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S J Hardy

Publications and source records attributed to S J Hardy.

At least 19 recordsLinked to original sources
PubMed2000

Direct demonstration that homotetrameric chaperone SecB undergoes a dynamic dimer-tetramer equilibrium.

We have shown here that the cytosolic bacterial chaperone SecB is a structural dimer of dimers that undergoes a dynamic equilibrium between dimer and tetramer in the native state. We demonstrated this equilibrium by mixing two tetrameric species of SecB that can be distinguished by size. We showed that the homotetrameric species exchanged dimers, because when the mixture was analyzed both by size exclusion chromatography and native polyacrylamide gel electrophoresis a third hybrid tetrameric species was detected. Furthermore, treatment of SecB with 5,5'-dithiobis-(2-nitrobenzoic acid), which modifies the sulfhydryl group on cysteines, caused irreversible dissociation to a dimer indicating that cysteine must be involved in the stabilizing interactions at the dimer interface. It is clear that the two dimer-dimer interfaces of the SecB tetramer are differentially stable. Dissociation at one interface allows for a dynamic dimer-tetramer equilibrium. Because only dimers were exchanged it is clear that the other interface between dimers is significantly more stable, otherwise oligomers should have formed with a random distribution of monomers.

Bacterial Proteins↗
PubMed2000

Microcystins (cyanobacterial toxins) in drinking water enhance the growth of aberrant crypt foci in the mouse colon.

Microcystis aeruginosa produces toxic cyclic peptides called microcystins, potent hepatotoxins that have been implicated in tumor promotion in skin and liver. The model used in this investigation was the azoxymethane (AOM)-induced aberrant crypt focus (ACF) in the male C57Bl/6J mouse colon. Three intraperitoneal (i.p.) injections of 5 mg/kg AOM were administered at 7-d intervals to mice; 19 d after the last AOM injection, drinking water containing Microcystis extract was commenced and continued for a further 212 d. The content of microcystins in the drinking water was determined by mouse bioassay, high-performance liquid chromatography (HPLC), capillary eletrophoresis, and protein phosphatase inhibition. The doses employed were 0, 382, and 693 micrograms/kg bodyweight/d at the midpoint of the trial. Following postmortem examination blood cells, serum enzymes and organ pathology were investigated. A significant microcystin dose-dependent increase in the area of aberrant crypt foci was observed. There was no marked increase in the number of crypts/colon. Two overt colonic tumors (approximately 30 mm3) were seen in microcystin-treated mice, and one microscopic colonic tumor in an AOM-alone-treated mouse. This investigation provides the first evidence for the stimulation of preneoplastic colon tumor growth by microcystin.

Animals↗
PubMed2000

Complexes between protein export chaperone SecB and SecA. Evidence for separate sites on SecA providing binding energy and regulatory interactions.

During localization to the periplasmic space or to the outer membrane of Escherichia coli some proteins are dependent on binding to the cytosolic chaperone SecB, which in turn is targeted to the membrane by specific interaction with SecA, a peripheral component of the translocase. Five variant forms of SecB, previously demonstrated to be defective in mediating export in vivo (Gannon, P. M., and Kumamoto, C. A. (1993) J. Biol. Chem. 268, 1590-1595; Kimsey, H. K., Dagarag, M. D., and Kumamoto, C. A. (1995) J. Biol. Chem. 270, 22831-22835) were investigated with respect to their ability to bind SecA both in solution and at the membrane translocase. We present evidence that at least two regions of SecA are involved in the formation of active complexes with SecB. The variant forms of SecB were all capable of interacting with SecA in solution to form complexes with stability similar to that of complexes between SecA and wild-type SecB. However, the variant forms were defective in interaction with a separate region of SecA, which was shown to trigger a change that was correlated to activation of the complex. The region of SecA involved in activation of the complexes was defined as the extreme carboxyl-terminal 21 aminoacyl residues.

Adenosine Triphosphatases↗
PubMed2000

Interactions of Neisseria meningitidis with cells of the human meninges.

The interaction of Neisseria meningitidis with the meninges that surround and protect the brain is a pivotal event in the progression of bacterial meningitis. Two models of the human meninges were established in vitro, using (i) sections of fresh human brain and (ii) cultures of viable cells grown from human meningiomas. Neisseria meningitidis showed a specific predilection for binding to the leptomeninges and meningeal blood vessels in human brain and not to the cerebral cortex. There was a close correlation between the adherence of different Neisseria species to leptomeninges and cultured cells. The major ligand that mediated adherence was the pilus, and pilin variation modulated the interactions. The presence of Opa protein increased the association of Cap+ meningococci that expressed low-adhesive pili, but did not influence the association of high-adhesive pili. In contrast, Opc did not influence the adherence of Cap+ meningococci, whereas loss of capsule was associated with a more intimate interaction between the bacteria and the meningioma cell that was not apparent with Cap+ meningococci. There was no evidence of internalization of meningococci by meningioma cells in vitro, an observation that is consistent with the barrier properties of the leptomeninges to N. meningitidis observed in vivo.

