ABSTRACT
Axial spondyloarthritis (axSpA) has been long classified as an autoimmune disease caused by a breakdown in the ability of the immune system to delineate self from foreign, resulting in self-reactive T cells. The strong genetic association of HLA-B27 supports this role for T cells. More recently, genetic and clinical studies indicate a prominent role of the environment in triggering axSpA, including an important role for microbes and the innate immune response. As an example, mutations in genes associated with innate immunity, including the anti-fungal signaling molecule Caspase recruitment domain-containing protein 9 (CARD9), have been linked to axSpA susceptibility. Thus, current thought classifies axSpA as a "mixed pattern condition" caused by both autoimmune and autoinflammatory mechanisms. The goal of this review is to convey.
ABSTRACT
INTRODUCTION: Beyond Compliance (BC) was introduced in 2012 to improve the monitoring and regulation of new medical devices and techniques, ensuring patient safety whilst promoting innovation through an evidence based appraisal of devices during their introduction. This study reports the 2 year outcomes of the first Total Knee Replacement (TKR) implant to be assessed through the BC process. METHODS: 100 consecutive patients undergoing primary knee arthroplasty were enrolled. All patients received a single radius cruciate retaining TKA (Unity, Corin), and the patella was resurfaced in all cases. Patients were followed up at 6 weeks, 3, 6, 12 and 24 months post operatively. Pre-and post-operative range of movement (ROM) as well as outcome scores including OKS, KOOS, EQ5D index and EQ5D VAS were recorded. RESULTS: 100 patients with a mean age 73.6 (SD = 8.7) were included. 2 patients died during the follow-up period due to unrelated reasons. Overall satisfaction rates were 96%. Complications included ongoing pain (5 patients), and a periprosthetic fracture (1 patient) nine months post-surgery (traumatic). No knees were revised during the follow-up period. Significant improvements were observed in all outcomes measures (OKS, KOOS, EQ5D, and EQ5D VAS). The mean added ROM was 13.2°. DISCUSSION: This knee prosthesis has been demonstrated to be safe and effective with excellent early outcomes. The careful regulated introduction of this device through BC has ensured patients safety while supporting innovation in knee arthroplasty. The success of BC requires surgeons to insist industry fully engage with the process for all new devices or techniques.
Subject(s)
Arthritis, Rheumatoid/surgery , Arthroplasty, Replacement, Knee/instrumentation , Knee Prosthesis , Osteoarthritis, Knee/surgery , Aged , Aged, 80 and over , Female , Follow-Up Studies , Humans , Male , Middle Aged , Patient Satisfaction , Prosthesis Design , Range of Motion, Articular , Time Factors , Treatment OutcomeABSTRACT
BACKGROUND: Longer operation times, poorer patient outcomes and increased early post-operative complications are reasons cited for not undertaking total knee arthroplasty (TKA) on morbidly obese patients. This study tests the hypothesis that there is no difference in intra-operative parameters between morbidly obese and non-obese patients, and no difference in patient outcome. METHODS: Intra-operative parameters, post-operative complications, patient outcomes and knee range of motion were compared between morbidly obese patients (BMI>40 kg/m2) and individually age and gender matched non-obese patients (BMI<30 kg/m2) undergoing cementless rotating platform TKA. RESULTS: Anaesthetic times and length of hospital stay were not significantly different between the morbidly obese and non-obese patients. Surgical time was significantly greater in morbidly obese patients. Improvements in patient outcomes following TKA were not significantly different between the morbidly obese and non-obese patients at early and short-term follow-up. CONCLUSIONS: In contrast to previous studies, post-operative complication rates within three months of surgery and up to one year post-operatively were not significantly higher for morbidly obese patients. LEVEL OF EVIDENCE: III.
