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PubMed1987

A short amino-terminal segment of microsomal cytochrome P-450 functions both as an insertion signal and as a stop-transfer sequence.

Co-translational insertion of liver microsomal cytochrome P-450 into the endoplasmic reticulum membrane is mediated by the signal recognition particle (SRP) and the presence in the cytochrome molecule of a signal sequence that can be recognized by SRP has been postulated. To locate this signal sequence, six hybrid cDNAs were constructed in which various segments of a cDNA for a rabbit liver cytochrome P-450 are fused with a cDNA or its fragment encoding yeast porin (an outer mitochondrial membrane protein) or with a cDNA for pre-interleukin 2 (a secretory protein) from which the 5'-terminal portion encoding most of its signal sequence had been removed. These hybrid cDNAs were inserted into an SP-6 transcription vector and transcribed in vitro. The mRNAs thus synthesized were translated in a cell-free system in the presence of rough microsomes. It was thus found that only those chimeric proteins containing (at their amino-terminal end) the amino-terminal cytochrome P-450 segments consisting of greater than or equal to 29 amino acid residues were co-translationally inserted into the membrane in an SRP-dependent fashion. These proteins were, however, neither processed nor translocated across the membrane. These findings, coupled with the observation that the major portion of these proteins, when inserted into the membrane, was degraded by trypsin, led to the conclusion that a short amino-terminal segment (less than 29 residues) of the cytochrome P-450 functions not only as an insertion signal but also as a stop-transfer sequence. This segment is, therefore, similar to the internal signal of type II plasma membrane proteins, but differs from the latter in the topogenic function.

Amino Acid Sequence↗

PubMed1990

Efficient translocation of positively charged residues of M13 procoat protein across the membrane excludes electrophoresis as the primary force for membrane insertion.

The coat protein of bacteriophage M13 is inserted into the Escherichia coli plasma membrane as a precursor protein, termed procoat, with a typical leader peptide of 23 amino acid residues. Its membrane insertion requires the electrochemical potential but not the cellular components SecA and SecY. Since the electrochemical gradients result in the periplasmic side of the membrane being positively charged, the membrane potential could contribute to the transfer of the negatively charged central region of procoat across the membrane. Here we demonstrate that the central domain following the leader peptide can be translocated across the membrane even when the net charge of the region is changed from -3 to +3. This rules out an electrophoresis-like insertion mechanism for procoat. We also show that the sec independence of procoat insertion is linked to the presence of the second apolar domain. The deletion of most of the second apolar domain from a procoat fusion protein results in sec dependent membrane insertion of the hybrid protein. Moreover, like other proteins that require the sec genes, translocation of this sec dependent procoat protein is inhibited when positively charged residues are introduced after the leader peptide. Loop models involving one or two hydrophobic regions are presented that account for the differences in tolerance of positively charged residues.

Amino Acid Sequence↗

PubMed1992

Insertion and assembly of the precursor of subunit II into the photosystem I complex may precede its processing.

The biogenesis and assembly of subunit II of photosystem I (PSI) (psaD gene product) were studied and characterized. The precursor and the mature form were produced in vitro and incubated with intact plastids or isolated thylakoids. Following import of the precursor into isolated plastids, mostly the mature form of subunit II was found in the thylakoids. However, when the processing activity was inhibited only the precursor form was present in the membranes. The precursor was processed by a stromal peptidase and processing could occur before or after insertion of the precursor into the thylakoids. Following insertion into isolated thylakoids, both the precursor and the mature form of subunit II were confined to the PSI complex. Insertion of the mature form of subunit II was much less efficient than that of the precursor. Kinetic studies showed that the precursor was inserted into the membrane. Only at a later stage, the mature form began to accumulate. These results suggest that in vivo the precursor of subunit II is inserted and embedded in the thylakoids, as part of the PSI complex. Only later, it is processed to the mature form through the action of a stromal peptidase.

Base Sequence↗

PubMed1976

Evaluation of trained midwives in a copper-T IUD insertion Program in Isfahan, Iran.

From four centers in Isfahan, data from 252 insertions of the Cu-T-200 made by midwives and 646 insertions of the same device made by doctors are compared. Although the net cumulative one-year continuation rate for women who had a copper-T inserted by a midwife is significantly lower than for women who had a copper-T inserted by a doctor there are no significant differences between the one-year event rates for the two groups of patients. These data suggest that an expanded role for midwives in IUD insertion programs would be an efficient use of health personnel.

Copper↗

PubMed1977

Postabortion insertions of the pleated membrane.

