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PubMed1998

Generalized transduction for genetic linkage analysis and transfer of transposon insertions in different Staphylococcus epidermidis strains.

Staphylococcus epidermidis phage 48 was used to efficiently transduce plasmid pTV1ts and a chromosomal Tn917 insertion M27 from S. epidermidis 13-1 to biofilm-producing clinical S. epidermidis isolates 1457, 9142, and 8400. The Tn917 insertion leading to the biofilm-negative phenotype of transposon mutant M10 was sequentially transduced to biofilm-producing S. epidermidis 1457 using S. epidermidis phage 48 and then, using the resulting biofilm-negative transductant 1457-M10 as a donor, into several unrelated biofilm-producing clinical S. epidermidis isolates using S. epidermidis phage 71. All resultant transductants displayed a completely biofilm-negative phenotype. In addition, S. epidermidis phage 71 was adapted to S. epidermidis 1457 and 8400, which allowed generalized transduction of transposon insertions in these wild-type strains. As Tn917 predominantly transposed into endogenous plasmids of all three strains used, an efficient system for chromosomal transposon mutagenesis was established by curing of S. epidermidis 1457 of a single endogenous plasmid p1457 by sodium dodecylsulfate treatment. After transduction of the resulting derivative, S. epidermidis 1457c with pTV1ts, insertion of transposon Tn917 to different sites of the chromosome of S. epidermidis 1457c was observed. Biofilm-producing S. epidermidis 1457c x pTV1ts was used to isolate a biofilm-negative transposon mutant (1457c-M3) with a chromosomal insertion apparently different from two previously isolated isogenic biofilm-negative transposon mutants, M10 and M11 (Mack, D., M. Nedelmann, A. Krokotsch, A. Schwarzkopf, J. Heesemann, and R. Laufs: Infect Immun 62 [1994] 3244-3253). S. epidermidis phage 71 was used to prove genetic linkage between transposon insertion and altered phenotype by generalized transduction. In combination with phage transduction, 1457c x pTV1ts will be a useful tool facilitating the study of bacterial determinants of the pathogenicity of S. epidermidis.

Blotting, Southern↗

PubMed1995

Risk factors for pneumothorax during percutaneous Hickman line insertion in patients with solid and haematological tumours.

The purpose of this study was to identify risk factors for pneumothorax during percutaneous subclavian Hickman line insertion in patients with haematological (HT) or solid tumours (ST). One hundred and twelve patients (55 HT, 57 ST) had 132 subclavian Hickman lines inserted under fluoroscopic control. Lines were inserted on the left on 116 occasions and the right in 16. Thirty-five single, 29 double and 68 triple lumen catheters were inserted. Variables included in the analysis were age, sex, side of insertion, catheter gauge and nutritional status as indicated by the body mass index (BMI kg/m2). Both univariate and logistic regression analyses were performed. There were ten pneumothoraces, all occurring in patients with STs. Univariate analysis revealed that patients with STs were older (median age 59 versus 36 years; P = 0.0001) and more cachectic (median BMI 21.9 versus 24.2 kg/m2; P = 0.03) than those with HTs, and the patients experiencing pneumothorax were older (median age 57 versus 44 years; P < 0.01) and more cachectic (median BMI 19.6 versus 24.0 kg/m2; P = 0.004) than those undergoing uncomplicated procedures. For patients with a BMI < 19 kg/m2 versus those with a BMI > 19 kg/m2, the pneumothorax rate was 5/8 versus 5/124 (P < 0.00001). Logistic regression analysis confirmed the significantly older age of the patients with STs (P < 0.001) and of those experiencing pneumothorax (P = 0.02). This analysis also confirmed the lower BMI of patients experiencing pneumothorax (P = 0.002). The patients' sex, the side of line insertion and the catheter gauge were not associated with pneumothorax on either the univariate or logistic regression analyses. From these data we conclude that pneumothorax complicating a subclavian Hickman line is significantly more likely in elderly patients with a low BMI. Such patients are more likely to have STs than HTs. In patients with a BMI < 19 kg/m2, the subclavian route should be used with caution.