Subject(s)
Arthroplasty, Replacement, Knee , Obesity, Morbid/complications , Operative Time , Postoperative Complications , Range of Motion, Articular , Adult , Aged , Case-Control Studies , Female , Follow-Up Studies , Humans , Male , Middle Aged , Osteoarthritis, Knee/surgery , Retrospective Studies , Young AdultABSTRACT
In an initial randomised controlled trial (RCT) we segregated 180 patients to one of two knee positions following total knee replacement (TKR): six hours of knee flexion using either a jig or knee extension. Outcome measures included post-operative blood loss, fall in haemoglobin, blood transfusion requirements, knee range of movement, limb swelling and functional scores. A second RCT consisted of 420 TKR patients randomised to one of three post-operative knee positions: flexion for three or six hours post-operatively, or knee extension. Positioning of the knee in flexion for six hours immediately after surgery significantly reduced blood loss (p = 0.002). There were no significant differences in post-operative range of movement, swelling, pain or outcome scores between the various knee positions in either study. Post-operative knee flexion may offer a simple and cost-effective way to reduce blood loss and transfusion requirements following TKR. We also report a cautionary note regarding the potential risks of prolonged knee flexion for more than six hours observed during clinical practice in the intervening period between the two trials, with 14 of 289 patients (4.7%) reporting lower limb sensory neuropathy at their three-month review.
Subject(s)
Arthroplasty, Replacement, Knee , Blood Loss, Surgical/prevention & control , Knee Joint/physiopathology , Posture , Range of Motion, Articular/physiology , Aged , Blood Loss, Surgical/physiopathology , Blood Transfusion/methods , Female , Follow-Up Studies , Humans , Male , Postoperative Period , Prospective Studies , Treatment OutcomeABSTRACT
AIMS: Recent NHS reforms have incentivised reduction in length of stay, with the UK department of health expecting health trusts to reduce bed days and ultimately reduce overall costs. The aim of this study was to identify avoidable causes for protracted hospital admission following total hip arthroplasty (THA) or total knee arthroplasty (TKA) within a fast-track unit. METHODS: During a 6-month period, 535 consecutive patients underwent primary THA or TKA under the care of a single surgeon. All patients with a post-operative stay of greater than 72 h were identified, and reasons for delayed discharge were determined. RESULTS: The majority of arthroplasty patients were discharged within 3 days post-operatively. Twenty-one per cent of THA patients and 25 % of TKA patients remained as inpatients for greater than 72 h. For the THA population, this equates to 43 % of bed days used by 21 % of patients, and for the TKA population, 44 % of bed days were used by 25 % of patients. The major factor within both groups for delayed discharge was attributed to inadequate social support. CONCLUSIONS: Delayed discharge can never be totally prevented. This unit aims to develop improvement in social work provision, with a greater focus on pre-admission discharge planning to reduce the number of delayed discharges and ultimately reduce the cost burden of joint replacement surgery. It is not conducive with the ethos of fast-track arthroplasty to only identify social circumstances upon admission.
Subject(s)
Arthroplasty, Replacement, Hip/statistics & numerical data , Arthroplasty, Replacement, Knee/statistics & numerical data , Length of Stay/statistics & numerical data , Patient Discharge/statistics & numerical data , Aged , Aged, 80 and over , Arthroplasty, Replacement, Hip/economics , Arthroplasty, Replacement, Knee/economics , Cost of Illness , Female , Health Services Research , Humans , Male , Middle Aged , Regression Analysis , Treatment OutcomeABSTRACT
The violent forces generated by the strong paraspinal muscles during generalised tonic-clonic seizures are known to be associated with numerous musculoskeletal injuries. Vertebral fractures following epileptic seizures are typically compression fractures, occurring in the mid thoracic region, usually without resultant neurological deficit. Despite the relative frequency of vertebral compression fractures complicating tonic-clonic seizures, burst fractures are rare. We report a case of a burst fracture resulting in conus medullaris syndrome following generalised motor seizures. Clinicians managing post-ictal patients should maintain a high degree of suspicion for secondary injury and conduct a detailed clinical examination. If there remains any doubt regarding diagnosis, appropriate imaging should be employed.
