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PubMed2001

Interaction between the P14 residue and strand 2 of beta-sheet B is critical for reactive center loop insertion in plasminogen activator inhibitor-2.

The molecular interactions driving reactive center loop (RCL) insertion are of considerable interest in gaining a better understanding of the serpin inhibitory mechanism. Previous studies have suggested that interactions in the proximal hinge/breach region may be critical determinants of RCL insertion in serpins. In this study, conformational and functional changes in plasminogen activator inhibitor-2 (PAI-2) following incubation with a panel of synthetic RCL peptides indicated that the P14 residue is critical for RCL insertion, and hence inhibitory activity, in PAI-2. Only RCL peptides with a P14 threonine were able to induce the stressed to relaxed transition and abolish inhibitory activity in PAI-2, indicating that RCL insertion into beta-sheet A of PAI-2 is dependent upon this residue. The recently solved crystal structure of relaxed PAI-2 (PAI-2.RCL peptide complex) allowed detailed analysis of molecular interactions involving P14 related to RCL insertion. Of most interest is the rearrangement of hydrogen bonding around the breach region that accompanies the stressed to relaxed transition, in particular the formation of a side chain hydrogen bond between the threonine at P14 and an adjacent tyrosine on strand 2 of beta-sheet B in relaxed PAI-2. Structural alignment of known serpin sequences showed that this pairing (or the equivalent serine/threonine pairing) is highly conserved ( approximately 87%) in inhibitory serpins and may represent a general structural basis for serpin inhibitory activity.

Amino Acids↗
PubMed2002

Chloroplast YidC homolog Albino3 can functionally complement the bacterial YidC depletion strain and promote membrane insertion of both bacterial and chloroplast thylakoid proteins.

A new component of the bacterial translocation machinery, YidC, has been identified that specializes in the integration of membrane proteins. YidC is homologous to the mitochondrial Oxa1p and the chloroplast Alb3, which functions in a novel pathway for the insertion of membrane proteins from the mitochondrial matrix and chloroplast stroma, respectively. We find that Alb3 can functionally complement the Escherichia coli YidC depletion strain and promote the membrane insertion of the M13 procoat and leader peptidase that were previously shown to depend on the bacterial YidC for membrane translocation. In addition, the chloroplast Alb3 that is expressed in bacteria is essential for the insertion of chloroplast cpSecE protein into the bacterial inner membrane. Surprisingly, Alb3 is not required for the insertion of cpSecE into the thylakoid membrane. These results underscore the importance of Oxa1p homologs for membrane protein insertion in bacteria and demonstrate that the requirement for Oxa1p homologs is different in the bacterial and thylakoid membrane systems.

Arabidopsis↗
PubMed2002

Specificity of Rho insert-mediated activation of phospholipase D1.

Members of the Rho subfamily of GTP-binding proteins regulate phospholipase D1 (PLD1) activity and signaling. In previous work, we demonstrated that binding of the Rho family member Cdc42 to PLD1 and the subsequent stimulation of its enzymatic activity are distinct events. Deletion of the insert helix from Cdc42 does not interfere with its switch I-mediated, GTP-dependent binding to PLD1 but inhibits Cdc42-stimulated PLD1 activity. To understand the mechanism of the insert-mediated activation of PLD1 by Cdc42 and to develop reagents to study Cdc42-activated PLD1 in cellular signaling events, we have undertaken a mutational analysis of the Rho insert region of Cdc42 and examined the specificity of the insert helix requirement in the other Rho family members, RhoA and Rac1. Here, we identify a critical residue, serine 124, in the Cdc42 insert helix central to its activation mechanism. Further, we examine this activation mechanism with respect to other members of the Rho family and demonstrate that each Rho protein activates PLD by distinct mechanisms, potentially allowing for unique signaling outcomes in the cell.

Amino Acid Substitution↗
PubMed2002

Functional consequences of insertions and deletions in the complementarity-determining regions of human antibodies.