Adult↗

PubMed2002

A study of release mechanisms of different ophthalmic drugs from erodible ocular inserts based on poly(ethylene oxide).

When topical controlled delivery of ophthalmic drugs is realised via erodible inserts, drug bioavailability is maximised, if release is controlled exclusively by insert erosion, since parallel mechanisms which increase the release rate, also increases the dose fraction cleared from the precorneal area by tear fluid draining. The respective contributions of diffusion and erosion to the release mechanism of different drugs, namely, prednisolone (PDS), oxytetracycline hydrochloride (OTH) and gentamicin sulfate (GTS), from erodible ocular inserts based on poly(ethylene oxide) (PEO) of molecular weight 400 or 900kDa was determined by an in vitro technique adequate to predict the release mechanism in vivo. PDS and OTH were released with erosion-controlled kinetics. With therapeutic doses of these drugs in the inserts (0.3mg, 1.5%), the possibility of a purely erosive mechanism was shown to rely upon drug-PEO molecular interactions, which limit drug diffusion in the swollen matrix. This was the case with OTH, for which strong interactions with PEO were measured, whereas some contribution from the parallel diffusive mechanism was evidenced for PDS, which showed weaker interactions with polymer. Such a contribution disappeared when the PDS concentration in the insert was increased to 6%, which suggested that the erosive mechanism is favoured by a drug concentration in the hydrated insert substantially higher than solubility. On the other hand, the release of about 50% GTS dose was controlled by diffusion, due to the high water solubility of this drug, accompanied by weak drug-PEO interactions. In this case the residence time of drug in the precorneal area is expected to be significantly shorter than that of the PEO carrier.

Benzocaine↗

PubMed1998

Silicone rubber/hydrogel composite ophthalmic inserts: preparation and preliminary in vitro/in vivo evaluation.

The present report describes the development and in vitro/in vivo testing of rod-shaped mucoadhesive ophthalmic inserts fitting the upper or lower conjunctival fornix. Cylindrical devices (diameter 0.9 mm, length 6-12 mm, weight 3-8 mg) all containing 0.8 mg oxytetracycline HCl (OXT) were prepared from appropriate mixtures of silicone elastomer, OXT and sodium chloride as release modifier. A stable polyacrylic acid (PAA) or polymethacrylic acid (PMA) interpenetrating polymer network (IPN; 30 or 46% w/w) was grafted onto the inserts' surface by treatment with a mixture of acrylic (or methacrylic) acid and ethylene glycol dimethacrylate in xylene at 100 degrees C. Mucoadhesion studies in vitro showed that the mucoadhesive properties increased significantly with increasing thickness of the IPN layer. The inserts were tested for drug release in vitro, and for drug release and retention in rabbit eyes. The presence of IPN, as well as of NaCl, in general increased the drug release rate. The PMA-grafted devices released OXT at lower rates when compared with the PAA-grafted ones. A nearly zero-order release rate for about 1 week was observed in vitro for some types of inserts. When tested in rabbits, some IPN-grafted inserts maintained in the lacrimal fluid a OXT concentration of 20-30 microg/ml for several days: the in vitro minimum inhibitory concentration values (MIC 90%) of OXT against micro-organisms responsible of common ocular infections range from 0.8 to 2.0 microg/ml, while MIC 90% values in the range 14-50 microg/ml have been indicated for Pseudomonas aeruginosa. The ocular retention of IPN-grafted samples was significantly higher with respect to ungrafted ones. The presently described mucoadhesive silicone inserts might prove efficient therapeutic systems for chemotherapy of ocular bacterial infections, such as trachoma.

Animals↗

PubMed1991

Incomplete opening of LGM (Vena Tech) filters inserted via the transjugular approach.

Over a 12-month period, 216 LGM vena caval filters were placed in 216 patients at four institutions. The transjugular approach was used in 31 of 216 insertions (14%); 185 of 216 filters (86%) were inserted via the femoral route. Incomplete opening of filters was encountered in 13 of 31 transjugular insertions (41%) and none of 185 transfemoral insertions. Delayed spontaneous filter opening occurred in three of 12 cases (25%) of incomplete opening (in which follow-up was available) at 5 minutes, 4 days, and 2 months after insertion. One filter opened completely after catheter manipulations. Several mechanisms explaining this complication are proposed. In its present form, the LGM filter should not be inserted via the jugular route. Since the filtering capabilities of the incompletely opened LGM device have been shown to be diminished in vitro, it may be advisable to place a second filter cephalad to an incompletely opened LGM filter.