Animals↗
PubMed1998

Calorimetric analyses of the interaction between SecB and its ligands.

SecB is a chaperone in Escherichia coli dedicated to export of proteins from the cytoplasm to the periplasm and outer membrane. It functions to bind and deliver precursors of exported proteins to the translocation apparatus before they fold into their native structures, thus maintaining them in a competent state for translocation across the membrane. The natural ligands of SecB are precursor proteins containing leader sequences. There are numerous reports in the literature indicating that SecB does not specifically recognize the leader peptides. However, two published investigations have concluded that the leader peptide is the recognition element (Watanabe M, Blobel G. 1989. Cell 58:685-705; Watanabe M, Blobel G. 1995. Proc Natl Acad Sci USA 92:10133-10136). In this work we use titration calorimetry to show that SecB binds two physiological ligands, which contain leader sequences, with no higher affinity than the same molecules lacking their leader sequences. Indeed, for one ligand the presence of the leader sequence reduces the affinity. Therefore, it can be concluded that the leader sequence provides no positive contribution to the binding energy.

ATP-Binding Cassette Transporters↗
PubMed1998

The interaction between the chaperone SecB and its ligands: evidence for multiple subsites for binding.

The chaperone protein SecB is dedicated to the facilitation of export of proteins from the cytoplasm to the periplasm and outer membrane of Escherichia coli. It functions to bind and deliver precursors of exported proteins to the membrane-associated translocation apparatus before the precursors fold into their native stable structures. The binding to SecB is characterized by a high selectivity for ligands having nonnative structure but a low specificity for consensus in sequence among the ligands. A model previously presented (Randall LL, Hardy SJS, 1995, Trends Biochem Sci 20:65-69) to rationalize the ability of SecB to distinguish between the native and nonnative states of a polypeptide proposes that the SecB tetramer contains two types of subsites for ligand binding: one kind that would interact with extended flexible stretches of polypeptides and the other with hydrophobic regions. Here we have used titration calorimetry, analytical ultracentrifugation, and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry to obtain evidence that such distinguishable subsites exist.

Aprotinin↗
PubMed1997

Binding of SecB to ribosome-bound polypeptides has the same characteristics as binding to full-length, denatured proteins.

The interaction of the chaperone SecB with ribosome-bound polypeptides that are in the process of elongation has been studied using an in vitro protein synthesis system. The binding is characterized by the same properties as those demonstrated for the binding of SecB to full-length proteins that are in nonnative conformation: it is readily reversible and has no specificity for the leader peptide. In addition, it is shown that the growing polypeptide chains must achieve a critical length to bind tightly enough to allow their isolation in complex with SecB. This explains the longstanding observation that, even when export is cotranslational, it begins late in synthesis. Furthermore, the required length is approximately the same as the length that defines the binding frame within denatured, full-length proteins bound to SecB.

Bacterial Proteins↗
PubMed1997

Determination of the binding frame of the chaperone SecB within the physiological ligand oligopeptide-binding protein.

Chaperone proteins demonstrate the paradoxical ability to bind ligands rapidly and with high affinity but with no apparent sequence specificity. To learn more about this singular property, we have mapped the binding frame of the chaperone SecB from E. coli on the oligopeptide-binding protein. Similar studies performed on the maltose-binding and galactose-binding proteins revealed centrally positioned binding frames of approximately 160 aminoacyl residues. The work described here shows that OppA, which is significantly longer than the previously studied ligands, has a binding frame that covers 460 amino acids, nearly the entire length of the protein. We propose modes of binding to account for the data.

Bacterial Proteins↗
PubMed1996

Reduced B subunit of heat-labile enterotoxin associates with membranes in vivo.

The B subunit of heat-labile enterotoxin, a periplasmic protein of Escherichia coli has an internal disulfide bond that forms after the protein has been exported. The presence of 2.5 mM dithiothreitol in the medium prevents the formation of the disulfide bond and this causes the protein to rapidly bind to membranes, preferentially but not exclusively to the cytoplasmic membrane. The binding is irreversible in vivo but chaotropic agents disrupt the association between the non-native B subunit and the membranes in vitro. The fact that the reduced B subunit binds to both the cytoplasmic and outer membranes that enclose the periplasm suggests that it is exported normally to the periplasm and then, because it is unable to form its native structure, adsorbs to membranes in the vicinity. This is confirmed by the finding that when synthesised by spheroplasts, in which the outer membrane is disrupted, the majority of reduced B subunit, which is not now confined in the periplasm, is exported to the medium and is not associated with membranes.