Insertions and deletions of nucleotides in the genes encoding the variable domains of antibodies are natural components of the hypermutation process, which may expand the available repertoire of hypervariable loop lengths and conformations. Although insertion of amino acids has also been utilized in antibody engineering, little is known about the functional consequences of such modifications. To investigate this further, we have introduced single-codon insertions and deletions as well as more complex modifications in the complementarity-determining regions of human antibody fragments with different specificities. Our results demonstrate that single amino acid insertions and deletions are generally well tolerated and permit production of stably folded proteins, often with retained antigen recognition, despite the fact that the thus modified loops carry amino acids that are disallowed at key residue positions in canonical loops of the corresponding length or are of a length not associated with a known canonical structure. We have thus shown that single-codon insertions and deletions can efficiently be utilized to expand structure and sequence space of the antigen-binding site beyond what is encoded by the germline gene repertoire.

Amino Acid Sequence↗
PubMed2002

Tail-anchored and signal-anchored proteins utilize overlapping pathways during membrane insertion.

Tail-anchored proteins are a distinct class of membrane proteins that are characterized by a C-terminal membrane insertion sequence and a capacity for post-translational integration. Although it is now clear that tail-anchored proteins are inserted into the membrane at the endoplasmic reticulum (ER), the molecular basis for their integration is poorly understood. We have used a cross-linking approach to identify ER components that may be involved in the membrane insertion of tail-anchored proteins. We find that several newly synthesized tail-anchored proteins are transiently associated with a defined subset of cellular components. Among these, we identify several ER proteins, including subunits of the Sec61 translocon, Sec62p, Sec63p, and the 25-kDa subunit of the signal peptidase complex. When we analyze the cotranslational membrane insertion of a comparable signal-anchored protein we find the nascent polypeptide associated with a similar set of ER components. We conclude that the pathways for the integration of tail-anchored and signal-anchored membrane proteins at the ER exhibit a substantial degree of overlap, and we propose that this reflects similarities between co- and post-translational membrane insertion.

Cell Membrane↗
PubMed2003

In vitro synthesis of lactose permease to probe the mechanism of membrane insertion and folding.

Insertion and folding of polytopic membrane proteins is an important unsolved biological problem. To study this issue, lactose permease, a membrane transport protein from Escherichia coli, is transcribed, translated, and inserted into inside-out membrane vesicles in vitro. The protein is in a native conformation as judged by sensitivity to protease, binding of a monoclonal antibody directed against a conformational epitope, and importantly, by functional assays. By exploiting this system it is possible to express the N-terminal six helices of the permease (N(6)) and probe changes in conformation during insertion into the membrane. Specifically, when N(6) remains attached to the ribosome it is readily extracted from the membrane with urea, whereas after release from the ribosome or translation of additional helices, those polypeptides are not urea extractable. Furthermore, the accessibility of an engineered Factor Xa site to Xa protease is reduced significantly when N(6) is released from the ribosome or more helices are translated. Finally, spontaneous disulfide formation between Cys residues at positions 126 (Helix IV) and 144 (Helix V) is observed when N(6) is released from the ribosome and inserted into the membrane. Moreover, in contrast to full-length permease, N(6) is degraded by FtsH protease in vivo, and N(6) with a single Cys residue at position 148 does not react with N-ethylmaleimide. Taken together, the findings indicate that N(6) remains in a hydrophilic environment until it is released from the ribosome or additional helices are translated and continues to fold into a quasi-native conformation after insertion into the bilayer. Furthermore, there is synergism between N(6) and the C-terminal half of permease during assembly, as opposed to assembly of the two halves as independent domains.

ATP-Dependent Proteases↗
PubMed2003

Is the Trypanosoma brucei REL1 RNA ligase specific for U-deletion RNA editing, and is the REL2 RNA ligase specific for U-insertion editing?

It was shown previously that the REL1 mitochondrial RNA ligase in Trypanosoma brucei was a vital gene and disruption affected RNA editing in vivo, whereas the REL2 RNA ligase gene could be down-regulated with no effect on cell growth or on RNA editing. We performed down-regulation of REL1 in procyclic T. brucei (midgut insect forms) by RNA interference and found a 40-50% inhibition of Cyb editing, which has only U-insertions, as well as a similar inhibition of ND7 editing, which has both U-insertions and U-deletions. In addition, both U-insertion and U-deletion in vitro pre-cleaved editing were inhibited to similar extents. We also found little if any effect of REL1 down-regulation on the sedimentation coefficient or abundance of the RNA ligase-containing L-complex (Aphasizhev, R., Aphasizheva, I., Nelson, R. E., Gao, G., Simpson, A. M., Kang, X., Falick, A. M., Sbicego, S., and Simpson, L. (2003) EMBO J. 22, 913-924), suggesting that the inhibition of both insertion and deletion editing was not due to a disruption of the L-complex. Together with the evidence that down-regulation of REL2 has no effect on cell growth or on RNA editing in vivo or in vitro, these data suggest that the REL1 RNA ligase may be active in vivo in both U-insertion and U-deletion editing. The in vivo biological role of REL2 remains obscure.