Subject(s)
Lumbar Vertebrae/injuries , Seizures/complications , Spinal Fractures/diagnosis , Spinal Fractures/etiology , Adult , Humans , Male , Spinal Cord Compression/diagnosis , Spinal Cord Compression/etiology , Spinal Cord Compression/therapy , Spinal Fractures/therapyABSTRACT
There have been few examples of the application of our growing knowledge of hormone action to crop improvement. In this review we discuss what is known about the critical points regulating auxin action. We examine auxin metabolism, transport, perception and signalling and identify genes and proteins that might be keys to regulation, particularly the rate-limiting steps in various pathways. Certain mutants show that substrate flow in biosynthesis can be limiting. To date there is little information available on the genes and proteins of catabolism. There have been several auxin transport proteins and some elegant transport physiology described recently, and the potential for using transport proteins to manage free indole-3-acetic acid (IAA) concentrations is discussed. Free IAA is very mobile, and so while it may be more practical to control auxin action through managing the receptor and signalling pathways, the candidate genes and proteins through which this can be done remain largely unknown. From the available evidence, it is clear that the reason for so few commercial applications arising from the control of auxin action is that knowledge is still limited.
Subject(s)
Indoleacetic Acids/biosynthesis , Plants/metabolism , Biological Transport , Gene Expression Regulation, Developmental , Homeostasis , Indoleacetic Acids/metabolism , Plant Development , Plants/genetics , Signal Transduction/physiologyABSTRACT
With a polyclonal antibody raised against calreticulin (CRT) the locations where the protein occurs in unpollinated and pollinated styles of Petunia hybrida were localized. The epitopes binding the CRT antibody were immunolocalized preferentially in pollen tubes. In transmitting tract cells, both before and after pollination, the level of CRT was low. The protein was mainly localized in the cytosol and around dictyosomes of transmitting-tract cells. In pollen tubes, a high level of CRT was found at their tips rich in endoplasmatic reticulum, cisternae piles of reticular and/or dictyosomal origin, and vesicles. Binding sites of the CRT antibody were also found in the internal callosic cell wall of the pollen tube. These results indicate a role of CRT in cells directly participating in pollen-pistil interaction.
Subject(s)
Calcium-Binding Proteins/analysis , Calcium-Binding Proteins/immunology , Immunohistochemistry/methods , Magnoliopsida/anatomy & histology , Magnoliopsida/chemistry , Plant Proteins/analysis , Plant Proteins/immunology , Pollen/chemistry , Ribonucleoproteins/analysis , Ribonucleoproteins/immunology , Antibodies/immunology , Blotting, Western , Calcium-Binding Proteins/chemistry , Calreticulin , Epitopes/immunology , Magnoliopsida/immunology , Magnoliopsida/ultrastructure , Microscopy, Electron , Plant Proteins/chemistry , Pollen/immunology , Pollen/ultrastructure , Ribonucleoproteins/chemistryABSTRACT
The hormone auxin is transported in plants through the combined actions of diffusion and specific auxin influx and efflux carriers. In contrast to auxin efflux, for which there are well documented inhibitors, understanding the developmental roles of carrier-mediated auxin influx has been hampered by the absence of specific competitive inhibitors. However, several molecules that inhibit auxin influx in cultured cells have been described recently. The physiological effects of two of these novel influx carrier inhibitors, 1-naphthoxyacetic acid (1-NOA) and 3-chloro-4-hydroxyphenylacetic acid (CHPAA), have been investigated in intact seedlings and tissue segments using classical and new auxin transport bioassays. Both molecules do disrupt root gravitropism, which is a developmental process requiring rapid auxin redistribution. Furthermore, the auxin-insensitive and agravitropic root-growth characteristics of aux1 plants were phenocopied by 1-NOA and CHPAA. Similarly, the agravitropic phenotype of inhibitor-treated seedlings was rescued by the auxin 1-naphthaleneacetic acid, but not by 2,4-dichlorophenoxyacetic acid, again resembling the relative abilities of these two auxins to rescue the phenotype of aux1. Further investigations have shown that none of these compounds block polar auxin transport, and that CHPAA exhibits some auxin-like activity at high concentrations. Whilst results indicate that 1-NOA and CHPAA represent useful tools for physiological studies addressing the role of auxin influx in planta, 1-NOA is likely to prove the more useful of the two compounds.
