Biomedical subjects

A D Miller

Publications and source records attributed to A D Miller.

At least 55 records · Page 3Linked to original sources

PubMed1999

Interaction with GroEL destabilises non-amphiphilic secondary structure in a peptide.

The Escherichia coli molecular chaperone GroEL can functionally interact with non-native forms of many proteins. An inherent property of non-native proteins is the exposure of hydrophobic residues and the presence of secondary structure elements. Whether GroEL unfolds or stabilises these structural elements in protein substrates as a result of binding has been the subject of extended debate in the literature. Based on our studies of model peptides of pre-formed helical structure, we conclude that the final state of a GroEL-bound substrate is dependent on the conformational flexibility of the substrate protein and the distribution of hydrophobic residues, with optimal association when these are able to present a cluster of hydrophobic residues in the binding interface.

Chaperonin 60↗

PubMed1999

Persistent, therapeutically relevant levels of human granulocyte colony-stimulating factor in mice after systemic delivery of adeno-associated virus vectors.

Adeno-associated virus (AAV) vectors have been shown to preferentially transduce hepatocytes after systemic administration in adult mice and to provide long-term expression of introduced genes. One application of this technology would be for the production of granulocyte colony-stimulating factor (G-CSF), which increases mature neutrophil numbers in humans and in animals, and has therapeutic effects in disorders featuring chronic neutropenia, including cyclic, severe congenital, and idiopathic neutropenia, and glycogen storage disease type Ib. We have treated mice by tail vein injection of AAV vectors encoding human G-CSF, and have detected high G-CSF levels and marked elevation of neutrophil counts for at least 5 months. A therapeutically relevant amount of G-CSF production was obtained when the liver-specific mouse albumin promoter-enhancer was used to drive G-CSF expression. In mice receiving higher amounts of vector, plasma levels of human G-CSF gradually increased over 3 weeks to high concentrations, whereas for lower amounts human G-CSF remained at initial, low levels. The previously observed effect of gamma irradiation, to increase AAV transduction rates, was diminished when large amounts of vector were used. Absolute neutrophil counts increased 10- to 50-fold for the period of observation to levels that would be therapeutic in the treatment of cyclic neutropenia. In conclusion, gene therapy with AAV vectors synthesizing G-CSF shows promise for the treatment of disorders featuring neutropenia.

Animals↗

PubMed1999

Secondary structure forming propensity coupled with amphiphilicity is an optimal motif in a peptide or protein for association with chaperonin 60 (GroEL).

The interactions of GroEL with six dansyl peptides were investigated by means of our previously established fluorescence binding assay [Hutchinson, J. P., Oldham, T. C., El-Thaher, T. S. H., and Miller, A. D. (1997) J. Chem. Soc., Perkin Trans. 2, 279-288]. Three peptides (AMPH series) were constructed with a hierarchy of alpha-helix-forming propensities and amphiphilic characteristics. The remaining three peptides (NON-AMPH series) were prepared with a reordered amino acid sequence designed to form peptides of differing non-amphiphilic alpha-helix-forming propensity. Of these six peptides, two (AMPH(+) and NON-AMPH(+)) were N-capped with an S-form alpha-helix-inducing template (Ro 47-1615, Hoffmann-La Roche), two (AMPH(-) and NON-AMPH(-)) were N-capped with an R-form non-inducing template (Ro 47-1614, Hoffmann-La Roche), and two (AMPH(R) and NON-AMPH(R)) were without N-cap modification. This paper describes how the known strength of interaction of an unfolded protein substrate with the molecular chaperone GroEL (K(d) micromolar to nanomolar) may be emulated with a single peptide (AMPH(+)) (apparent K(d) 5 nM) which has a high propensity to form an amphiphilic alpha-helical structure in solution. Secondary structure forming propensity is not, in and of itself, an important contributor to the strength of interaction with GroEL. However, secondary structure forming propensity coupled with amphiphilicity may be sufficient to account for most, if not all, of the interaction strength between GroEL and an unfolded peptide or protein substrate.