The pleated or intrauterine membrane (IUM) was designed to fit a range of uterine sizes and shapes and to adjust to transient changes resulting from uterine motility. The retention and contraceptive abilities of postabortion IUM insertions are analyzed in this study of 154 IUM insertions made within 3 days of treatment for an incomplete or inevitable abortion. Results indicate low 1-year net cumulative event rates for pregnancy (1.7 per 100 users), expulsion (7.2 per 100 users), and removal for bleeding and/or pain (3.9 per 100 users). These rates were similar to those reported in a study of interval insertions of the IUM; moreover, these results also compared favorably with corresponding rates of postabortion insertions reported in studies using other devices. Thus, the postabortion period is indicated as being an effective time for IUM insertion.

Abortion, Induced↗

PubMed1995

Thermal response and torque resistance of five cortical half-pins under simulated insertion technique.

A model was developed that can quantitate heat generation during placement of half-pins in cortical bone. Five half-pins were tested to assess differences in insertion torque, heat generation, and micro-damage at the pin-bone interface. Thin thermocouple probes were placed 0.5 mm from the track of the pin and within the pin to measure its temperature during insertion. Scanning electron microscopy was used to view the pin-bone interface to assess the microdamage during placement. The design of the tip of the pin influenced insertion torque and heat generation. Higher heat generation was measured when a thermocouple was placed within the pin itself and less was measured when thermocouple probes were placed within bone samples 0.5 mm from the impending pin track. Furthermore, insertion torque and thermal responses were related, but there were no significant differences in microdamage to bone when different pins and drilling/tapping techniques were used. Due to the significant heat generation at the pin-bone interface, proper cooling with saline irrigation should be applied during pin insertion regardless of the design of the pin. The microdamage observed at the surface of the pin track may have significant implications with regard to loosening of pins, but such effects must be studied with in vivo models.

Animals↗

PubMed2006

Medial collateral ligament insertion site and contact forces in the ACL-deficient knee.

The objectives of this research were to determine the effects of anterior cruciate ligament (ACL) deficiency on medial collateral ligament (MCL) insertion site and contact forces during anterior tibial loading and valgus loading using a combined experimental-finite element (FE) approach. Our hypothesis was that ACL deficiency would increase MCL insertion site forces at the attachments to the tibia and femur and increase contact forces between the MCL and these bones. Six male knees were subjected to varus-valgus and anterior-posterior loading at flexion angles of 0 degrees and 30 degrees. Three-dimensional joint kinematics and MCL strains were recorded during kinematic testing. Following testing, the MCL of each knee was removed to establish a stress-free reference configuration. An FE model of the femur-MCL-tibia complex was constructed for each knee to simulate valgus rotation and anterior translation at 0 degrees and 30 degrees, using subject-specific bone and ligament geometry and joint kinematics. A transversely isotropic hyperelastic material model with average material coefficients taken from a previous study was used to represent the MCL. Subject-specific MCL in situ strain distributions were used in each model. Insertion site and contact forces were determined from the FE analyses. FE predictions were validated by comparing MCL fiber strains to experimental measurements. The subject-specific FE predictions of MCL fiber stretch correlated well with the experimentally measured values (R2 = 0.95). ACL deficiency caused a significant increase in MCL insertion site and contact forces in response to anterior tibial loading. In contrast, ACL deficiency did not significantly increase MCL insertion site and contact forces in response to valgus loading, demonstrating that the ACL is not a restraint to valgus rotation in knees that have an intact MCL. When evaluating valgus laxity in the ACL-deficient knee, increased valgus laxity indicates a compromised MCL.

Aged↗

PubMed1990

Potential of hypertonic medium treatment for embryo micromanipulation: II. Assessment of nuclear transplantation methodology, isolation, subzona insertion, and electrofusion of blastomeres to intact or functionally enucleated oocytes in rabbits.