Animals↗
PubMed2005

NarJ chaperone binds on two distinct sites of the aponitrate reductase of Escherichia coli to coordinate molybdenum cofactor insertion and assembly.

Understanding when and how metal cofactor insertion occurs into a multisubunit metalloenzyme is of fundamental importance. Molybdenum cofactor insertion is a tightly controlled process that involves specific interactions between the proteins that promote cofactor delivery, enzyme-specific chaperones, and the apoenzyme. In the assembly pathway of the multisubunit molybdoenzyme, membrane-bound nitrate reductase A from Escherichia coli, a NarJ-assisted molybdenum cofactor (Moco) insertion step, must precede membrane anchoring of the apoenzyme. Here, we have shown that the NarJ chaperone interacts at two distinct binding sites of the apoenzyme, one interfering with its membrane anchoring and another one being involved in molybdenum cofactor insertion. The presence of the two NarJ-binding sites within NarG is required to ensure productive formation of active nitrate reductase. Our findings supported the view that enzyme-specific chaperones play a central role in the biogenesis of multisubunit molybdoenzymes by coordinating subunits assembly and molybdenum cofactor insertion.

Binding Sites↗
PubMed2006

Insertion of the enteropathogenic Escherichia coli Tir virulence protein into membranes in vitro.

Insertion of the enteropathogenic Escherichia coli Tir protein into the plasma membrane of intestinal epithelial cells is a crucial event in infection because it provides a receptor for intimate bacterial adherence. This interaction with the bacterial outer membrane protein intimin is also essential in generating a number of signaling activities associated with virulence. Tir can be modified at various sites by phosphorylation and functionally interacts with multiple host proteins. To investigate the mechanism of membrane insertion and to establish a model system in which the multiple interactions/functions of Tir can be uncoupled and independently characterized, we used intrinsic tryptophan fluorescence, surface plasmon resonance, and protease digestion assays to show that Tir can insert directly into phospholipid vesicles in a composition-dependent manner to generate the topology reported in vivo. This is the first time that Tir has been shown to insert into membranes in a simple model system in the absence of chemical modification or other factors. These data are consistent with the protein interacting with lipids through two sites. The major site is localized to the transmembrane/intimin-binding domain region and includes Trp235, which is shown to be an effective reporter of interaction. The minor site is located within the C-terminal domain. Together, these data support a model in which Tir is released into the cytoplasm by the type III translocon and then independently inserts into the plasma membrane from a cytoplasmic location. A thorough understanding of this mechanism will be crucial to understand the subtleties of enteropathogenic E. coli pathogenesis.

Bacterial Outer Membrane Proteins↗
PubMed2006

Randomized study on removable PGE2 vaginal insert versus PGE2 cervical gel for cervical priming and labor induction in low-Bishop-score pregnancy.

