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At least 433 records · Page 24Linked to original sources
PubMed2002

Experience with GyneFIX insertions in Spain: favorable acceptance of the intrauterine contraceptive implant with some limitations.

This study evaluates the incidents associated with GyneFIX insertion and first-year expulsion and continuity rates within the usual intrauterine contraceptive practice of a working group of Spanish professionals (GESEG), formed specifically with this aim. It is a prospective, multicenter, observational study of GyneFIX insertion in 1684 women. Data were prospectively collected on a structured form and processed centrally. Interest was focused on difficulties encountered during the insertion procedure and symptoms experienced during insertion. All terms were defined by consensus. Among the total, 18.6% of the women were nulliparous. GyneFIX insertion was rated easy in 92%, with more difficulty in nulliparous women, who showed significantly more symptoms during insertion of the device. First-year expulsion and continuity rates were 5.6 and 88 per 100 women, respectively. The pregnancy rate was 0.3 per 100 women/years. The GyneFIX system is an interesting alternative to standard IUDs for intrauterine contraception with copper, particularly in women who have experienced expulsion of other types of IUDs. Experienced professionals in IUD insertion quickly acquire familiarity with the GyneFIX insertion system, but proper implantation does not completely eliminate the risk of expulsion. Thus, the insertion system should be further modified to achieve a simpler, safer technique.

Adult↗
PubMed2001

Studies on indomethacin inserts prepared by water-soluble polymers. II. The relation between dissolution rate and swelling behaviour.

Inserts containing indomethacin were prepared using water-soluble polymers such as hydroxypropyl cellulose, methylcellulose, hydroxypropyl methylcellulose and polyvinyl alcohol by the film casting method. According to the different characteristics of the polymer used, these inserts exhibit different release kinetics and swelling behaviour. In this study, we examined the relation between swelling behaviour of the polymer and the release of the indomethacin from inserts. Thus an electrical device for measuring the thickness of the hydrated inserts was developed. The thickness of the hydrated inserts was measured by this electrical device at selected time intervals for release studies. The results were interpreted from normalised increases in thickness of the hydrated insert. The mechanism of drug release was identified by means of the value of the ratio R/F, calculated according to the equation developed by Peppas. When the ratio R/F of the insert decreased, drug release from the insert became diffusive. As the normalised thickness of the insert increased, the rate of drug release decreased.

Algorithms↗
PubMed1987

Broviac catheter insertion: operating room or neonatal intensive care unit.

We reviewed the records of all infants hospitalized in the neonatal intensive care unit (NICU) who underwent insertion of a Broviac catheter from July 1, 1984 through August 30, 1985. Eighty-six catheters were inserted in 81 patients. Thirty-one catheters were inserted in the NICU and the remainder were inserted in the operating room (OR). The patient's average weight at the time of catheter insertion was the same in both groups. Fifty-two of the 55 OR catheters (95%) were inserted in the external or internal jugular vein while only 68% of the NICU catheters were placed in the jugular veins. Six of the NICU catheters (19%) and 11 of the OR catheters (20%) developed catheter-associated sepsis with positive blood cultures. The infection rate per catheter day was similar in both groups as was the incidence of catheter occlusion. The NICU catheters were in place for an average of 51 days, and there was an average 46 day lifespan for the OR inserted catheters. Broviac catheter insertion can safely be performed in the NICU without an increase in morbidity. Broviac catheter insertion in the NICU is less costly and saves transportation of the sick neonate to the operating room.

California↗
PubMed2006

[Efficacy and efficiency of preoperative mydriasis: drops versus ocular insert].

OBJECTIVE: The primary objective of this study was to compare eye drops and inserts in terms of the quality of preoperative mydriasis. The secondary objective was to evaluate the safety and ease of use of each modality. PATIENTS AND METHODS: Prospective randomized and simple blinded study. Patients were randomized into two groups: the insert protocol (atropine 1%, diclofenac 0.1% collyrium and Mydriasert) or the eye drop protocol (atropine 1%, diclofenac 0.1%, tropicamide 0.5%, and phenylephrine 10% collyrium). Fifty-one patients were included: 25 in the insert protocol and 26 in the drop protocol. Mydriasis quality was evaluated by nurses and surgeons. The ease of use was estimated by the number of gestures required to obtain mydriasis and the difficulties encountered. Safety was evaluated at the patient's bedside. RESULTS: Mydriasis of at least 6 mm was obtained in 92% of the patients in the insert protocol (23/25) and 85% of the patients in the drop protocol (22/26) (NS). Four patients lost their insert during the study. Of the patients in the insert group, 16% (4/25) experienced stinging in their eyes, as did 23% (6/26) of patients in the group using drops (NS). To reach effective mydriasis, the insert protocol required fewer nurse gestures per patient than the drop protocol (3.0 versus 8.3; p<0.001). CONCLUSION: With the protocol using an insert, preoperative mydriasis is not inferior to that obtained with drops only and is more efficient in terms of nurse time and number of gestures. Nurses should be trained in appropriate handling of the insert and avoiding the loss of the device.