Binding, Competitive↗

PubMed1999

The RD114/simian type D retrovirus receptor is a neutral amino acid transporter.

The RD114/simian type D retroviruses, which include the feline endogenous retrovirus RD114, all strains of simian immunosuppressive type D retroviruses, the avian reticuloendotheliosis group including spleen necrosis virus, and baboon endogenous virus, use a common cell-surface receptor for cell entry. We have used a retroviral cDNA library approach, involving transfer and expression of cDNAs from highly infectable HeLa cells to nonpermissive NIH 3T3 mouse cells, to clone and identify this receptor. The cloned cDNA, denoted RDR, is an allele of the previously cloned neutral amino acid transporter ATB0 (SLC1A5). Both RDR and ATB0 serve as retrovirus receptors and both show specific transport of neutral amino acids. We have localized the receptor by radiation hybrid mapping to a region of about 500-kb pairs on the long arm of human chromosome 19 at q13.3. Infection of cells with RD114/type D retroviruses results in impaired amino acid transport, suggesting a mechanism for virus toxicity and immunosuppression. The identification and functional characterization of this retrovirus receptor provide insight into the retrovirus life cycle and pathogenesis and will be an important tool for optimization of gene therapy using vectors derived from RD114/type D retroviruses.

3T3 Cells↗

PubMed1999

A human cell-surface receptor for xenotropic and polytropic murine leukemia viruses: possible role in G protein-coupled signal transduction.

Although present in many copies in the mouse genome, xenotropic murine leukemia viruses cannot infect cells from laboratory mice because of the lack of a functional cell surface receptor required for virus entry. In contrast, cells from many nonmurine species, including human cells, are fully permissive. Using an expression library approach, we isolated a cDNA from HeLa cell RNA that conferred susceptibility to xenotropic envelope protein binding and virus infection when expressed in nonpermissive cells. The deduced product is a 696-aa multiple-membrane spanning molecule, is widely expressed in human tissues, and shares homology with nematode, fly, and plant proteins of unknown function as well as with the yeast SYG1 protein, which has been shown to interact with a G protein. This molecule also acts as a receptor for polytropic murine leukemia viruses, consistent with observed interference between xenotropic and polytropic viruses in some cell types. This xenotropic and polytropic retrovirus receptor (XPR1) is the fourth identified molecule having multiple membrane spanning domains among mammalian type C oncoretrovirus receptors and may play a role in G protein-coupled signal transduction, as do the chemokine receptors required for HIV entry.

3T3 Cells↗

PubMed1999

Keratinocyte growth factor stimulates transduction of the respiratory epithelium by retroviral vectors.

Cell proliferation is required for transduction by standard retrovirus vectors derived from viruses in the murine leukemia virus (MuLV) group. Since proliferation rates are low in the mature pulmonary epithelium, we tested the hypothesis that the efficiency of retrovirus-mediated transduction of respiratory epithelial cells can be enhanced by stimulation of cell proliferation with recombinant human keratinocyte growth factor (rhKGF). A marked increase in proliferation of bronchiolar and alveolar epithelial cells was observed after intratracheal administration of rhKGF (30 mg/kg) to adult FVB/N mice. Two days after rhKGF or saline treatment, 10(7) AP+ FFU of LAPSN, a recombinant amphotropic retrovirus that expresses human placental alkaline phosphatase (AP), was instilled intratracheally into the mice. Transduction efficiency, measured 2 days after infection, was increased approximately 70-fold by rhKGF pretreatment. However, even after KGF treatment the total numbers of AP-expressing cells were few. Transduction efficiency was similar using either LAPSN packaged by amphotropic host range packaging cells or LAPSN pseudotyped with 10A1 MuLV envelope protein (0.091 +/- 0.006 versus 0.094 +/- 0.028 transduction events/mm2, respectively). Amphotropic vectors use Pit-2 for cell entry, while 10A1 MuLV vectors can use Pit-1 or Pit-2 for cell entry. By in situ hybridization the retroviral receptor Pit-2 (Ram-1) mRNA was expressed only in the pulmonary vasculature, and Pit-1 (Glvr-1) mRNA was expressed at low levels throughout the lung. In vitro studies demonstrated that retrovirus was inactivated by pulmonary surfactant. Stimulating proliferation of the respiratory epithelium increased retroviral transduction in vivo, but the paucity of retroviral receptors and inactivation by surfactant are additional barriers to high-level retroviral gene transfer in the lung.