Subject(s)
Arabidopsis Proteins , Glycolates/pharmacology , Indoleacetic Acids/antagonists & inhibitors , Mutation , Phenylacetates/pharmacology , Plant Proteins/genetics , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis/physiology , Biological Transport , Gravitropism , Indoleacetic Acids/metabolism , Phenotype , Plant Roots/growth & developmentABSTRACT
To explore the role of auxin-binding protein (ABP1) in planta, a number of transgenic tobacco (Nicotiana tabacum) lines were generated. The wild-type KDEL endoplasmic reticulum targeting signal was mutated to HDEL, another common retention sequence in plants, and to KEQL or KDELGL to compromise its activity. The auxin-binding kinetics of these forms of ABP1 were found to be similar to those of ABP1 purified from maize (Zea mays). To test for a physiological response mediated by auxin, intact guard cells of the transgenic plants were impaled with double-barreled microelectrodes, and auxin-dependent changes in K(+) currents were recorded under voltage clamp. Exogenous auxin affected inwardly and outwardly rectifying K(+) currents in a dose-dependent manner. Auxin sensitivity was markedly enhanced in all plants overexpressing ABP1, irrespective of the form present. Immunogold electron microscopy was used to investigate the localization of ABP1 in the transgenic plants. All forms were detected in the endoplasmic reticulum and the KEQL and KDELGL forms passed further across the Golgi stacks than KDEL and HDEL forms. However, neither electron microscopy nor silver-enhanced immunogold epipolarization microscopy revealed differences in cell surface ABP1 abundance for any of the plants, including control plants, which indicated that overexpression of ABP1 alone was sufficient to confer increased sensitivity to added auxin. Jones et al. ([1998] Science 282: 1114-1117) found increased cell expansion in transgenic plants overexpressing wild-type ABP1. Single cell recordings extend this observation, with the demonstration that the auxin sensitivity of guard cell K(+) currents is mediated, at least in part, by ABP1.
Subject(s)
Indoleacetic Acids/metabolism , Nicotiana/metabolism , Plant Proteins/metabolism , Plants, Toxic , Receptors, Cell Surface/metabolism , Blotting, Northern , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Immunohistochemistry , Microscopy, Immunoelectron , Mutagenesis, Site-Directed , Mutation , Patch-Clamp Techniques , Plant Leaves/metabolism , Plant Leaves/ultrastructure , Plants, Genetically Modified , Polymerase Chain Reaction , Potassium/metabolism , RNA, Messenger/metabolism , RNA, Plant/metabolism , Receptors, Cell Surface/genetics , Nicotiana/ultrastructureABSTRACT
Auxin-binding protein (ABP1) is an important receptor for the plant hormone auxin that is involved with many growth and developmental responses in plants. The maize ABP1 gene has been expressed in insect cells, purified and crystallized. Type II crystals are monoclinic, with two glycosylated homodimers in the asymmetric unit, and diffract to 1.9 A using synchrotron radiation.
Subject(s)
Plant Growth Regulators , Plant Proteins , Receptors, Cell Surface/chemistry , Crystallization , Crystallography, X-Ray , Protein ConformationABSTRACT
Using a polyclonal antibody raised against calreticulin purified and sequenced from maize, we performed an immunocytological study to characterize putative domain-specific subcellular distributions of endoplasmic reticulum (ER)-resident calreticulin in meristematic cells of maize root tip. At the light microscopy level, calreticulin was immunolocalized preferentially at cellular peripheries, in addition to nuclear envelopes and cytoplasmic structures. Punctate labelling at the longitudinal walls and continuous labelling at the transverse walls was characteristic. Immunogold electron microscopy revealed plasmodesmata as the most prominently labelled cell periphery structure. In order to further probe the ER-domain-specific distribution of maize calreticulin at plasmodesmata, root apices were exposed to mannitol-induced osmotic stress. Plasmolysis was associated with prominent accumulations of calreticulin at callose-enriched plasmodesmata and pit fields while the contracting protoplasts were depleted of calreticulin. In contrast, other ER-resident proteins recognized by HDEL peptide and BiP antibodies localized exclusively to contracted protoplasts. This finding reveals that, in plasmolysed cells, calreticulin enriched ER domains at plasmodesmata and pit fields are depleted of other ER-resident proteins containing the HDEL retention peptide.