Equipment Failure↗

PubMed2000

Anatomy of the left upper quadrant for cannula insertion.

STUDY OBJECTIVE: To determine the anatomy of the left upper quadrant (LUQ) of the abdomen in women with different body mass indexes. DESIGN: Review of computed tomographic (CT) scans and medical records (Canadian Task Force classification II-2). SETTING: Large tertiary care medical center. PATIENTS: Sixty-three women over age 18 years who had scans performed for any indication. Nine women were excluded because of contraindication to LUQ laparoscopic cannula insertion and five because of incomplete records. Intervention. None. MEASUREMENTS AND MAIN RESULTS: The closest organs to the LUQ insertion site were the liver and stomach. There was significantly more subcutaneous fat at the umbilicus than at the insertion site. A positive correlation was found between body mass index and distance between structures and the site. CONCLUSION: Cannulas should not be inserted in the LUQ in women with hepatomegaly or splenomegaly. Because the stomach is so close to the insertion site, gastric drainage should be performed before cannula insertion. The site is likely safe in obese women whose abdominal organs are far away from it, and who have less subcutaneous fat there than at the umbilicus.

Abdomen↗

PubMed2003

Measurement of trocar insertion force using a piezoelectric transducer.

We attempted to establish a model to measure the force required for trocar insertion at laparoscopy. A 3-cm, circular transducer was constructed from piezoresistive material that changes its impedance as force is exerted on its surface. The transducer is connected by an interface box to a personal computer to record surface contact pressure digitally (pressure = force/area) profile continuously during trocar insertion. Each subject had three trocars inserted: a 10-mm trocar at the umbilicus after creation of pneumoperitoneum, and 5-mm trocars at corresponding sites on the left and right sides of the lower abdomen. All insertions were performed by the same operator using reusable trocar with a conical tip. Each subject acted as her own control. Recordings were successfully obtained from eight women. There was no instance of transducer failure. The mean (SE) peak contact surface pressure for the 10-mm and 5-mm left and right trocars were 5.3 (0.32), 6.4 (0.51), and 6.81 (0.27) pounds/square inch, respectively. Placement of the 10-mm trocar required less insertion force than placement of the 5-mm trocars. There was a strong negative correlation (r = -0.97, p < 0.001) between body weight and peak insertion force for the 10-mm trocar.

Adult↗

PubMedDate not supplied

Characteristics of pullout failure in conical and cylindrical pedicle screws after full insertion and back-out.

BACKGROUND CONTEXT: Biomechanical studies show that bone-mineral density, pedicle morphology, and screw thread area affect pedicle screw pullout failure. The current literature is based on studies of cylindrical pedicle screw designs. Conical screws have been introduced that may provide better "fit and fill" of the dorsal pedicle as well as improved resistance to screw bending failure. However, there is concern about loss of fixation if conical screws must be backed out after insertion. PURPOSE: To determine that conical screws have comparable initial stiffness and fixation strength compared with standard, cylindrical screws, and to assess whether conical screw fixation deteriorates when screws are backed out from full insertion. STUDY DESIGN/SETTING: This biomechanical analysis compared pullout strength of cylindrical and conical pedicle screw designs, using porcine lumbar vertebrae in a paired testing format. METHODS: Porcine lumbar vertebrae were instrumented with conical and cylindrical pedicle screws with the same thread pitch, area and contour, and an equivalent diameter at the pedicle isthmus, 1.2 cm distal to the hub. Axial pullout was performed at 1.0 mm/minute displacement. Pullout loads, work and stiffness were recorded at 0.02-second intervals. Conical versus cylindrical screws were tested using three paired control configurations: fully inserted, backed out 180 degrees and backed out 360 degrees. Fully inserted values were compared with each set of back-out values to determine relative loss of fixation strength. Screw pullout data were analyzed using a Student's t test. RESULTS: Pullout loads in these porcine specimens were comparable to data from healthy human vertebrae. Conical screws provided a 17% increase in the pullout strength compared with cylindrical screws (P<.10) and a 50% increase in initial stiffness (P<.05) at full insertion. There was no loss in pullout strength, stiffness or work to failure when conical or cylindrical screws were backed out 180 or 360 degrees from full insertion. CONCLUSIONS: Conical screws offer improved initial fixation strength compared with cylindrical screws of the same size and thread design. Our results suggest that appropriately designed conical screws can be backed out 180 to 360 degrees for intraoperative adjustment without loss of pullout strength, stiffness or work to failure. Intraoperative adjustments of these specific conical screws less than 360 degrees should not affect initial fixation strength. These results may not hold true for screws with a smaller thread area or larger minor diameter.