Animals↗

PubMed1999

Molecular chaperones stimulate bone resorption.

Molecular chaperones, also known as heat shock proteins (hsp), are intracellular proteins found in all cells that catalyze protein folding. We have discovered that one class of bacterial molecular chaperone, the chaperonins, are potent inducers of bone resorption. To address the question of whether the osteolytic activity of the chaperonins was unique to this protein class, or was a common attribute of molecular chaperones generally, we have examined a number of bacterial and mammalian molecular chaperones for activity in the murine calvarial bone resorption assay. All the Escherichia coli molecular chaperones (groEL, groES, and dnaK) were active. The osteolytic activity of groEL was inhibited by indomethacin and the natural antagonist of interleukin-1 receptor antagonist (IL-1ra) but was unaffected by neutralization of tumor necrosis factor (TNF) or inhibition of 5-lipoxygenase. Mammalian molecular chaperones of molecular mass 27, 47, 70, and 90 kDa were also tested and, with the exception of the 47 kDa protein, all showed activity in the murine calvarial assay. Molecular chaperones appear, therefore, to have the capacity to modulate the cellular processes in bone explant cultures, resulting in resorption of the calcified matrix. The possibility that these proteins could play a role in the normal or pathological remodeling of bone is discussed.

Animals↗

PubMed1999

A search within the IL-1 type I receptor reveals a peptide with hydropathic complementarity to the IL-1beta trigger loop which binds to IL-1 and inhibits in vitro responses.

In previous research, we were able to demonstrate that a seven amino acid residue peptide (VITFFSL), designed as an antisense peptide of the beta-bulge trigger loop region of interleukin 1beta (IL-1beta) (QGEESND; residues 48-54 [mature protein sequence]), was able to interact with IL-1 specifically and inhibit the response to IL-1 in an in vitro bioassay. The evidence was consistent with a specific interaction ocurring between antisense peptide and the trigger loop region. On the basis that antisense peptides are able to interact with their corresponding sense peptide sequences as a result of their mutually complementary hydropathic profiles (Fassina G., Verdoliva, A., Cassani, G., Melli, M., 1994. Binding of type I IL-1 receptor fragment 151-162 to IL-1. Growth Factors 10, 99-106; Maier, C.C., Moseley, H.N.B., Zhou, S., Whitaker, J.N., Blalock, J.E., 1994. Indentification of interactive determinants on idiotypic-anti-idiotypic antibodies through comparison of their hydropathic profiles. Immunomethods 5, 107-113), we devised a computer program (FINDH) to search the amino acid residue sequence of interleukin-1 type 1 receptor (IL-1 R1) for peptide motifs possessing hydropathic complementarity to the trigger loop sequence. The most complementary "best-fit peptide" motif (LITVLNI) was located in the third extracellular domain of IL-1 R1. A best-fit peptide corresponding to this motif was synthesised and found to bind to IL-1beta as well as inhibit the response to IL-1 in two independent in vitro bioassays (monitoring IL-1 dependent serum amyloid A synthesis and IL-1 dependent alkaline phosphatase activity, respectively). A second peptide motif (VIEFITL) was identified and the corresponding peptide synthesised along with a reordered version (LTILINV) of the best fit peptide. Both failed to bind measurably with IL-1beta or inhibit the response to IL-1 in the two bioassays. This best fit peptide behaved very similarly, in terms of IL-1 binding and inhibition behaviour, to the original trigger loop antisense peptide. Reference to the recently released X-ray crystal structure of IL-1beta and the IL1-R1 extracellular domain shows that the best fit peptide motif in IL-1 R1 is not apparantly interacting with the IL-1 trigger loop, although both are close in space. The intriguing possibility exists that the best fit peptide motif could represent an alternative site for IL-1beta receptor interaction which has not thus far been identified.