Subject(s)
Calcium-Binding Proteins/analysis , Intercellular Junctions/chemistry , Plant Root Cap/cytology , Ribonucleoproteins/analysis , Zea mays/cytology , Antibodies , Blotting, Western , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/physiology , Calreticulin , Cell Wall/chemistry , Cytoplasm/chemistry , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/drug effects , Fluorescent Antibody Technique , Intercellular Junctions/drug effects , Intercellular Junctions/ultrastructure , Mannitol/pharmacology , Microscopy, Electron , Molecular Weight , Nuclear Envelope/chemistry , Oligopeptides/genetics , Osmotic Pressure/drug effects , Plant Root Cap/chemistry , Plant Root Cap/drug effects , Plant Root Cap/ultrastructure , Protein Sorting Signals , Protoplasts/chemistry , Protoplasts/cytology , Protoplasts/drug effects , Ribonucleoproteins/chemistry , Ribonucleoproteins/physiology , Zea mays/chemistry , Zea mays/drug effects , Zea mays/ultrastructureABSTRACT
The localisation of maize (Zea mays L.) auxin-binding protein (ABP1) has been studied using a variety of techniques. At the whole-tissue level, tissue printing indicated that ABP1 is expressed to similar levels in all cells of the maize coleoptile and in the enclosed leaf roll. Within cells, the signals from immunofluorescence and immunogold labelling of ultrathin sections both indicated that ABP1 is confined to the endoplasmic reticulum (ER), none being detected in either Golgi apparatus or cell wall. This distribution is consistent with targeting motifs in its sequence. These observations are discussed with reference to the various reports which place a population of ABP1 on the outer face of the plasma membrane, including those suggesting that it is necessary on the cell surface for rapid, auxin-mediated protoplast hyperpolarisation. We have tested the ER, namely that auxin binding induces a conformational change in ABP1 leading to concealment of the KDEL retention motif. Using double-label immunofluorescence the characteristic auxin-induced rise in Golgi-apparatus signal was found, yet no change in the distribution of the ABP1 signal was detected. Maize suspension cultures were used to assay for auxin-promoted secretions of ABP1 into the medium, but secretion was below the limit of detection. This can be ascribed at least partly to the very active acidification of the medium by these cells and the instability of ABP 1 in solution below pH 5.0. In the insect-baculovirus expression system, in which cell cultures maintain pH 6.2, a small amount of ABP1 secretion, less than 1% of the total, was detected under all conditions, Insect cells were shown to take up auxin and no inactivation of added auxin was detected, but auxin did not affect the level of ABP1 in the medium. Consequently, no evidence was found to support the model for auxin promotion of ABP1 secretion. Finally, quantitative glycan analysis was used to determine what proportion of ABP1 might reach the plasma membrane in maize coleoptile tissue. The results suggest that less than 15% of ABP1 ever escapes from the ER as far as the cis-Golgi and less than 2% passes further through the secretory pathway. Such leakage rates probably do not require a specialised mechanism allowing ABP1 past the KDEL retrieval pathway, but we are not able to rule out the possibility that some ABP1 is carried through associated with other proteins. The data are consistent with the presence of ABP1 both on the plasma membrane and in the ER. The relative sizes of the two pools explain the results obtained with immunofluorescence and immunogold labelling and illustrate the high efficiency of ER retention in plants.
Subject(s)
Endoplasmic Reticulum/metabolism , Indoleacetic Acids/metabolism , Plant Growth Regulators , Plant Proteins , Receptors, Cell Surface/metabolism , Zea mays/metabolism , Animals , Polysaccharides/metabolism , RabbitsABSTRACT
High level expression of the major auxin-binding protein (ABP1) from maize (Zea mays L.) has been used to demonstrate that the machinery for retaining proteins in the endoplasmic reticulum (ER) of insect cells functions efficiently throughout the baculovirus infection cycle. Immunolocalization showed wild-type ABP1 (ABP1-KDEL) to be targeted to the lumen of the ER, in accordance with its signal peptide and carboxyterminal KDEL ER-retention signal. The protein accumulated in dilations of the ER, and none was detected at the cell surface. Immunoblotting of concentrated culture medium confirmed that ABP1-KDEL was not secreted at a detectable level. In contrast, when the carboxyterminus was mutated to KEQL, secretion of the baculovirus-expressed protein was readily detected. Immunolocalization and immunoblotting demonstrated that a high proportion of the ABP1-KEQL protein was secreted at the cell surface and into the culture medium. The data demonstrate that the ER of insect cells has a great capacity to retain proteins and that this property is largely unaffected by the cellular disruption caused by baculovirus replication.