Animals↗

PubMed2006

Optimal depth of insertion of left-sided double-lumen endobronchial tubes cannot be predicted from body height in below average-sized adult patients.

BACKGROUND AND OBJECTIVE: The optimal depth of insertion of left-sided double-lumen endobronchial tubes is strongly correlated with body height in average-sized adults. However, this relationship has not been studied in below average-sized adult patients. We investigated whether or not there is a clinically useful relationship in below average-sized adult patients. METHODS: One hundred and ninety six consecutive adult patients undergoing thoracic surgery under one-lung anaesthesia (body height < or = 155 cm) were included in this study. Left-sided double-lumen tubes were inserted under the guidance of a fibre-optic bronchoscope. Optimal depth was defined as the proximal surface of the bronchial cuff positioned just below the carina. RESULTS: There was a statistically significant positive correlation between body height and the optimal depth of insertion (r = 0.61, P < 0.0001); however, the correlation coefficient was low. The actual optimal depth of insertion of one patient was even 4.5 cm shorter than that obtained from the equation. CONCLUSION: Although there was a statistically significant correlation between body height and the optimal depth of insertion of left sided double lumen tubes in adult patients of short stature (< or = 155 cm), clinical application of the equation is not warranted and these tubes should be inserted under direct vision with a fibre-optic bronchoscope.

Aged↗

PubMed1993

Accommodation of insertions in helices: the mutation in hemoglobin Catonsville (Pro 37 alpha-Glu-Thr 38 alpha) generates a 3(10)-->alpha bulge.

Hemoglobin Catonsville is a mutation of human hemoglobin (an alpha 2 beta 2 tetramer) in which a glutamate residue is inserted into the first turn of a highly conserved 3(10) helix (the C helix) of each alpha subunit. In theory, amino acid insertions (or deletions) in protein helices can be accommodated via two distinct mechanisms. One, termed the register shift mechanism, preserves the geometry of the helix while requiring all of the residues on one flank of the insertion site to rotate by 100 degrees in the case of an alpha helix or by 120 degrees in the case of a 3(10) helix. The other, termed the bulge (or indentation) mechanism, distorts the local geometry of the helix but does not alter the helix register. High-resolution X-ray diffraction analysis of deoxyhemoglobin Catonsville shows that the inserted residue is accommodated as a bulge, demonstrating that this is a viable mechanism. (In contrast, no such evidence is yet available for the register shift mechanism.) More specifically, the insertion converts one turn of the C helix from 3(10) geometry to alpha helix-like geometry, raising the possibility that a common mechanism for accommodating insertions and deletions within helices may involve localized interconversions between 3(10), alpha, and pi helical structures.

Amino Acid Sequence↗

PubMed1991

Characterization of lipid insertion into monomolecular layers mediated by lung surfactant proteins SP-B and SP-C.