Alkaline Phosphatase↗

PubMed1999

Behaviors of bulbar respiratory interneurons during fictive swallowing and vomiting.

Behaviors of the same individual medullary respiratory interneurons were examined during both swallowing and vomiting. In 8 decerebrated and paralyzed cats, 18 neurons having either augmenting expiratory (E-AUG), decrementing expiratory (E-DEC), decrementing inspiratory (I-DEC), or constant inspiratory (I-CON) firing patterns were recorded near the most rostral part of the nucleus ambiguus. All neurons exhibited elementary reflexes to single-shock stimulation of the superior laryngeal nerve. During fictive swallowing elicited by superior laryngeal nerve stimulation, all neurons were basically inactive. During fictive vomiting induced by vagal stimulation and/or emetic drugs, all E-AUG and E-DEC neurons tested either were silent or fired weakly between successive retches, whereas I-DEC and I-CON neurons tested exhibited burst activity during the retching and early-expulsion phases. These results indicate that these bulbar respiratory neurons, which may be involved in respiratory rhythmogenesis, are multifunctional neurons that could also be involved in vomiting but not likely in swallowing.

Animals↗

PubMed1999

Cationic liposome-mediated DNA transfection in organotypic explant cultures of the ventral mesencephalon.

We have examined the potential of cationic liposomes as a tool for approaches to gene therapy in the CNS. Our previous work has shown that cationic liposomes formulated from 3 beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) and dioleoyl-L-alpha-phosphatidylethanolamine (DOPE) could achieve high transfection levels in a neuronal cell line (McQuillin et al. Neuroreport 1997; 8: 1481-1484). We therefore wished to assess transfection efficiencies in organotypic cultures from the brain with a reporter plasmid expressing E. coli beta-galactosidase in order to mimic an in vivo model. Explant cultures were generated according to the method of Stoppini et al (J Neurosci Meth 1991; 37: 173-182) with slight modifications. Brain slices were maintained on transparent porous membranes and were observed to maintain their intrinsic connectivity and cytoarchitecture to a large degree over periods of up to 6 weeks in culture. CNS tissue was obtained from rats at birth or 5 days after birth. After transfection beta-galactosidase expression was detected in cells of both neuronal and non-neuronal morphology. Control cultures were exposed to liposome alone and a plasmid that had the beta-galactosidase gene insert removed. Only low levels of endogenous beta-galactosidase reactivity were seen in these control cultures. DC-Chol/DOPE-mediated transfection was confirmed using a RT-PCR protocol capable of differentiating between untranscribed plasmid DNA and RNA generated from the transfected vector. These results suggest that cationic liposomes, particularly DC-Chol/DOPE liposomes, will be useful as delivery agents for gene transfer to CNS cells in vitro and possibly in vivo.

Animals↗

PubMed1999

Improved gene transfer into canine hematopoietic repopulating cells using CD34-enriched marrow cells in combination with a gibbon ape leukemia virus-pseudotype retroviral vector.