The objective of this research was to study efficiency of embryo development following transfer of blastomeres into the perivitelline space of oocytes. Single blastomeres from 8-, 16-, and 32-cell embryos were obtained following mucin coat and zona pellucida removal by combined treatments with pronase and acidic phosphate-buffered saline (PBS, pH = 2.5). Blastomeres were separated by pipetting with a fire-polished micropipette following incubation in Ca+(+)-free PBS for 15 min at 39 degrees C. This procedure resulted in over 97% blastomere separation. For ease of blastomere insertion, oocytes were placed in droplets of 0.5 M sucrose in PBS (SPBS) during micromanipulation. To functionally enucleate oocytes some were stained with Hoechst 33342 DNA stain and irradiated. A single 8- or 16-cell blastomere was aspirated into an injection pipette (35 microns or 25 microns at the tip, respectively) and inserted into the perivitelline space of an irradiated or non-irradiated oocyte, but not fused with the oocyte. This micromanipulation procedure did not affect development of individual blastomeres into blastocysts or trophectoderm vesicles when compared with cultured control single blastomeres (P greater than .05). When the inserted blastomere was induced to fuse with an intact non-irradiated oocyte under an electric field, 56-57% were fused and 39-45% of the fused and activated oocytes developed to morulae or blastocysts. When an inserted blastomere (from 8-32-cell embryos) was induced to fuse with a functionally enucleated oocyte treated by Hoechst 33342 staining, followed by washing and UV-light irradiation, 63-66% of them were fused, but only 15-22% developed to the morula or blastocyst stage. This research demonstrated that the use of hypertonic medium treated oocytes greatly improved the ease and success rate of blastomere subzona insertion, but the value of functionally enucleated oocytes as recipient cells for nuclear transfer requires further investigation.

Animals↗

PubMed1995

S-ovalbumin, an ovalbumin conformer with properties analogous to those of loop-inserted serpins.

Most serpins are inhibitors of serine proteinases and are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the reactive center loop into a beta-sheet of the inhibitor. Ovalbumin, although a serpin, is not an inhibitor of serine proteinases. It has been proposed that this deficiency arises from the presence of a charged residue, arginine, at a critical point (P14) in the reactive center region, which prevents loop insertion into the beta-sheet and thereby precludes inhibitory properties. To test whether loop insertion is prevented in ovalbumin we have examined the properties of two forms of ovalbumin: the native protein and S-ovalbumin, a form that forms spontaneously from native ovalbumin and has increased stability. Calorimetric measurements showed that S-ovalbumin was more stable than ovalbumin by about 3 kcal mol-1. CD spectra, which indicated that S-ovalbumin had less alpha-helix than native ovalbumin, and 1H NMR spectra, which indicated very similar overall structures, suggest limited conformational differences between the two forms. From comparison of the susceptibility of the reactive center region of each protein to proteolysis by porcine pancreatic elastase and by subtilisin Carlsberg, we concluded that the limited native-to-S conformational change specifically affected the reactive center region. These data are consistent with a structure for S-ovalbumin in which part of the reactive center loop has inserted into beta-sheet A to give a more stable structure, analogously to other serpins. However, the rate of loop insertion appears to be very much lower than for inhibitory serpins.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed2005

Folding is not required for bilayer insertion: replica exchange simulations of an alpha-helical peptide with an explicit lipid bilayer.

We implement the replica exchange molecular dynamics algorithm to study the interactions of a model peptide (WALP-16) with an explicitly represented DPPC membrane bilayer. We observe the spontaneous, unbiased insertion of WALP-16 into the DPPC bilayer and its folding into an alpha-helix with a transbilayer orientation. The free energy surface suggests that the insertion of the peptide into the DPPC bilayer precedes secondary structure formation. Although the peptide has some propensity to form a partially helical structure in the interfacial region of the DPPC/water system, this state is not a productive intermediate but rather an off-pathway trap for WALP-16 insertion. Equilibrium simulations show that the observed insertion/folding pathway mirrors the potential of mean force (PMF). Calculation of the enthalpic and entropic contributions to this PMF show that the surface bound conformation of WALP-16 is significantly lower in energy than other conformations, and that the insertion of WALP-16 into the bilayer without regular secondary structure is enthalpically unfavorable by 5-10 kcal/mol/residue. The observed insertion/folding pathway disagrees with the dominant conceptual model, which is that a surface-bound helix is an obligatory intermediate for the insertion of alpha-helical peptides into lipid bilayers. In our simulations, the observed insertion/folding pathway is favored because of a large (>100 kcal/mol) increase in system entropy that occurs when the unstructured WALP-16 peptide enters the lipid bilayer interior. The insertion/folding pathway that is lowest in free energy depends sensitively on the near cancellation of large enthalpic and entropic terms. This suggests the possibility that intrinsic membrane peptides may have a diversity of insertion/folding behaviors depending on the exact system of peptide and lipid under consideration.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2006

The effect of the levonorgestrel intrauterine system on uterine artery blood flow 1 year after insertion.