Humans↗
PubMed2002

Effect of chitosan on in vitro release and ocular delivery of ofloxacin from erodible inserts based on poly(ethylene oxide).

The effects of chitosan hydrochloride (CH-HCl) on in vitro release of ofloxacin (OFX) from mucoadhesive erodible ocular inserts and on the relevant ocular pharmacokinetics have been studied both to contribute evidence of the ability of CH-HCl to enhance transcorneal penetration of drugs and to increase the therapeutic efficacy of topically applied OFX. Circular inserts of 6 mm in diameter, 0.8-0.9 mm in thickness and 20 mg in weight, medicated with 0.3 mg drug, were prepared by powder compression. The addition of 10, 20 or 30% medicated CH-HCl microparticles, obtained by spray-drying, to formulations based on poly(ethylene oxide) of MW 900 kDa (PEO 900) or 2000 kDa (PEO 2000) produced changes in the insert microstructure which accelerated both insert erosion and OFX release from inserts. The effect was stronger with higher CH-HCl fractions. Of the CH-HCl-containing formulations based on either PEO 900 or PEO 2000, PEO 900-CH-HCl (9:1 w/w) was more suitable for a prolonged OFX release. Following insertion in the lower conjunctival sac of the rabbit's eye, such an insert produced no substantial increase of AUC(eff) (AUC in the aqueous humour for concentrations >MIC(90%)) with respect to inserts based on plain PEO; however, it produced a concentration peak in the aqueous significantly higher than that produced by any of the CH-HCl-free PEO inserts, and well higher than the MIC(90%) for the more resistant ocular pathogens (7 microg/ml vs. 4 microg/ml). It has been argued that the increase was due to the ability of CH-HCl to enhance the transcorneal permeability of the drug.

Animals↗
PubMed1987

Intraaortic balloon pump insertion: a randomized study comparing percutaneous and surgical techniques.

To compare the percutaneous and surgical techniques of intraaortic balloon pump insertion, 101 patients referred for this procedure were randomly assigned to either percutaneous or surgical insertion. Insertion using the designated technique was successful in 45 (88%) of 51 patients with percutaneous insertion and 48 (96%) of 50 patients with surgical insertion (difference not statistically significant). The time from the beginning of the insertion procedure to the initiation of counterpulsation was 13 +/- 8 minutes for the percutaneous technique versus 31 +/- 16 minutes for the surgical technique (p less than 0.001). In the percutaneous group, 10 patients required Fogarty thrombectomy after balloon pump removal, and 1 patient developed severe leg ischemia requiring immediate termination of balloon pump support. In the surgical group, one patient developed leg ischemia requiring surgical intervention, three patients developed sepsis with bacteremia (including one patient who required vein patch repair of the femoral artery), one patient developed a wound infection requiring debridement and one patient had a cerebral embolus. Aortic dissection, aortoiliac perforation or amputation did not occur in either group. Major vascular complications occurred in 11 patients (22%) with percutaneous insertion versus 2 patients (4%) with surgical insertion (p less than 0.05). It is concluded that although the percutaneous technique for intraaortic balloon pump insertion is faster than the surgical technique and is technically easy, it is associated with a higher incidence of vascular complications.

Blood Pressure↗
PubMed1992

SecA insertion into phospholipids is stimulated by negatively charged lipids and inhibited by ATP: a monolayer study.