Subject(s)
Endoplasmic Reticulum/metabolism , Proteins/metabolism , Spodoptera/metabolism , Amino Acid Sequence , Animals , Baculoviridae/genetics , Baculoviridae/ultrastructure , Base Sequence , Cell Line , DNA, Complementary/genetics , Endoplasmic Reticulum/ultrastructure , Microscopy, Immunoelectron , Mutagenesis, Site-Directed , Plant Proteins/genetics , Plant Proteins/metabolism , Plasmids/genetics , Proteins/genetics , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spodoptera/ultrastructure , Spodoptera/virology , Zea mays/geneticsABSTRACT
To achieve continuous expression of the major maize auxin-binding protein (ABP1) in insect cells, the ABP1 gene coding region was placed under control of a baculovirus immediate-early gene promoter and transfected into Spodoptera frugiperda Sf9 cells. The ABP1 gene was detected in twelve cell lines, one of which was selected for detailed analysis. Immunolocalisation demonstrated that ABP1 was targeted to and retained in the endoplasmic reticulum (ER), in accordance with its signal peptide and carboxy-terminal KDEL ER-retention signal. We discuss the advantages of stable-transformation over transient expression systems for characterising proteins targeted to the secretory system of insect cells.
Subject(s)
Indoleacetic Acids , Plant Growth Regulators , Plant Proteins/genetics , Receptors, Cell Surface/genetics , Zea mays/genetics , Animals , Baculoviridae/genetics , Base Sequence , Cell Line, Transformed , Gene Expression Regulation, Plant , Genetic Vectors , Molecular Sequence Data , Oligodeoxyribonucleotides , Plant Proteins/biosynthesis , Receptors, Cell Surface/biosynthesis , Spodoptera , Transformation, GeneticABSTRACT
Electrophysiological experiments have indicated that a fraction of the major auxin-binding protein (ABP1) of maize (Zea mays L.) might be a receptor on the outer surface of the plasma membrane. The predominant location of ABP1 is in the lumen of the endoplasmic reticulum (ER), in accord with its C-terminal KDEL retention signal. Little is known about the biology of the protein in vivo or the rate at which it might pass to the cell surface. We have examined the turnover of ABP1 by in vivo labelling of maize coleoptile sections. After different chase times, ABP1 was immunoprecipitated from detergent-solubilised membrane preparations. Two polypeptides coprecipitated with ABP1. Neither was recognised by any ABP1 antibodies nor by monoclonals to ER retention sequences. The possible significance of these coprecipitating polypeptides is discussed. In addition, we have used a monoclonal antibody to precipitate HDEL proteins from the same membrane preparations. Two dimensional electrophoresis and N-terminal sequencing showed that the major HDEL protein precipitated was a member of the heat-shock-protein 70 family, a homologue of BiP (immunoglobulin-binding protein). We have investigated the turnover of this BiP homologue for comparison with ABP1 and found that both had extended lifetimes, with half-lives greater than 24 h. Use of cordycepin to inhibit transcription indicated that ABP1 mRNA was also long-lived. Synthesis of ABP1 was strongly reduced by heat stress, was reduced a little in response to dithiothreitol and was not markedly changed by tunicamycin. In contrast, BiP synthesis increased markedly in response to tunicamycin and dithiothreitol and increased a little after heat stress.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Carrier Proteins/metabolism , Plant Proteins/metabolism , Receptors, Cell Surface/metabolism , Amino Acid Sequence , Arabidopsis Proteins , Cell Membrane/metabolism , Endoplasmic Reticulum/metabolism , Heat-Shock Proteins/metabolism , Molecular Sequence Data , Plant Growth Regulators/metabolism , Precipitin Tests , RNA, Messenger/metabolism , Time Factors , Zea maysABSTRACT
The major auxin-binding protein (ABP1) from maize (Zea mays L.) has been expressed in insect cells using the baculovirus expression system. The recombinant protein can be readily detected in total insect cell lysates by Coomassie blue staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Our data suggest that ABP1 is processed similarly in both insect cells and maize. The signal peptide is cleaved at the same position as in maize and the mature protein undergoes tunicamycin-sensitive glycosylation, yielding a product with the same mobility on SDS-PAGE as authentic maize ABP1. On immunoblots the expressed protein is recognized by anti-KDEL monoclonal antibodies. Immunofluorescence localization demonstrates that it is targeted to and retained in the endoplasmic reticulum of insect cells in accordance with its signal peptide and KDEL retention sequence. The expressed ABP1 also appears to be active, since extracts of insect cells expressing ABP1 contain a saturable high-affinity 1-naphthylacetic acid-binding site, whereas no saturable auxin-binding activity is detected in extracts from control cells.