BACKGROUND: Dinoprostone vaginal insert has been compared to Dinoprostone cervical gel in few studies, whose cases presented different Bishop scores and gestational ages at admission, and various treatment strategies in control arms. The present study compares the vaginal insert to the cervical gel in patients with low Bishop score at term. METHODS: Prospective multicenter randomized trial, with parity-based randomization. Admission criteria: single pregnancy with Bishop score of 0-4, gestational age of 37-41 weeks, intact membranes, no previous cesarean section, no bleeding or abnormal cardiotocography at admission. RESULTS: Vaginal prostaglandins were required as a second-line induction procedure in 25% of study patients versus 47.1% of controls (p < 0.03, chi2). Study patients experienced shorter induction-to-delivery time (920 +/- 428 versus 1,266 +/- 740 min, p <0,01), with a mean difference of 5 h and 46 min between the groups. Even though patients that received vaginal insert showed a trend of increased incidence of abnormal cardiotocography during labor (12% versus 6.3%) and hyperkinetic labor (11.8% versus 2.1%), the incidence of cesarean sections (21.4% versus 21.6%), cesareans for fetal distress (12.5% versus 11.8%), and umbilical artery pH <7.10 (4.9% versus 2.5%) was comparable between the two groups. CONCLUSIONS: Dinoprostone vaginal insert is more efficient than cervical gel in promoting cervical priming and labor induction in low-Bishop-score patients at term. The vaginal insert placement seems to be safe for the mother and the newborn, although larger studies are required to investigate uterine hyperstimulation incidence.

Adult↗
PubMed2004

Computer-assisted fluoroscopic navigation of pedicle screw insertion: an in vivo feasibility study.

BACKGROUND: Accurate placement of pedicle screws is difficult. PATIENTS AND METHODS: We evaluated the feasibility and accuracy of pedicle screw insertion assisted by a real-time, 2-dimensional (2D) image-guided navigation system in 12 patients who underwent thoraco-lumbar and/or lumbar stabilization. 66 pedicle screws were inserted either by senior spine surgeons or residents. The accuracy of positioning of the screws was evaluated using postoperative plain radiographs and thin-cut CT. RESULTS: 61 of the 66 screws were inserted successfully. 5 screw insertions showed structural violations: 4 on the medial and 1 on the lateral pedicle wall. The accuracy was higher in the sagittal plane than in the axial plain. There was no difference between the surgical error rates caused by the senior surgeons and the residents. INTERPRETATION: Using computer-assisted 2D fluoroscopic image navigation, it is possible to achieve reliable and accurate pedicle screw insertion during low thoracic and lumbar spinal surgery.

Adult↗
PubMed2000

Effect of N-acetylcysteine on the incidence of recurrence of otitis media with effusion and re-insertion of ventilation tubes.

Previous studies have demonstrated the anti-inflammatory, anti-oxidant, and mucolytic nature of N-acetylcysteine (NAC). Theoretically, these properties make the substance ideal for therapeutic use against otitis media with effusion (OME). The disease is characterized as a sustained non-specific inflammation of the middle ear mucosa with secretory transformation of the epithelium resulting in accumulation of fluid in the middle ear space. To investigate the effects of instillation of NAC in the middle ear, a double-blind, placebo-controlled, randomized trial was carried out. A total of 75 children who were undergoing their first bilateral insertion of ventilation tubes (VT) due to OME were randomized to Mucomyst (NAC) or placebo (the vehicle) on one ear in relation to the VT insertion. The contralateral ear underwent VT insertion exclusively. Instillation of Mucomyst or placebo was repeated 3 and 7 days afterwards. The children were followed regularly for 11-39 months. Episodes of otorrhea, recurrence of OME after VT extrusion and re-insertion of VTs were registered as primary outcome parameters. The results demonstrated that Mucomyst significantly reduced the recurrence of OME and re-insertion of VTs (p < 0.025) and significantly increased the time until VT extrusion (p < 0.0167). In addition, the number of episodes of ear problems and visits at the ENT clinic were reduced significantly by NAC (p < 0.0383).

Acetylcysteine↗
PubMed2006

Combining perimodiolar electrode placement and atraumatic insertion properties in cochlear implantation -- fact or fantasy?