Bacterial Toxins↗
PubMed1996

Formation of disulfide bonded dimer of mutated heat-labile enterotoxin in vivo.

One of the two cysteines in the B subunit of heat-labile enterotoxin has been changed to a serine by site-directed mutagenesis so that the internal disulfide bond cannot form. The mutant protein, like the wild-type protein synthesised in the presence of the reducing agent dithiothreitol, does not form pentamers in the periplasm but binds to available membranes. Binding to membranes is disrupted by chaotropic agents but not by salt. More than half the molecules of mutant protein form disulfide-bonded dimers when exported to the periplasm but no dimer is detected when the protein is exported to the medium by spheroplasts.

Bacterial Toxins↗
PubMed1995

Polyenoic very-long-chain fatty acids mobilize intracellular calcium from a thapsigargin-insensitive pool in human neutrophils. The relationship between Ca2+ mobilization and superoxide production induced by long- and very-long-chain fatty acids.

Fatty acids with more than 22 carbon atoms (very-long-chain fatty acids; VLCFAs) are normal cellular components that have been implicated in the pathophysiology of a number of peroxisomal disorders. To date, however, essentially nothing is known regarding their biological activities. Ca2+ mobilization is an important intracellular signalling system for a variety of agonists and cell types. Given that several polyunsaturated long-chain fatty acids mobilize intracellular Ca2+ and that we have postulated that the VLCFAs may be involved in signal transduction, we examined whether the tetraenoic VLCFA induced Ca2+ mobilization in human neutrophils. We report that fatty acid-induced intracellular Ca2+ mobilization declined for fatty acid species of more than 20 carbon atoms, but increased again as the carbon chain length approached 30. This Ca2+ mobilization occurred independently of inositol 1,4,5-triphosphate production and protein kinase C translocation and involved both the release of Ca2+ from the intracellular stores and changes to the influx or efflux of the ion. We further observed that triacontatetraenoic acid [30:4(n-6)] mobilized Ca2+ from a thapsigargin-insensitive intracellular pool distinct from the thapsigargin-sensitive pools affected by arachidonic acid [20:4(n - 6)] or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). 20:4 (n - 6) induced strong superoxide production (chemiluminescence) which was inhibited by thapsigargin pretreatment. In contrast, fatty acid-induced superoxide production progressively declined as the carbon chain length increased beyond 20-22 carbon atoms. Further studies suggested that the thapsigargin-insensitive Ca2+ mobilization elicited by 30:4 (n - 6) was not related to oxyradical formation, while the thapsigargin-sensitive Ca2+ mobilization induced by 20:4 (n - 6) may be involved in the initiation but not necessarily the maintenance of superoxide production. In conclusion, this is the first report to demonstrate a biological activity for the VLCFA and indicates that 30:4 (n - 6) influences second messenger systems in intact cells that differ from those affected by long-chain fatty acids such as 20:4 (n - 6).

Calcium↗
PubMed1995

High selectivity with low specificity: how SecB has solved the paradox of chaperone binding.

Fundamental to the function of all molecular chaperones is their amazing ability to selectively and rapidly bind proteins in non-native states. Chaperones modulate a kinetic partitioning among the alternative pathways open to polypeptides within a cell, so that the proper pathway is taken. Here we review studies of SecB, a chaperone in Escherichia coli dedicated to facilitation of protein export, and emphasize the features that enable it to bind rapidly with high affinity and selectivity in the absence of consensus in sequence. The concepts discussed are likely to be generally applicable to chaperones.

Bacterial Proteins↗
PubMed1995

Accidental epinephrine auto-injector-induced digital ischemia reversed by phentolamine digital block.

The use of epinephrine-containing auto-injectors as a prescription medication for treating routine to severe anaphylactic reactions is now widely accepted. Associated with this trend is an increasing number of accidental injections of epinephrine into digits, causing severe vasoconstriction and the risk of ischemic necrosis. When epinephrine is accidentally discharged into a digit, ischemic skin necrosis resulting from the alpha-adrenergic blocking effects of this agent can lead to the need for multiple operations, wound infection, and even loss of the digit. The alpha-adrenergic blocking characteristics of phentolamine administered by a variety of methods have proved effective in reversing the effects of epinephrine in these cases. The authors urge that the described treatment protocol become more widely disseminated among primary care and emergency physicians.