Pulmonary surfactant proteins, SP-B and SP-C, if present in preformed monolayers can induce lipid insertion from lipid vesicles into the monolayer after the addition of (divalent) cations [Oosterlaken-Dijksterhuis, M. A., Haagsman, H. P., van Golde, L. M. G., & Demel, R. A. (1991) Biochemistry 30, 8276-8287]. This model system was used to study the mechanisms by which SP-B and SP-C induce monolayer formation from vesicles. Lipid insertion proceeds irrespectively of the molecular class, and PG is not required for this process. In addition to lipids that are immediately inserted from vesicles into the monolayer, large amounts of vesicles are bound to the monolayer and their lipids eventually inserted when the surface area is expanded. SP-B and SP-C are directly responsible for the binding of vesicles to the monolayer. By weight, the vesicle binding capacity of SP-B is approximately 4 times that of SP-C. For vesicle binding and insertion, the formation of close contacts between monolayer and vesicles is essential. SP-B and SP-C show very similar surface properties. Both proteins form extremely stable monolayers (collapse pressures 36-37 mN/m) of alpha-helical structures oriented parallel to the interface. In monolayers consisting of DPPC and SP-B or SP-C, an increase in mean molecular area is observed, which is mainly attributed to the phospholipid. This will greatly enhance the insertion of new lipid material into the monolayer. The results of this study suggest that the surface properties and the hydrophobic nature of SP-B and SP-C are important for the protein-mediated monolayer formation.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1992

Brominated phospholipids as a tool for monitoring the membrane insertion of colicin A.

The intrinsic fluorescence of the colicin A thermolytic fragment does not change after insertion into normal phospholipid vesicles and is thus an unsuitable probe for monitoring the membrane insertion process. In this paper, we report the results of studies on the quenching of this fluorescence by brominated dioleoylphosphatidylglycerol (Br-DOPG) vesicles. Bromine atoms located at the midpoint of the phospholipid acyl chain quench the tryptophan fluorescence, indicating contact between fluorophores of the protein and the bilayer's hydrophobic core. Addition of Br-DOPG vesicles to a protein solution quenches the tryptophan fluorescence in a time-dependent manner. This quenching can be fitted to a single-exponential function, and thus interpreted as a one-step process. This allows calculation of an apparent rate constant of protein insertion into the membrane. Parameters known to affect the insertion of the thermolytic fragment into phospholipid monolayers or vesicles (pH and negative charge density) also affect the rate constant in comparable ways. In addition to the information gained concerning membrane exposure in the steady state, this approach provides the first real-time method for measuring the insertion of colicin into membranes. It is highly quantitative and can be used on all versions of the protein, e.g., full size, proteolytic fragments, and mutants. Brominated lipids provide experimental conditions identical to normal lipids and allow for great flexibility in protein/lipid ratios and concentrations. The kinetic analysis shows clearly the existence of a two-step process involving a rapid adsorption of the protein to the lipid surface followed by a slow insertion.

Citrobacter freundii↗

PubMed1991

Structure-function studies of human cholesteryl ester transfer protein by linker insertion scanning mutagenesis.

Human plasma cholesteryl ester transfer protein (CETP) enhances transfer and exchange of cholesteryl ester (CE) and triglyceride (TG) between high-density lipoprotein and other lipoproteins. To define regions responsible for the neutral lipid transfer activities at the molecular level, a total of 27 linker insertion mutants at 18 different sites along the CETP molecule were prepared and transiently expressed in a mammalian cell line (COS). The inserted linkers were small (usually 6 bp) and did not interrupt the translational reading frame of the CETP cDNA. Although secretion of each mutant protein was less than that of wild-type CETP, the majority of the mutants had normal cholesteryl ester transfer activity (transfer activity per nanogram of CETP in media). However, insertional alterations in three regions severely impaired CE transfer activity: (1) in the region of amino acids 48-53; (2) at amino acid 165; and (3) in the region of amino acids 373-379. Although the impaired activities could also be a result of globally incorrect folding of these CETP mutants, hydrophobicity analysis and secondary structure predictions tended to exclude this possibility for most of the insertion sites at which insertions resulted in inactivation. The insertion at amino acid 379 occurs immediately after a triplet of lysine residues, suggesting that this region might be involved in an essential step in the mechanism of CE and TG transfer, such as the binding of CETP to phosphatidylcholine molecules in the lipoprotein surface. Effects on TG transfer activity were generally similar to those on CE transfer activity, suggesting a similar structural requirement for both neutral lipid transfer activities.