OBJECTIVE: To assess whether the morphological alterations of the endometrium induced by the levonorgestrel intrauterine system affect the uterine artery vasculature. METHODS: Forty-eight premenopausal women attending our gynecology clinic because of menorrhagia were enrolled into the study. Clinical measures of menstrual bleeding, endometrial thickness and Doppler flow of the uterine artery (resistance (RI) and pulsatility (PI) indices) were evaluated before and 1 year after insertion of the levonorgestrel intrauterine system. Thirty-three (69%) women were available for re-evaluation at the end of the first year and the continuation rate was 76% (n = 25). A paired samples t-test and the Mann-Whitney U-test were used for comparison of values between groups. RESULTS: The mean age of the thirty-three patients was 44.3 +/- 7.6 years. Nine patients (27.3%) were amenorrheic and three patients were in menopause at the end of the year. After excluding the women in menopause, the increase in mean RI 1 year after insertion was statistically significant (0.86 +/- 0.07 vs. 0.81 +/- 0.08; P = 0.046), as was the decrease in uterine volume (736.9 +/- 276.9 mL vs. 972.75 +/- 465.71 mL; P = 0.029). The mean endometrial thickness decreased 1 year after insertion, but not significantly (4.6 +/- 1.6 mm vs. 6.6 +/- 3.5 mm; P = 0.07) and there was no statistically significant difference in mean PI between the groups (2.00 +/- 0.63 and 2.03 +/- 0.64 pre- and post-insertion, respectively; P = 0.83). CONCLUSION: The increase in mean RI of the uterine artery and the decrease in uterine volume 1 year after insertion of the levonorgestrel intrauterine system may be related to its progestational effect. This increase in RI may have a role in cessation of menstruation.

Adult↗

PubMed1997

A common 844INS68 insertion variant in the cystathionine beta-synthase gene.

Mildly elevated plasma homocysteine has been shown to be associated with an elevated risk for cardiovascular disease. In this study, we analyzed the frequency of a common 844ins68 insertion variant in the cystathionine beta-synthase gene (CBS) in patients with arterial occlusive disease and in controls and assessed the association between the insertion variant and plasma homocysteine concentrations. The insertion variant was equally distributed between both study groups. Furthermore, the presence of this insertion variant, either in the heterozygous or the homozygous state, is not associated with hyperhomocysteinemia. We therefore conclude that this common 844ins68 variant is a neutral insertion variant.

Arterial Occlusive Diseases↗

PubMed2001

Alternative splicing of Rh blood group polypeptide mRNA produces a novel transcript containing a short nucleotide insertion on human erythroleukemia K562 cells.

A novel isoform of the human Rh blood group polypeptide cDNA was isolated from human erythroleukemic K562 cells. This isoform was produced by deletion of the sequences derived from exons 2 and 3 of the RHCE gene and insertion of 44-bp into the resulting junction between exon 1 and 4 derived sequence. The deduced amino acid sequence revealed that the 44-bp insertion sequence contains an in-frame stop codon that causes premature chain termination. A sequence homology search using GenBank showed that the inserted sequence was derived from the intron between exons 1 and 2 of the RHCE gene. Moreover, analysis of the region surrounding the inserted sequence indicated that the insert was a cryptic exon flanked by consensus donor and acceptor splice sequences. This novel transcript was most likely produced by alternative splicing.

Alternative Splicing↗

PubMed1999

A mouse chromosome 19 genetic map including the Lvis1 viral insertion site.

Somatic insertion mutations, such as those caused by the insertion of a proviral element, can contribute to abnormal cell growth by activating cellular proto-oncogenes or inactivating tumor suppressor genes. Lvis1 is a genomic locus frequently disrupted by viral insertion in AKXD B-cell lymphomas (manuscript submitted for publication). To determine whether insertion mutations at Lvis1 affect a known proto-oncogene or tumor suppressor gene, we mapped Lvis1 to distal mouse chromosome 19. A detailed molecular genetic map of this region was constructed, localizing Lvis1 relative to 20 gene and microsatellite markers, 3 of which have not been mapped in the mouse (Nfkb2, Nlz, and Wnt8b). This analysis revealed that Lvis1 maps between two previously identified viral insertion sites, His2 and Frat1, and does not cosegregate with known gene markers. In addition, our study refines the gene order for distal mouse chromosome 19 and expands the comparative map between mouse chromosome 19 and the human chromosome 10q23-q26 homology region.

Animals↗

PubMed1995

Functional map of the alpha subunit of Escherichia coli RNA polymerase: insertion analysis of the amino-terminal assembly domain.