Adult↗
PubMed1994

Effect of exogenous fatty acids with greater than 22 carbon atoms (very long chain fatty acids) on superoxide production by human neutrophils.

The effects of exogenous long chain fatty acids (LCFA) and very long chain fatty acids (VLCFA) on superoxide production by human neutrophils were compared. Superoxide production was greater and more rapid in response to arachidonic (20:4 (n-6)), eicosapentanoic (20:5 (n-3)), and docosahexanoic (22:6 (n-3)) acids than for triacontatetranoic (30:4 (n-6)), dotriacontatetranoic (32:4 (n-6)), and tetratriacontahexanoic (34:6 (n-3)) acids, although all of these fatty acids gave responses larger than FMLP. A similar decline in activity with increasing carbon chain length was observed for the monoenoic VLCFA (22:1 (n-9) to 34:1 (n-9)). 32:4 (n-6) did not affect responses to a maximally stimulatory concentration of 20:4 (n-6). However, the simultaneous addition of 20:4 (n-6) and 30:4 (n-6) gave additive responses if suboptimal dosages of 20:4 (n-6) were used. This suggests that the LCFA and VLCFA may use the same signal transduction systems. In addition, 30:4 (n-6) was only 10% as effective as was 20:4 (n-6) at gaining access to the organic solvent extractable cellular fraction. This figure correlated with the relative biologic potency of 20:4 (n-6) and 30:4 (n-6), suggesting that the extent of association with the cell may regulate the biologic activity of the fatty acids. The saturates, arachidic (20:0) and cerotic (26:0) acids, were either inactive or poor activators in all assay systems examined. The failure of 20:0 to induce superoxide production and the lower responses to 30:4 (n-6) and 34:6 (n-3) were not because of extracellular Ca2+, because the biologic potency of these fatty acids was not greatly enhanced by removing Ca2+ from the extracellular medium. In contrast, 20:4 (n-6)- and 22:6 (n-3)-induced superoxide production was markedly increased under Ca(2+)-free conditions.

Calcium↗
PubMed1994

In vitro activation of rat brain protein kinase C by polyenoic very-long-chain fatty acids.

A variety of fatty acids including the cis-polyunsaturated very-long-chain fatty acids (VLCFA) (> 22 carbon atoms) common in retina, spermatozoa, and brain were examined for their ability to activate protein kinase C (PKC) purified from rat brain. Arachidonic [20:4(n-6)], eicosapentaenoic [20:5(n-3)], and docosahexaenoic [22:6(n-3)] acids as well as the VLCFA dotriacontatetraenoic [32:4(n-6)] and tetratriacontahexaenoic [34:6(n-3)] were equally capable of activating PKC in vitro with maximal activity being between 25 and 50 microM. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate further enhanced the in vitro activation of PKC when added to the protein kinase assay system with the fatty acids. The fully saturated arachidic acid (20:0) was inactive in both assay systems. The potential significance of the in vitro activation of PKC by the VLCFA is discussed.

Animals↗
PubMed1994

Neutrophil migration inhibitory properties of polyunsaturated fatty acids. The role of fatty acid structure, metabolism, and possible second messenger systems.

The n-3 polyunsaturated fatty acids (PUFA) appear to have antiinflammatory properties that can be partly explained by their biological activity on leukocytes. Since leukocyte emigration is an essential component of the inflammatory response, we have examined the effects of the n-3 PUFA (eicosapentaenoic and docosahexaenoic acids) on neutrophil random and chemotactic movement. Preexposure of neutrophils for 15-30 min to 1-10 micrograms/ml PUFA reduced the random and chemotactic migration to both FMLP- and fungi-activated complement. The inhibitory effect diminished with increasing saturation and carbon chain length, and methylation abolished this activity. Arachidonic and docosahexaenoic acids were the most active fatty acids. The PUFA concentration required to inhibit migration was dependent on cell number, suggesting that the fatty acid effects on leukocyte migration in vivo may be governed by the stage of the inflammatory response. It was concluded that the PUFA rather than their metabolites were responsible for the inhibition since: (a) antioxidants did not prevent the PUFA-induced migration inhibition and the hydroxylated intermediates were less active, and (b) inhibitors of the cyclooxygenase and lipoxygenase pathways were without effect. Inhibitors of protein kinases and calmodulin-dependent enzyme system did not prevent the PUFA-induced migration inhibition, which was also independent of phospholipase D-catalyzed hydrolysis of phospholipids. It is also shown that PUFA decrease the FMLP-induced Ca2+ mobilization.

Calcium↗