CONCLUSIONS: Except for basal cochlear traumatization, all specimens implanted into scala tympani showed atraumatic insertion properties and good perimodiolar electrode positioning. Cochleostomy preparation and placement can have a significant impact on levels of basal cochlear trauma. OBJECTIVE: In the past, perimodiolar cochlear implant electrodes increased the risk for intracochlear traumatization when compared to free-fitting arrays. Recently, however, clinical evidence for atraumatic perimodiolar implantations with preservation of residual hearing has been described. The aim of this paper was to histologically evaluate a perimodiolar cochlear implant array for its insertion properties in cadaver human temporal bones. Surgical and electrode factors, as well as preparation artifacts influencing intracochlear trauma, were considered in the evaluation. MATERIALS AND METHODS: Sixteen human temporal bones were harvested up to 24 hours post mortem and implanted immediately with the Nucleus 24 Contour Advance cochlear implant electrode array. Implantations were either performed using a regular caudal approach cochleostomy or through the round window membrane. After implantation, all bones underwent special histological processing, which allowed sectioning of undecalcified bone. Insertion properties were evaluated according to a grading system. RESULTS: Fourteen specimens were implanted into scala tympani and only two exhibited basal trauma attributable to electrode insertion characteristics. Two bones were implanted into scala vestibuli after causing trauma in the region of the cochleostomy. Insertion depths ranged from 180 degrees to 400 degrees. All bones showed good perimodiolar electrode positioning. Basal trauma due to surgical issues and histological artifacts was present in 10 of 16 bones.

Auditory Threshold↗
PubMed2005

Trans-tympanic silicone plug insertion for chronic patulous Eustachian tube.

CONCLUSION: Trans-tympanic insertion of a new silicone plug seems to be useful for controlling the distressing symptoms of patients with a chronic patulous Eustachian tube (PET). OBJECTIVE: To evaluate the effectiveness of a new silicone plug for blocking the isthmus of a PET in patients whose symptoms were resistant to other therapies for > 6 months. MATERIAL AND METHODS: The silicone plug (total length 23-25 mm; tip diameter 1.0-2.0 mm) was inserted in 44 ears of 37 patients with chronic PET. It was inserted through the tympanic orifice of the ET to obstruct the isthmus of the tube via an incision in the anterosuperior portion of the tympanic membrane. RESULTS: Insertion of the plug was possible in all except two ears, in which it failed because of a narrow tympanic orifice of the ET. In 11 ears of 10 patients, the plug was replaced by a larger one using the same approach to improve efficacy. Of the 42 ears in which the silicone plug was successfully inserted, 30 (71.4%) achieved relief from symptoms of PET without additional treatment. In > 60% of these cases, the symptoms of PET were well controlled with an aerated middle ear. The follow-up period ranged from 6 to 68 months (mean 38.9 months).

Adult↗
PubMed2002

Insertion of depth electrodes with or without subdural grids using frameless stereotactic guidance systems--technique and outcome.

Over recent years frameless stereotactic systems have begun replacing framed systems for many neurosurgical procedures. However, little has been published regarding the use of these systems to guide intracranial electrode implantation for epilepsy surgery patients. Here we report our experience utilising such a system to insert depth electrodes and subdural grid electrodes. The SteathStation Image-Guided System (SSIGS) (Sofamor Danek, Memphis TN.) was used to insert bilateral depth electrodes in 13 patients, of whom 5 also underwent the insertion of subdural grids or strip electrodes. Initially, a surgical plan based on an entry and target point on axial and sagittal images was performed for the insertion of electrodes. Navigational views, using three-planar images, were then performed to determine which structures the electrodes would pass through to be correctly placed in the amygdala and hippocampus. The correct site of electrode implantation was confirmed post-operatively by spiral CT scans in 4 patients (which were then co-registered to the pre-implantation MRI using a surface matching technique) and the other 9 patients by post-implantation MRI. The SSIGS was found to have a mean registration error of 2.0 mm (range 1.8-2.5) in 10 cases; in the 3 cases where the error was greater than 2.5 mm a surfacemerge technique was used with a mean error 0.9 (0.8-1.00). The post-implantation MRI or CT-MRI co-registration confirmed an accurate electrode placement in the mesial temporal region in all cases. Seizure onset lateralisation was achieved in 11 patients, all of whom went onto formal resections based on these results. The only long-term complication was a case of osteomyelitis which required removal of the bone flap. 73% of patients had an excellent seizure outcome. Frameless stereotactic systems can be safely used to intracranial electrodes, avoid the disadvantages of the framed system and have the added advantage of the surgeon being able to visualise the trajectory and to adjust this to avoid vital structures. As well they eliminate surgical obstruction to the insertion of subdural grids at the same operation, which may be caused by a framed system.

Adult↗
PubMed1985

Insertion and internalization of acetylcholine receptors at clustered and diffuse domains on cultured myotubes.