The alpha subunit of Escherichia coli RNA polymerase plays a key role in assembly of the core enzyme. Deletion analysis of alpha indicated that the amino-terminal domain consisting of 215 amino acid residues between positions 21 and 235 is involved in this assembly. For fine mapping of the site(s) within this region required for subunit-subunit contacts, we constructed a set of insertion mutants of the rpoA gene, each encoding mutant alpha with two extra amino acid residues, Ala (A) and Ser (S), inserted at 20 residue intervals. The over-expressed alpha derivatives were purified to apparent homogeneity and examined for their ability to form dimers and to assemble beta and beta' subunits into core enzymes in vitro. Among a total of 11 alpha insertion derivatives tested, four mutants having the insertion at dispersed positions retained the ability to form active core enzymes. Other mutants showed defects in core enzyme assembly at various steps depending on the position of AS insertion: one mutant formed an unstable alpha 2 beta complex; one mutant exhibited decreased binding of beta' subunit; and five mutants did not form stable alpha dimers, of which one formed an alpha 2 beta complex and another formed an alpha beta complex. These results suggest that alpha dimerization involves multiple contact sites. Among alpha mutants with dimer formation ability, the mutation at amino acid residue 80 interfered with the binding of both beta and beta' subunits, and the mutation at position 200 made the alpha dimer inactive in beta' binding.

DNA-Directed RNA Polymerases↗

PubMed1998

The diffusion of molecules in axonal plasma membranes: the sites of insertion of new membrane molecules and their distribution along the axon surface.

The neuronal cell surface consists of two domains, the somatodendritic and axonal plasma membranes. Each domain serves different functions, and has a different complement of membrane molecules. Since membrane molecules are able to diffuse in the plane of the plasma membrane lipid bilayer, with diffusion coefficients ranging from 10-8 cm 2 s-1 for lipids to 10-10 cm 2 s-1 for proteins, mechanisms must exist to prevent as intermixing of membrane molecules from each domain by diffusion. Presented here is a theoretical analysis of the distribution of axonal molecules in both growing and non-growing axons based on two models for the insertion of these molecules into the axonal membrane, namely insertion exclusively at the distal end of the axon, or insertion with equal probability all along the axon. In all cases, assuming that the molecules have a finite half-life in the axonal membrane, compositional differences between the axonal and somatodendritic membranes can be obtained that are similar to those observed in other polarized cells, even in the absence of a physical barrier to prevent the intermixing of membrane molecules. Moreover, our analyses demonstrate that the diffusion of membrane molecules in the plane of the axonal lipid bilayer is a sufficiently slow process to preclude the possibility that membrane molecules are inserted into axonal membranes at a site remote from their final location, and then move to their final locations for diffusion. Thus, in long axons, for membrane molecules that are localized all along the length of the axon, mechanisms must exist for their insertion in the axonal membrane at sites all along the axon, and not just at the distal end.

Animals↗

PubMed1998

An IS4 insertion at the glnA control region of Escherichia coli creates a new promoter by providing the -35 region of its 3'-end.

An insertion element (IS)4 insertion selected as suppressor of the rpoN73::Tn5 alelle was located inside the control region of the glnA gene in Escherichia coli. In the rpoN73::Tn5 background the IS4 insertion promotes glnA transcription at a low constitutive level sufficient to sustain glutamine-independent growth. The IS4 insertion mutation in either rpoN73::Tn5 or wild-type backgrounds promotes glnA transcription from a new start site located two bases downstream of the glnAp2 start site. Analysis of sequences flanking the insertion point showed a promoter sequence whose -35 region was located inside the IS4 sequence and the -10 region was inside the glnA control region. Site-directed mutagenesis of relevant nucleotide residues of the newly created promoter impaired transcription of a reporter gene. The results support our contention that IS4 carries a -35 promoter region that is able to create functional hybrid promoters. We propose that this mechanism could be one of the molecular reasons of the suppressor activity previously reported for IS4.

DNA Transposable Elements↗

PubMed1993

Insertional editing in mitochondria of Physarum.

RNA produced from a number of genes on the mitochondrial (mt) DNA of Physarum polycephalum have nucleotides inserted at specific sites in their sequence. These insertions are spaced at approximately 25 nucleotide intervals and create open reading frames in mRNA and functional structure in tRNAs and rRNAs. Although most of the insertions at a site are single cytidines; single uridines and certain dinucleotides containing adenosine and guanosine as well as cytidine and uridine are also occasionally inserted at certain sites. This mixed nucleotide insertional RNA editing is unique among currently characterized editing systems.

Amino Acid Sequence↗