be similar to the concentrations in plasma after either iontophoresis or passive dermal application, a result indicating that direct penetration of salicylic acid occurs only to a depth of 3-4 mm.

Administration, Cutaneous↗
PubMed2006

Cooperative alpha-helix unfolding in a protein-DNA complex from hydrogen-deuterium exchange.

We present experimental evidence for a cooperative unfolding transition of an alpha-helix in the lac repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogen-deuterium (H-D) exchange experiments monitored by NMR spectroscopy. In the EX1 limit, observed exchange rates become pH-independent and exclusively sensitive to local structure fluctuations that expose the amide proton HN to exchange. Close to this regime, we measured decay rates of individual backbone HN signals in D2O, and of their mutual HN-HN NOE by time-resolved two-dimensional (2D) NMR experiments. The data revealed correlated exchange at the center of the lac headpiece recognition helix, Val20-Val23, and suggested that the correlation breaks down at Val24, at the C terminus of the helix. A lower degree of correlation was observed for the exchange of Val9 and Ala10 at the center of helix 1, while no correlation was observed for Val38 and Glu39 at the center of helix 3. We conclude that HN exchange in the recognition helix and, to some extent, in helix 1 is a cooperative event involving the unfolding of these helices, whereas the HN exchange in helix 3 is dominated by random local structure fluctuations.

Bacterial Proteins↗
PubMed2004

Theoretical Debye-Waller factors of alpha-MoO(3) estimated by an equation-of-motion method.

EXAFS oscillations of MoO(3), which has a highly asymmetric local structure, have been calculated using backscattering amplitudes and phase shifts derived from the FEFF8 code and using Debye-Waller factors from an equation-of-motion method. They were compared with polarization-dependent empirical EXAFS data of the alpha-MoO(3) single crystal at various temperatures. The theoretical EXAFS oscillations of Mo-O bonds for the [001] direction of the single crystal, where two symmetric Mo-O bonds exist, reproduced well the experimental data. On the other hand, the calculated data for the [100] direction, which contain two asymmetric Mo-O bonds with different bond lengths, agree well with the experimental data only after adjustment of the amplitude reduction factors for different Mo-O bonds. EXAFS oscillations of MoO(3) powder were also calculated by the same method, and theoretical parameters that could reproduce the experimental data were found. These results suggest that the equation-of-motion method can evaluate the Debye-Waller factors efficiently in molecules with asymmetric local structures and can reduce curve-fitting parameters.

Journal Article↗
PubMed2000

Distinct effects of Vibrio cholerae haemagglutinin/protease on the structure and localization of the tight junction-associated proteins occludin and ZO-1.

Vibrio cholerae produces a little-studied cytotoxin, haemagglutinin/protease (HA/P), in addition to several better-characterized enterotoxins, i.e. cholera toxin (CT), zonula occludens toxin (ZOT) and accessory cholera enterotoxin (Ace). We have found recently that HA/P perturbs the barrier function of Mardin-Darby canine kidney epithelial cell line I (MDCK-I) by affecting the intercellular tight junctions (TJs) and the F-actin cytoskeleton. In the present study we have assessed more specifically how TJs are affected by HA/P by investigating the cellular localization and biochemical integrity of two well-characterized TJ-associated proteins, occludin and ZO-1. Western blot analysis showed that occludin bands of 66-85 kDa were digested by HA/P to two predominant bands of around 50 kDa and 35 kDa, and that this degradation was greatly attenuated when the specific bacterial metalloproteinase inhibitor Zincov was co-administered. Trypsin, on the other hand, did not degrade occludin when it was applied in the same way, suggesting that the degradation of occludin by HA/P is an early and specific event. The other TJ-associated protein ZO-1 was not degraded by HA/P in parallel experiments, suggesting the selectivity of HA/P-associated protein degradation. Moreover, immunofluorescence labelling and confocal microscopy showed that ZO-1, but not occludin, around cell-cell boundaries was rearranged by HA/P treatment. Since ZO-1 is located on the inside of the plasma membrane and is directly associated with occludin, the results indicate that breakdown of occludin may send signals to ZO-1 that affect its organization and the structure of the F-actin cytoskeleton. Our finding that the zinc-containing metalloprotease of V. cholerae specifically degraded occludin suggests that specific degradation of important host proteins by bacterial zinc-containing metalloproteases may be an important mechanism in microbial pathogenesis.

Animals↗