SecA-lipid interactions are believed to be important for the translocation of precursor proteins across the inner membrane of Escherichia coli [Lill, R., Dowhan, W., & Wickner, W. (1990) Cell 60, 271-280]. SecA insertion into the phospholipid bilayer could a role in this process. We investigated this possibility by studying the interactions between SecA and different phospholipids using the monolayer technique. It was established that SecA is surface-active and can insert into lipid monolayers. This insertion was greatly enhanced by the negatively charged lipids DOPG and Escherichia coli cardiolipin. Insertion of SecA into these negatively charged lipids could be detected up to initial surface pressures of 34 mN/m for DOPG and 36 mN/m for Escherichia coli cardiolipin, implying a possible role for negatively charged lipids in the insertion of SecA in biological membranes. High salt concentrations did not inhibit the SecA insertion into DOPG monolayers, suggesting not only an electrostatic but also a hydrophobic interaction of SecA with the lipid monolayer. ATP decreased both the insertion (factor 2) and binding (factor 3) of SecA to DOPG monolayers. ADP and phosphate gave a decrease in the SecA insertion to the same extent as ATP, but the binding of SecA was only slightly reduced. AMP-PNP and ATP-gamma-S did not have large effects on the insertion or on the binding of SecA to DOPG monolayers. The physiological significance of these results in protein translocation is discussed.

Adenosine Triphosphatases↗
PubMed2006

Topography of the hydrophilic helices of membrane-inserted diphtheria toxin T domain: TH1-TH3 as a hydrophilic tether.

After low pH-triggered membrane insertion, the T domain of diphtheria toxin helps translocate the catalytic domain of the toxin across membranes. In this study, the hydrophilic N-terminal helices of the T domain (TH1-TH3) were studied. The conformation triggered by exposure to low pH and changes in topography upon membrane insertion were studied. These experiments involved bimane or BODIPY labeling of single Cys introduced at various positions, followed by the measurement of bimane emission wavelength, bimane exposure to fluorescence quenchers, and antibody binding to BODIPY groups. Upon exposure of the T domain in solution to low pH, it was found that the hydrophobic face of TH1, which is buried in the native state at neutral pH, became exposed to solution. When the T domain was added externally to lipid vesicles at low pH, the hydrophobic face of TH1 became buried within the lipid bilayer. Helices TH2 and TH3 also inserted into the bilayer after exposure to low pH. However, in contrast to helices TH5-TH9, overall TH1-TH3 insertion was shallow and there was no significant change in TH1-TH3 insertion depth when the T domain switched from the shallowly inserting (P) to deeply inserting (TM) conformation. Binding of streptavidin to biotinylated Cys residues was used to investigate whether solution-exposed residues of membrane-inserted T domain were exposed on the external or internal surface of the bilayer. These experiments showed that when the T domain is externally added to vesicles, the entire TH1-TH3 segment remains on the cis (outer) side of the bilayer. The results of this study suggest that membrane-inserted TH1-TH3 form autonomous segments that neither deeply penetrate the bilayer nor interact tightly with the translocation-promoting structure formed by the hydrophobic TH5-TH9 subdomain. Instead, TH1-TH3 may aid translocation by acting as an A-chain-attached flexible tether.

Boron Compounds↗
PubMed1998

The insertion of human apolipoprotein H into phospholipid membranes: a monolayer study.

Apolipoprotein H (ApoH) is a plasma glycoprotein isolated from human serum. The interactions of ApoH with lipid membrane were reported to be essential for its physiological and pathogenic roles. In this paper we studied the ability of ApoH to insert into phospholipid membranes using the monolayer approach. The results show that ApoH is surface active and can insert into the lipid monolayers. The insertion ability of ApoH is stronger when a higher content of negatively charged lipids is present in the membrane. The acidic-pH and low-ionic-strength conditions will also enhance ApoH insertion, but these factors may not have much influence on the final insertion ability of ApoH, suggesting that, in the mechanism of ApoH insertion, not only electrostatic forces, but also hydrophobic interactions, are evidently involved. Modification by heat inactivation and reduction/alkylation does not change the critical insertion pressure (pic) of ApoH, suggesting a stable domain, maybe a linear sequence motif, but not the native three-dimensional structure of ApoH, is responsible for its insertion. The extent to which insertion of ApoH into phospholipid membranes may facilitate the 'immune cleaning' of plasma liposomes is discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2003

Influence of stainless steel inserts on the resistance to sliding of esthetic brackets with second-order angulation in the dry and wet states.