We have used dogs to study gene transfer into hematopoietic stem cells, because of the applicability of results in dogs to human transplantation and the availability of canine disease models that mimic human diseases. Previously we reported successful gene transfer into canine marrow repopulating cells, however, gene transfer efficiency was low, usually below 0.1% (Kiem et al, Hum Gene Ther 1996; 7: 89). In this study we have used CD34-enriched marrow cells to study different retroviral pseudotypes for their ability to transduce canine hematopoietic repopulating cells. Cells were divided into two equal fractions that were cocultivated for 72 h with irradiated packaging cells producing vector with different retroviral pseudotypes (GALV, amphotropic or 10A1). The vectors used contained small sequence differences to allow differentiation of cells genetically marked by the different vectors. Nonadherent and adherent cells from the cultures were infused into four dogs after a myeloablative dose of 920 cGy total body irradiation. Polymerase chain reaction (PCR) analysis of DNA from peripheral blood and marrow after transplant showed that the highest gene transfer rates (up to 10%) were obtained with the GALV-pseudotype vector. Gene transfer levels have remained stable now for more than 18 months. Southern blot analysis confirmed the high gene transfer rate. Interference studies on canine D17 cells revealed that 10A1 virus behaved like an amphotropic virus and was not able to use the GALV receptor. In summary, our results show improved gene transfer into canine hematopoietic repopulating cells when CD34-enriched cells are transduced by cocultivation on a GALV-pseudotype packaging cell line in combination with a GALV-pseudotype vector. Furthermore, these results demonstrate that the monoclonal antibody to canine CD34 used in this study is able to enrich for hematopoietic repopulating cells.

Animals↗

PubMed1999

Efficient transduction by an amphotropic retrovirus vector is dependent on high-level expression of the cell surface virus receptor.

Transduction by murine leukemia virus-based retrovirus vectors is limited in certain cell types, particularly in nondividing cells. But transduction can be inefficient even in cells that divide rapidly. For example, exposure of 208F rat embryo fibroblasts to an excess of an amphotropic retrovirus vector encoding alkaline phosphatase results in a transduction efficiency of only about 10%, even though these cells divide rapidly. Here we show that transduction of 208F cells is limited by cell surface retrovirus receptor levels; overexpression of the amphotropic retrovirus receptor Pit2 markedly improved the transduction efficiency to 50%. To characterize receptor levels and binding affinity, we synthesized a fusion protein that joins the amino terminus of the amphotropic envelope protein to the Fc region of a human immunoglobulin G1 molecule for use in binding assays. In comparison to the parental cell line, the modified cell line showed an order of magnitude increase in binding sites of from 18,000 to 150,000 per cell. Thus, efficient transduction by an amphotropic retrovirus vector requires high-level expression of the retrovirus receptor Pit2. These results provide the rationale for further examination of the role of receptor levels in inefficient transduction, especially with regard to target cells for gene therapy, where a high transduction rate is often crucial.

Animals↗

PubMed1999

Replication of Mus dunni endogenous retrovirus depends on promoter activation followed by enhancer multimerization.

Mus dunni endogenous virus (MDEV) is an apparently intact retrovirus that normally lies transcriptionally silent in cultured M. dunni cells, but the provirus can be activated by treatment of the cells with hydrocortisone or 5-iodo-2'-deoxyuridine. Sequence analysis of a molecular clone of the replicating virus revealed a simple retrovirus with a chimeric VL30/GALV-like structure. Interestingly, in the region of the long terminal repeat (LTR) that typically contains the retroviral transcription enhancers, we found over six 80-bp repeats with only a single mismatch, indicating that acquisition of the repeats was a recent event. Here we provide evidence for the following model of MDEV activation and replication. The MDEV provirus in M. dunni cells has a chimeric structure similar to that of the molecular clone but has only 1.15 copies of the 80-bp repeat sequence found in the molecular clone. Activating chemicals directly stimulate transcription from the LTR, allowing a low level of virus replication. Copying errors made during reverse transcription allow multimerization of the 80-bp enhancer region, resulting in viruses with higher transcriptional rates and improved fitness, but increased enhancer copy number is likely balanced by the natural instability of retroviral repeats and constraints imposed by virion packaging limits. The resultant population of replicating MDEV is widely heterogeneous, having from 2.15 to 13.15 enhancer repeats in the LTR. These results reveal a novel mechanism for regulation of transcription and replication of an endogenous retrovirus, in terms of both activation of the virus by the steroid hydrocortisone and the large number and variation in enhancer repeats observed.