Subject(s)
Plant Growth Regulators , Plant Proteins/biosynthesis , Receptors, Cell Surface/biosynthesis , Zea mays/metabolism , Amino Acid Sequence , Animals , Baculoviridae , Base Sequence , DNA Primers , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique , Genetic Vectors , Indoleacetic Acids/metabolism , Molecular Sequence Data , Molecular Weight , Polymerase Chain Reaction , Protein Sorting Signals/metabolism , Receptors, Cell Surface/isolation & purification , Receptors, Cell Surface/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spodoptera , TransfectionABSTRACT
The major auxin-binding protein in maize membranes is thought to function as a physiological receptor. From earlier information, including the use of site-directed irreversible inhibitors, several of the amino acids likely to form part of the active auxin-binding site were provisionally assigned. Inspection of the amino acid sequence of the auxin-binding protein showed a short region containing all but one of these amino acids. We find that antisera raised against a synthetic peptide encompassing this region recognize all isoforms of the maize auxin-binding protein together with homologous polypeptides in other species. We further find that the antibodies hyperpolarize protoplast transmembrane potential in an auxin-like manner. We conclude that these antibodies display auxin agonist activity and that we have identified an essential portion of the auxin-binding site.
Subject(s)
Antibodies , Indoleacetic Acids/metabolism , Plant Growth Regulators , Plant Proteins , Receptors, Cell Surface/metabolism , Zea mays/physiology , Amino Acid Sequence , Intracellular Membranes/drug effects , Intracellular Membranes/physiology , Membrane Proteins/isolation & purification , Microsomes/drug effects , Microsomes/physiology , Molecular Sequence Data , Molecular Weight , Naphthaleneacetic Acids/pharmacology , Peptides/chemical synthesis , Peptides/immunology , Plant Physiological Phenomena , Protoplasts/physiology , Receptors, Cell Surface/immunology , Receptors, Cell Surface/isolation & purificationABSTRACT
The epitopes of two monoclonal antibodies raised to a putative auxin receptor have been mapped. Carboxy-peptidase A digestion of the antigen, auxin-binding protein (ABP) purified from maize, completely abolished binding of antibody MAC 256 and impaired binding of MAC 259, suggesting that they both recognise C-terminal epitopes. Published sequences of ABP showed that the C terminus was KDEL, a tetrapeptide used for targeting proteins to the ER in animal cells. We have used this short homology to confirm that the two monoclonals recognise C-terminal KDEL, showing that animal KDEL proteins and synthetic KDEL peptides are recognised and that animal cell ER is stained strongly and specifically. Sucrose density gradient fractionation of maize microsomal membranes showed that plant KDEL proteins, including ABP, fractionated with markers for the endoplasmic reticulum. However, few proteins are stained by anti-KDEL monoclonals in plants. For comparison, a monoclonal antibody raised to a synthetic HDEL peptide was also used and found to stain a set of proteins in all plant species tested. The anti-HDEL and anti-KDEL monoclonals were sequence specific, staining different proteins. On density gradient fractionation HDEL proteins also banded with ER marker activities. However, the intracellular distribution of HDEL and KDEL proteins determined by immunofluorescence was different. Whereas HDEL proteins showed a distribution characteristic of plant ER, and this localisation was confirmed by immunogold labelling of ultrathin sections and electron microscopy, KDEL proteins showed strong fluorescence in discrete parts of the cell cortex. These observations are discussed in terms of the potential these monoclonal antibodies have as markers for ER and of the role ABP plays in plant cell signalling.