Amino Acid Sequence↗

PubMed2005

Topography of diphtheria toxin A chain inserted into lipid vesicles.

The membrane-inserting T domain of diphtheria toxin aids the low-pH-triggered translocation of the catalytic A chain of the toxin across endosomal membranes. To evaluate the role of the isolated A chain in translocation, the topography of isolated A chain inserted into model membrane vesicles was investigated using a mixture either of dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG) or of dimyristoleoylphosphatidylcholine (DMoPC) and DOPG. The latter mixture was previously found to promote deep insertion of the T domain. A series of single Cys mutants along the A chain sequence were labeled with bimane or BODIPY groups. After A chain insertion into model membranes, the location of these groups within the lipid bilayer was determined via bimane fluorescence emission lambda(max), binding of externally added anti-BODIPY antibodies, and a novel technique involving the comparison of the quenching of bimane fluorescence by aqueous iodide and membrane-associated 10-doxylnonadecane. The results show that in both DOPC- and DMoPC-containing bilayers, membrane-inserted residues all along the A chain sequence occupy shallow locations that are relatively exposed to the external solution. There were only small differences between A chain topography in the two different types of lipid mixtures. However, the behavior of the A chain in the two different lipid mixtures was distinct in that it strongly oligomerized in DMoPC-containing vesicles as judged by Trp fluorescence. In addition, A chain selectively induced fusion of the DMoPC-containing vesicles, and this may aid oligomerization by increasing the A chain/vesicle ratio. Fusion may also explain why A chain also selectively induced leakage of the contents of DMoPC-containing vesicles. We propose that isolated A chain is unlikely to be inserted in a transmembrane orientation, and thus its interaction with the T domain is likely to be critical for properly orienting the A chain within the bilayer in a fashion that allows translocation.

4-Chloro-7-nitrobenzofurazan↗

PubMed1997

Serpin conformational change in ovalbumin. Enhanced reactive center loop insertion through hinge region mutations.

Ovalbumin is a noninhibitory member of the serpin superfamily that does not spontaneously undergo the loop-to-sheet conformational change upon cleavage of its reactive center that is characteristic of inhibitory serpins. We tested the hypothesis that ovalbumin could be turned into a proteinase inhibitor by increasing the rate of loop insertion through hinge region mutations alone. We found that none of the three variants examined showed any detectable proteinase inhibitory properties. However, replacement of the P14 arginine residue of ovalbumin by serine, either alone or in combination with changes of P12-P10 to alanine, resulted in a large increase in the rate of loop insertion into beta-sheet A following cleavage at the P1-P1' bond by porcine pancreatic elastase (PPE), as shown by the spontaneous formation of a loop-inserted form upon cleavage that has increased the thermal stability. From the magnitude of the increase in stability of the cleaved, loop-inserted forms of the P14 ovalbumin variants, as well as the accessibility of the P1-P1'-cleaved reactive center loop to further proteolysis at P8-P7, we concluded that the reactive center loop can only partially insert into beta-sheet A and therefore that ovalbumin is also defective in the ability of beta-sheet A to expand to fully accommodate the whole of the reactive center loop. This defect, through its effect on the extent and/or rate of loop insertion, is likely to be a principal reason for ovalbumin not being a proteinase inhibitor.

Animals↗

PubMed1997

Inhibition of preprotein translocation and reversion of the membrane inserted state of SecA by a carboxyl terminus binding mAb.