Animals↗

PubMed1999

Central mechanisms of vomiting.

Nausea and vomiting (emesis) occur under a variety of conditions in response to activation of one or more emetic triggers. The act of vomiting is coordinated by neuronal circuitry located in the brain stem between the obex and the retrofacial nucleus, including the region extending from the nucleus of the solitary tract through the lateral tegmental field of the reticular formation to the ventrolateral medulla. The area postrema, medullary midline, and certain higher brain centers are also important for vomiting. The sensation of nausea is thought to involve the cerebral cortex. The most effective near-term treatment for combating nausea and vomiting associated with cyclic vomiting syndrome may come from experimental drugs (NK-1 receptor antagonists, 5-HT1A receptor agonists) or P6 acustimulation, which have been shown to combat nausea and vomiting in response to a broad spectrum of emetic challenges and thus presumably act on central emetic mechanisms.

Acupuncture Points↗

PubMed1998

Physiological basis and pharmacology of motion sickness: an update.

Motion sickness can occur when sensory inputs regarding body position in space are contradictory or are different from those predicted from experience. Signals from the vestibular system are essential for triggering motion sickness. The evolutionary significance of this malady is unclear, although it may simply represent the aberrant activation of vestibuloautonomic pathways that typically subserve homeostasis. The neural pathways that produce nausea and vomiting during motion sickness are presumed to be similar to those that generate illness after ingestion of toxins. The neural substrate of nausea is unknown but may include neurons in the hypothalamus and inferior frontal gyrus of the cerebral cortex. The principal motor act of vomiting is accomplished through the simultaneous contractions of inspiratory and expiratory respiratory muscles and is mediated by neurons in the lateral medullary reticular formation and perhaps by cells near the medullary midline. Cocontraction of the diaphragm and abdominal muscles increases pressure on the stomach, which causes gastric contents to be ejected through the mouth. Effective drugs for combating motion sickness include antihistamines, antimuscarinics, 5-HT1A (serotonergic) receptor agonists and neurokinin type 1 receptor antagonists. However, considerable information concerning the physiological basis and pharmacology of motion sickness is unknown; future research using animal models will be required to understand this condition.

Antiemetics↗

PubMed1998

Improved gene transfer into baboon marrow repopulating cells using recombinant human fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor.

We have used a competitive repopulation assay in baboons to develop improved methods for hematopoietic stem cell transduction and have previously shown increased gene transfer into baboon marrow repopulating cells using a gibbon ape leukemia virus (GALV)-pseudotype retroviral vector (Kiem et al, Blood 90:4638, 1997). In this study using GALV-pseudotype vectors, we examined additional variables that have been reported to increase gene transfer into hematopoietic progenitor cells in culture for their ability to increase gene transfer into baboon hematopoietic repopulating cells. Baboon marrow was harvested after in vivo administration (priming) of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF). CD34-enriched marrow cells were divided into two equal fractions to directly compare transduction efficiencies under different gene transfer conditions. Transduction by either incubation with retroviral vectors on CH-296-coated flasks or by cocultivation on vector-producing cells was studied in five animals; in one animal, transduction on CH-296 was compared with transduction on bovine serum albumin (BSA)-coated flasks. The highest level of gene transfer was obtained after 24 hours of prestimulation followed by 48 hours of incubation on CH-296 in vector-containing medium in the presence of multiple hematopoietic growth factors (interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor). Using these conditions, up to 20% of peripheral blood and marrow cells contained vector sequences for more than 20 weeks, as determined by both polymerase chain reaction and Southern blot analysis. Gene transfer rates were higher for cells transduced on CH-296 as compared with BSA or cocultivation. In one animal, we have used a vector expressing a cell surface protein (human placental alkaline phosphatase) and have detected 10% and 5% of peripheral blood cells expressing the transduced gene 2 and 4 weeks after transplantation as measured by flow cytometry. In conclusion, the conditions described here have resulted in gene transfer rates that will allow detection of transduced cells by flow cytometry to facilitate the evaluation of gene expression. The levels of gene transfer obtained with these conditions suggest the potential for therapeutic efficacy in diseases affecting the hematopoietic system.