Stainless steel (SS) inserts have been added to plastic, ceramic, and composite brackets to improve their frictional characteristics while preserving their esthetic appeal. When coupled with SS archwires, the resistances to sliding of esthetic brackets with and without SS inserts were compared with control brackets of SS. The resistances to sliding were measured in both the dry and wet (saliva) states at 32 second-order angles between -12 and + 12 degrees. When clearances existed between the walls of the brackets and the archwires, the resistances to sliding for the esthetic brackets without inserts were between 38 cN in the dry state and 73 cN in the wet state; those of the esthetic brackets with inserts ranged from 42 cN in the dry state to 65 cN in both states. The resistances to sliding of the SS brackets equaled 38 and 52 cN in the dry and wet states, respectively. When clearances no longer existed, the resistances to sliding for the esthetic brackets with and without inserts generally increased with angulation at a rate equal to or greater than that of the SS brackets--except for the polycarbonate (PC) brackets in the dry state. Because PC brackets without inserts elastically deformed, they had lower resistances to sliding when deformation occurred. For the polycrystalline alumina brackets without inserts, the resistances to sliding increased rapidly and nonlinearly as angulation increased above 4.8 degrees. Upon examination, the presence of scratches on the archwires and SS debris on the brackets was observed. The addition of these particular SS inserts did not considerably improve the resistance to sliding over those esthetic brackets without inserts.

Aluminum Oxide↗
PubMed2002

The rotational technique with a partially inflated laryngeal mask airway improves the ease of insertion in children.

BACKGROUND: This study compared the ease of insertion of the laryngeal mask airway (LMA) with a partially inflated cuff using the standard 'nonrotational' technique versus the rotational technique. METHODS: One hundred and forty-five children undergoing anaesthesia using the LMA were randomly assigned to either method. The cuff was partially inflated in both groups. The ease of insertion was assessed by the time taken to complete the LMA insertion, the number of attempts before successful placement and the occurrence of complications. RESULTS: The success rate of insertion at the first attempt was higher in the rotational technique group (99% versus 79%, P < 0.05). All patients in the rotational group had the mask inserted within two attempts. On the other hand, three patients had the mask inserted on the first attempt with the rotational technique after three unsuccessful attempts by an anaesthesiologist with the standard 'nonrotational' technique. Insertion technique made no difference on insertion time. CONCLUSIONS: The rotational technique was associated with a higher success rate for insertion and a lower incidence of complications in children. Using the rotational technique with a partially inflated cuff could be the first-choice approach in paediatric patients.

Anesthesia↗
PubMed1998

Antenatal diagnosis of velamentous umbilical cord insertion and vasa previa with color Doppler imaging.

OBJECTIVE: To determine whether cord insertion can be consistently visualized and whether velamentous cord insertion and vasa previa can be consistently identified with color Doppler imaging during routine sonography in the mid-trimester. DESIGN: A prospective study. SUBJECTS: A total of 587 fetuses at 18-20 weeks' gestation. METHODS: During routine ultrasound examinations, the sonographer was instructed to take additional time and to image the placental cord insertion with color Doppler imaging and classify this as normal, velamentous or 'not seen'. When the insertion was velamentous, the sonographer was instructed to indicate whether or not it was vasa previa. The sonogram obtained at 18-20 weeks' gestation was used for comparison with outcome data. RESULTS: Cord insertion was visualized by color Doppler imaging in 99.8% (586/587) of the fetuses in our study. The mean time required for examination was 20 s and, in 95% of the cases, cord insertion was visualized within 1 min. The sonographic identification of velamentous cord insertion had a sensitivity of 100% (5/5), a specificity of 99.8% (580/581), a positive predictive value of 83% (5/6) and a negative predictive value of 100% (580/580). In our study, vasa previa was diagnosed at 18 gestational weeks in two cases and, in one of the cases, vasa previa was confirmed at delivery. CONCLUSIONS: We could consistently identify cord insertion and velamentous cord insertion with color Doppler imaging during routine sonography in the mid-trimester. Transvaginal color Doppler imaging and serial scans were needed to identify vasa previa.

Female↗
PubMed1999

Depth of insertion at flexible sigmoidoscopy: implications for colorectal cancer screening and instrument design.