SecA is the peripheral subunit of the preprotein translocase of Escherichia coli. SecA consists of two independently folding domains, i.e., the N-domain bearing the high-affinity nucleotide binding site (NBS-I) and the C-domain that harbors the low-affinity NBS-II. ATP induces SecA insertion into the membrane during preprotein translocation. Domain-specific monoclonal antibodies (mAbs) were developed to analyze the functions of the SecA domains in preprotein translocation. The antigen binding sites of the obtained mAbs were confined to five epitopes. One of the mAbs, i.e., mAb 300-1K5, recognizes an epitope in the C-domain in a region that has been implicated in membrane insertion. This mAb, either as IgG or as Fab, completely inhibits in vitro proOmpA translocation and SecA translocation ATPase activity. It prevents SecA membrane insertion and, more strikingly, reverses membrane insertion and promotes the release of SecA from the membrane. Surface plasmon resonance measurements demonstrate that the mAb recognizes the ADP- and the AMP-PNP-bound state of SecA either free in solution or bound at the membrane at the SecYEG protein. It is concluded that the mAb actively reverses a conformation essential for membrane insertion of SecA. The other mAbs directed to various epitopes in the N-domain were found to be without effect, although all bind the native SecA. These results demonstrate that the C-domain plays an important role in the SecA membrane insertion, providing further evidence that this process is needed for preprotein translocation.

Adenosine Triphosphatases↗

PubMed1998

Membrane insertion of cytochrome P450 1A2 promoted by anionic phospholipids.

The role of phospholipids in the membrane binding and subsequent insertion of the microsomal protein rabbit cytochrome P450 (P450) 1A2 into phospholipid bilayers was investigated. The insertion of P450 1A2 into phospholipid bilayers was measured by the quenching of Trp fluorescence of P450 1A2 by pyrene and brominated and doxyl-labeled phospholipids. When the phosphatidylcholine (PC) matrix was replaced with acidic phospholipids [phosphatidic acid (PA), phosphatidylserine, and phosphatidylinositol] and phosphatidylethanolamine (PE), the extent of insertion into lipid bilayers was strictly dependent on the type of acidic phospholipids. All anionic phospholipids caused the penetration of P450 1A2 into lipid bilayers, but PA was the most efficient in facilitating deep penetration of P450 1A2 into bilayers. On the other hand, binding of P450 1A2 to liposomes was increased by acidic phospholipids to the same degree regardless of the type of acidic phospholipids. PE was found to act as an inert matrix phospholipid, similar to PC, as it exerted very little effect on the insertion of P450 1A2 into lipid bilayers and the binding of P450 1A2 to membranes. It was also found that the phospholipid-dependent membrane insertion of P450 1A2 was associated with altered enzyme activity, increased alpha-helix content, and increased Trp fluorescence of P450 1A2. These results indicate that negative charges on the acidic phospholipids are important for the initial binding of P450 1A2 to membranes, but the penetration of P450 1A2 into lipid bilayers is regulated by the type of acidic phospholipids, and that phospholipid-dependent insertion of P450 1A2 is accompanied by a structural change of P450 1A2.

Animals↗

PubMed1998

Insertion of magainin into the lipid bilayer detected using lipid photolabels.

We investigated the interaction of the antimicrobial peptides Ala19-magainin 2 amide and magainin 2 amide with lipid using two lipid photolabels, azidobenzoyl galactosylceramide (GalCer-PL) and azidobenzoylamido capryloyl galactosylceramide (GalCer-C8-PL), which position their photosensitive groups near the apolar-polar interface and near the center of the bilayer, respectively. Magainins have been postulated to permeabilize membranes either by inserting in a transmembrane fashion into the bilayer and forming a channel or by binding to the surface of the bilayer and disturbing lipid packing. Evidence for channel formation has been difficult to obtain, possibly because only a fraction of the peptide may form a channel at any one time and because the channels may have a short lifetime. Both photolabels significantly labeled the peptides when bound to acidic phospholipid vesicles. The extent of labeling by GalCer-C8-PL was at least 70% of that by GalCer-PL, indicating that some of the peptide was inserted deeply into the bilayer at least transiently. The extent of labeling of Ala19-magainin 2 amide increased significantly with an increase in the peptide to lipid mole ratio, indicating cooperativity and supporting the channel model. The extent of labeling of this peptide was maximal by 30 s and did not change over 30 min, indicating that peptide insertion is rapid and either that the peptide remains inserted for at least 30 min or that equilibrium between inserted and noninserted peptide is achieved by 30 s. The latter is supported by other studies in the literature. Use of this hydrophobic photolabeling technique has permitted detection of peptide monomers which inserted into the bilayer and/or formed a channel at some time during the labeling procedure.

Animals↗