Animals↗

PubMed1998

The expression of constitutively active isotypes of protein kinase C to investigate preconditioning.

The role of protein kinase C (PKC) in ischemic preconditioning remains controversial because of difficulties with both its measurement and pharmacological manipulation. We investigated preconditioning in isolated neonatal rat cardiocytes by expressing constitutively active isotypes of PKC. Observations at differing durations of simulated ischemia suggested beta-galactosidase (beta-gal) activity reflected viability within transfected myocytes. Preconditioning with 90 min of ischemia significantly increased beta-gal activity and myocyte survival after 6 h of ischemia; an effect abolished by PKC inhibitors. After co-transfection with plasmids encoding beta-gal and either constitutively active mutants of PKC-delta, PKC-alpha, wild type PKC-delta, or empty vector, cardiocytes were subjected to 6 h of ischemia. Only PKC-delta, rendered constitutively active by a limited deletion within the pseudosubstrate domain, consistently increased resistance to simulated ischemia (beta-gal activity was 85.6 +/- 11.9% versus 53.7 +/- 6.5% (p </= 0.01) and dead myocytes 46.8 +/- 3.4% versus 68.7 +/- 2.8% (p </= 0.01)). Since transfection was apparent in only 5-12% of cells, the results suggested a protective bystander effect that was confirmed by co-culture of transfected myocytes with untransfected myocytes. In neonatal cardiocytes expression of active PKC-delta increases resistance to simulated ischemia. This observation may provide further insight into the mechanism and possible avenues for therapeutic exploitation of preconditioning.

Animals↗

PubMed1998

Homogeneous Escherichia coli chaperonin 60 induces IL-1 beta and IL-6 gene expression in human monocytes by a mechanism independent of protein conformation.

Escherichia coli chaperonin (cpn) 60 (groEL) is a protein-folding oligomer lacking tryptophan residues that copurifies with tryptophan-containing proteins and peptides. Cpn 60 is a major immunogen in infectious diseases, and evidence suggests that groEL and mycobacterial cpn 60s can induce cytokine synthesis, stimulate cytokine-dependent bone resorption, and up-regulate expression of vascular endothelial cell adhesion molecules. Whether such activities are due to the cpn 60 or to the copurifying/contaminating proteins/peptides has not been determined. Here we report a method for removing the protein contaminants of groEL and demonstrate that this, essentially homogeneous, groEL remains a potent inducer of human monocyte IL-1beta and IL-6 production. Contaminating peptides had no cytokine-inducing activity and did not synergize with purified groEL. The LPS inhibitor polymyxin B and the CD14-neutralizing Ab MY4 had no inhibitory action on groEL demonstrating that activity is not due to LPS contamination. Heating groEL had no effect on its capacity to stimulate human monocytes to secrete IL-6. Proteolysis of groEL with trypsin, sufficient to produce low molecular mass peptides, also had no inhibitory effect. Thus, we conclude that groEL is a potent inducer of monocyte proinflammatory cytokine production, which acts through the binding of nonconformational peptide domains that are conserved after proteolysis. These data suggest that if groEL was released from bacteria it could induce prolonged tissue pathology by virtue of its cytokine-inducing activity and its resistance to proteolytic inhibition of bioactivity.

Antibodies, Monoclonal↗