BACKGROUND AND STUDY AIMS: The depth of insertion at flexible sigmoidoscopy is variable, depending upon bowel preparation, patient tolerance and distal colonic anatomy. Many endoscopists routinely aim to insert the 60 cm flexible sigmoidoscope to the splenic flexure; however internal endoscopic markers are unreliable, making the true anatomical extent of the examination difficult to assess. The aim of this study was to assess the depth of insertion at flexible sigmoidoscopy. PATIENTS AND METHODS: Two separate studies were done. In the first (study 1), magnetic endoscopic imaging was used to determine the final depth of insertion at non-sedated, screening flexible sigmoidoscopy. In the second (study 2), "real-time" imaging was utilized to determine sigmoid looping and the anatomical location of the endoscope tip after 60 cm of instrument had been inserted during total or limited colonoscopy. A total of 117 consecutive average-risk patients, aged 55-65 years participated in study 1, and 136 patients underwent either limited, (33) or attempted total colonoscopy (103) in study 2. RESULTS: In study 1 the median insertion distance was 52 cm, range 20-58. In 61 % of patients the imaging system showed that the descending colon had not been visualized by the end of the procedure. Failure to reach the sigmoid/descending junction occurred in 29 (24%) patients. Reasons for failure included poor tolerance of the procedure due to pain (23 patients) inadequate preparation (3 patients) and, excessive looping (3 patients). In study 2, after 60 cm of instrument had been inserted, the splenic flexure or beyond was reached in 29% and the descending colon in 9%, whilst in 62 % the endoscope tip had not passed beyond the sigmoid/descending colon junction. A sigmoid loop formed in 70% of patients, and unusual loops such as the alpha, reverse alpha and reverse sigmoid spiral loop occurred more frequently in women compared to men (P = 0.0249). In those 104 patients where the splenic flexure was reached the mean maximum length of instrument inserted prior to reaching the flexure was 75.4 cm, (SD = 21.9). CONCLUSIONS: Examination of the entire sigmoid was not achieved in approximately one-quarter of patients undergoing screening flexible sigmoidoscopy, mainly because of discomfort. The descending colon is intubated in a minority of cases (using standard instruments), even after 60 cm has been inserted. Alternative instruments with different shaft characteristics (floppy, narrow calibre, 80-100 cm in length) may be necessary to ensure deeper routine intubation in nonsedated patients.

Aged↗
PubMed2001

Use of an overtube for enteroscopy--does it increase depth of insertion? A prospective study of enteroscopy with and without an overtube.

BACKGROUND AND STUDY AIMS: Although a stiffening overtube is commonly used with push enteroscopy, in the belief that this will allow increased insertion into the small intestine, there is no prospective data to support this view. The aim of this study is to prospectively study the depth of insertion into the small intestine at enteroscopy with and without an overtube. PATIENTS AND METHODS: A total of 38 patients referred for enteroscopy were prospectively studied. Alternate enteroscopies were performed with or without an overtube; therefore 19 patients had enteroscopy with and 19 without an overtube. The groups were well matched for age, sex, indication, use of fluoroscopy, and dedicated anesthetic assistance. Depth of insertion was assessed by advancing the enteroscope as far as possible, then straightening the enteroscope until the tip began withdrawing. The difference between the straightened insertion depth and the distance from the incisors to the pylorus was recorded as the insertion depth beyond the pylorus. This was considered the major end point. Statistical analysis was performed using the Mann-Whitney test for nonparametric data. RESULTS: The median straightened total insertion depth from the incisors was greater when enteroscopy was performed with an overtube compared with enteroscopy without an overtube (125 cm vs. 110 cm, P=0.05). The median straightened insertion depth beyond the pylorus was significantly greater with overtube use (70 cm vs. 50 cm, P = 0.01). No significant difference between the groups was observed in terms of the likelihood of significant findings at enteroscopy. CONCLUSIONS: Use of an overtube for push enteroscopy results in significantly deeper insertion into the small intestine. Although a larger study would be needed to demonstrate an increase in diagnostic yield and to confirm the safety of overtube use, this study does provide the first objective evidence of an advantage in terms of insertion depth.

Adult↗
PubMed1977

Sequences homologous to ribosomal insertions occur in the Drosophila genome outside the nucleolus organizer.

Many repeating units of Drosophila melanogaster rDNA contain a DNA sequence within the gene for 28S rRNA that does not code for rRNA. This sequence has been called the ribosomal insertion [Wellauer, P. K. & Dawid, I. B. (1977) Cell 10, 193-212]. We report here that members of the same sequence family occur outside the ribosomal locus. "Non-rDNA insertion DNA" was separated from rDNA by density gradient centrifugation, and sequences homologous to the ribosomal insertion were detected by hybridization with restriction endonuclease fragments derived from a cloned rDNA repeating unit. Pure insertion sequences from cloned rDNA separated from main band DNA and behaved like a component with high G + C content. Non-rDNA components hybridizing to the insertion also separated from main band DNA but less so than pure insertion sequences, suggesting that non-rDNA insertion sequences are linked to DNA of different nucleotide composition. Restriction endonuclease analysis of non-rDNA insertion DNA showed many fragments of different sizes. The patterns obtained were similar in embryonic, larval, pupal, and adult DNA and DNA from cultured cells (Schneider cell line 3). Non-rDNA insertion sequences account for about 0.2% of the genome or about 400 kbases of DNA per haploid complement.