Two populations of acetylcholine receptors (AChRs) are present in cultured myotubes. One forms large aggregates or clusters and the other has a much lower density of AChRs, which are diffusely distributed. Both clustered and diffuse AChRs are inserted and removed (internalized) from the sarcolemma. To determine the insertion and removal rates of AChRs in these two plasma membrane domains, we used a double label technique to distinguish and quantitate newly inserted and "old" AChRs. Application of our method revealed that the rate of AChR internalization is the same at the clustered and diffuse regions of the plasma membrane, whereas the rate of insertion is threefold greater at the clusters than elsewhere in the plasma membrane. Thus, the increase in AChR number at the clusters is not due to an increase in their half-life, but to an increase in their rate of insertion.

Animals↗
PubMed1987

Isolation and characterization of cDNA clones for rat ribophorin I: complete coding sequence and in vitro synthesis and insertion of the encoded product into endoplasmic reticulum membranes.

Ribophorins I and II are two transmembrane glycoproteins that are characteristic of the rough endoplasmic reticulum and are thought to be part of the apparatus that affects the co-translational translocation of polypeptides synthesized on membrane-bound polysomes. A ribophorin I cDNA clone containing a 0.6-kb insert was isolated from a rat liver lambda gtll cDNA library by immunoscreening with specific antibodies. This cDNA was used to isolate a clone (2.3 kb) from a rat brain lambda gtll cDNA library that contains the entire ribophorin I coding sequence. SP6 RNA transcripts of the insert in this clone directed the in vitro synthesis of a polypeptide of the expected size that was immunoprecipitated with anti-ribophorin I antibodies. When synthesized in the presence of microsomes, this polypeptide, like the translation product of the natural ribophorin I mRNA, underwent membrane insertion, signal cleavage, and co-translational glycosylation. The complete amino acid sequence of the polypeptide encoded in the cDNA insert was derived from the nucleotide sequence and found to contain a segment that corresponds to a partial amino terminal sequence of ribophorin I that was obtained by Edman degradation. This confirmed the identity of the cDNA clone and established that ribophorin I contains 583 amino acids and is synthesized with a cleavable amino terminal insertion signal of 22 residues. Analysis of the amino acid sequence of ribophorin I suggested that the polypeptide has a simple transmembrane disposition with a rather hydrophilic carboxy terminal segment of 150 amino acids exposed on the cytoplasmic face of the membrane, and a luminal domain of 414 amino acids containing three potential N-glycosylation sites. Hybridization measurements using the cloned cDNA as a probe showed that ribophorin I mRNA levels increase fourfold 15 h after partial hepatectomy, in confirmation of measurements made by in vitro translation of liver mRNA. Southern blot analysis of rat genomic DNA suggests that there is a single copy of the ribophorin I gene in the haploid rat genome.

Amino Acid Sequence↗
PubMed1979

Requirements for the insertion of the Sindbis envelope glycoproteins into the endoplasmic reticulum membrane.

Previous work has shown that the Sindbis structural proteins, core, the internal protein, and PE2 and E1, the integral membrane glycoproteins are synthesized as a polyprotein from a 26S mRNA; core PE2 and E1 are derived by proteolytic cleavage of a nascent chain. Newly synthesized core protein remains on the cytoplasmic side of the endoplasmic reticulum while newly synthesized PE2 and E1 are inserted into the lipid bilayer, presumably via their amino-termini. PE2 and E1 are glycosylated as nascent chains. Here, we examine a temperature-sensitive mutant of Sindbis virus which fails to cleave the structural proteins, resulting in the production of a polyprotein of 130,000 mol wt in which the amino-termini of PE2 and E1 are internal to the protein. Although the envelope sequences are present in this protein, it is not inserted into the endoplasmic reticulum bilayer, but remains on the cytoplasmic side as does the core protein in cells infected with wild-type Sindbis virus. We have also examined the fate of PE2 and E1 in cells treated with tunicamycin, an inhibitor of glycosylation. Unglycosylated PE2 and E1 are inserted normally into the lipid bilayer as are the glycosylated proteins. These results are consistent with the notion that a specific amino-terminal sequence is required for the proper insertion of membrane proteins into the endoplasmic reticulum bilayer, but that glycosylation is not required for this insertion.

Animals↗