Animals↗
PubMed1992

Torsional stress generated by RecA protein during DNA strand exchange separates strands of a heterologous insert.

Previous studies have shown that the helical RecA nucleoprotein filament formed on a circular single strand of DNA causes the progressive, directional transfer of a complementary strand from naked linear duplex DNA to the nucleoprotein filament, even when the duplex contains a sizable heterologous insertion. Since RecA protein lacks demonstrable helicase activity, the mechanism by which it pushes strand exchange through long heterologous inserts has been a quandary. In the present study, a linear duplex substrate with an insertion of 110 base pairs in its middle yielded the expected products, whereas much less of the heteroduplex product was seen when the insertion was located at either end of the duplex substrate or 160 base pairs from the far end of the duplex substrate. In an ongoing reaction of the substrate with an insertion in its middle, P1 nuclease cleaved intermediates from the point of the insertion to various distal sites. Acting on a duplex substrate that contained a single nick located in the complementary strand just beyond the insertion, RecA protein formed joint molecules but failed to complete strand exchange. These data show that negative torsional stress is generated by distant homologous interactions that occur beyond the heterologous insertion and that such stress is essential for unwinding a heterologous insertion that otherwise halts strand exchange.

Base Composition↗
PubMed2004

Functional mapping of Cre recombinase by pentapeptide insertional mutagenesis.

Cre is a site-specific recombinase from bacteriophage P1. It is a member of the tyrosine integrase family and catalyzes reciprocal recombination between specific 34-bp sites called loxP. To analyze the structure-function relationships of this enzyme, we performed large scale pentapeptide insertional mutagenesis to generate insertions of five amino acids at random positions in the protein. The high density of insertion mutations into Cre allowed us to identify an unexpected degree of functional tolerance to insertions into the 4-5 beta-hairpin and into the loop between helices J and K (both of which contact the DNA in the minor groove) and also into helix A. The phenotypes of the majority of inserts allowed us to confirm a variety of predictions made on the basis of sequence conservation, known three-dimensional structure, and proposed catalytic mechanism. In particular, most insertions into conserved regions or secondary structure elements inactivated Cre, and most insertions located in nonconserved, unstructured regions preserved Cre activity. Less expectedly, the non-conserved and poorly structured loops and linkers between helices A-B, E-F, and M-N did not tolerate insertions, thus identifying these as critical regions for recombinase activity. We purified and characterized in vitro several representatives of these "unexpected" Cre insertion mutants. The role of those regions in the recombination process is discussed.

Amino Acid Sequence↗
PubMed2004

Unfolding events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membrane-inserted pore channel.

The insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis undergo several conformational changes from crystal inclusion protoxins to membrane-inserted channels in the midgut epithelial cells of the target insect. Here we analyzed the stability of the different forms of Cry1Ab toxin, monomeric toxin, pre-pore complex, and membrane-inserted channel, after urea and thermal denaturation by monitoring intrinsic tryptophan fluorescence of the protein and 1-anilinonaphthalene-8-sulfonic acid binding to partially unfolded proteins. Our results showed that flexibility of the monomeric toxin was dramatically enhanced upon oligomerization and was even further increased by insertion of the pre-pore into the membrane as shown by the lower concentration of chaotropic agents needed to achieve unfolding of the oligomeric species. The flexibility of the toxin structures is further increased by alkaline pH. We found that the monomer-monomer interaction in the pre-pore is highly stable because urea promotes oligomer denaturation without disassembly. Partial unfolding and limited proteolysis studies demonstrated that domains II and III were less stable and unfold first, followed by unfolding of the most stable domain I, and also that domain I is involved in monomer-monomer interaction. The thermal-induced unfolding and analysis of energy transfer from Trp residues to bound 1-anilinonaphthalene-8-sulfonic acid dye showed that in the membrane-inserted pore domains II and III are particularly sensitive to heat denaturation, in contrast to domain I, suggesting that only domain I may be inserted into the membrane. Finally, the insertion into the membrane of the oligomeric pre-pore structure was not affected by pH. However, a looser conformation of the membrane-inserted domain I induced by neutral or alkaline pH correlates with active channel formation. Our studies suggest for the first time that a more flexible conformation of Cry toxin could be necessary for membrane insertion, and this flexible structure is induced by toxin oligomerization. Finally the alkaline pH found in the midgut lumen of lepidopteran insects could increase the flexibility of membrane-inserted domain I necessary for pore formation.

Anilino Naphthalenesulfonates↗